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EPFL School of Life Sciences - 2009 Annual Report Table Of Contents Introduction ............................................................................................................................. 3

Main Scientific Events ........................................................................................................................................ Public-Oriented Events ....................................................................................................................................... Honors-Awards-Announcements .......................................................................................................................... The Undergraduate Studies ................................................................................................................................. The Graduate Studies......................................................................................................................................... Our Core Facilities & Technology Platforms ............................................................................................................

4 4 5 6 6 7

BMI - The Brain Mind Institute ................................................................................................. 9

Aebischer Lab ..................................................................................................................................................10 Blanke Lab ......................................................................................................................................................12 Fraering Lab ....................................................................................................................................................14 Gerstner Lab ....................................................................................................................................................16 Hadjikhani Group..............................................................................................................................................18 Herzog Lab ......................................................................................................................................................20 Lashuel Lab .....................................................................................................................................................22 Luthi-Carter Lab ...............................................................................................................................................24 Magistretti Lab .................................................................................................................................................26 Markram Lab ...................................................................................................................................................28 Blue Brain Project .............................................................................................................................................30 Moore Lab .......................................................................................................................................................32 Petersen Lab ....................................................................................................................................................34 Sandi Lab ........................................................................................................................................................36 Schneggenburger Lab .......................................................................................................................................38

IBI - Institute of Bioengineering & Co-Affiliates ..................................................................... 41

Auwerx Lab .....................................................................................................................................................42 Barrandon Lab .................................................................................................................................................44 Dal Peraro Lab .................................................................................................................................................46 Deplancke Lab .................................................................................................................................................48 Hubbell Lab .....................................................................................................................................................50 Lutolf Lab ........................................................................................................................................................52 Naef Lab .........................................................................................................................................................54 Swartz Lab ......................................................................................................................................................56 Wurm Lab .......................................................................................................................................................58

IBI - Co-affiliated Research Groups ........................................................................................ 60

Aminian Lab ....................................................................................................................................................60 Guiducci Lab ....................................................................................................................................................61 Hatzimanikatis Lab ...........................................................................................................................................62 Ijspeert Lab .....................................................................................................................................................63 Johnsson Lab ...................................................................................................................................................64 Jolles-Haeberli Lab............................................................................................................................................65 Maerkl Lab.......................................................................................................................................................66 Millán Lab ........................................................................................................................................................67 Pioletti Lab ......................................................................................................................................................68 Psaltis Lab .......................................................................................................................................................69 Radenovic Lab..................................................................................................................................................70 Stergiopulos Lab ..............................................................................................................................................71

GHI - Global Health Institute .................................................................................................. 73

Blokesch Lab....................................................................................................................................................74 Cole Lab ..........................................................................................................................................................76 Harris Lab .......................................................................................................................................................78 Lemaître Lab....................................................................................................................................................80 McKinney Lab...................................................................................................................................................82 Trono Lab ........................................................................................................................................................84 Van Der Goot Lab .............................................................................................................................................86

ISREC - Swiss Institute for Experimental Cancer Research .................................................... 89

Aguet Lab ........................................................................................................................................................90 Beard Lab........................................................................................................................................................92 Brisken Lab .....................................................................................................................................................94 Constam Lab....................................................................................................................................................96 Duboule Lab ....................................................................................................................................................98 Gönczy Lab .................................................................................................................................................... 100 Grapin-Botton Lab .......................................................................................................................................... 102 Hanahan Lab ................................................................................................................................................. 104 Huelsken Lab ................................................................................................................................................. 106 Kühn Lab....................................................................................................................................................... 108 Lingner Lab ................................................................................................................................................... 110 Radtke Lab .................................................................................................................................................... 112 Simanis Lab ................................................................................................................................................... 114 Beermann Group ............................................................................................................................................ 116 Bucher Group................................................................................................................................................. 118

External Adjunct Professor ................................................................................................... 120

Molinari Group ............................................................................................................................................... 120 Rainer Group ................................................................................................................................................. 122 Schorderet Group ........................................................................................................................................... 124 Marcel Tanner ................................................................................................................................................ 126


EPFL School of Life Sciences - 2009 Annual Report

Introduction

Preamble The EPFL School of Life Sciences was launched with the strong belief that biomedical research will increasingly rely on quantitative approaches and high-end technologies, and that its future lies at the crossroads of biology, basic sciences, informatics and engineering. Accordingly, we train a new breed of researchers whose combined skills in these various fields are set to address fundamental biological questions and to attack the major medical problems of our time with the true spirit of systems biologists. Our School now hosts some forty-five research groups that apply this philosophy to broad questions including cancer, diabetes, infectious diseases and mental or neurological disorders, pushing for integrated approaches that span a range of disciplines from functional genomics to high-tech bio-engineering, and from computational neurosciences to structural modeling. A bachelor’s degree (Life Sciences and Technology), two master’s degrees (Life Sciences and Technology, Bioengineering), and three Ph.D. programs (Biotechnology and Bioengineering, Neurosciences, Molecular and Systems Life Sciences), constitute the educational arms of our school, hosting some six hundred students from all geographic and scientific horizons. 2009 was another spectacular year! It witnessed the inauguration of our latest building which immediately welcomed close to 300 researchers, the graduation of our second class of Engineers, and the granting of prestigious awards to our faculty, including the Marcel Benoist Prize to Gisou van der Goot (the first woman ever to receive this award!), the Danone Prize to Johan Auwerx, and the Kochon Prize to Stewart Cole. In addition to these awards, several of our professors were elected to the European Molecular Biology Organization, and the new ISREC director, Douglas Hanahan, was elected to the National Academy of Sciences of the United States of America. We also had the great pleasure of welcoming Carlotta Guiducci, Nicola Harris, Melanie Blokesch, Jonathan Knowles, Emmanuel Baetge and Christian Doerig as new members of our faculty, and of developing further fruitful interactions with many colleagues at EPFL and in other institutions in Switzerland and abroad. The EPFL School of Life Sciences is maturing into a beautiful young adult!

Professor Didier Trono Professor & Dean of the School of Life Sciences Ecole Polytechnique Fédérale de Lausanne (Switzerland) http://sv.epfl.ch


EPFL School of Life Sciences - 2009 Annual Report

Main Scientific Events February 11-13: BMI hosted the 2009 EPFL-LATSIS Symposium: “Understanding Violence”. February 25-26: EPFL Neuroprosthetics Workshop June 10: EPFL Stem Cell Day, hosted by IBI, featured young scientists at the forefront of this field. July 6 to August 28: The forth International Summer Research Program for undergraduate students offered intensive research training opportunities to 25 talented students interested in life science research careers. http://ssv.epfl.ch/summer-research September 2 -4: The new SV building was inaugurated during the School of Life Sciences Inaugural Symposium (LSS09). An exceptional group of high-ranking speakers covering a wide range of areas participated. As a part of the program, the Debiopharm Life Sciences Award was given to Dr. Lluis Quintana-Murci from the Instiut Pasteur in France for his outstanding innovative research in the emerging field of evolutionary genetics of infection. September 10-11: The 2nd Swiss-Japanese Symposium on Bio-Nanotechnology (BioNano2009) took place in the beautiful medieval Aigle castle.

La Science Appelle les Jeunes

Public-Oriented Events February: Prof. Hadjikhani gave a public conference at UNIL on “Social and Emotional Competencies and Autism” March 9-13: For the first time, the SV labs welcomed 6 high school students to spend one week gaining hands-on lab experience under the framework of La Science Appelle les Jeunes! (Schweizer Jugend forscht!) March 4: Visiting artist in residence, Luca Forcucci, presented a talk on “Music for Brain Waves”. October 3: The 2nd generation of Life Sciences and Technology Engineers received their Master’s degree at the EPFL Dies Academicus.


EPFL School of Life Sciences - 2009 Annual Report

Honors-Awards-Announcements January: Four SV Scientists received the 2009 Pfizer Award for Medical Research: Bigna Lenggenhager & Tej Taki (BMI) for their work studying mechanisms underlying bodily self consciousness and Ilaria Malanchi & Joerg Huelsken (ISREC) for their work identifying some tissue-specific stem cell tumors. February: Douglas Hanahan was recognized for his contributions to cancer science and biotechnology by his election to the Institute of Medicine of the U.S. National Academy of Sciences. March: Carl Petersen and Hilal Lashuel (BMI) were awarded Human Frontiers Science Program Grants which supports innovative and interdisciplinary basic research. April: IBI’s Melody Swartz and Jeffrey A. Hubbell received grants from the Bill & Melinda Gates Foundation to explore the use of nanotechnology in triggering the immune responses against tuberculosis. May: Gisou van der Goot (GHI) along with Stefan Kunz (UNIL/CHUV) won the Leenaards Prize 2009 for their work on pathogen mechanisms. August: Hilal Lashuel (BMI) was one of 8 EPFL scientists to be awarded an ERC (European Research Council) Starting Grant. October: Johan Auwerx received the Danone International Prize for Nutrition for his research in molecular nutrition focused on preventing and combating obesity and cardiometabolic diseases. This is in addition to his prestigious ERC Advanced Grant that he received at the end of 2008. October: The EMBO (European Molecular Biology Organization) elected Gisou van der Goot, Yann Barrandon and Didier Trono as new members for their scientific excellence. October: Peter Beard received the Polysphères 2009 award delivered by the EPFL student association -the AGEpoly- in recognition of his outstanding teaching skills and personal investment in the students. November: GHI professor, Gisou van der Goot, was the first woman to receive the 2009 Marcel Benoist Prize. December: Stewart Cole and his team were awarded the Kochon Prize for their significant contribution to combating tuberculosis. December: Pierre Magistretti, BMI Director was endorsed by the Governing Council of the International Brain Research Organization (IBRO).

Summer Research Program Symposium


EPFL School of Life Sciences - 2009 Annual Report

The Undergraduate Studies The Life Sciences curriculum aims to educate a new generation of engineers who can master the technical and scientific skills needed for studying life processes and developing the biomedical technologies of tomorrow. This educational program, established under the direction of Prof. William F. Pralong, M. D., is unique in Switzerland and Europe.

Bachelor’s program (3 years)

The first two years provide basic courses followed throughout the EPFL, such as analysis, linear algebra, physics, chemistry (general and organic), and numerical methods. Specific courses in Life Sciences begin with biochemistry, cellular, molecular and developmental biology. In the first two years, life sciences courses make up less than 20% of the total academic load. In the third year, engineering courses (signals and systems, electronic and electrical systems) and typical life sciences courses such as genetics, immunology, and functional genomics applied to biological development, bio-computing, neuroscience, molecular biology, systems biology via the study of human physiology are integrated. Physiology training also gives the opportunity to apply the engineering and biological knowledge acquired up to this point. During this year, the students also fine tune their training by choosing some of their credits from one of the specializations offered in the masters’ program. This includes a bachelor project either in bioengineering and biotechnology or in neurosciences and molecular medicine.

Master’s programs (2 years)

Master’s in Life Science and Technology includes several specializations. Among these are neurosciences, molecular medicine, and bio-computing. Each specialization is made up of 15 credits of required courses plus 15 credits of optional courses. Master’s in Biotechnology and Bioengineering, includes two specializations in Biotechnology and Bioengineering. Each one requires taking 15 specific and obligatory credits together with 15 optional credits. An additional specialization is offered as a minor in Biomedical engineering consisting of 22 optional credits with a mandatory project worth 8 credits. This specialization is organized with the School of Engineering. Both degree programs share a common basic curriculum that aims to provide students with the knowledge of the modern technologies used in the life sciences such as imaging, bio-computing and optical systems applied to biology. In addition, courses in management, economics, applied laws and ethics for the life sciences are offered. A large portion of the master’s program (60 credits) is dedicated to laboratory work and projects.

Rolex Learning Center

The Graduate Studies All three graduate programs comprise a combination of coursework, laboratory-based research, in-house seminars, and national or international conferences.

The Doctoral Program in Biotechnology and Bioengineering aims at providing doctoral students with the education necessary to be leaders in the fast-growing industrial and academic biotechnology and bioengineering sectors, i.e. a depth of knowledge and competence in their specific research area as well as a breadth of knowledge in biology, bioengineering and biotechnology. These program themes include: genomics and proteomics, biomolecular engineering and biomaterials, stem cell biotechnology, cell and process engineering, biochemical engineering, orthopaedic engineering, biomechanics, mechanobiology, cell biophysics, computational biology, biomedical imaging as well as molecular, cell and tissue engineering. ‘http://phd.epfl.ch/edbb’ The Doctoral Program in Neuroscience

provides its students with training from the genetic to the behavioural level including molecular, cellular, cognitive, and computational neuroscience. Students matriculate in the highly dynamic and interdisciplinary environment of the BMI-EPFL of the SV. The program is further strengthened by research and training opportunities in collaboration with the Universities of Lausanne and Geneva. ‘http://phd.epfl.ch/edne’

The Doctoral Program in Molecular Biology of Cancer and Infection is a joint program between the Swiss Institute for Experimental Cancer Research (ISREC-EPFL) and the Global Health Institute (GHI-EPFL). The program provides training and research opportunities to highly motivated doctoral students in key areas of modern biology in Lausanne, Switzerland. Highly qualified applicants worldwide are chosen twice a year through a competitive selection procedure. ‘http://www.international-phd.ch/index2.html’


EPFL School of Life Sciences - 2009 Annual Report

Our Core Facilities & Technology Platforms To enhance the training and research capabilities of its students and scientists, EPFL and the School of Life Sciences have made a significant investment over the past few years to establish state-of-the-art technology platforms and core facilities. These facilities are directed and managed by dedicated teams of highly trained and experienced staff and are run on a fee-for-service basis. They offer training, access to technology, assistance with experimental design and high level data analysis, and collaborations. In addition, scientists from our School of Life Sciences closely collaborate with other services in the Lemanic region, including the ‘Center for Biomedical Imaging’ (http://www.cibm.ch) and the ‘Lake Geneva Area Genomics Core Facility’ (http://unil.ch/dafl). Currently, the following Life Sciences related core facilities and technology platforms are available at the EPFL School of Life Sciences:

BIOIMAGING & OPTICS http://biop.epfl.ch Head: Dr Arne Seitz

BIOMOLECULAR SCREENING http://bsf.epfl.ch Head: Dr Gerardo Turcatti

FLOW CYTOMETRY http://fccf.epfl.ch Head: Dr Miguel Garcia

PROTEIN EXPRESSION http://pecf.epfl.ch Head: Dr David Hacker

PROTEOMICS http://pcf.epfl.ch

Head: Dr Marc Moniatte

HISTOLOGY http://hcf.epfl.ch

Head: Dr Jessica Dessimoz

PROTEIN CRYSTALLOGRAPHY CORE FACILITY http://pcrycf.epfl.ch Head: Dr. Florence Pojer

BIOINFORMATICS & BIOSTATISTICS http://bbcf.epfl.ch Head: Dr Jacques Rougemont

BIO-ELECTRON MICROSCOPY http://cime.epfl.ch

Head of Bio-EM at CIME: Dr Graham Knott

CENTER FOR PHENOGENOMICS http://sv.epfl.ch Head: Dr Xavier Warot

TRANSGENIC CORE FACILITY http://tcf.epfl.ch Heads: Dr Friedrich Beermann Dr Isabelle Barde


BMI

EPFL School of Life Sciences - 2009 Annual Report

BMI - The Brain Mind Institute The mission of the Brain Mind Institute is to understand the fundamental principles of brain function in health and disease, by using and developing unique experimental, theoretical, technological and computational approaches. The scientific challenge addressed by the BMI consists of connecting different levels of analysis of brain activity, such that cognitive functions can be understood as a manifestation of specific brain processes; specific brain processes as emerging from the collective activity of thousands of cells and synapses; synaptic and neuronal activity in turn as emerging properties of the biophysical and molecular mechanisms of cellular compartments. Understanding information processing in the brain and its higher emerging properties is arguably one of the major challenges in the life sciences. Research at the BMI focuses on three main areas: i) Molecular neurobiology and mechanisms of neurodegeneration ii) Molecular and cellular mechanisms of synapse and microcircuit function up to the behavioural level and including metabolic aspects; iii) Sensory perception and cognition in humans. In all areas, the BMI strives to integrate knowledge gained by multidisciplinary approaches and across different disciplines and research laboratories. Finally, underlying all levels of analysis, research at BMI is characterized by a sustained interest in pathological processes. In order to achieve these scientific goals, the Brain Mind Institute benefits from a unique academic environment: • An institute organized as a network of independent laboratories reflecting complementary technological approaches; each laboratory collaborates with several others within the institute in addition to cross-disciplinary interactions on campus. • A campus that stands out as a premier technological university in engineering, computer science and basic sciences. • An intimate collaboration with the Blue Brain Project which stands out as one of the most challenging neuroscience simulation and data basing projects worldwide. • A proximity to and joint affiliations of our faculty with top university hospitals in Lausanne and Geneva in particular for projects related to cognition and neurodegenerative diseases. • A new initiative in neuroprosthetics to which the BMI is strongly committed that will further the collaboration with engineering sciences by a host of inspiring common projects. A feature of the Brain Mind Institute is that several faculty members have strong expertise in physics or mathematics; this holds not only for theoretical but also for experimental neuroscience. In this way, the Brain Mind Institute reflects the mission of the School of Life Science: to provide a life science curriculum with a strong emphasis on quantitative approaches. As far as teaching is concerned, the BMI Faculty is committed to provide a comprehensive and formal training in neuroscience from the undergraduate to the graduate levels. ‘http://bmi.epfl.ch’

© Co opyright 2004-2010 0 EPFL for or a ma ater eria ia al pu publ blis ishe he ed iin n this rep port rt Copyright alllll m material published report info f .sv@ v@e epfl.c ..ch ch info.sv@epfl

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EPFL School of Life Sciences - 2009 Annual Report

BMI - Brain Mind Institute

Aebischer Lab Head of lab (PI) - http://len.epfl.ch Team members

Patrick Aebischer Full Professor

President of EPFL

Ursula Alves-Zwahlen, Administrative Assistant, 80% Jean-Charles Bensadoun, Senior Scientist Nicolas Bouche, Engineer, 10% Matthias Cacquevel, Post-Doc Carine Ciron, Post-Doc Philippe Colin, Technician Philippe Coune, PhD student Elisabeth Dirren, PhD student Julien Dusonchet, PhD student Meret Gaugler, PhD student David Genoux, Post-Doc Dairin Kieran, Post-Doc Aurélien Lathuilière, PhD student Karin Löw, Post-Doc Osiris Marroquin Belauzaran, PhD student left October 2009 Safa Mohanna, Master student Vivianne Padrun, Chief technician Fabienne Pidoux, Technician, 90% Emilda Pino, PhD student Eugene D. Redmond, visiting Professor Aline Rolaz, Technician Christel Sadeghi, Technician Bernard Schneider, Senior Scientist Veronica Setola, Post-Doc Christopher Towne, PhD student Josephine Uldry, Master student

Introduction

During the last decade, our laboratory has primarily focused on the use of viral vectors to mimic in a reasonable time frame both neuropathological and behavioral features of diverse human conditions such as Parkinson’s disease, amyotrophic lateral sclerosis and Alzheimer’s disease. The biological characteristics of viral vectors have also been investigated to assess various therapeutic concepts, playing with transgene and capsid characteristics, as well as the route of administration. Altogether, the recent developments of viral vectors for both modeling and treating CNS disorders have widely helped understanding critical interactions between genetic defects, cellular dysfunctions and environmental factors leading to the demise of specific neuronal populations.

Keywords

Disease modelling, gene therapy, animal models, Parkinson’s disease, Amyotrophic lateral sclerosis, Alzheimer’s disease, viral vectors, lentivirus, adenoassociated virus, cell encapsulation

Results Obtained in 2009

Alpha-synuclein has been widely documented as a key player in the pathogenicity of both familial and sporadic forms of Parkinson’s disease. However, molecular mechanisms associated with the specific neuronal dysfunction still need to be investigated. Using adeno-associated viral vectors (AAVs), we have explored in vivo the effect of alpha-synuclein in the nigrostriatal pathway. More specifically, overexpression of wild-type α-synuclein in the substantia nigra of adult rats leads to a behavioral phenotype attributable to disturbances in dopamine transmission rather than cell loss. These changes seem tied to

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biomembrane-binding properties of α-synuclein, as they fail to appear upon overexpression of bindingdeficient A30P mutant. Behavioral deficits are also correlated to a significant depletion of dopaminergic vesicles in axon terminals projecting to the striatum. Our results show that alterations in α-synuclein function and deficits in neurotransmission might represent a primary step towards the demise of nigral neurons. Viral vectors such as AAVs are promising tools for gene therapy in the context of neuromuscular disorders. With the aim of maximizing (scale-up) the delivery of therapeutic proteins to motor neurons, the spread and infectivity of AAV serotype 6 (AAV6) were assessed using non-invasive routes of administration. The intravenous injection of AAVs resulted in a systemic transduction profile, corresponding to transduction of the entire skeletal musculature as well as heart and liver. However, motor neurons at all levels of the spinal cord and brain stem were scarcely transduced, amounting to only 3-5% of the lower motor neuron pool. Taking advantage of the retrograde transport skills of the AAV6, intramuscular injections (intercostals, masseter, tongue, thoracic cavity, forearm and hind limb muscles) gave rise to up to 40% local transduction of motor neurons in the spinal cord (lumbar level). As expected, the targeted muscles were almost entirely transduced. Recently, we have investigated the intra-cerebroventricular (i.c.v.) route in newborn mice. Interestingly, AAV6 injection in the ventricles allowed to transduced cells in various spread areas such as the cortex, the hippocampus, the cerebellum and the spinal cord. More specifically, in the spinal cord, both sensory and motor pathways were positive. At the level of the ventral horn, the pattern of transduction over the entire

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mouse spinal cord was variable, but reached up to 80-90% in some parts, with an overall average of approximately 60% of the spinal motor neuron pool. Together with selected genetic strategies, the choice of a specific route of administration should allow to better understanding the role of specific cell populations in motor disorders such as amyotrophic lateral sclerosis and the spino-muscular atrophy.

neurotrophin-3 gene therapy prevents motor decline in an X-linked adrenoleukodystrophy mouse model. Annals of Neurology, 66: 117-122.

In order to develop a robust animal model of Alzheimer’s disease (AD) rapidly presenting neuropathology, neuronal cell loss and behavioural deficits similar to AD, we have injected AAV vectors carrying various combinations of tau and Aβ mutations in mice. Preliminary results showed that the intra-cerebroventricular injection of AAV6 vectors carrying 4 different Aβ mutations in mouse pups led to amyloid deposits in both entorhinal cortex and hippocampus at 2 months post-injection. Similarly, AAV6-tau vectors led to typical tangle-like structures in the cortex at the same time point. Further experiments to fully characterize this model are under investigation.

Azeredo da Silveira S., Schneider B.L., Cifuentes-Diaz C., Sage D., Abbas-Terki T., Iwatsubo T., Unser M. and Aebischer A. (2009). Phosphorylation does not prompt, nor prevent, the formation of alpha-synuclein toxic species in a rat model of Parkinson’s disease. Human Molecular Genetics, 18: 872-887.

Following the positive results of the encapsulationbased passive immunotherapy approach on the production and aggregation of Aβ as well as on the behavioural deficits in AD mice (Marroquin et al., in submission), we have initiated a program on the development of new high-capacity flat encapsulation device for non-invasive systemic application. Together with a strong emphasis on genetically modified cell selection, these new capsules should allow the secretion of various full or fragment Aβ antibodies to reach significant antibody plasma levels compatible with therapeutic benefits in both mice models and humans.

Selected publications

Mastroeni R., Bensadoun J.C., Charvin D., Aebischer P., Pujol A. and Raoul C.(2009). Insulin-like growth factor-1 and

Dusonchet J., Bensadoun J.C., Schneider B.L. and Aebischer P. (2009). Targeted overexpression of the parkin substrate Pael-R in the nigrostriatal system of adult rats to model Parkinson’s disease. Neurobiology of Disease, 35: 32-41.

Towne C, Pertin M., Beggah A.T., Aebischer P. and Decosterd I. (2009). Recombinant adeno-associated virus serotype 6 (rAAV2/6)-mediated gene transfer to nociceptive neurons through different routes of delivery. Molecular Pain, 5: 52. Ulusoy A., Shain G., Björklund T., Aebischer P. and Kirik D.(2009). Dose optimization for long-term rAAV-mediated RNA interference in the nigrostriatal projection neurons. Molecular Therapy, 17: 1574-1584. Szulc J. and Aebischer P. (2008). Conditional gene expression and knockdown using lentivirus vectors encoding shRNA. Methods in Molecular Biology, 434: 291-309. Lo Bianco C., Shorter J., Régulier E., Lashuel H., Iwatsubo T., Lindquist S. and Aebischer P. (2008). Hsp104 antagonizes alpha-synuclein aggregation and reduces dopaminergic degeneration in a rat model of Parkinson disease. The Journal of Clinical Investigation, 118: 3087-3097. Towne C., Raoul C., Schneider B.L. and Aebischer P. (2008). Systemic AAV6 delivery mediating RNA interference against SOD1: neuromuscular transduction does not alter disease progression in fALS mice. Molecular Therapy, 16: 10181025.

Targeting the rat substantia nigra (SN). Sequential double infection of adeno-associated vectors serotype 9 and 6 in the nigrostriatal pathway. AAV9green fluorescent protein (GFP) was injected in the SN and AAV6-red fluorescent protein (RFP) in the striatum 4 weeks later.

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BMI

EPFL School of Life Sciences - 2009 Annual Report


EPFL School of Life Sciences - 2009 Annual Report

BMI - Brain Mind Institute

Blanke Lab Head of lab (PI) - http://lnco.epfl.ch Team members

Olaf Blanke Associate Professor

Shahar Arzy, Visiting scientist Jane Aspell, Postdoctoral fellow Sebastian Dieguez, PhD student Nathan Evans, PhD student Luca Forcucci, Artist (Swiss AiL-scholarship) Pär Halje, PhD student Bruno Herbelin. Postdoctoral fellow Lucas Heydrich, PhD student Silvio Ionta, Postdoctoral fellow Oliver Kannape, PhD student Gordana Kokorus, Administrative assistant Tom Lavanchy, BA-student Bigna Lenggenhager, PhD student Christophe Lopez, Postdoctoral fellow Roberto Martuzzi, Postdoctoral fellow Manuel Mercier, PhD student Leila Overney, Postdoctoral fellow Estelle Palluel, Postdoctoral fellow Danilo Rezende, PhD student Tej Tadi, PhD student

Introduction

We focus our investigations on the functional and neural mechanisms of body perception (somatosensory, vestibular, visual, auditory), corporeal awareness and self-consciousness. For this we apply paradigms from cognitive science, neuroscience, neuroimaging, robotics, and virtual reality. Our main goal is to investigate the human brain mechanisms involved in the multisensory own body perception in order to develop a neurobiological model of self-consciousness and to apply these findings to the investigation and treatment of neurological and psychiatric patients

Keywords

Multisensory perception, bodily awareness, self-consciousness, intracranial human electrophysiology, neuroimaging, fMRI, EEG, neuropsychology, cognitive neurology, epileptology, optical body tracking, virtual reality, neuroscience robotics, vestibular system, mental imagery

Results Obtained in 2009

The major achievement in 2009 was the full installation of a 3D human centrifuge including an onboard 192-channel EEG and evoked potential recording system which will form LNCO’s major research platform over the coming years. The research will allow us to describe in a fine-grained temporal and anatomical manner the last missing sensory system: the vestibular or balance system. With several publications in major neuroscience and biology journals we extended our investigation of the cognitive and neurobiological mechanisms of body

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perception, bodily self-consciousness, and subjectivity using techniques such as fMRI, high-density EEG, virtual reality and robotics.

Selected publications

Dieguez S, Mercier MR, Newby N, Blanke O (2009). Feeling numbness for someone else’s finger. Curr Biol. 19(24):R1108-9. Arzy S, Collette S, Ionta S, Fornari E, Blanke O (2009). Subjective mental time: the functional architecture of projecting the self to past and future. Eur J Neurosci. 30(10):2009-17. Blanke O, Metzinger T (2009) Full-body illusions and minimal phenomenal selfhood. Trends Cogn Sci 13: 7-13. Blanke O, Forcucci L, Dieguez S (2009). Don’t forget the artists when studying perception of art. Nature 462(7276):984. Blanke O, Gassert R (2009) Total control: Virtual reality and robotics. Frontiers Neurosci 3 (1). Lopez C, Bachofner C, Mercier M, Blanke, O (2009). Gravity and observer’s body orientation influence the visual perception of human body postures. Journal of Vision, 9(5):1, 1-14. Aspell JE, Lenggenhager B, Blanke O (2009). Keeping in touch with one’s self: multisensory mechanisms of selfconsciousness. PLoS One. (8):e6488 Blanke O, Aspell JE (2009). Brain technologies raise unprecedented ethical challenges. Nature 458(7239):703 Schwabe L, Lenggenhager B, Blanke O (2009). The timing

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of temporoparietal and frontal activations during mental own body transformations from different visuospatial perspectives. Hum Brain Mapp 30(6):1801-12.

Farivar R, Blanke O, Chaudhuri A (2009) Dorsal-ventral integration in the recognition of motion defined unfamiliar faces. J Neurosci 29: 5336-5342.

Ionta S, Blanke O (2009). Differential influence of hands posture on mental rotation of hands and feet in left and right handers. Exp Brain Res. 195(2):207-17

Tadi T, Overney LS, Blanke O (2009). Three sequential brain activations encode mental transformations of upright and inverted human bodies: A high resolution evoked potential study. Neuroscience 159(4):1316-25

Arzy S, Adi_Japha E, Blanke O (2009) The mental time line: An analogue of the mental number line in the mapping of life events. Conscious Cogn 18: 7-15. Blanke O, Heydrich L, Lenggenhager B (2009) KĂśrperliches Ich-Bewusstsein. Geist und Gehirn. 12: 60-63. Overney L, Blanke O (2009) Impaired mental imagery for body parts but not external objects. Brain Topography 22:27-43. Blanke O, Morgenthaler FD, Brugger, P, Overney SO (2009) Preliminary evidence for a fronto-parietal dysfunction in able-bodied subjects with a desire for limb amputation. J Neuropsychology 3: 181-200.

Mercier M, Schwartz S, Michel CM, Blanke O (2009). Motion direction tuning in human visual cortex. Eur J Neurosci. 29(2):424-34. Lenggenhager B, Mouthon M, Blanke O (2009). Spatial aspects of bodily self-consciousness. Conscious Cogn. 18(1):110-7 Overney LS, Arzy S, Blanke O (2009). Deficient mental own-body imagery in a neurological patient with out-ofbody experiences due to cannabis use. Cortex. 45(2):22835

Brain activity in temporo-parietal cortex during a manipulation of bodily selfconsciousness (from Tadi et al., 2009).

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BMI

EPFL School of Life Sciences - 2009 Annual Report


EPFL School of Life Sciences - 2009 Annual Report

BMI - Brain Mind Institute

Fraering Lab Head of lab (PI) - http://fraering-lab.epfl.ch

Team members

Patrick Fraering

Tenure Track Assistant Professor Merck-Sorono Chair in Neuroscience

Lorene Aeschbach, Technician Jean-René Alattia, Postdoctoral fellow Saoussen Ben Halima, Master student (until July 2009) Tristan Bolmont, Postdoctoral fellow Nathalie Bot, Postdoctoral fellow Isabelle Chang, Technician (start December 2009) Jose Luis Galbete, visiting scientist (start June 2009) Mitko Dimitrov, PhD student (start February 2009) Jemila Houacine, PhD student Isabelle, Magold PhD student Sylviane Reymond, Technician (until December 2009) Caroline Rheiner, Administrative Assistant Claude Schweizer, Postdoctoral fellow Fang Wu, Postdoctoral fellow

Introduction

The Laboratory of Molecular and Cellular Biology of Alzheimer’s Disease is focusing on better understanding the molecular, cellular and biochemical mechanisms of γ-secretase and Alzheimer’s disease. Our laboratory is also implicated in the design and development of new therapeutic strategies to slow down the pathogenesis of Alzheimer’s disease.

Keywords

Molecular and Cellular Biology of Alzheimer’s disease, γ-Secretase, Amyloid-beta peptides (Aβ), intramembrane-cleaving proteases, therapeutic targets

Results Obtained in 2009

By far the most frequent age related neurological disorder is Alzheimer’s diseases (AD), a devastating disorder of the brain’s nerve cells that impairs memory, thinking, and behavior and leads, ultimately to death. The progressive accumulation, aggregation and deposition in senile plaques and microvessels of amyloid-β peptides (Aβ) in brain regions subserving memory and cognition are essential processes in the pathogenesis of AD. Three-dimensional structure of γ-secretase at 12Å resolution γ-Secretase is an integral membrane protein complex catalyzing the final intramembrane cleavage of the β-amyloid precursor protein (APP) during the neuronal Aβ production. As such, the protease has emerged as a key target for developing agents to treat and prevent Alzheimer’s disease. Despite this evidence, the structure of the protease remains poorly understood, mainly because the limited amount of purified γ-secretase that can be produced has prevented the achievement of a high-resolution crystal structure by X-ray crystallography or 2D crystallization. Because of this observation, we first developed a rapid protocol for the high-grade purification of

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proteolytically active γ-secretase (Cacquevel et al., 2008). Next, we used these cells and purification methods to solve the 3D structure of the γ-secretase complex at 12Å resolution as obtained by cryoelectron microscopy and single-particle image reconstruction (Osenkowski et al., 2008). The structure revealed several domains on the extracellular side, three solvent-accessible low-density cavities, and a potential substrate-binding surface groove (an important target for drug design) in the transmembrane region of the complex. Gene expression profiling in cells with enhanced γ-secretase activity Yet, processing by γ-secretase of many type-I membrane protein substrates triggers signaling cascades by releasing intracellular domains which, following nuclear translocation, modulate the transcription of genes regulating a diverse array of cellular and biological processes. This includes intestinal stem cell differentiation, induction of satellite cells after injury and neural specification of embryonic stem cells. Because γ-secretase is a cancer and AD therapeutic target, the mapping of γ-secretase activity susceptible gene transcription is important to sharpen our view of affected genes, molecular functions and biological pathways. We analyzed by cDNA micro-array the cellular transcriptomes of mammalian cells with enhanced and inhibited γ-secretase activity. This revealed several affected clusters of molecular functions, several key players of the three Wnt pathways to be transcriptionally altered in response to enhanced γ-secretase, and more specifically 21 genes that hold significant potential for a better understanding of the biology of γ-secretase and its roles in Alzheimer’s disease pathology (Magold et al, 2009). Substrate-targeting γ-secretase modulators Collectively, it is accepted that strategies that decrease the accumulation of Aβ, might be therapeutically beneficial to slow down the progression of AD,

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if they do not interfere with the other γ-secretase functions (e.g. severe gut toxicity has been observed in clinical trials with γ-secretase inhibitors because of interference with the Notch signaling pathway and intestinal stem cell differentiation). Thus, selective lowering of Aβ peptides (without affecting the processing of the Notch receptor) with small molecule γ-secretase modulators (GSMs) is a promising therapeutic approach for AD. By using novel biotinylated photo-activatable GSMs, we were able to show that these compounds reduced Aβ production by labelling the γ-secretase substrate APP (Kukar et al, 2008). We further demonstrated that substrate targeting by GSMs results in two therapeutic actions: (1) alteration in Aβ42 production, and (2) inhibition of Aβ aggregation, that may synergistically reduce Aβ deposition in AD (Kukar et al, 2008).

Osenkowski P, Li H, Ye W, Li D, Aeschbach L, Fraering PC, Wolfe MS, Selkoe DJ, Li H (2008). Cryo-Electron Microscopy Structure of γ-Secretase at 12 Å Resolution. Journal of Molecular Biology. 16;385(2):642-52. Kukar TL, Ladd TB, Bann MA, Fraering PC, Narlawar R, Maharvi GM, Healy B, Chapman R, Welzel AT, Price RW, Moore B, Rangachari V, Cusack B, Eriksen J, Jansen-West K, Verbeeck C, Yager D, Eckman C, Ye W, Sagi S, Cottrell BA, Torpey J, Rosenberry TL, Fauq A, Wolfe MS, Schmidt B, Walsh DM, Koo EH, Golde TE (2008). Substrate-targeting γ-secretase modulators. Nature. 12;453(7197):925-9. Cacquevel M, Aeschbach L, Osenkowski P, Li D, Ye W, Wolfe MS, Li H, Selkoe DJ and Fraering PC (2008). A rapid protocol for the purification of active γ-secretase, an intremembrane-cleaving protease involved in Alzheimer’s disease. Journal of Neurochemistry, 104(1): 210-20.

Selected publications

Magold, A.I., Cacquevel, M. and Fraering PC. (2009). Gene expression profiling in cells with enhanced gammasecretase activity. PLoSONE. 2009 Sep 18;4(9):e6952.

Several key players of the Wnt signalling pathways are increasingly (red quadrangles) or decreasingly (blue quadrangles) transcribed in cells with enhanced γ-secretase activity. β-Catenin is a central node connecting Wnt – Frizzled – Dishevelled to a downstream effect influencing the cell cycle.

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15

BMI

EPFL School of Life Sciences - 2009 Annual Report


EPFL School of Life Sciences - 2009 Annual Report

BMI - Brain Mind Institute

Gerstner Lab Head of lab (PI) - http://lcn.epfl.ch

Team members

Wulfram Gerstner

Full Professor: Life Sciences and Computer & Communication Sciences

Nicolas Fremaux, PhD student Felipe Gerhard, PhD student Guillaume Hennequin, PhD student Danilo Jimenez Rezende, PhD student Nicolas Marcille, PhD student Chantal Mellier, Administrative Assistant Skander Mensi, PhD student Richard Naud, PhD student Christian Pozzorini, PhD student Dr. Henning Sprekeler, PostDoc Christian Tomm, PhD student Dr. Tim Vogels, PostDoc, start date 01.04.2010 Dr. Daan Wierstra, PostDoc Friedemann Zenke, PhD student Lorric Ziegler, PhD student

Introduction

The Laboratory of Computational Neuroscience uses theoretical methods from mathematics, computer science, and physics to understand brain function. Questions addressed are: What is the code used by neurons in the brain? How can changes of synapses lead to learning?

Keywords

Modeling, Hebbian Learning, Spike-Timing Dependent Plasticity, Simulation, Spiking Neuron models

Results Obtained in 2009

We have been active in three different, but connected areas: Single-Neuron Modeling. We have shown that the electrical behaviour of neurons under somatic current or conductance injection can be well described by simplified neuron models with only one or two equations. The parameters of these neuron models can be directly extracted from experimental data. Our work has answered in this context two questions: first, what is the best simplified neuron model – the answer is exponential integrate-and fire model combined with adaptation and/or refractoriness (Badel et al 2008). Second, is there a way to quantify the heterogeneity of neurons – the answer is yes, since model parameters can be estimated reliably and on a neuron-by-neuron data from a few seconds of electrophysiological data (Badel et al. 2008c). The mathematical properties of such neuron models have been analysed (Naud et al 2008). To compare our approach with other approaches, we have organized an international competition – and indeed the simplified neuron models from our and one other lab were the winners (Jolivet et al. 2008, 2008b). The work on single-neuron modeling involves collaborations with the labs of Henry Markram and Carl Petersen. In 2009, the official organisation of the competition was transferred to the International Neuroinformatics Coordinating Facility (INCF). R. Naud from the

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EPFL-LCN and Dr. R. Ritz from the INCF worked out the details of the competition. Since the idea of competitions in neural modelling is a rather novel idea in Neuroscience, it led to a “perspective” article in Science (Gerstner & Naud). Modeling synaptic plasticity. We have developed a model that combines induction of synaptic plasticity with consolidation of synapses. The model of induction accounts for induction of Long-Term Potentiation under protocols of voltage-dependent and Spike-Timing Dependent Plasticity and leads to the tagging of the synapse. The model of consolidation combines a bistable dynamics with a triggering process for protein synthesis (Clopath et al. 2008). The model accounts for a large variety of tagging protocols. We also studied consequences of plasticity in a recurrent network (Nature Neuroscience 2010). Network Simulation. We have analysed the dynamics of visual illusions in the framework of a neural field model of cortical activity (Hermens et al. 2008). This project which is finished now involves collaboration with the laboratory of Michael Herzog. The collaboration with the Herzog lab is continued with a PhD student who connects models of decision processes with psychophysical data on rapid visual processing

Selected publications:

Clopath, Claudia ; Büsing, Lars ; Vasilaki, Eleni ; Gerstner, Wulfram, Connectivity reflects coding: a model of voltagebased STDP with homeostasis, In: Nature Neuroscience, On Line, 2010 Vasilaki, Eleni ; Frémaux, Nicolas ; Urbanczik, Robert ; Senn, Walter et al., Spike-Based Reinforcement Learning in Continuous State and Action Space: When Policy Gradient Methods Fail, PLoS Comput Biol, vol. 5, num. 12, 2009 Gerstner, Wulfram ; Naud, Richard, How Good are Neuron Models? In: Science, vol. 326, 2009, p. 379

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Hines, Michael ; Davison, Andrew P. ; Muller , Eilif NEURON and Python, Frontiers in Neuroinformatics, vol. 3, num. 1, 2009

and-fire models from experimental data using dynamic I-V curves, Biological Cybernetics, vol. 99, num. 4-5, 2008, p. 361-370

Lefort, Sandrine; Tomm, Christian; Sarria, J.-C. F. ; Petersen, Carl C.H. The Excitatory Neuronal Network of the C2 Barrel Column in Mouse Primary Somatosensory Cortex, Neuron, vol. 61, num. 2, 2009, p. 301-316

Claudia Clopath, Lorric Ziegler, Eleni Vasilaki, Lars Busing, Wulfram Gerstner (2008) Tag-Trigger-Consolidation: A Model of Early and Late Long-Term-Potentiation and Depression, PLoS Comput Biol, vol. 4, num. 12, 2008

Sheynikhovich, Denis; Chavarriaga, Ricardo; Strösslin, Thomas; Arleo, Angelo et al. Is there a geometric module for spatial orientation? Insights from a rodent navigation model. Psychological Review, vol. Vol 116, num. 3, 2009, p. 540-566

Jolivet, Renaud ; Schürmann, Felix ; Berger, Thomas K. ; Naud, Richard et al. The quantitative single-neuron modeling competition. Biological Cybernetics, vol. 99, num. 4-5, 2008, p. 417-426

Luksys, G. ; Gerstner, W. ; Sandi, C. Stress, genotype and norepinephrine in the prediction of mouse behavior using reinforcement learning, Nature Neuroscience, vol. 12, num. 9, 2009, p. 1180-1186

Naud, Richard ; Marcille, Nicolas ; Clopath, Claudia ; Gerstner, Wulfram, Firing patterns in the adaptive exponential integrate-and-fire model, Biological Cybernetics, vol. 99, num. 4-5, 2008, p. 335-347

Badel L.; Lefort S.; Berger T. K.; Petersen C. C. H.; Gerstner W. and Richardson M. J. E, Extracting non-linear integrate-

From spike to movements: learning the code

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17

BMI

EPFL School of Life Sciences - 2009 Annual Report


EPFL School of Life Sciences - 2009 Annual Report

BMI - Brain Mind Institute

Hadjikhani Group Group leader - http://bmi.epfl.ch/page64718.html

Team members

Reyhaneh Bakhtiari, Visiting Scientist Sandra Da Costa, Master Student Juliana Iranpour, Master Student Cristina Granziera, Postdoctoral Fellow Loyse Hippolyte, Scientific Collaborator Karine Metrailler, Research Coordinator Anthony Lissot, Scientific Collaborator Ophelie Rogier, Postdoctoral Fellow Carole Schwab, Administrative Assistant Britt Russo Scientific Collaborator Nicole ZĂźrcher, PhD Student

Nouchine Hadjikhani SFN Professor

Introduction

The theme of our research is the neuro-anatomical bases of emotional, social and cognitive difficulties in autism. Our lab is also interested in examining the pathophysiology and the possible role of the cerebellum in migraine.

Keywords

Functional and anatomical brain imaging, cognition, emotion, autism, plasticity, migraine, cerebellum.

Results Obtained in 2009

Our lab has been working at not only performing research on autism spectrum disorders, but also on informing the public about this condition, and organizing parents’ association meetings to provide a better understanding of autism. We have been publishing a number of papers examining the mechanisms of social cognition in normal controls, as well as underlining the potential involvement of the different brain mechanisms in autism. We have also published papers related to our research in migraine. Observing that the prevalence of autism has increased dramatically during the last decade, we decided to examine which could be the various causes leading to that fact. Among the potential environmental factors, hyperserotonemia during pregnancy and its effect on brain development could be playing a role in this rising prevalence. In the rodent model developed by Whitaker-Azmitia and colleagues, hyperserotonemia during fetal development results in a dysfunction of the hypothalamo-pituitary axis, affecting the amygdala as well as pro-social hormone oxytocin regulation. We emitted the hypothesis that selective Serotonin Reuptake Inhibitors (SSRI), which are the most widely used class of antidepressants drugs, and are not contra-indicated during pregnancy, could be involved in the increase of autism prevalence. If our hypothesis is confirmed, it will not only shed light on one of the possible reason

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for autism prevalence, but also offer new preventive and treatment options. Autism spectrum disorders (ASD) are characterized by difficulties in social interactions, and in emotion expression. We had reported in the past that perception of body expression of emotion triggers a series of brain activation suggesting contagious fear. In a study using the same stimuli (pictures of actors expressing fear vs. pictures of actors performing neutral actions, all with face blurred), we demonstrated that in ASD, this fear contagion is absent, pointing one more time to our main working hypothesis: a deficient mirror neurons system in ASD. Rapid categorization of visual scenes is still not completely understood. Using high-temporal and spatial magneto-encephalography (MEG), in collaboration with colleagues from the Netherland and from the Martinos Center in Boston, we showed that highlevel processing could be observed already 70ms after stimulus presentation, as demonstrated by the presence of an inversion effect for faces and bodies. Our results provide the first direct evidence that category-specific processing in high-level categorysensitive cortical areas already takes place within the first 100-ms of visual processing, significantly earlier than previously thought, and suggesting the existence of fast category-specific neocortical routes in the human brain. We also used MEG to examine the specificity of face perception, and the basis of the human tendency to perceive faces in random patterns. We used pictures of objects that look like faces, and showed that they activated face-specific cortex at very early stages of face processing, showing that this was an early process and not a late re-interpretation cognitive phenomenon. Facial expression and direction of gaze are two important sources of social information, and what message each conveys may ultimately depend on how

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the respective information interacts in the eye of the perceiver. Differences in culture may have influence on how we perceive emotional expressions. In collaboration with Japanese and American teams, we examined the differences in neural activation in response to direct- versus averted fear expression, and found clear evidence for a culturally determined role of gaze in the processing of fear. In parallel, we are pursuing, in collaboration with our group at the Harvard Medical School in Boston, our research on migraine, its physiopathology and its long-term effect on the brain.

Selected publications

Adams RB Jr, Franklin RG Jr, Rule NO, Freeman JB, Kveraga K, Hadjikhani N, Yoshikawa S, Ambady N. Culture, Gaze and the neural processing of fear expressions. Soc Cogn Affect Neurosci 2009 Dec 17 [epub ahead of print] Hadjikhani N. Serotonin, pregnancy and increased autism prevalence: is there a link? Medical Hypotheses 2009, Dec 15 [epub ahead of print] Granziera C, Schmahmann JD, Hadjikhani N, Heiko M, Meuli R, Wedeen VJ, Krueger G. Diffusion Spectrum imaging shows the structural basis of functional cerebellar circuits in the human cerebellum in vivo. PLoSONE 2009;4(4):e5101. Epub 2009 Apr 2, Hadjikhani N, Kveraga K, Naik P, Ahlfors S. Early activation of face-specific cortex by face-like objects. NeuroReport 2009, 20:403-407.

Dahlem M, Hadjikhani N. Migraine aura: retracting particlelike waves in weakly susceptible cortex. PLoSONE 2009 4(4):e5007. Epub 2009 Apr 1. Meeren HK, Hadjikhani N, Ahlfors S, Hämäläinen MS, de Gelder B. Early category-specific cortical activation revealed by visual stimulus inversion. PLoSONE 2008 2008;3(10) e3503. Hadjikhani N, Joseph RM, Maoach DS, Naik P, Snyder J, Dominick K, Hoge R, Van den Stock J, Tager-Flusberg H, de Gelder B. Body expressions of emotion do not trigger fear contagion in autism. Soc Cog Affect Neurosci 2009 Mar;4(1):70-8. Kaaro J, Partonen T, Naik P, Hadjikhani N. Is migraine a lateralization defect? NeuroReport 2008;19(13)1351-1353. Knaus T, Silver A, Lindgren K, Hadjikhani N, Tager-Flusberg H. fMRI activation during a language task in adolescents with autism spectrum disorder” The Journal of the International Neuropsychological Society 2008;14(16):967979. Thakkar K, Polli F, Joseph R, Tuch D, Hadjikhani N, Barton J, Manoach D. Response monitoring, repetitive behavior, and anterior cingulate abnormalities in autism spectrum. Brain 2008; 2008;131:2464-2478. Hadjikhani N. Relevance of cortical thickness in migraine sufferers. Expert Rev. Neurotherapeutics 2008; 8(3):327-329. Hadjikhani N, Hoge R, Snyder J, de Gelder B. Pointing with the eyes: the role of gaze in communicating danger. Brain and Cognition 2008;68(1)1-8.

Diffusion spectrum imaging of the cerebellum.

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19

BMI

EPFL School of Life Sciences - 2009 Annual Report


EPFL School of Life Sciences - 2009 Annual Report

BMI - Brain Mind Institute

Herzog Lab Head of lab (PI) - http://lpsy.epfl.ch

Team members

Michael Herzog

Kristoffer Aberg, Phd Student Marco Boi, Phd Student Laure Dayer, Administrative Assistant Anne Dehnert, Phd Student Adriana De Pesters, Master Student Mauro Manassi , Phd Student Gijs Plomp, Postdoctoral Fellow Marc Repnow, Engineer Johannes Rüter, Phd Student Toni Saarela, Phd Student Bilge Sayim, Phd Student Elisa Tartaglia, Phd Student Evelina Thunnel, Phd Student

Associate Professor

Introduction

In humans, vision is the most important sensory modality. Surprisingly, the mechanisms of even the simplest forms of visual processing, such as spotting a pen on a cluttered desk, are largely unknown. Our research aims to understand how and why humans can cope with visual tasks so remarkably well whereas, interestingly, the most sophisticated robots cannot. The main goal of our research is to characterize the interplay of spatial integration and temporal binding processes with the help of psychophysics, TMS, EEG, mathematical modelling, and clinical investigations. Main topics of research are: feature integration, contextual modulation, time course of information processing, and perceptual learning. In clinical studies, deficits of visual information processing are investigated in schizophrenic patients

Keywords

Vision Research, Spatio-temporal Vision, Schizophrenia, Psychophysics, TMS, EEG, Modelling.

Results Obtained in 2009

Spatial and temporal visual processing are usually considered to be largely independent research fields. Consequently, there is very little cross talk between the two research areas. We have shown that this assumption is not justified in most vision research fields including contrast vision (Saarela & Herzog, 2009), crowding (Saarela, Sayim, Westheimer & Herzog. 2009), and visual masking (Dombrowe, Hermens, Francis & Herzog, 2009; Hermens, Herzog & Francis, 2009; Hermens, Scharnowski & Herzog, 2009). Instead, we propose that Gestalt factors determine visual processing to a larger extent than previously thought. With computer simulations, we could show that these Gestalt factors may operate on visual areas even as early as V1 (Hermens, Scharnowski & Herzog, 2009). These Gestalt processes seem to be

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intact in schizophrenic patients and adolescents with psychosis (Herzog & Brand, 2009, Holzer, Jaugey, Chinet, & Herzog, 2009). Gestalt factors seem also to underlie non-retinotopic processing which also seems to occur more often than previously thought (Boi, Ogmen, Krummenacher, Otto & Herzog, 2009; Aydin, Herzog & Ogmen, 2009; Otto, Ogmen & Herzog, 2009). EEG and TMS recordings show that both retinotopic and non-retinotopic integration last for up to 400600ms even though stimuli are presented only for 60-180ms (Plomp, Mercier, Otto, Blanke & Herzog, 2009; Scharnowski, Rüter, Jolij, Hermens, Kammer & Herzog, 2009). Perceptual learning is usually thought to be driven mainly by stimulus presentation. We have shown that perceptual learning can, surprisingly, occur in the absence of stimulus presentation (Tartaglia, Bamert, Mast & Herzog, 2009; see also Tartaglia, Aberg & Herzog, 2009a). We proposed that the conscious mind constitutes itself through perceptual learning (Herzog & Esfeld, 2009). In line with this proposal, perceptual learning did not occur during anaesthesia (Aberg, Albrecht, Tartaglia, Farron, Soom, & Herzog, 2009). On the other hand, stimulus presentation seems to induce automatic neural changes because we found that a minimal number of stimuli per session is needed to induce learning (Aberg, Tartaglia & Herzog, 2009) and that learning interferes when neural representations of stimuli interfere (Tartaglia, Aberg & Herzog, 2009b; Aberg & Herzog, 2009).

Selected publications

Tartaglia E. M., Bamert L., Mast F.W., Herzog M.H. (2009). Human perceptual learning by mental imagery. Current Biology, 19, (24) 2081-2085. Saarela T. P., Sayim, B, Westheimer, G., Herzog, M. H. (2009). Global stimulus configuration modulates crowding. Journal of Vision, 9(2):5, 1-11.

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Holzer L., Jaugey L., Chinet L., Herzog M.H. (2009). Deteriorated visual backward masking in the shine-through effect in adolescents with psychosis. Journal of Clinical and Experimental Neuropsychology, 31, 641-647.

tegration by transcranial magnetic stimulation. Journal of Vision, 9(6):1, 1-10. Faculty of 1000 Biology Evaluation: http://www.f1000biology.com/article/id/1161357/evaluation

Tartaglia E. M., Aberg K.C., Herzog M.H. (2009). Perceptual learning and roving: stimulus types and overlapping neural populations. Vision Research, 49, 1420–1427.

Plomp, G., Mercier, M.R., Otto, T.U., Blanke, O., Herzog, M.H. (2009). Non-retinotopic feature integration decreases response-locked brain activity as revealed by electrical neuroimaging. NeuroImage, 48, 405-414.

Herzog M.H., Esfeld M. (2009). How the mind constitutes itself through perceptual learning. Learning & Perception, 1, 147-154. Herzog M.H., Brand A. (2009). Pitting temporal against spatial integration in schizophrenia. Psychiatry Research, 168, 1-10. Hermens F., Scharnowski F., Herzog M.H. (2009). Spatial grouping determines temporal integration. Journal of Experimental Psychology: Human Perception and Performance, 35(3), 595-610.

Aberg, K. C., Albrecht, E., Tartaglia, E. M., Farron, A., Soom, P., Herzog, M. H. (2009). Anesthesia prevents perceptual learning. Anesthesiology, 111, 1010-5. Boi M., Ogmen H., Krummenacher J., Otto T.U., Herzog M.H. (2009). A (fascinating) litmus test for human retinovs. non-retinotopic processing. Journal of Vision, 9(13): 5, 1-11.

Scharnowski F., Rüter J., Jolij J., Hermens F., Kammer T., Herzog M.H. (2009). Long lasting modulation of feature in-

Dynamics of non-retinotopic feature integration. Leading up to the behavioral response, the red areas showed decreased activity when two features integrated in a sequential metacontrast paradigm. The timeline in the center of the figure indicates time relative to the button press. The lower leftmost plot shows the integration effect in the Middle Temporal Gyrus (MT) at 530 ms before the response. Activity in all areas decreased when the two features integrated. The interaction closely resembled behavior: with integration, discrimination accuracy was around chance. This resemblance is corroborated by linear regression of estimated current densities on accuracy, depicted in the second plot from the left, green line. The only area where activity increased with feature integration was the Middle Frontal Gyrus (MFG), at around -300 ms. In this area the interaction changed sign, as can be seen in the two lower right plots. Here too, a strong correlation with accuracy was observed. Error bars denote 95% confidence intervals (adapted from Plomp et al., 2009).

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21

BMI

EPFL School of Life Sciences - 2009 Annual Report


EPFL School of Life Sciences - 2009 Annual Report

BMI - Brain Mind Institute

Lashuel Lab Head of lab (PI) - http://nmnf.epfl.ch Team members

Carole Schwab, Administrative Assistant Saviana, Di Giovanni, Postdoctoral associate Farah El-Turk, PhD student Bruno, Fauvet, PhD student Filip Vercruysse, PhD student Michel Pruddent, Postdoctoral associate Valerie, Grimmiinger, Postdoctoral associate Martial, Mbefo Kamdem, PhD student Hajer, Ouertatani-Sakouhi, PhD student Abid, Oueslati, Postdoctoral associate Ekaterini, Paleologou, Postdoctoral associate Asad Jan Qureshi, PhD student Sara, Butterfield, Postdoctoral associate Nathalie, Jordan, Research Associate John, Perinn, Research Associate

Hilal Lashuel Tenure Track Assistant Professor

Introduction

Research in the Lashuel’s laboratory is focused on applying chemical, biophysical, structural and molecular biology approaches to understanding molecular and structural basis of protein misfolding and self-assembly and the mechanisms by which these processes contribute to the pathogenesis of neurodegenerative diseases.

Keywords

Neurodegeneration, Parkinson’s disease, Alzheimer’s disease, amyloid, aggregation, toxicity, protofibrils, phosphorylation, polo like kinases, macrophage migration inhibitory factor, Hsp104,

Results Obtained in 2009

α-Synuclein phosphorylation: Increasing evidence suggests that phosphorylation at serine 129 (S129) may play an important role in modulating of α-syn aggregation, Lewy body (LB) formation and neurotoxicity in PD and related synucleinopathies. In addition to elucidating the effect of this modification on the structure, aggregation, and membrane binding of monomeric α-syn, our work over the last two years has led to the identification and characterization of a new phosphorylation site (Serine S87) and the identification of the polo like kinases PLK2 and PLK3 as the major kinases responsible for a-synuclein phosphorylation in vivo. The specificity and efficiency with which these kinases phosphorylate α-syn in vitro and in vivo provide unique opportunities to elucidate the molecular mechanisms and functional consequences of α-syn phosphorylation in vivo and validate the potential of PLKs as therapeutic targets for treating PD and related synucleinopathies. Together, our findings have significant implications for developing strategies to model and elucidate the role of phosphorylation in modulating protein structure and function in health and disease.

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Covalent Cross-linking of a-syn by tissue transglutaminase: Tissue transglutaminase (tTG) has been implicated in the pathogenesis of Parkinson disease (PD). However, the precise role of tTG in modulating the structural and functional properties of alpha-synuclein (alpha-syn) and the pathogenesis of PD remain unknown. Using site-directed mutagenesis combined with detailed biophysical and mass spectrometry analyses, we identified, for the first time, the exact glutamine and lysine residues involved in tTG-catalyzed cross-linking of wild-type and α-syn mutants associated with PD. We demonstrated that 1) tTGcatalyzed cross-linking of monomeric α-syn involves multiple cross-links (specifically 2-3) and block α-syn fibrillization and 2) Gln(79) and Gln(109) serve as the primary tTG reactive sites. To further elucidate the sequence and structural basis underlying these effects, we identified the lysine residues that form isopeptide bonds with Gln(79) and Gln(109). Macrophage Migration Inhibitory factor (MIF): Macrophage migration inhibitory factor (MIF) is a homotrimeric multifunctional proinflammatory cytokine that has been implicated in the pathogenesis of several inflammatory and autoimmune diseases. Current therapeutic strategies for targeting MIF focus on developing inhibitors of its tautomerase activity or modulating its biological activities using anti-MIF neutralizing antibodies. We identified a new class of isothiocyanate (ITC)-based irreversible inhibitors of MIF. Modification by benzyl isothiocyanate (BITC) and related analogues occurred at the N-terminal catalytic proline residue without any effect on the oligomerization state of MIF. Biophysical and structural studies demonstrated that modification of Pro1 alters the tertiary, but not the secondary or quaternary, structure of the trimer. These changes in tertiary structure and the loss of catalytic activity translated into a reduction in MIF receptor binding activity, MIFmediated glucocorticoid overriding, and MIF-induced

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Akt phosphorylation. Together, these findings highlight the role of tertiary structure in modulating the biochemical and biological activities of MIF and present new opportunities for modulating MIF biological activities in vivo.

Selected publications

Mbefo MK, Paleologou KE, Boucharaba A, Oueslati A, Olschewski D, Schell H, Fournier M , Zweckstetter M., Kahle PJ, Masliah E Hirling H, and Lashuel HA* (2010). “Phosphorylation of synucleins (α, β and γ) by members of the Polo Like family of Kinases, PLKs 1-4”, 2010, J. Biol. Chem, 285(4):2807-22. Paleologou KE., Ouselti Abid, Kim H-Y, Lamberto GR., Rospigliosi CC., Schmid A., Chiappe D., Moniatte M., Eliezer D., Zweckstetter M., Masliah E. Lashuel HA*.(2010)” In vivo and in vitro characterization of α-synuclein phosphorylation at Serine 87”. J. Neuroscience, 2010, 3;30(9):3184-98. Schmid A.,Chiappe D., Pignet V., Chiappe D., Grimminger V., Hang, I., Moniatt M., Lashuel HA* (2009). “Dissecting the mechanisms of tissue transglutaminase induced crosslinking of α-synuclein: Implications for the pathogensis of Parkinson’s disease”. J. Biol. Chem. 284(19):13128-42.

Lashuel HA*and Pappu RA. (2009) “Amyloid go genomics: “Insights regarding the sequence determinants of prion formation from genome-wide studies” ChemBiochem, 10(12):1951-1954. Ouertatani-Sakouhi H., El-turk F., Lashuel HA* (2009).. “Novel classes of small molecule inhibitors of the catalytic and biological activity of macrophage migration inhibitory factor (MIF)”, Biochemistry, 2009, 48, 9858-9870. Kumar KA, Haj-Yahya M, Olschewski D, Lashuel HA, Brik A. (2009). “Highly efficient and chemoselective peptide ubiquitylation”. Angew Chem Int Ed, 2009, 48(43):8090-4. Grimminger V and Lashuel HA*. (2009). “Structure and function of the molecular chaperone Hsp104 from yeatst”, (2009), Bioploymers, 93(3):252-76. Cho MK., Kim HY., Kumar A., Maier E., Siebenhaar C., Becker S., Fernandez CO., Lahsuel HA., Benz R., Lang A., and Zweckstetter M. (2009). “Micro-mole scale pore forming oligomers by cold-induced dissociation of amyloid”. J. Amer Chem Soc, 2009, 131, 17482 - 17489

High Throughput Screening provides drug targets and tools for hypothesis testing and drug discovery.

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23

BMI

EPFL School of Life Sciences - 2009 Annual Report


EPFL School of Life Sciences - 2009 Annual Report

BMI - Brain Mind Institute

Luthi-Carter Lab Lab Luthi-Carter Head of lab (PI) - http://lngf.epfl.ch Team members

Ruth Luthi-Carter

Tenure Track Assistant Professor

Laure Dayer, Administrative Assistant Ozgün Gokce, Phd Student Lely Feletti, Intern Marc Forrest, Lab Technician Ana Jovicic, Phd Student Elena Katsyuba, Student Assistant Irina Krier, Phd Student Alexandre Kuhn, Postdoctoral Fellow Aline Monin, Intern Anne-Véronique Planchamp, Postdoctoral Fellow Maria De Fatima Rey, Lab Technician Nikita Rudinskiy, Phd Student Heike Runne, Postdoctoral Fellow Tamara Seredenin, Postdoctoral Fellow David Taylor, Postdoctoral Fellow Doris Chu Voo Thu, Postdoctoral Fellow

Introduction

The Laboratory of Functional Neurogenomics (LNGF) uses high-throughput gene expression profiling and other molecular approaches to elucidate new aspects of brain function and neurodegenerative disease. In this manner we are able to study the diverse effects of pharmacologic agents, environment, and disease-causing proteins on the expression of the entire mammalian genome. This information is then used to understand which molecules are responsible for a particular brain-related process. Currently our efforts focus on the molecular study of hereditary polyglutamine neurodegenerative diseases, primarily Huntington’s disease, and the normal function of the brain regions involved in Huntington’s disease.

Keywords

Neurodegenerative disease, Huntington’s disease, striatum, cerebral cortex, motor cortex

Results Obtained in 2009

Gene regulatory effects of brain-derived neurotrophic factor (BDNF) in striatal neurons. Combining pharmacology and gene expression readouts, we have elucidated the importance of specific arms of the trkB-dependent signaling pathway for acute gene expression responses to BDNF. BDNF-responsive genes in striatal cells were first determined globally by DNA microarray analysis. We then used parallel comparisons of the effects of specific pharmacologic inhibitors to assess the signaling pathways which were most important for the expression of particular mRNAs (measured by quantitative realtime PCR) and particular transcriptional regulatory pathways (using western blotting and enhancer-reporter assays). These results show that MEK/ERK and calcium are universal regulators of the acute BDNF gene expression response, whereas PI3K/Akt, NOS, and CAMK have gene-specific effects. We also determined that MEK/ERK-dependent regulation by BDNF acts via phosphorylation and activation of multiple transcriptional regulators, including Elk and CREB.

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We further demonstrate that the induction of acute MEK/ERK-dependent gene expression responses are important for the structural plasticity of striatal neurons, as demonstrated by their requirement for BDNF-induced axonal outgrowth (Gokce et al., 2009). Striatal expression and potential Huntingtin’s disease-modulating effects of the neuronal calcium sensor hippocalcin. We determined that hippocalcin, the predominant striatally expressed neuronal calcium sensor protein, is dramatically down regulated in animal models of Huntington’s disease and in human Huntington’s disease brain. Using yeast-2-hybrid screening and immunoprecipitation combined with LC-MS detection, we identified novel hippocalcin interacting proteins that might comprise effectors for its striatal neuron activities. We further assessed whether restoring hippocalcin levels had the potential to modify the neurodegenerative effects of mutant huntingtin, mitochondrial electron transport chain inhibition, or excitotoxicity in striatal cells. (Rudinskiy et al., 2009, J. Neurochem.) Negative regulation of AP-2- and clatrhin-dependent endocytosis by calpain and its potential role in neurodegeneration. We discovered that the alpha and beta- subunits of the AP2-clathrin adaptor complex are in vivo substrates for calpains, a small family of calcium-activated proteases whose activity may be regulated in neurons as a consequence of normal signaling and pathologically upregulated in neurodegenerative diseases. The primary beta-adaptin cleavage site was identified as the peptide bond between phenylalanine 691 and alanine 692. Using heterologous expression studies, we further showed that the presence of calpain-generated beta-2-adaptin fragments can decrease plasma membrane recruitment of other endocytic proteins. At the functional level, we have shown that adaptin cleavage decreases receptor endocytosis and can sensitize neurons to excitotoxic cell death (Rudinskiy et al.,2009; J. Biol. Chem.).

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Selected publications

Rudinskiy, N., Grishchuk, Y., Vaslin, A., Puyal, J., Delacourte, A., Hirling, H., Clarke, P.G.H. and Luthi-Carter, R. (2009) Calpain hydrolysis of alpha- and beta2-adaptins decreases clathrin-dependent endocytosis and may promote neurodegeneration. J. Biol. Chem. 284: 12447-12458. Rudinskiy, N., Kaneko, Y., Beesen, A.A., Gokce, O., Régulier, E., Déglon, N. and Luthi-Carter, R. (2009) Diminished hippocalcin expression in Huntington’s disease brain does not account for increased striatal neuron vulnerability as assessed in primary neurons. J. Neurochem. 111:460-472. Gokce, O., Kuhn, A., Runne, H., Taylor, D. and Luthi-Carter, R. (2009) Acute striatal gene expression responses to brain-derived neurotrophic factor require MEK and ERK activation. PLoS ONE 4, e5292. Drouet, V., Perrin, V., Hassig, R., Dufour, N., Auregan, G., Alves, S., Brouillet, E., Luthi-Carter, R., Hantraye, P., Déglon, N. (2009) Sustained effects of nonallele-specific huntingtin silencing. Ann. Neurol. 65:278-285. Perrin, V., Dufour, N., Raoul, C., Aebischer, P., Luthi-Carter, R., Déglon, N. (2009) Activation and pathological implica-

tion of the JNK pathway in a rat model of HD. Exp. Neurol. 215:191-200. Runne, H., Régulier, E., Kuhn, A., Zala, D., Gokce, O., Perrin, V., Sick, B., Aebischer, P., Déglon, N. and Luthi-Carter, R. (2008) Dysregulation of gene expression in primary neuron models of Huntington’s disease shows that polyglutaminerelated effects on the striatal transcriptome may not be dependent on brain circuitry. J. Neurosci. 28:9723-9731. Willi-Monnerat, S., Migliavacca, E., Surdez, D., Delorenzi, M., Luthi-Carter, R., and Terskikh, A. (2008) Comprehensive spatiotemporal transcriptomic analyses of the ganglionic eminences demonstrate the uniqueness of its caudal subdivision. Mol. Cell. Neurosci. 37:845-856. Jan, A., Gokce, O., Luthi-Carter, R. and Lashuel, H. (2008) The ratio of monomeric to aggregated forms of Abeta 40 and Abeta 42 is an important determinant of Abeta aggregation, fibrillogenesis, and toxicity. J. Biol. Chem. 283: 28176-28189. Taylor, D., Maxwell, M., Luthi-Carter, R. and Kazantsev, A. (2008) Biological and potential therapeutic roles of sirtuin deacetylases. Cell. Mol. Life Sci. 65(24):4000-4018.

Proposed model showing how calpain-mediated regulation of clathrin-dependent endocytosis may affect transcellular communication pathways in neuronal synapses. A major determining aspect of the specific functional effects of such regulation is whether calpain activation occurs presynaptically or postsynaptically, as may be illustrated for neurotransmission-related effects. In the presynaptic terminal, a given rate of synaptic vesicle endocytosis is maintained when calpain activity is low, ensuring a given depolarization-dependent release of neurotransmitter. When calpains are activated presynaptically (illustrated as top bouton), decreased endocytosis may lead to a decrease of releasable pools of synaptic vesicles and to an inhibition of neurotransmitter release. The net effect on synaptic communication may be the opposite when considering the postsynaptic terminal (illustrated as bottom bouton), however. Neurotransmitter receptors undergo persistent clathrin-dependent internalization and thus their surface population depends on the rates of endo- and exocytosis. Here, inhibition of endocytosis via elevated calpain activity may result in an increased presence of cell surface receptors, leading to increased neurotransmission, or, under pathological circumstances, to excitotoxicity. As illustrated by other symbols, effects on the surface presence and internalization of other receptors and their ligands can also be envisaged; for instance, cells may be deprived of nutrients and growth factor signals that depend on receptor internalization under conditions of persistently high calpain activation.

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BMI

EPFL School of Life Sciences - 2009 Annual Report


EPFL School of Life Sciences - 2009 Annual Report

BMI - Brain Mind Institute

Magistretti Lab Head of lab (PI) - http://bmi.epfl.ch/page61215.html Team members

Pierre Magistretti

Full Professor Joint Chair EPFL/UNIL-CHUV Director BMI

Igor Allaman, Senior Postdoctoral Fellow Nicolas Aznavour, 1st Assistant Cendrine Barrière Borgioni, Technician Maxime Baud, MD/PhD Student Mireille Bélanger, Postdoctoral Fellow Daniel Boss, PhD Student Sophie Burlet Godinot, Postdoctoral Fellow Elsy Dunand, Technician Gabriele Grenningloh, Senior Scientist Joël Gyger, Technician Annick Hoffmann, Trainee Pascal Jourdain, Postdoctoral Fellow Sylvain Lengacher, Senior Postdoctoral Fellow Pierre Marquet, Senior Postdoctoral Fellow Maude Marti Favre, Technician Elena Migacheva, PhD Student Corinne Moratal, Technician Julia Parafita, PhD Student Hélène Perreten, PhD Student Jean-Marie Petit, Senior Postdoctoral Fellow Emilie Pralong, Administrative assistant Evelyne Ruchti, Scientific Assistant Monika Saxena, Trainee

Introduction

Our laboratory has two main lines of research: the principal one is to try to understand the cellular and molecular mechanisms of the interactions between neurons and glial cells (astrocytes), termed neurometabolic coupling and to investigate this coupling in other aspects of brain function and dysfunction, such as the sleep-wake cycle, learning and memory as well as neurodegeneration. The second line of research, which is a joint effort with the laboratory of Christian Depeursinge, is represented by the neurophotonics project to develop a new type of microscope for the study of dynamic cellular processes by non-invasive, on-line, three-dimensional visualization of cells with a spatial resolution in the order of the nanometer and a millisecond temporal resolution

Keywords

Neuroenergetics, neuro-glia interactions, brain metabolism, neuronal plasticity, glial plasticity, highresolution optical imaging, digital holographic microscopy, cell dynamics, neurodegeneration, sleep, functional brain imaging, dialogue between neurosciences and psychoanalysis

Results Obtained in 2009

Neuroenergetics and neurodegeneration (Igor Allaman, Mireille Belanger) Alterations of brain energy metabolism and oxidative stress are key features of Alzheimer’s disease (AD). We could show that amyloid-beta (Aβ) peptides, key mediators in AD pathogenesis, alter astrocytic phenotype with regards to glucose metabolism and oxidative-stress status. In particular, Aβ increased glucose uptake, and its various metabolic fates, as well as glutathione and H2O2 release. Further studies showed that aggregation and binding of Aβ peptides to class A

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scavenger receptors are critical elements of Aβ action which induce a deleterious cascade leading to neurotoxicity. These results indicate that modifications of energetic and oxidative stress profiles of astrocytes induced by aggregated Aβ are linked to neurodegeneration in AD. Assessing the importance of the anaplerotic cycle of glucose metabolism in the CNS in vivo: A combination of molecular and Magnetic Resonance Spectroscopy approaches (Sylvain Lengacher, Nicolas Aznavour, Maude Marti) Glutamine synthetase and pyruvate carboxylase are found almost exclusively in astrocytes. RNA interference (RNAi) was used to reduce pyruvate carboxylase (PC) expression in rat brain. RNAi was delivered in the ventricle or cortex and siRNA effects were measured in the hippocampus and cortex. The spectra were acquired on a 9.4T magnet. Cerebral glucose consumption was assessed by quantitative autoradiography. In the hemisphere injected with the PC siRNA, we observed an increase in aspartate and lactate concentrations in the cortex, compared to the controlateral side. In parallel to this NMR approach, we evaluate the knock down of these enzymes by mapping changes in glucose consumption in rat brain. Metabolic plasticity during learning (Julia Parafita, Sylvain Lengacher, Elsy Dunand) The mapping of glucose utilization on 2-deoxyglucose autoradiograms allowed us to identify brain regions engaged in a learning. Two of these regions, hippocampus and retrosplenial cortex, were dissected by laser capture microscopy to study gene expression related to neuron-glia metabolic coupling, glucose metabolism and synaptic plasticity by quantitative PCR. Several genes showed a higher expression in the hippocampus at day 1 of the learning task

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compared to day 9, while an increased expression was found in the retrosplenial cortex at day 9. Brain energy metabolism and sleep-waking cycle (Jean-Marie Petit, Sophie Burlet, Maxime Baud, Joël Gyger) Sleep fragmentation, a common feature accompanying sleep apnoeas syndrome or diseases with chronic pain, impairs cognitive function in humans and animals. To investigate its impact on brain energy metabolism thought to be linked to synaptic plasticity, we developed an instrumental sleep fragmentation model in mice. Our protocol induced an increase in sleep bouts number and a decrease in their duration. In addition to the decrease of the slow-wave sleep (SWS) duration of 15%, spectral analysis of the EEG indicate that the energy spectrum during SWS was increased in the theta, beta, alpha and gamma frequency bands (from 7 to 40 Hz) compared to baseline. This increase in the most rapid frequency bands might be related to a less deep sleep. In addition, we assessed the impact of a 14 days-sleep fragmentation protocol on the brain metabolic profile using spectroscopy 1H-NMR in collaboration with the CIBM. Lactate and GABA levels displayed a significant decrease in hippocampus whereas no significant change was observed in the cortex. These results stress on the brain metabolic impact of sleep fragmentation. Neurophotonic project (P. Marquet, P. Jourdain, B. Rappaz, D. Boss, A. Hoffmann, C. Moratal) We also apply an optical digital holographic microscopy technique (DHM) to visualize dynamic cellular processes, including those involved in plasticity. This technique allows observing non-invasively and in real time the 3D cellular dynamics with an axial resolution of a few tens of nanometers.

Selected publications

Allaman I., Papp M., Kraftsik R., Fiumelli H. and Magistretti P. J. et al. Expression of brain-derived neurotrophic factor is not modulated by chronic mild stress in the rat hippocampus and amygdala.. Pharmacological reports : PR, vol. 60, num. 6, p. 1001-7, 2009.

Morgenthaler F. D. , Lanz B. R., Petit J.-M., Frenkel H. and Magistretti P. J. et al. Alteration of brain glycogen turnover in the conscious rat after 5h of prolonged wakefulness. Neurochemistry international, vol. 55, num. 1-3, p. 4551, 2009. Jolivet R., Magistretti P. J. and Weber B. Deciphering neuron-glia compartmentalization in cortical energy metabolism.. Frontiers in neuroenergetics, vol. 1, p. 4, 2009. Berthet C., Lei H., Thevenet J., Gruetter R. and Magistretti P. J. et al. Neuroprotective role of lactate after cerebral ischemia.. Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism, vol. 29, num. 11, p. 1780-9, 2009. Breuillaud L., Halfon O., Magistretti P. J., Pralong F. P. and Cardinaux J.-R. Mouse fertility is not dependent on the CREB coactivator Crtc1.. Nature medicine, vol. 15, num. 9, p. 989-90; author reply 991, 2009. Magistretti P. J. Neuroscience. Low-cost travel in neurons. Science (New York, N.Y.), vol. 325, num. 5946, p. 134951, 2009. Brunet J. F., Allaman I., Magistretti P. J. and Pellerin L. Glycogen metabolism as a marker of astrocyte differentiation. Journal of cerebral blood flow and metabolism : official journal of the International Society of Cerebral Blood Flow and Metabolism, vol. 30, num. 1, p. 51-5, 2009. Wyss MT, Weber B, Treyer V, Heer S, Pellerin L, Magistretti PJ, Buck A. “Stimulation-induced increases of astrocytic oxidative metabolism in rats and humans investigated with 1-11 C-acetate.” J Cereb Blood Flow Metab. 2009 Jan;29(1): 44-56. Allaman I, Papp M, Kraftsik R, Fiumelli H, Magistretti PJ, Martin JL. Expression of brain-derived neurotrophic factor is not modulated by chronic mild stress in the rat hippocampus and amygdala. Pharmacol Rep. 2008, 60:1001-7. Rappaz B, Charrière F, Depeursinge C, Magistretti PJ, Marquet P. Simultaneous cell morphometry and refractive index measurement with dual-wavelength digital holographic microscopy and dye-enhanced dispersion of perfusion medium. Opt Lett. 2008 Apr 1; 33(7):744-6.

3D perspective image of a living neuron obtained with DHM

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BMI

EPFL School of Life Sciences - 2009 Annual Report


EPFL School of Life Sciences - 2009 Annual Report

BMI - Brain Mind Institute

Markram Lab Head of lab (PI) - http://bmi.epfl.ch/page61216.html Team members

Henry Markram Full Professor Director BBP

Debono Christiane, secretary, Berger Thomas, PhD student, left 31.10.09 Coelho Monica, PhD student, arrived 01.05.2009 Delattre Vincent, PhD student, arrived 01.03.2009 Gambazzi Luca, PhD student, Gobril Jean Pierre, PhD student, arrived 01.04.2009 Khazen Georges, PhD student, Muralidhar Shruti, PhD student, Perin Rodrigo, PhD student, Reiman Michael, PhD student, arrived 15.03.2009 Riachi Imad, PhD student, Gatri Mondher, engineer, left 14.12.09 La Mendola Deborah, lab technician, Langlet Delphine, lab technician, left 01.03.2009 Meystre Julie, lab technician, Ducret Audrey, apprentice lab technician, left 21.07.09 La Mendola Rachel, intern apprentice lab technician, arrived 02.11.09 Lecoultre Antoine, apprentice lab technician, left 07.08.09 Suter Diego, apprentice lab technician, left 27.08.09 Simon Sidney, Visiting Professor, left 31.10.0’9 Catsiyannis Maximilien, student-assistant, Lachaut Thibaut, student-assistant, 01.07.2008 Mato Pau, student-assistant, 15.10.2008 Pulcrano Marisa, intern, left 31.08.09

Keywords

Neurons, synapses, synaptic transmission, synaptic plasticity, emergent properties, neural microcircuits, ion channels, receptors, signaling, the neural code.

Results Obtained in 2009

The Laboratory of Neural Microcircuitry is dedicated to understanding the structure, function and plasticity of the microcircuitry of the neocortex. The neocortex constitutes nearly 80% of the human brain and is made of a repeating stereotypical microcircuit of neurons. This neural microcircuit lies at the heart of the information processing capability of the neocortex, the capability of mammals to adapt to a rapidly changing environment, memory, and higher cognitive functions. LNMC specializes in the structure and function of the neocortical microcircuitry. We have derived the blue prints for this microcircuit and together with the Blue Brain Project have reconstructed the elementary microcircuitry of the neocortex called the neocortical column. Specifically, the laboratory conducts studies on the morphological and electrophysiological characteristics of different types of neurons, the types of synapses that can form between different types of pre-postsynaptic neurons, the microanatomical and physiological characteristics of the various synaptic connections, principles of micro-connectomics, synaptic and microcircuit plasticity, single-cell gene expression in specific morpho-electrical types of neocortical neurons, and the emergent behavior of the microcircuitry when stimulated. LNMC also hosts a group studying the neuropathology of animal models of autism that studies changes in gene expression using DNA microarrays, and changes in levels of proteins expressed using western blot. This group also performs sterology analysis to assess changes

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in cell density, immunohistochemistry to detect protein distributions (ion channels, receptors, etc) and a battery of behavioral studies. In its research the LNMC regularly uses a broad range of standard, state-of-the-art and one-of-a-kind technologies. These include (i) multi-neuron patch-clamp setups (41, 62, 71 & 121 electrodes) making it possible to patch-clamp up to many neurons simultaneously - allows rapid dissection of synaptic pathways, principles of functional connectomics, and the dynamics of small networks; (ii) single-cell patch-clamp specialized in multiplex RT-PCR - allows studying expression of up to 96 genes in single identified cells; (iii) a 2 patch-clamp system combined with a dynamic clamp apparatus including a DSP on which HH ion channel kinetics can be programmed for virtually real-time (10 nanosecond) feedback current injection as a function of recorded voltage – allows electrical lesioning of single or combinations of ion channels; (iv) a multi-electrode array (MEA) setup capable of 64 or 128 channel 3D-electrode stimulation and recording system combined with dual patch-clamp to study emergent behavior of neurocircuits, (v) an inverted microscope setup with dual patch-clamp system to study long-term changes in functional connectivity in neuronal cultures; (vi) a Flyion inverted patch robot for automated ion channel screening in CHO cell lines together with facilities for stable and transient transfection of cell lines; (vi) a variety of imaging systems including fast CCD imaging for measuring Ca2+ dynamics, 2 confocal microscopes to identify retrogradely labels neurons and trace anterogradely labeled fibers and a 2P laser scanning microscope used to image the fine structure and changes in structure in axons and dendrites; (vii) 1 neurolucida system for 3D anatomical reconstructions and 2 offline reconstruction systems running Microbrighfield’s automorph, (viii) an in vivo surgery and tract tracer

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EPFL School of Life Sciences - 2009 Annual Report

Using multi-neuron patch-clamp (12 neurons) we have studied principles of connectivity. We found the long sought after Hebbian assemblies (densely coupled neurons) are predetermined and proposed for the first time a neural substrate for innate prerepresentations. We further proposed that these pre-representations serve as the foundation on which acquired learning takes place by combining these pre-representations in different ways – a form of “lego memory”. All neurological and psychiatric disorders involve the neocortex at some stage and at some level. We propose that autism is caused by the convergence of three factors; a polygenetic predisposition (sets the threshold for autism), an epigenetic insult (triggers autism) and environmental surprise after birth (drives the progression). We are conducting experiments at each level to test this hypothesis. If correct, we will be able to predict which insults to avoid during pregnancy, develop early diagnostics for autism if triggered, and provide a pharmacological and behavioral strategy to minimize the severity and potentially heal autism. We have also used carbon nanotube coated multi-electrode arrays to demonstrate a new interface technology for neuroprosthetics and used this to reveal neuropathological changes in Hungtington’s disease.

Selected publications

Multiquantal release underlies the distribution of synaptic efficacies in the neocortex. Loebel A, Silberberg G, Helbig D, Markram H, Tsodyks M, Richardson MJ. Front Comput Neurosci. 2009;3:27. Epub 2009 Nov 24. Frequency-dependent disynaptic inhibition in the pyramidal network: a ubiquitous pathway in the developing rat neocortex. Berger TK, Perin R, Silberberg G, Markram H.

J Physiol. 2009 Nov 15;587(Pt 22):5411-25. Epub 2009 Sep 21. Fixing the location and dimensions of functional neocortical columns. Markram H. HFSP J. 2008 Jun;2(3):132-5. Epub 2008 May 23. Carbon nanotubes might improve neuronal performance by favouring electrical shortcuts. Cellot G, Cilia E, Cipollone S, Rancic V, Sucapane A, Giordani S, Gambazzi L, Markram H, Grandolfo M, Scaini D, Gelain F, Casalis L, Prato M, Giugliano M, Ballerini L. Nat Nanotechnol. 2009 Feb;4(2):12633. Epub 2008 Dec 21. Substrate arrays of iridium oxide microelectrodes for in vitro neuronal interfacing. Gawad S, Giugliano M, Heuschkel M, Wessling B, Markram H, Schnakenberg U, Renaud P, Morgan H. Front Neuroengineering. 2009;2:1. Epub 2009 Jan 22. Hyper-connectivity and hyper-plasticity in the medial prefrontal cortex in the valproic Acid animal model of autism. Rinaldi T, Perrodin C, Markram H. Front Neural Circuits. 2008;2:4. Epub 2008 Oct 29. Petilla terminology: nomenclature of features of GABAergic interneurons of the cerebral cortex. Petilla Interneuron Nomenclature Group, Ascoli et al.. Nat Rev Neurosci. 2008 Jul;9(7):557-68. Slow oscillations in neural networks with facilitating synapses. Melamed O, Barak O, Silberberg G, Markram H, Tsodyks M. J Comput Neurosci. 2008 Oct;25(2):308-16. Epub 2008 May 16. Inferring connection proximity in networks of electrically coupled cells by subthreshold frequency response analysis. Calì C, Berger TK, Pignatelli M, Carleton A, Markram H, Giugliano M. J Comput Neurosci. 2008 Jun;24(3):33045. Epub 2007 Nov 28. Hyperconnectivity of local neocortical microcircuitry induced by prenatal exposure to valproic acid. Rinaldi T, Silberberg G, Markram H. Cereb Cortex. 2008 Apr;18(4):763-70. Epub 2007 Jul 17.

Figure 1: 12 Neuron Patch-Clamp Recording System: (a) 12 neurons layer 5 pyramidal neurons recorded and stained with biocytin. (b) Region of the rat brain studied (somatosensory cortex, S1). (c) 3D reconstruction of 12 neurons recorded in a single experiment. (d) 12 patch-clamp system with Luigs-Newman high precision mini patch clamp manipulators. (e) Synaptic responses triggered by each cell in succession represented by the diagnonal 5-spike presynaptic stimulation. The color code represents different neurons. (f) a network diagram of functional synaptic connections observed from the 12 neuron patch-clamp.

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BMI

system for anterograde and retrograde tract tracing and (ix) and a 64 in vivo Plexon multichannel system for in vivo recording during behavior.


EPFL School of Life Sciences - 2009 Annual Report

BMI - Brain Mind Institute

Blue Brain Project Head of lab (PI) - http://bluebrain.epfl.ch Team members

Henry Markram Full Professor Director BBP

Demiri Barhdyl, administrative assistant Hill Sean, project manager for computational neuroscience Markram Kamila, project manager medical diagnostics, 01.10.09 Schürmann Felix, general project manager Keller Dan, postdoc Reid Michael, postdoc, (01.06.2009 - 31.10.2009) Sfyrakis Konstantinos, postdoc (until 31.01.2009) Kenyon John, engineer King James, engineer Walker Richard, administrative specialist Lasserre Sebastien, PhD student Ramaswamy Srikanth, PhD student Ranjan Rajnish, PhD student Tauheed Farhan, affiliated PhD student Druckmann Shaul, affiliated PhD student (Thesis October 2009) Gidon Albert, affiliated PhD student Hay Etay, affiliated PhD student Anastassiou Costas, visiting researcher, 31.03.2009 - 18.05.2009 Manninen Tiina, visiting researcher (01.03.2009 - 30.04.2009) Akiki Shadi, visiting student, student assistant Auhing Ricardo, visiting student Gapal Vasunder, visiting student McColgan Thomas, visiting student Melanson Michael, visiting student Menze Denis, visiting student Moor Ruben, visiting student Sreepathi Sarat, visiting student Ryckx Nick, civil servant Agarwal Piyush, summer student Geetanjali Saha, summer student Katiki Meghana, summer student Medina Felix Julian, student assistant Saxena Shreya, student assistant

Introduction

The combination of experiment and theory has long formed the basis of the scientific method. As computers become faster, computer simulations – combining experimental measurements and theoretical models – that capture the biological complexity of the brain become possible as demonstrated in the first phase of the Blue Brain Project. Four years into the project it is now possible to perform rapid “in silico” experiments on a virtual piece of cortex of a neonatal rat that would require complex technical apparatuses and years to carry out on biological tissue. We found that many biological findings can be reproduced using the virtual tissue and that even deeper insights can be obtained than possible on biological tissue.

Keywords

Neocortex, simulation-based research, reverse engineering, high performance computing

Results Obtained in 2009

The year 2008 marked the successful proof-of-concept for the Blue Brain Project that a novel modelling facility can be set up that automatically creates cellular models of neural circuitry in a completely data-driven fashion. In this, the electrophysiological, anatomical and genetic data of the Laboratory of Neural Microcircuitry plays a crucial role in the model

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generation and validation and a tight revision cycle between the modelling and experimentation has been established. In 2009 we started to use the Blue Brain facility for extensive “in silico” experimentation: on the one hand, we explored published protocols from other research groups covering from the single cell level to thalamocortical network dynamics and, on the other hand, we used the facility to complement experimental insights into the construction principles of the cortical column that wouldn’t be achievable by other means. In particular the question how each neuron precisely places thousands of synapses distributed onto different branches of many neurons: We found that when 3D morphologically reconstructed model neurons, obtained from many different experiments and animals, were placed in 3D in their laminar positions, that both the numbers and distributions of synapses formed along the presynaptic axon and postsynaptic dendrite closely matched experimental measurements for many different types of pre and postsynaptic pairs of neurons. This finding was independent of the precise positioning and orientation of the neurons within a neocortical layer. This finding indicates that synapse numbers and locations are mostly determined by only the geometrical characteristics of different types of neurons. We further found that the joint neurite density between

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EPFL School of Life Sciences - 2009 Annual Report

Beyond the extensive exploitation of the cellular level facility and model, extensions towards subcellular as well as macroscopic detail have been brought on the way. On EPFL’s side, for example, we have adopted several molecular simulators (MCell, STEPS) and started research of various subcellular systems (e.g., in collaboration with Ralf Schneggenburger’s LSYM laboratory). On large scale data basing of spatial data, a collaboration with Anastasia Ailamaki has been started. Outside of EPFL, the newly founded Cajal Blue Brain initiative of twelve Spanish principle investigators from Consejo Superior de Investigaciones Cientificas (CSIC) and the Technical University of Madrid (UPM) is supporting these efforts through BBP-aligned agendas in electron microscopy, data extraction and visualization. A collaboration with Christof Koch, on the other hand, explores the biophysical basis of local field potentials. The increased computing demands of exploitation and extension are met with an upgrade to a new IBM BlueGene/P supercomputer acquired by the cantons of Vaud and Geneva hosted in the multi-institution compute center CADMOS (Center for advanced modelling and simulation). The BlueGene/P has 16384 processors, 16Terabytes of RAM and 1 Petabytes of filesystem.

Selected publications

King, J., Hines, M., Hill, S., Goodman, P., Markram, H., and Schürmann, F. 2009. A Component-Based Extension Framework for Large-Scale Parallel Simulations in NEURON, Frontiers in Neuroinformatics, available online, doi:10.3389/ neuro.11.010.2009 Anwar, H., Riachi, I., Hill, S., Schürmann, F. and Markam, H., 2009. An approach to capturing neuron morphological diversity. In: Computational modeling methods for neuroscientists. DeSchutter, E. (Ed.), Neuronal Modeling. MIT Press. Druckmann, S., Berger, T., Hill, S., Schürmann, F., Markram, H., and Segev, I., 2008. Evaluating automated parameter constraining procedures of neuron models by experimental and surrogate data, Biol Cybern., 99(4-5):371-9 Hernando Vieites, J., Schürmann, F., Markram, H., and de Miguel Anasagasti, P. 2008: RTNeuron, an application for interactive visualization of detailed cortical column simulations. Jornadas de Paralelismo conference, Spain Hines, M. L., Markram, H. and Schürmann, F., 2008b. Fully implicit parallel simulation of single neurons. J Comput Neurosci., 25(3) :439-48 Hines, M. L., Eichner, H. and Schürmann, F., 2008a. Neuron splitting in compute-bound parallel network simulations enables runtime scaling with twice as many processors. J Comput Neurosci. 25(1), 203-210. Kozloski, J., Sfyrakis, K., Hill, S., Schürmann, F., Peck, C. and Markram, H., 2008. Identifying, tabulating, and analyzing contacts between branched neuron morphologies. IBM Journal of Research and Development. 52. Druckmann, S. et al., A Novel Multiple Objective Optimization Framework for Constraining Conductance-Based Neuron Models by Experimental Data, Frontiers in Neuroscience, Vol. 1, Issue 1, 2007

The Blue Brain Project has access to a number of powerul computer and IT resources. Of note are two powerful and complementary computational resources: a massively parallel, highly interconnected IBM BlueGene/P and a large sharedmemory SMP SGI Prism Extreme. Access to the BlueGene/P is provided by CADMOS and the installed configuration is a 4-rack system with 4094 nodes (16384 cores), 56TeraFlops (peak performance), 16TeraByte of distributed memory, 1PetaByte file system. The SGI system, directly owned by the BBP features 32 Itanium2 processors, 300GigaBytes sharedmemory and 16 graphics card, and is used for data analysis and visualization. The two systems are interconnected through dedicated 100GB fibre optic cables for real-time visualization of simulation results. Shown on the right is the reconstructed neocortical column.

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BMI

the morphological classes is the key parameter that predicts both the innervation profile and mean number of synaptic contacts for a given synaptic connection. Since spine lengths, neurite diameter, tortuosity and branching frequency and angles determine joint neurite density, the statistical properties of neuron morphology determine the wiring diagram of neural circuits. An entire circuit with the full diversity of neuron morphologies found in the neocortical column therefore emerges with the “correct” synaptic numbers and locations without any higher level or plasticity-dependent specification.


EPFL School of Life Sciences - 2009 Annual Report

BMI - Brain Mind Institute

Moore Lab Head of Lab (PI) - http://moorelab.epfl.ch

Team members

Liliane Glauser, Lab technician / lab manager Agata Kosinska, PhD student David Ramonet, Postdoctoral fellow Caroline Rheiner, Administrative assistant Klodjan Stafa, PhD student Alzbeta Trancikova, Postdoctoral fellow

Darren Moore

Tenure Track Assistant Professor

Introduction

The Laboratory of Molecular Neurodegenerative Research (LMNR) investigates the pathophysiology of Parkinson’s disease, a chronic neurodegenerative movement disorder. Our laboratory investigates the normal biological function and pathological dysfunction of various proteins, that when genetically mutated, cause an inherited (familial) form of Parkinson’s disease. Our mission is to understand the molecular mechanisms and pathways through which diseaseassociated mutations in these proteins cause neuronal damage and neurodegeneration. Our research employs a range of molecular, cellular and biochemical approaches to understand Parkinson’s disease and uses baker’s yeast, cultured cells and neurons, human brain tissue and genetically modified model organisms to create disease models. We aim to use this information to develop novel therapies and neuroprotective strategies to delay or prevent this devastating disease.

Keywords

Parkinson’s disease, parkinsonism, neurodegeneration, genetic mutations, disease models, neuronal cell death, leucine-rich repeat kinase 2 (LRRK2), parkin, α-synuclein, ATP13A2, therapeutic targets

Results Obtained in 2009

The Moore laboratory focuses its investigations on a number of proteins that when mutated cause familial Parkinson’s disease. These proteins include leucinerich repeat kinase 2 (LRRK2), α-synuclein, parkin and ATP13A2. Mutations in the LRRK2 and α-synuclein genes cause autosomal dominant forms of disease, whereas parkin and ATP13A2 mutations cause autosomal recessive disease. Mutations in the LRRK2 gene were first discovered in 2004 and we have been working over the years to model the pathogenic effects of these dominant mutations. In 2009, we continued to develop and phenotype a collection of novel transgenic mice that we recently created to over express mutated forms of human LRRK2 protein in the brain. These transgenic models develop some features of Parkinson’s disease with advanced age and will prove extremely useful for understanding how

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LRRK2 mutations cause neurodegenerative disease. At the same time, we have developed a basic model in the baker’s yeast, Saccharomyces cerevisiae, to further understand the molecular pathobiology of LRRK2 and we have used this model to identify genes that can modify LRRK2-dependent phenotypes. We are actively investigating the mechanisms through which the proteins derived from these genes interact with the LRRK2 protein in neuronal models. Finally, our research is attempting to clarify the mechanisms underlying neuronal cell death induced by mutated LRRK2 and here we continue to focus on the role of mitochondrial dysfunction, neuronal morphology and proteins or protein complexes that interact with, or are phosphorylated by, LRRK2. Our research into α-synuclein has focused on the development of new and improved genetic animal models to recreate the key features of Parkinson’s disease caused by mutations in this gene. We developed conditional transgenic mice that selectively over express mutated forms of human α-synuclein protein in dopaminergic neurons of the nigrostriatal pathway, the principal neurons that are destroyed in Parkinson’s disease and cause abnormal movement. These transgenic mice exhibit reduced levels of dopamine in the striatum, similar to Parkinson’s disease patients, and will prove useful for further dissecting the pathological properties of α-synuclein. In 2009 we also began a new project on the ATP13A2 protein. Genetic mutations in ATP13A2 cause familial Parkinson’s disease and Kufor-Rakeb syndrome, a juvenile-onset, autosomal recessive familial disorder characterized by pallido-pyramidal neurodegeneration with severe parkinsonism and dementia. ATP13A2 is a novel P-type ATPase protein which is thought to use ATP hydrolysis to transport cations across intracellular vesicular membranes such as lysosomes. We are attempting to understand the normal function of ATP13A2 in neurons, and we are creating disease models based upon gene disruption or silencing to replicate the effects of recessive “lossof-function” mutations.

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Selected publications

Xiong, Y., Coombes, C.E., Kilaru, A., Li, X., Gitler, A.D., Bowers, W.J., Dawson, V.L., Dawson, T.M., Moore, D.J. (2009). GTPase activity plays a key role in the pathobiology of LRRK2. PLoS Genetics. In press. Vives-Bauza, C., Zhou, C., Huang, Y., Cui, M., de Vries, R.L., Kim, J., May, J., Tocilescu, M.A., Liu, W., Ko, H.S., Magrane, J., Moore, D.J., Dawson, V.L., Grailhe, R., Dawson, T.M., Li, C., Tieu, K., Przedborski, S. (2009). PINK1-dependent recruitment of Parkin to mitochondria in mitophagy. Proc. Natl. Acad. Sci. U. S. A. 107(1):378-83. Daher, J-P.L., Ying, M., Banerjee, R., McDonald, R.S., Hahn, M.D., Yang, L., Beal, M.F., Thomas, B., Dawson, V.L., Dawson, T.M., Moore, D.J. (2009). Conditional transgenic mice expressing C-terminally truncated human α-synuclein (Syn119) exhibit reduced striatal dopamine without loss of nigrostriatal pathway dopaminergic neurons. Mol. Neurodegeneration 4:34. Higashi, S., Moore, D.J., Yamamoto, R., Minegishi, M., Sato, K., Togo, T., Katsuse, O., Uchikado, H., Furukawa, Y., Hino, H., Kosaka, K., Emson, P.C., Wada, K., Dawson, V.L., Dawson, T.M., Arai, H., Iseki, E. (2009). Abnormal localization of leucine-rich repeat kinase 2 to the endosomal-lysosomal compartment in Lewy body disease. J. Neuropathol. Exp. Neurol. 68(9):994-1005.

Dawson, T.M., Moore, D.J., West, A.B. (2009). Revelations and revolutions in the understanding of Parkinson’s disease. Biochim. Biophys. Acta 1792:585-6. Ko, H.S., Bailey, R., Smith, W.W., Liu, Z., Shin, J-H., Lee, Y-I., Zhang, Y-J., Jiang, H., Ross, C.A., Moore, D.J., Patterson, C., Petrucelli, L., Dawson, T.M., Dawson, V.L. (2009). CHIP regulates leucine-rich repeat kinase-2 ubiquitination, degradation and toxicity. Proc. Natl. Acad. Sci. U.S.A. 106:2897-902. Moore, D.J., Dawson, T.M. (2008). Value of Genetic Models in Understanding the Cause and Mechanisms of Parkinson’s Disease. Curr. Neurol. Neurosci. Rep. 8:288-96. Moore, D.J. (2008). The Biology and Pathobiology of LRRK2: Implications for Parkinson’s Disease. Parkinsonism Relat. Disord. 14:S92-98. Wang, L., Xie, C., Greggio, E., Parisiadou, L., Shim, H., Sun, L., Chandran, J., Lin, X., Lai, C., Yang, W.J., Moore, D.J., Dawson, T.M., Dawson, V.L., Chiosis, G., Cookson, M.R., Cai, H. (2008). The chaperone activity of heat shock protein 90 is critical for maintaining the stability of leucine-rich repeat kinase 2. J. Neurosci. 28:3384-91. Moore, D.J., West, A.B., Dikeman, D.A., Dawson, V.L., Dawson, T.M. (2008). Parkin mediates the degradation-independent ubiquitination of Hsp70. J. Neurochem.105:1806-19

LRRK2 biology and pathophysiology. Top left image: LRRK2 in dementia with Lewy bodies (DLB). Co-localization of LRRK2 with the lysosomal marker, LAMP2, and the endosomal marker, Rab7B, in abnormal neuronal vacuoles in affected regions of post-mortem brains from human subjects with DLB. Top right image: A yeast model of LRRK2 cytotoxicity. Inducible expression LRRK2 protein domains in Saccharomyces cerevisiae reduces cell viability. Lower image: Domain architecture of the LRRK2 protein highlighting functional domains, familial disease-associated mutations (below), and key functional residues and motifs (above).

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BMI

EPFL School of Life Sciences - 2009 Annual Report


EPFL School of Life Sciences - 2009 Annual Report

BMI - Brain Mind Institute

Petersen Lab Head of Lab (PI) - http://lsens.epfl.ch

Team members

Carl Petersen

Rachel Aronoff, Postdoctoral fellow Michael Avermann, PhD student Sylvain Crochet, Visiting scientist (permanent position is at the University of Lyon) Luc Gentet, Postdoctoral fellow Gordana Kokorus, Administrative assistant Natalya Korogod, Postdoctoral fellow (collaboration with Graham Knott) Yves Kremer, Postdoctoral fellow Céline Matéo, PhD student Ferenc Mátyás, Postdoctoral fellow Aurelie Pala, PhD student James Poulet, Postdoctoral fellow Shankar Sachidhanandam, Postdoctoral fellow Nadia Urbain, Postdoctoral fellow

Associate Professor (since January 2010)

Introduction

Sensory perception is an active process in which neurons in the brain construct an internal representation of the world. Our goal is to obtain causal and mechanistic explanations for simple forms of sensory perception in mice at the level of individual neurons and their synaptic connections.

Keywords

Neocortex, Synaptic transmission, Sensory perception, Learning, Whisker behaviour, Barrel cortex, Cortical circuits, Whole-cell recording, Voltage-sensitive dye Imaging, Calcium-sensitive dye imaging, Two photon microscopy, Lentivirus, Adeno-associated virus

Results Obtained in 2009

Tactile sensory information is processed in the primary somatosensory cortex. In mice, a large part of the primary somatosensory cortex is devoted to processing information from the mystacial vibrissae. Each whisker on the snout of the mouse is individually represented as an anatomically defined barrel in the mouse primary somatosensory neocortex. We focus our investigations on the processing of sensory information from one specific well-defined whisker, the ‘C2 whisker’. Sensory information relating to deflection of the C2 whisker is processed in the C2 barrel column of primary somatosensory neocortex. The C2 barrel column is composed of approximately 6,500 neurons, some of which interact with each other through synaptic connections. The internal computations taking place in the microcircuits of the C2 barrel column might underlie the first steps in generating a sensory percept for the mouse derived from tactile information from the C2 whisker. In order to understand how information is processed in the C2 barrel column, we clearly need to know how the individual neurons transmit information to each other. In order to directly measure the functional synaptic connectivity of the C2 barrel column, we made multiple simultaneous whole-cell recordings from neurons

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specifically located in the C2 barrel column (Lefort et al., 2009). The C2 barrel column was functionally mapped using in vivo intrinsic optical imaging and subsequently the C2 barrel column was specifically labelled with red fluorescent dye. The brain was then sectioned in order to prepare brain slices for in vitro electrophysiological analyses. The fluorescently labelled slice was identified and whole-cell recordings were obtained from excitatory neurons in different cortical layers. By carrying out many experiments, we obtained statistical data on the layer-specific patterns of synaptic connectivity in the mouse C2 barrel column. We found that neurons in layer 4 made prominent synaptic connections with neurons in all other layers. Layer 4 is the layer in which thalamic afferents terminate most densely and it is thus often considered the input layer where sensory information first reaches the neocortex. The high output connectivity of layer 4 therefore provides a signalling pathway, which can rapidly distribute sensory information to other cortical layers. In addition, we identified prominent excitatory connectivity from supragranular to infragranular layers. Perhaps of more general importance, we found an interesting distribution of the amplitudes of unitary excitatory postsynaptic potentials, which had a long tail indicating the presence of a few very large synaptic connections in the neocortex. Computational modelling suggested that these few large excitatory connections might dominate the activity of the neocortex. These strong synaptic connections were also found to be reliable transmitters of information with low variance. Sparse but strong connections in the neocortex might therefore form a backbone for reliable sensory processing. The measurements of excitatory synaptic connectivity in the mouse C2 barrel column have therefore begun to provide interesting information, which might help us understand how sensory processing occurs in this brain area. In future studies, it will be of great interest to further define the excitatory and inhibitory connectivity of genetically defined neocortical populations of neurons.

© Copyright 2004-2010 EPFL for all material published in this report info.sv@epfl.ch


Selected publications

Crochet S, Petersen CCH (2009) Cortical dynamics by layers. Neuron 64: 298-300.

Aronoff R, Petersen CCH (2008) Layer, column and celltype specific genetic manipulation in mouse barrel cortex. Front Neurosci 2: 64-71.

Petersen CCH (2009) Genetic manipulation, whole-cell recordings and functional imaging of the sensorimotor cortex of behaving mice. Acta Physiol 195: 91-99.

Poulet JFA, Petersen CCH (2008) Internal brain state regulates membrane potential synchrony in barrel cortex of behaving mice. Nature 454: 881-885.

Lefort S, Tomm C, Sarria JCF, Petersen CCH (2009) The excitatory neuronal network of the C2 barrel column in mouse primary somatosensory cortex. Neuron 61: 301-316.

Badel L, Lefort S, Brette R, Petersen CCH, Gerstner W, Richardson MJ (2008) Dynamic I-V curves are reliable predictors of naturalistic pyramidal-neuron voltage traces. J Neurophysiol 99: 656-666.

Gersbach M, Boiko DL, Niclass C, Petersen CCH, Charbon E (2009) Fast-fluorescence dynamics in nonratiometric calcium indicators. Optics Letters 34: 362-364. Helmchen F, Petersen CCH (2008) New views into the brain of mice on the move. Nat Methods 5: 925-926. Badel L, Lefort S, Berger TK, Petersen CCH, Gerstner W, Richardson MJ (2008) Extracting non-linear integrate-andfire models from experimental data using dynamic I-V curves. Biol Cybern 99: 361-370.

Ascoli GA, Alonso-Nanclares L, Anderson SA, Barrionuevo G, Benavides-Piccione R, Burkhalter A, Buzsáki G, Cauli B, DeFelipe J, Fairén A, Feldmeyer D, Fishell G, Fregnac Y, Freund TF, Gardner D, Gardner EP, Goldberg JH, Helmstaedter M, Hestrin S, Karube F, Kisvárday ZF, Lambolez B, Lewis DA, Marin O, Markram H, Muñoz A, Packer A, Petersen CCH, Rockland KS, Rossier J, Rudy B, Somogyi P, Staiger JF, Tamas G, Thomson AM, Toledo-Rodriguez M, Wang Y, West DC, Yuste R (2008) Petilla terminology: nomenclature of features of GABAergic interneurons of the cerebral cortex. Nature Rev Neurosci 9: 557-568.

An illustration of some elements of a neocortical column: upper face shows red fluorescent labelling of the functionally mapped location of the C2 barrel column; right face shows DAPI labelling of nuclei throughout the depth of the cortical layers; and front face shows a few specific excitatory pyramidal neurons filled with biocytin and stained to reveal their dendritic and axonal arborisations.

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35

BMI

EPFL School of Life Sciences - 2009 Annual Report


EPFL School of Life Sciences - 2009 Annual Report

BMI - Brain Mind Institute

Sandi Lab Head of Lab (PI) - http://lgc.epfl.ch Team members

Carmen Sandi

Associate Professor

Reto Bisaz, PhD Student Pere Boadas, Trainee Jorge Eduardo Castro Cifuentes, PhD Student Lisa Conboy, Postdoctoral Fellow Maria Isabel Cordero Campana, External Employee Shanaz Diessler, Trainee Mark Fajans, Trainee Martina Fantin, Postdoctoral Fellow Virginia Gao, Trainee David Genoux, Postdoctoral Fellow Barbara Goumaz, Administrative Assistant Keerthana Iyer, Trainee Marianne Larsen, Trainee Wiktor Lisowski, Trainee Gediminas Luksys, PhD Student Cristina Marquez, Postdoctoral Fellow Grégoire Parchet, Technician Nicolas Perret, Trainee Guillaume Poirier, Postdoctoral Fellow Clara Rossetti, Techician Basira Salehi, PhD Student Yannick Sevelinge, Postdoctoral Fellow Coralie Siegmund, Technician Ioanna Steinmann, Administrative Assistant Marjan Timmer, PhD Student Stamatina Tzanoulinou, PhD Student Angélique Vaucher, Technician Vandana Veenit, PhD Student Philip Vercruysse, Trainee Fabienne Vionnet, Administrative Assistant

Introduction

The Laboratory of Behavioural Genetics investigates the impact and mechanisms whereby stress affects brain function and cognition, with a focus on learning and memory processes and on psychiatric disorders - such as anxiety, depression, and violence.

Keywords

Stress – Learning – Memory – Violence - Neuroplasticity – Cell adhesion molecules – Glutamate receptors

Results Obtained in 2009 Neural and molecular correlates of abnormal aggression

Intensive research is devoted to unravel the neurobiological mechanisms mediating adult hippocampal neurogenesis, its regulation by antidepressants, and its behavioral consequences. Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine that is expressed in the CNS, where its function is unknown. We have shown, for the first time, the relevance of MIF expression for adult hippocampal neurogenesis. We have identified MIF expression in neurogenic cells (in stem cells, cells undergoing proliferation, and in newly proliferated cells undergoing maturation) in the subgranular zone of the rodent dentate gyrus. A causal function for MIF in cell proliferation was shown using genetic (MIF gene

36

deletion; in collaboration with the group of Thierry Calandra at the CHUV, Lausanne) and pharmacological (treatment with the MIF antagonist Iso-1; in collaboration with the group of Hilal Lashuel at the BMI) approaches. Behaviorally, genetic deletion of MIF resulted in increased anxiety- and depressionlike behaviors, as well as of impaired hippocampusdependent memory. Our study provides evidence supporting a pivotal function for MIF in both basal and antidepressant-stimulated adult hippocampal cell proliferation. Moreover, loss of MIF results in a behavioral phenotype that, to a large extent, corresponds with alterations predicted to arise from reduced hippocampal neurogenesis. Furthermore, these findings underscore MIF as a potentially relevant molecular target for the development of treatments linked to deficits in neurogenesis, as well as to problems related to anxiety, depression, and cognition (Conboy et al., in press).

Stress at learning facilitates memory formation by regulating AMPA receptor trafficking through a glucocorticoid action

Stress and glucocorticoids can facilitate memory formation. However, the molecular mechanisms mediating their effects are largely unknown. AMPA receptor (AMPAR) trafficking has been implicated in the changes in synaptic strength at central glutamatergic synapses associated with memory formation.

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Using the water maze spatial task involving different stress levels, mice trained under more stressful conditions (water at 22°C) showed better learning and memory, and higher post-training corticosterone levels, than mice trained under lower stress (water at 30°C). Strikingly, this facilitated learning by stress was accompanied by enhanced synaptic expression of GluR2 AMPARs that was not observed in mice trained under less stressful conditions. We also found evidence for the involvement of the neural cell adhesion molecule N-cadherin in interaction with GluA2. These findings underscore a new mechanism whereby stress can improve memory function (Conboy and Sandi, in press).

Modelling stress effects on behaviour

In collaboration with the group of Wulfram Gerstner at the BMI, we used a reinforcement learning approach for model-based analysis of rodent learning in two tasks: the Morris water maze and the holebox conditioning. Using two inbred strains of mice (C57BL/6 and DBA/2), we investigated how different stressors – extrinsic (e.g. prior exposure to an elevated platform) and intrinsic (e.g. food deprivation in the hole-box or water temperature in the water maze) – affect animal learning and behavioural performance. In the hole-box study, we also pharmacologically manipulated the noradrenergic system. Then, we interpreted the effects of experimental manipulations using best-fitting parameters of reinforcement learning models, such as the learning rate, the exploration-exploitation balance, and the future reward discounting. We observed that stress and noradrenaline led to higher exploitation of current knowledge and smaller consideration of future rewards, although its effects were dependent on genetic strain, stress type, and other factors (Luksys et al., 2009).

Selected publications

Conboy L., Varea E., Castro J.E., Sakouhi-Ouertatani H., Calandra T., Lashuel H. and Sandi C. Macrophage migration inhibitory factor (MIF) is critically involved in basal and fluoxetine-stimulated adult hippocampal cell proliferation and in anxiety, depression and memory related behaviours. Mol. Psychiatry, in press.

Conboy L. and Sandi C. Stress at learning facilitates memory formation by regulating AMPA receptor trafficking through a glucocorticoid action. Neuropsychopharmacology, in press. Luksys G., Gerstner W., and Sandi C. (2009) Stress, genotype and norepinephrine in prediction of mouse behavior using reinforcement learning. Nat. Neurosci. 12:11801186. Sandi C. and Richter-Levin G. (2009) From high anxiety trait to depression: A neurocognitive hypothesis, Trends Neurosci. 32:312-320. Bisaz R., Conboy L. and Sandi C. (2009) Learning under stress: A role for the neural cell adhesion molecule NCAM. Neurobiol. Learn, Mem. 91:333-342. Luethi M, Meier B., and Sandi C. (2009) Stress effects on working memory, explicit memory, and implicit memory for neutral and emotional stimuli. Front. Behav. Neurosci., DOI:10.3389/neuro 08.005.2008. Conboy L., Tanrikut C., Zoladz P.R., Campbell A.M., Park C.R., Gabriel C., Mocaer E., Sandi C. and Diamond D.M. (2009) The antidepressant agomelatine blocks the adverse effects of stress on memory and enables spatial learning to rapidly increase neural cell adhesion molecule (NCAM) expression in the hippocampus of rats. Int. J. Neuropsychopharmacol. 12:329-341. Jakobsson J., Cordero M.I., Bisaz R., Groner A.C., Busskamp V., Bensadoun J.C., Cammas F., Losson R., Mansuy I.M., Sandi C. and Trono D. (2008) KAP1-mediated epigenetic repression in the forebrain modulates behavioral vulnerability to stress. Neuron 60:818-831. Sandi C., Cordero M.I., Ugolini A., Varea E., Caberlotto L. and Large C. (2008) Chronic stress-induced alterations in amygdala responsiveness and behavior: Modulation by trait anxiety and CRF systems. Eur. J. Neurosci. 28:18361848. Markram K., Rinaldi T., La Mendola D., Sandi C. and Markram H. (2008) Abnormal fear conditioning and amygdala processing in an animal model of autism. Neuropsychopharmacology 33:901-912.

Distribution of Macrophage migration inhibitory factor (MIF) in the brain. (a) Panoramic view of the hippocampus. (b) Detailed view of the dentate gyrus of the hippocampus. (c) A more detailed view shows the presence of MIF immunoreactive cells in the SGZ, a neurogenic area of the adult brain. Asterisks mark cells MIF IR in the subgranular zone with the unipolar characteristic morphology; fd = fascia dentata. Examples of expression of MIF in other brain regions: Rostral migratory stream (d), Neo-Cortex (e) and white matter (f). Scale bar: 1 mm (a), 200 μm (b,d,e,f) and 50 μm (c).

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37

BMI

EPFL School of Life Sciences - 2009 Annual Report


EPFL School of Life Sciences - 2009 Annual Report

BMI - Brain Mind Institute

Schneggenburger Lab Head of Lab (PI) - http://lsym.epfl.ch

Team members

Laure Dayer, Administrative Assistant Zhizhong Dong, Postdoctoral Fellow Özgür Genc, Phd Student Juan Goutman, Postdoctoral Fellow Yunyun Han, Phd Student Olexiy Kochubey, Postdoctoral Fellow Nicolas Michalski, Postdoctoral Fellow Heather Murray, Lab Technician David Perkel, Invited Professor Le Xiao, Phd Student

Ralf Schneggenburger Associate Professor

Introduction

In the brain, nerve cells are arranged in intricate neuronal networks, and communicate with each other at synapses, in a process called “synaptic transmission”, which involves the Ca2+-dependent fusion of transmitter-loaded vesicles with the plasma membrane. Synaptic transmission is the only means of fast information transfer between neurons. Therefore, a detailed understanding of the signalling mechanisms in synaptic transmission is an important pre-requisite to understand how information is processed in neuronal circuits.

Keywords

Synaptic transmission, nerve terminal, Ca2+, second messenger, short-term plasticity, Synaptogenesis, auditory system

Results Obtained in 2009

At synapses, vesicles fuse with the presynaptic plasma membrane following the entry of Ca2+ through voltage-gated Ca2+ channels. In order to study transmitter release under direct electrical control of the presynaptic nerve terminal, we use a large glutamatergic model synapse in the auditory brainstem circuit, the calyx of Held. At this synapse, whole-cell patch-clamp recordings can be routinely performed at the presynaptic nerve terminal, and Ca2+ indicator dyes as well as light-sensitive Ca2+ chelators can be loaded via the patch-pipette directly into the presynaptic nerve terminal. This allow us to measure and control the intracellular Ca2+ concentration ([Ca2+]i) in the nerve terminal by near-UV light; which is not possible at most other synapses. Using these techniques of Ca2+-imaging and Ca2+uncaging, we could show in 2009 that the intrinsic Ca2+ sensitivity of vesicle fusion remains unchanged during developmental refinement at the calyx of Held synapse. On the other hand, when using brief presynaptic depolarizations to open voltage-gated

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Ca2+ channels, we found that Ca2+ entry through Ca2+ channels became significantly more effective in triggering transmitter release (see Figure). These results suggests that the physical distance between Ca2+ channels and docked vesicles, which is in the range of 10’s of nanometers, becomes reduced upon developmental maturation of the synapse (Kochubey, Han & Schneggenburger, 2009). In 2009, we have started to address the molecular mechanisms that determine the distance between Ca2+ channels and vesicles. We are especially interested in RIM proteins (for Rab3a-interacting molecules), a family of presynaptic scaffolding proteins. To enable the conditional removal of presynaptic proteins with the Cre-lox system, we have identified several Cre mouse lines which express Cre recombinase specifically in the auditory brainstem (Yunyun Han, Le Xio; Nicolas Michalski, ongoing). These Cre mouse lines will allow us to remove presynaptic proteins specifically at the calyx of Held, using floxed alleles of presynaptic proteins. The availability of specific Cre mouse lines will also enable us to study the function of signalling pathways in the development of the large calyx of Held synapses (Le Xiao; Nicolas Michalski; ongoing). Lastly, we have been studying the role of Synaptotagmin-2, the likely Ca2+ sensor for the fast phase of transmitter release at the calyx of Held. Syt-2, like its close homologue Syt-1 in forebrain synapses, is a vesicular transmembrane protein, with two C2 domains (C2A and C2B), which bind phospholipids in a Ca2+-dependent manner. To study the function of these proteins at the calyx of Held, we have established a «rescue» approach of Syt-2 function using Syt-2 k.o. mice and adenovirus-vector mediated overexpression of proteins. We have studied the effects of mutating separate residues in the C2B domain on the regulation of Ca2+-evoked and spontaneous release (Kochubey and Schneggenburger, in preparation).

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Selected publications

Kochubey O., Han Y., Schneggenburger R. (2009) Developmental regulation of the intracellular Ca2+ sensitivity of vesicle fusion and Ca2+ -secretion coupling at the rat calyx of Held. J Physiology 587: 3009-3023. Lou, X., Korogod, N., Betz, A., Brose, N., Schneggenburger, R. (2008) Phorbol esters modulate spontaneous and Ca2+ -evoked transmitter release via acting on both munc-13 and protein kinase C. J. Neuroscience 28: 8257 - 8267.

Müller, M., Felmy, F., Schneggenburger, R. (2008) A limited contribution of Ca2+-current facilitation to paired-pulse facilitation of transmitter release at the rat calyx of Held. J. Physiology 586: 5503 - 5520. Schneggenburger, R. (2008) “Exocytosis: Ca2+-sensitivity”. The New Encyclopedia of Neuroscience, edited by Larry Squire et al., 2008.

Developmental refinement of Ca2+ - secretion coupling at the calyx of Held synapse. A, Presynaptic Ca2+ currents following brief voltage-clamp depolarizations to 0 mV (upper panel), and the resulting excitatory postsynaptic currents, EPSCs (lower panel). B, Plot of EPSC amplitude, as a measure of transmitter release, versus the Ca2+ current charge in the presynaptic nerve terminal. Note the highly nonlinear relationship between both quantities (double-logarithmic plot). The data from the more mature synapses (black data points; postnatal days 12 - 14) are leftward-shifted, showing that presynaptic Ca2+ currents become more efficient in driving transmitter release upon developmental maturation. Taken, with permission, from Kochubey, Han & Schneggenburger 2009, J. Physiology 587: 3009 - 3023.

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39

BMI

EPFL School of Life Sciences - 2009 Annual Report


EPFL School of Life Sciences - 2009 Annual Report

External Adjunct Professor

Molinari Group Head of Lab (PI) - http://www.irb.ch/

Team Members

Riccardo Bernasconi, PhD student Siro Bianchi, Technician Verena Calanca, Technician Carmela Galli, Scientific collaborator Danilo Presotto, PhD student Tatiana SoldĂ , Scientific collaborator

Maurizio Molinari

External Adjunct Professor Institute for Research in Biomedicine Bellinzona

Introduction

Cell and organism homeostasis relies on a balanced activity of the protein folding, quality control and degradation machineries operating in the lumen of the endoplasmic reticulum (ER). The capacity to manipulate protein folding, quality control and degradation will be instrumental to delay progression or even to cure diseases caused by inefficient functioning of the cellular protein factory such as cystic fibrosis and several types of tumor and of neurodegenerative diseases. It will also increase productivity of recombinant proteins to be employed in the clinics and in the industry (reviewed in Hebert, D.N. and Molinari, M. (2007) Physiol. Rev. 87, 1377-1408).Results obtained in 2009 Recognition and dislocation across the ER membrane of terminally misfolded polypeptides. The endoplasmic reticulum of eukaryotic cells is the site of maturation for proteins destined to intracellular organels of the endocytic and secretory pathway, to the plasma membrane and to the extracellular space. Part of our work aims at the characterization of mechanisms that regulate clearance from the ER of terminally misfolded polypeptides (ER-associated degradation, ERAD). Most polypeptides entering the ER lumen are covalently modified at asparagine side chains with glucose3-mannose9-N-acetylglucosamine2- oligosaccharides. Their maturation is assisted by a dedicated folding machinery comprising the oligosaccharide-binding chaperones calnexin and calreticulin and the oxidoreductase ERp57. Processing of oligosaccharides displayed on misfolded conformers by ER-resident Îą1,2-mannosidases irreversibly extracts folding-defective polypeptides from the lectin-operated folding machinery. We have characterized the function in ER quality control of few proteins derived from alternative splicing of the OS9 and the XTP3-B genes. These proteins are ubiquitously expressed in human tissues, are amplified in tumors and are transcriptionally induced upon activation of

the Ire1/Xbp1 ER-stress pathway. They do not associate with folding-competent proteins. Rather, they selectively bind folding-defective ones thereby inhibiting transport of non-native conformers through the secretory pathway. Their intralumenal level inversely correlates with the fraction of folding-defective glycoproteins that escapes retention-based ER quality control. OS9 and XTP3-B play a dual role in mammalian ER quality control: firstly as crucial retention factors for misfolded conformers, and secondly as promoters of protein disposal from the ER lumen. We have defined OS-9 and XTP3-B as ERAD shuttles because they transport soluble, extensively demannosylated terminally misfolded glycopolypeptides (ERAD-Ls substrates) from the ER lumen to the site of dislocation across the ER membrane. The ERAD shuttles deliver ERAD-Ls substrates to a multiprotein complex comprising the membrane receptor SEL1L, the associated E3 ubiquitin ligase HRD1 and an elusive dislocation (retro-translocation) channel. Extensively de-mannosylated membrane-tethered polypeptides with the same luminal defects (ERADLm substrates) are cleared from the ER lumen even upon complete inactivation of the ERAD-Ls pathway. ERAD tuning Another focus of our research is the characterization of mechanisms that regulate the content in the ER lumen of molecular chaperones assisting rate-limiting steps in the processes of clearance of misfolded polypeptides from the ER lumen. This regulation is crucial for maintenance of cell and organism homeostasis because hyper-activation of the ER disposal machinery causes premature interruption of ongoing folding programs (folding-intermediates are improperly recognized as misfolded end-products and are cleared from the folding environment before they have a chance to attain the native structure). EDEM1 is a crucial ERAD regulator that extracts non-native glycopolypeptides from the folding ma-


EPFL School of Life Sciences - 2009 Annual Report

chinery that has been characterized in our lab (Science 299, 1397). Under normal growth conditions, the intralumenal level of EDEM1 must be low to prevent premature interruption of ongoing folding programs. Our studies showed that in unstressed cells, EDEM1 is segregated from the bulk ER into LC3-Icoated vesicles and is rapidly degraded. The rapid turnover of EDEM1 (and of other ERAD regulators) has been named ERAD tuning. It is regulated by a novel mechanism that shows similarities but is clearly distinct from macroautophagy. Cells with defective EDEM1 turnover contain unphysiologically high levels of EDEM1, show enhanced ERAD activity and are characterized by impaired capacity to efficiently complete maturation of model glycopolypeptides.

Selected publications

Hebert, D.N. Bernasconi, R. and Molinari, M. (2010) ERAD Substrates: Which Way Out? Semin. Cell Dev. Biol. IN PRESS Aebi, M., Bernasconi, R., Clerc, S. and Molinari, M. (2010) N-Glycan Structures: Recognition and Processing in the ER. TIBS 35, 74-82. Bernasconi, R., Galli, C., Calanca, V., Nakajima, T. and Molinari, M. (2010) Stringent requirement for HRD1, SEL1L and

OS-9/XTP3-B for disposal of ERAD-Ls substrates. J. Cell Biol. 188, 223-235.- Highlights in J. Cell Biol. Calì, T., Galli, C., Olivari, S. and Molinari, M. (2008) Segregation and Rapid Turnover of EDEM1 Modulates Standard ERAD and Folding Activities. Biochem. Biophys. Res. Commun. 371, 405-410. Bernasconi, R., Pertel, T., Luban, J. and Molinari M. (2008) A Dual Task for the Xbp1-Responsive OS-9 Variants in the Mammalian ER: Inhibiting Secretion of Misfolded Protein Conformers and Enhancing Their Disposal. J. Biol. Chem. 283, 16446-16454. Vanoni, O., Paganetti, P. and Molinari, M. (2008) Consequences of Individual N-Glycan Deletions and of Proteasomal Inhibition on Secretion of Active BACE. Mol. Biol. Cell 19, 4086-4098. Bernasconi, R. and Molinari M. (2008) ER-Associated Folding and Degradation: Learning From Yeast? In Protein misfolding: New Research O’Doherty, C.B. and Byrne, A.C. Eds, Nova Science Publishers, Inc., Hauppauge, NY, 113-123. Calì, T., Vanoni, O. and Molinari, M. (2008) The Endoplasmic Reticulum: Crossroads for Newly Synthesized Polypeptide Chains. Progress in Mol. Biol. Transl. Sci. 83, 135-179.

The ERAD mechanism as depicted in this figure, can be divided in four steps: First, the folding-incompetent glycoprotein is extracted from the calnexin cycle through the concerted activity of several mannose-trimming enzymes (GH47); Second, the lectin ERAD shuttles OS-9 and XTP3-B transport the misfolded glycopolypeptide from the ER lumen to the membrane-anchored adaptor protein SEL1L, which is associated with the E3 ubiquitin ligase HRD1; Third, the misfolded glycopolypeptide has to be unfolded (e.g., reduction of aberrant disulfide bonds and cis/trans isomerization of peptidyl-prolyl bonds) in order to; Fourth, allow dislocation across the ER membrane through an elusive channel, poly-ubiquitylation and degradation by the 26S proteasome. The different mannose composition of the N-glycan is depicted in color code: gray, oligosaccharide with nine mannose (Man9); red, five to seven mannose residues (Man5-7); and white, oligosaccharide with an undefined number of mannose residues.


EPFL School of Life Sciences - 2009 Annual Report

External Adjunct Professor

Rainer Group Head of Lab (PI) - http://www.unifr.ch/inph/vclab

Team Members

Xiaozhe Zhang, Postdoctoral fellow Laetitia Fouillen, Postdoctoral fellow Anwesha Bhattacharyya, PhD student Sara Falasca, PhD student Julia Veit, PhD student Abbas Khani, PhD student Filomena Petruzziello, PhD student Pierre Joris, PhD student (joint supervision with Prof. Philippe Renaud)

Gregor Rainer External Adjunct Professor University of Fribourg

Introduction

The primate visual system is comprised of numerous brain regions, connected to each other in a highly recurrent fashion. Our research aims at understanding how visual information is processed and stored, enabling us to recognize objects and interact with our environment. Understanding the neural code of the visual system is a fundamental requirement for advancing sensory brain-machine interfaces. We combine sophisticated behavioural psychophysical methods with multi-channel electrophysiological recordings, electrical microstimulation and pharmacological manipulations. We are particularly interested in studying how cholinergic neuromodulation and associated neuropeptides affect visual processing mechanisms and behavioural performance. Laminar recordings in the visual cortex We are examining neural activity at the level of single neurons and local field potentials in the primary visual cortex using tetrodes as well laminar probes that are designed at EPFL in a collaboration funded by the Stoicescu program in Neurotechnology. We are developing and refining these probes for different applications including laminar cortical recordings, deep brain stimulation and epidural recording. We employ a variety of visual stimuli, designed to probe particular computational functions of the visual cortex, allowing us to describe layer-specific relationships between cortical oscillatory activity at the mesoscopic level and information transmission by single neurons. We apply cholinergic receptor agonists and antagonists as well as electrical microstimulation in the basal forebrain to up- and downregulate cholinergic neuromodulation, and study how these manipulations affect behaviour as well as neural activations in the visual system. Cholinergic

neuromodulation has linked to a number of neurological disorders including Alzheimer’s disease and Schizophrenia. Neurochemical Dynamics We are developing methods based on Liquid Chromatography and Mass Spectrometry (LC-MS) as well as electrochemical detection (ECD) to track the concentration of amino acids, small molecule neurotransmitters such as Acetylcholine, Acetylcarnitine, Dopamine or Serotonin and their metabolites and neuropeptides with high temporal resolution. Our work includes the development of direct brain-mass spectrometer interface for fast and automated quantitation of neurochemicals, as well as characterization of brain-region specific neuropeptides that are linked to visual cognition and cholinergic neuromodulation. Recent advances in neurochemical monitoring have shown that the cholinergic system, as well as other neuromodulators, can act at very fast time scales. These systems are thus significantly more specific than has previously been assumed. Exploring this fast action is thus likely to provide important novel insights into their mechanisms of action during cognition.

Selected publications

Nielsen, K., N. K. Logothetis and G. Rainer: Object features used by humans and monkeys to identify rotated shapes. Journal of Vision 8(2): 9, 1-15 (2008) Zhang, X., A. Rauch, H. Xiao, G. Rainer and N. K. Logothetis: Mass spectrometry-based neurochemical analysis: perspectives for primate research. Expert Review of Proteomics 5(5), 641-652 (2008) Rainer, G.: Localizing cortical computations during visual search. Neuron 57, 480-81 (2008)


EPFL School of Life Sciences - 2009 Annual Report

Liebe, S., Fischer, E., Logothetis, N. K., Rainer, G. Color and shape interactions in the recognition of natural scenes by human and monkey observers. Journal of Vision, 9(5):14, 1-16 (2009)

Bießmann F., Meinecke F.C., Gretton A., Rauch A., Rainer G., Logothetis N.K., Müller K.R. Temporal Kernel CCA and its Application in Multimodal Neuronal Data Analysis. Machine Learning (2009)

On the left, a polyimide laminar neural recording probe is shown with contact spacing of 100μm and three different contact sizes. An example single trial local field potential recording in the primary visual cortex is shown in the center panel; the vertical line represents the onset of the visual stimulus. On the left, action potentials recorded on one of the probe channels are shown.


IBI IB B

EPFL School of Life Sciences - 2009 Annual Report

IBI - Institute of Bioengineering & Co-Affiliates The Institute of Bioengineering sits at the interface of the life sciences and engineering, being situated in both the School of Life Sciences and the School of Engineering and reporting to both deans. This dual affiliation allows great diversity in hiring faculty from different backgrounds and with different research perspectives, all focused on basic biological sciences using quantitative and systems analyses, as well as translating the biological and biochemical sciences into therapeutics and diagnostics. The dual affiliation also provides a rich educational environment, both at the BS/MS and PhD levels; at present, a joint MS in Bioengineering is being developed between the two Schools (due to come into effect in the Fall 2010). In pursuit of basic biological mechanisms, IBI faculty investigate questions such as: How the cellular microenvironment controls cellular differentiation and morphogenetic processes; How stem cell processes, such as self-renewal and differentiation, are determined; How cell migration and trafficking in complex environments is modulated; How complex biological networks such as metabolism, gene expression and protein trafficking are regulated; and How biophysical and biomolecular signals interact in controlling cellular behavior. Our goal is to transform knowledge gained from our studies into clinical applications. To that end, the IBI faculty develop novel technologies in areas including: interventional and diagnostic biomedical microdevices, synthetic and biosynthetic biomaterials for delivery of small molecule drugs, proteins and DNA, materials in bionanotechnology, immunotherapy based on active biomolecules and nanomaterials, novel molecules for photodynamic therapy, and tissue engineering for therapeutics as well as physiological modelling based on biomolecular and stem cell approaches. ‘http://ibi.epfl.ch’

© Co opyright 2004-2010 0 EPFL for or a ma ater eria ia al pu publ blis ishe he ed iin n this rep port rt Copyright alllll m material published report info f .sv@ v@e epfl.c ..ch ch info.sv@epfl

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EPFL School of Life Sciences - 2009 Annual Report

IBI - Institute of Bioengineering

Auwerx Lab Head of Lab (PI) - http://auwerx-lab.epfl.ch/

Team Members

Johan Auwerx MD, PhD Full Professor Nestle Chair in Energy Metabolism

Pénélope Andreux, Ph.D student Sabrina Bichet, lab technician Carles Canto, Postdoctoral associate Marie-Laure Dénéreaz, lab technician Pablo Fernandez-Marcos Postdoctoral associate Riekelt Houtkooper, Postdoctoral associate Ellen Jeninga, Postdoctoral associate Chikage Mataki, Postdoctoral associate Norman Moullan, lab technician Mitsonura Nomura, Ph.D. student Lilia Noriega, Postdoctoral associate Eliane Pierrel, lab technician Thijs Pols, Postdoctoral associate Genevieve Rydlo, Master student Kristina Schoonjans, Senior group leader Valérie Stengel, Administrative Assistant Raffaele Teperino, Postdoctoral associate Evan Williams, Master Student exchange USA Hiroyasu Yamamoto, Postdoctoral associate Jiujiu Yu, Postdoctoral associate

Introduction

The research of the Laboratory of Integrative and Systems Physiology (LISP) aims to understand how regulatory proteins, including nuclear receptors, membrane receptors and transcriptional cofactors, act as sensors for molecules of nutritional, metabolic or pharmacological origin, and translate this into altered gene expression and protein patterns affecting metabolic function.

Keywords

Diabetes, genetics, metabolism, metabolic disease, phenogenomics, transcription

Results Obtained in 2009

The Auwerx/Schoonjans laboratory was amongst the pioneers to unravel the wide-ranging implications of the three PPARs, PPARα, PPARβ/δ, and PPARγ, in metabolic control. Perhaps most striking in this context was our discovery of an association between the PPARγ Pro12Ala gene variant with type 2 diabetes and obesity, identified long before the era of genome-wide association studies, and as such the first gene tied with these common complex diseases. We established how the enterohepatic nuclear receptors, LRH-1 and SHP, govern hepatic lipid and bile acid metabolism, regulate mucosal immune homeostasis, and control fertility via their commanding role on steroid production. We furthermore identified bile acids as endocrine regulators of energy expenditure, through the activation of a novel membrane receptor, TGR5. Finally, we established that transcriptional cofactors, such as the acetyltransferases (SRC2/TIF2 and SRC-3) and the deacetylases (such as SIRT1), fine-tune energy homeostasis by changing the acetylation status of PGC-1α, the master regulator of mitochondria. Since altered signaling by nuclear receptors and cofactors, contributes to the

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pathogenesis of type 2 diabetes, obesity and atherosclerosis, our research paved the way for novel preventive and therapeutic strategies for these common diseases. The importance of these discoveries is testified by the fact that several compounds targeting these receptors and/or cofactors have made it into the clinic. Examples of drugs for which our research contributed to clinical development are the fibrates (that target PPARα), thiazolidinediones (that target PPARγ), PPARβ/δ agonists, bile acids and bile acid derivatives (that target both the TGR5 and FXR), and resveratrol and SRT1720 (which activate SIRT1).

Selected publications

Thomas C., Gioiello A., Noriega L., Strehle A., Oury J., Rizzo G., Machiarullo A, Yamamoto H., Mataki C., Pruzanski M., Pelliciari R., Auwerx J., Schoonjans K. TGR5-mediated bile acid sensing controls glucose homeostasis. Cell Metabolism, 2009, 167-177. Koutnikova H., Laakso M., Lu L., Combe R., Paananen J., Kuulasmaa T., Kuusisto J., Haring H. U., Hansen T., Pedersen O., Smith U., Hanefeld M., Williams R. W., Auwerx J. Identification of the UBP1 locus as a critical blood pressure determinant using a combination of mouse and human genetics. Plos Genetics, 2009, e1000591. Argmann C., Dobrin R., Heikkinen S., Auburtin A, Pouilly L., Cock T. A., Koutnikova H., Zhu J., Schadt E. E., Auwerx J. PPARg is a key driver of longevity in the mouse. Plos Genetics, 2009, e1000752 Heikkinen S., Argmann C., Feige J. N., Koutnikova H., Champy M. F., Dali-Youcef N., Schadt E. S., Laakso M., Auwerx J. The Pro12Ala PPARg2 variant determines metabolism at the gene-environment interface. Cell Metabolism, 2009, 9, 88-98. Canto C., Gerhart-Hines Z., Feige J. N., Lagouge M., Noriega L., Millne J. C., Puigserver P., Auwerx J. AMPK regulates

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EPFL School of Life Sciences - 2009 Annual Report

Heikkinen S., Argmann C., Feige J.N., Koutnikova H., Champy M.F., Dali-Youcef N., Schadt E. S., Laakso M., Auwerx J. The Pro12Ala PPARg2 variant determines metabolism at the gene-environment interface. Cell Metabolism, 2009, 9, 88-98. Duggavathi R., Volle D.H., Mataki C., Antal M. C., Messadeq N., Auwerx J., Murphy B. D., Schoonjans K. Liver receptor homolog 1 is essential for ovulation. Genes Dev., 2008, 22, 1871-1876. Coste A., louet J. F., Lagouge M., Lerin C., Antal M. C., H. Meziane, Schoonjans K., Puigserver P., O’Malley B. W.,

Auwerx J. The genetic ablation of SRC-3 protects against obesity and improves insulin sensitivity by reducing the acetylation of PGC-1a. Proc. Natl. Acad. Sci. USA, 2008, 105, 17187-17192. Feige J.N., Lagouge M., Canto C., Strehle A., Houten S., Millne J, Lambert P. D., Mataki C., Elliot P.J., Auwerx J. Specific SIRT activation mimics low energy levels and protects against diet-induced metabolic disorders by enhancing fat oxidation. Cell Metabolism, 2008, 8, 347-358. Gofflot F., Chartoire N., Vasseur L., Heikkinen S., Dembele D., Le Merre J., Auwerx J. Systematic gene expression mapping clusters nuclear receptors on the basis of their function in the brain. Cell, 2007, 131, 405-418.

Mice treated with resveratrol are protected from obesity.

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IBI

energy expenditure by modulating NAD+ metabolism and SIRT1 activity. Nature, 2009, 458, 1056-1060.


EPFL School of Life Sciences - 2009 Annual Report

IBI - Institute of Bioengineering

Barrandon Lab Head of Lab (PI) - http://ldcs.epfl.ch/

Team Members

Yann Barrandon

Full Professor, EPFL-CHUV Head of Exp. Surgery - CHUV

Amici Alessandro, PhD student Bon Anne-Charlotte, trainee Bonfanti Paola, MD, Post-doctoral fellow Brouard Michel, MD, Post-doctoral fellow Claudinot Stéphanie, Post-doctoral fellow Gorostidi François, MD, PhD student Graber Julien, scientist collaborator Grasset Nicolas, MD, Post-doctoral fellow Guex Nathalie, administrative assistant Lathion Droz Georget Stéphanie, Post-doctoral fellow Maggioni Melissa, PhD student Mercier Louis, technician Mosig Johannes, PhD student Namba Daisuke, Post-doctoral fellow (left 30.11.2009) Petermann Katrin, master student Rochat Ariane, PhD, Senior researcher Salmon Basile, master student Savioz-Dayer Emmanuelle, clinical trial collaborator Schnell Liliane, technician (left 30.11.2009) Stolf Daiana, PhD Student (begin 09.2009) Teisanu Roxana, Post-doctoral fellow (begin 1.7.2009) Toki Fujio, Post-doctoral fellow (left 30.11.2009) Vermot Steeve, technician Volorio Christelle, Post-doctoral fellow

Introduction

The laboratory of Stem Cell Dynamics at EPFL and Experimental Surgery at the CHUV has three main objectives: first, to understand the relationship between stem/progenitors cells of stratified epithelia, second to understand the impact of the environment on stem cell behavior and third to comprehend stem cell engraftment. All projects ultimately aim at improving cell and gene therapy using epithelial stem/ progenitor cells. The laboratory is a partner in three stem cell consortia within the EEC 7th framework program, aiming at the fundamentals of stem cells (EuroSyStem) and stem cell therapy (OptiStem and Betacelltherapy).

Keywords

Stem cell, micro-environment, epithelia, skin, thymus, cell and gene therapy

Results Obtained in 2009

Maintenance of organ function relies on the same basic mechanisms involved during morphogenesis; in those tissues and organs undergoing extensive remodeling, there are cells termed stem cells that are responsible for long-term renewal, tissue regeneration and repair. Stem cells have two fundamental properties, the capacity to self-renew and to generate a differentiated progeny for an extended period of time (theoretically for a lifetime). Within tissue stem cells, the skin is privileged because its stem cells (epithelial and mesenchymal) can be exten-

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sively cultivated and cloned, genetically manipulated and transplanted in laboratory animals, but also in human. We have demonstrated that all stratified epithelia of the rat, independent of their primary germ line origin (e.g. the endodermal esophagus or the ectodermal cornea), contain clonogenic stem cells that can respond to skin morphogenetic signals by forming epidermis, sebaceous glands and functional hair follicles in serial transplantation. Furthermore, we have demonstrated that the thymus, which has a unique 3D structure that does not resemble that of a simple or stratified epithelium, contains a population of clonogenic epithelial cells with astonishing capabilities. These clonogenic cells maintain a thymic identity in vitro or in a reconstituted thymus in vivo, but adopt the fate of bona fide multipotent stem cells of the hair follicle when exposed to skin morphogenetic signals, a property maintained in serial transplantation. Gene profiling experiments have demonstrated that several transcription factors important for thymic identity were either down regulated or silenced in thymic epithelial cells recovered from skin. This clearly represents a crossing of lineage boundaries, an increase in potency and the demonstration that adult stem/progenitor cells can be robustly reprogrammed by microenvironmental cues. Engraftment is the quintessence of stem cell behavior as it draws on all stem cell basic functions, i.e. homing, attachment, migration, proliferation, fate choice, renewal, differentiation and death. In a normal situation, these decisions are tightly controlled and influenced by the microenvironment (the niche), but

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EPFL School of Life Sciences - 2009 Annual Report

Selected publications

Bonfanti, P., Claudinot, S., Amici, A.W., Farley, A., Blackburn, C.C, Barrandon, Y. (2010). Microenvironmental reprogramming of thymic epithelial cells to skin multipotent stem cells. Nature in press.

Majo, F., Rochat, A., Nicolas, M., Abou JaoudĂŠ, G., Barrandon, Y. (2008). Oligopotent stem cells are distributed throughout the ocular surface. Nature 456: 250-254. Epub 2008 Oct 1. Research Highlights in Nature Reports Stem Cells Epub Oct 9, 2008 release Gurtner, G. C., Werner, S., Barrandon, Y., Longaker, M. T. (2008). Wound repair and regeneration. Nature 453, 314321. Nakamura, T., Ohtsuka, T., Sekiyama, E., Cooper, L. J., Kokubu, H., Fullwood, N. J, Barrandon, Y., Kageyama, R. (2008). Hes1 Regulates Corneal Development and the Function of Corneal Epithelial Stem/progenitor Cells. Stem Cells 26(5): 1265-74. Feutz, A.C., Barrandon, Y., Monard, D. (2008). Control of thrombin signaling through PI3K is a mechanism underlying plasticity between hair follicle dermal sheath and papilla cells. J.Cell Sci. 121: 1435-1443.

Clone of hair follicle multipotent stem cells expressing FUCCI reporter genes. Bar: 100 microns

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IBI

in therapy, the microenvironment may be diseased, damaged by the preconditioning treatment, or even completely missing as in third degree burns or limbal deficiency. Hence, transplanted stem cells have to adapt to an environment that is far from ideal, if not hostile. Surprisingly, little is known on engraftment that remains more a lottery than a scientifically controlled process. We have demonstrated that transplanted stem cells respond to adverse conditions by favoring differentiation rather self-renewal or death. We are now using state-of-the art architecture, informatics and visualization technology to construct models that will allow us to virtually manipulate stem cell behavior and predict the consequences on organ function using the skin, the thymus and the cornea as model systems.


EPFL School of Life Sciences - 2009 Annual Report

IBI - Institute of Bioengineering

Dal Peraro Lab Head of Lab (PI) - http://lbm.epfl.ch/

Team Members

Davide Alemani, Postdoctoral fellow Matteo Degiacomi, PhD student Thomas Lemmin, PhD student Enrico Spiga, PhD student Marie-France Radigois, Administrative Assistant Shantanu Roy, Postdoctoral fellow Marco Stenta, Postdoctoral fellow

Matteo Dal Peraro

Tenure Track Assistant Professor

Introduction

We use advanced molecular modeling techniques combined with high-performance computing to investigate biological systems, in particular their function emerging from structure. Our main targets are bacterial and viral systems and their mechanism of resistance towards natural and clinical drugs. We develop new multi-scale schemes and models to extend the power of current molecular simulations to tackle problems such as the assembly of large macromolecular complexes and the design of remedies for pathogenic infections.

Keywords

Computational biophysics, biochemistry, and structural biology; bacteria and viruses; multi-scale molecular simulations; macro-molecular assembly; protein and drug design; high-performance computing.

Results Obtained in 2009

In the past years, computational structural biology has greatly improved our knowledge of biological function at the molecular level shedding light on features that are often experimentally inaccessible. Nonetheless, it still remains very difficult by using current simulation techniques (all-atom simulations, AA) to reach dimensions and dynamical scales that are significant to most of the biological processes both in vitro and in vivo. Thus, we are currently engaged in the development of novel schemes and models to enable a more consistent overlap of quantities one can derived from the computational and experimental setting. We are building a new coarsegrained (CG) description of proteins, nucleic acids and membranes in molecular simulations, which will allow us to tackle complex problems such as proteinligand recognition and protein-protein interactions in large macromolecular networks with unprecedented sampling power and accuracy. This new framework

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will also permit to efficiently integrate experimental input to guide the computational modeling of the biological systems of interest [Chimia 2009]. We are currently using this approach to understand the function of biological complexes from bacteria and viruses. In particular, in 2009 we introduced a non-radial potential term for coarse-grained (CG) molecular simulations of proteins. This term mimics the backbone dipole−dipole interactions and accounts for the needed directionality to form stable folded secondary structure elements. We show that α-helical and β-sheet peptide chains are correctly described in dynamics without the need of introducing any a priori bias potentials or ad hoc parametrizations, which limit broader applicability of CG simulations for proteins. Moreover, our model is able to catch the formation of supersecondary structural motifs, like transitions from long single α-helices to helix−coil−helix or β-hairpin assemblies. This novel scheme requires the structural information of Cα beads only; it does not introduce any additional degrees of freedom to the system and has a general formulation, which allows it to be used in synergy with various CG and hybrid AA/CG protocols (Figure 1), leading to an improved description of the structural and dynamic properties of protein assemblies and networks [Journal of Chemical Theory and Computation 2010].

Selected publications

Alemani D, Collu F., Cascella M. and Dal Peraro M, (2010) A nonradial coarse-grained potential for proteins produces naturally stable secondary structure elements, Journal of Chemical Theory and Computation, 6(1), 315-324 Simona F., Magistrato A., Dal Peraro M., Cavalli A., Vila A. J. and Carloni P. (2009) Common mechanistic features among metallo-β-lactamases: a computational study of Aeromonas hydrophila CphA enzyme, The Journal of biological chemistry, 284(41), 28164-28171

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EPFL School of Life Sciences - 2009 Annual Report

Ho M.-H., De Vivo M., Dal Peraro M. and Klein M. L. (2009) Unraveling the Catalytic Pathway of Metalloenzyme Farnesyltransferase through QM/MM Computation, Journal of Chemical Theory and Computation, (6), 1657-1666 Khurana, E., Dal Peraro, M., DeVane, R.,Vemparala, S., DeGrado, W.F., Klein, M.L. (2009) Molecular dynamics calculations suggest a conduction mechanism for the M2 proton channel from influenza A virus. Proc Natl Acad Sci USA, 106(4), 1069 –1074. Wagner, S., Sorg, I., Degiacomi, M., Journet, L., Dal Peraro, M., Cornelis, G.R. (2009) The helical content of the YscP

molecular ruler determines the length of the Yersinia injectisome. Mol Microbiol, 71(3), 692-701 De Vivo, M., Dal Peraro, M., Klein, M.L. (2008) Phosphodiester Cleavage in Ribonuclease H Occurs via an Associative Two-Metal-Aided Catalytic Mechanism. J Am Chem Soc, 130(33), 10955–10962. Cascella, M., Neri, M.A., Carloni, P., Dal Peraro, M. (2008) Topologically based multipolar reconstruction of electrostatic interactions in multiscale simulations of proteins. J Chem Theory Comput, 4(8), 1378–1385. Calhoun, J. R., Liu, W., Spiegel, K., Dal Peraro, M., Klein, M. L., Valentine, K. G., Wand, A. J. and DeGrado W. F. (2008). Solution NMR structure of a designed metalloprotein and complementary molecular dynamics refinement. Structure. 16(2): 210-215.

Resolution models for molecular simulations. Pictorial views of the mechanosensitive channel (MscS, PDB code entry: 2OAU) at different resolution schemes: All-atom resolution (AA), two-bead coarse-grained (CG) and, hybrid all-atom/coarse-grained (AA/CG) models. Different colors correspond to the subunits of the channel. Atoms are drawn in ball-and-sticks, CG-beads in spheres. In AA, ribbon representation of the channel is superimposed to better clarify the secondary structure elements of the protein complex (adapted from Chimia 2009).

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Cascella M. and Dal Peraro M. (2009) Challenges and perspectives in biomolecular simulations: from atomistic picture to multiscale modeling, CHIMIA International Journal for Chemistry, 63(1-2), 14-18


EPFL School of Life Sciences - 2009 Annual Report

IBI - Institute of Bioengineering

Deplancke Lab Head of Lab (PI) - http://deplanckelab.epfl.ch/

Team Members

Korneel Hens, Postdoctoral fellow Sunil Raghav, Postdoctoral fellow Carine Gubelmann, Ph.D. student Alina Isakova, Ph.D. student Irina Krier, Ph.D. student Andreas Massouras, Ph.D. student Jovan Simicevic, Ph.D. student Julien Bryois, Master’s student Antonina Iagovitina, Master’s student Jean-Daniel Feuz, Technician Marie-France Divorne, Administrative Assistant

Bart Deplancke

Tenure Track Assistant Professor

Introduction

Gene regulatory networks play a vital role in the development and function of multicellular organisms since they control the spatio-temporal expression of genes. Consequently, deregulation of these networks has been implicated in several diseases including cancer, diabetes and neurodegenerative pathologies. The interactions between genes and their respective regulatory transcription factors (TFs) that form the basis of gene regulatory networks have however been poorly characterized. The overall goal of our laboratory is to reverse engineer and model the gene regulatory networks that control metazoan development and function to make predictions on how specific regulatory networks will behave under different physiological or pathological conditions.

Keywords

Systems Biology, Gene Regulatory Network, Transcription, Quantitative Genetics, Mouse, Drosophila, Yeast, Genetic Engineering

Results Obtained in 2009

Key for most experimental projects in the lab is the availability of a high-quality TF clone resource. We spent considerable efforts in the last two years to clone the majority of mouse and Drosophila TFs. As of today, we have completed the cloning of 95% of all Drosophila TFs (~700) as well as 70% of all mouse TFs (~1,000). Since these clones will be used to exogenously express TF proteins, it is critical that they are fully sequence-verified. Hence, we developed a novel tool, WebPrInSeS, allowing automated full-length clone sequence identification and verification using high-throughput sequencing data as a cost-effective and efficient alternative to conventional Sanger sequencing. WebPrInSeS encompasses two separate software applications. The first is WebPrInSeS-C which performs automated sequence verification of user-defined open-reading frame (ORF)

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clone libraries. The second is WebPrInSeS-E, which identifies positive hits in cDNA or ORF-based library screening experiments such as yeast one-, two-, or three-hybrid assays. Both tools perform de novo assembly using high-throughput sequencing data from any of the three major sequencing platforms. We subsequently used the same algorithm principle to explore our ability to not only identify but also assemble sequence variants up to the nucleotide level in whole eukaryotic genomes. Specifically, we used PrInSeS to detect structural variation in Drosophila melanogaster whole genomes. For this purpose, we used data from the Drosophila Genetic Reference Panel, which features over 200 inbred phenotyped D. melanogaster lines. We selected one line out of the forty for which high-throughput sequencing data has been released. In total, we detected more than 35,000 non-SNP variants. Importantly and in contrast to other structural variation mapping programs, we were also able to assemble these sequence variants. The latter uniquely enables us to validate our results by plotting the overall read alignment improvement as measured by average read depth for each re-assembled variant. When re-aligning the reads to the detected variants, we observed that the coverage of these variants improved in 93% of the cases, thus providing a confidence measure for the majority of detected D. melanogaster structural variants (Fig. 1a-c) as well as for our method in general.

Selected publications

Simicevic J., Deplancke B. DNA-centered approaches to characterize regulatory protein-DNA interaction complexes. Molecular Biosystems, 2010 (DOI: 10.1039/B916137F). Reece-Hoyes J. S.*, Deplancke B.*, Barrasa I., Hatzold I., Smit R., Arda E., Pope P., Gaudet J., Conradt B., Walhout A. J. M. The C. elegans Snail homolog CES-1 can activate gene expression by binding to an extended binding site, and share targets with bHLH transcription factors, Nucleic

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EPFL School of Life Sciences - 2009 Annual Report

Acid Research, 37: 3689-3698, 2009. (*, shared first authorship)

IBI

Deplancke B. Experimental advances in the characterization of metazoan gene regulatory networks. Briefings in Functional genomics and Proteomics, 8:12-27, 2009.

Mukhopadhyay A.*, Deplancke B.*, Walhout A. J. M., Tissenbaum H. A. Chromatin immunoprecipitation (ChIP) coupled to detection by quantitative real-time PCR to study transcription factor binding to DNA in Caenorhabditis elegans. Nature Protocols, 3:698-709, 2008. (*, shared first authorship)

Figure 1 ¦ (a-c) Validation of detected whole genome D. melanogaster RAL-304 variants via analysis of Maq alignment improvement. Each panel shows the average read depth before (blue) and after (red) sequence adjustment of each region with the respective variant. For each region, a segment equal to the sequencing read length is included up- and downstream of the variant after which the read depth is averaged for all regions that either show alignment (a) improvement (93%), (b) deterioration (4%), or (c) no change (3%). The gaps at position 0 are caused by the fact that the variant itself is not included in order to allow direct comparison to the reference sequence. (d) Bar graph depicting the number of non-SNP substitutions (green), deletions (red), and insertions (blue) per Mb per chromosome (4 and X) or chromosome arm (2L, 2R, 3L, and 3R). (e) Bar graph depicting the number of non-SNP sequence variants per Mb in “coding” (here defined as all exons including UTRs totaling 33.2 Mb) and “non-coding” (86.8 Mb) regions. (f) Box plot depicting the distribution of broad-sense heritability values (H2) derived from for genes with or without variations. The number of genes in each group is indicated. Variants were considered to be associated with a gene if they fall within the region spanning 500 bp up- to 500 bp downstream of the respective genes. A Wilcoxon rank-sum test was used to calculate the P value.

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EPFL School of Life Sciences - 2009 Annual Report

IBI - Institute of Bioengineering

Hubbell Lab Head of Lab (PI) - http://lmrp.epfl.ch/ Team Members

Jeffrey A. Hubbell

Full Professor Director of IBI Merck-Serono Chair in Drug Delivery

Ahmadloo Hamideh, PhD student Bonzon Carol Anne, Secretary Ciapala Karine, PhD student De LaPorte Laura, visiting postdoctoral fellow Dessibourg Céline, HES diploma student Dane Karen, Postdoctoral fellow Eby Jackson, PhD student Engelhardt, Eva-Maria, Postdoctoral fellow Frey Peter, Professor, Adjunct professor Hasegawa Urara, Postdoctoral fellow Kontos Stephane, PhD student Kourtis Iraklis, PhD student Kraehenbuehl Thomas, PhD student until 30.06.2009 Larsson Hans Mattias, PhD student Lee Seung Tae, postdoctoral fellow Lorentz Kristen, PhD student Losada Ricardo, PhD student until 31.05.2009 Mahou Redouan, PhD student Martino Mikaël, PhD student Micol Lionel, PhD student Mochizuki Mayumi, postdoctoral fellow Nembrini Chiara, postdoctoral fellow O’Neil Conlin, PhD student Pasquier Miriella, Laboratory assistant, 25% Patterson Jennifer, Postdoctoral fellow Pisarek Rubin Berek, PhD student Quaglia Xavier, Laboratory assistant Raghunathan Sandeep, PhD student Rice Jeffrey, Postdoctoral fellow Rothenfluh Dominique André, PhD student until 30.09.2009 Schütz Catherine, PhD student Simeoni Eleonora, Scientist Stano Armando, PhD student Tomei Alice, Postdoctoral fellow Traub Stephanie, Trainee until 30.06.2009 Van der Vlies André, Postdoctoral fellow Velluto Diana, Postdoctoral fellow Wandrey Christine PhD, Maître d’enseignement et de recherche Yang Lirong, PhD student Yun JungIm, Postdoctoral fellow

Introduction

We design novel materials for investigation of basic cell biological phenomena such as stem cell self-renewal and differentiation and applications in medicine such as drug delivery, regenerative medicine, and vaccination. We focus on examples where novel materials are necessary to solve the problem, thus working at the interface between materials science and biology.

Keywords

Biomaterials, tissue engineering, protein engineering, drug and gene delivery, vaccines

Results Obtained in 2009

Regenerative medicine: The laboratory made exciting advances in engineering matrix-bound morphogens for conjugation in biomaterial matrices for tissue repair and regeneration: IGF-1 variants for use in smooth muscle, VEGF-A variants for inducing angiogenesis, fibronectin variants for supporting stem cell differentiation in bone repair, and FGF-18 variants for cartilage repair. We have discovered that a domain of fibronectin displays very promiscuous binding to a very large number of

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growth factors, and we are exploring its use as a very generic tool in tissue engineering. We explored synthetic materials as matrices in embryonic stem cell self-renewal, and demonstrated that signalling through four specific integrins is critical in this process. Drug and gene delivery: Chemists, biologists and bioengineers collaborated to push our approaches with block copolymers forward in delivering small molecule drugs (such as paclitaxel in cancer, cyclosporine A in immunosuppression) and gene-based drugs (both siRNA and plasmid DNA in vitro and in vivo). Polymers have been developed to carry these diverse payloads. Vaccines and immunotherapeutics: In collaboration with the Laboratory for Mechanobiology and Morphogenesis (Prof. M.A. Swartz), the laboratory demonstrated that nanoparticles can be used as a vaccine platform for targeting cells in the lymph nodes. This, combined with advanced design of the polymeric nanoparticle surface, may enable a new generation of vaccines, highly stable and very economical, for use in both the developing and the

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developed world. The team has demonstrated that ultra-small particles, smaller than biological particles, can be swept into the lymphatics within a few minutes of injection, drain to the lymph nodes, and are collected there for antigen presentation. Novel approaches for vaccination of the oral and airway mucosae have been demonstrated.

Hubbell, J.A., Thomas, S.N. & Swartz, M.A. Materials engineering for immunomodulation. Nature 462, 449-460 (2009).

Selected publications

Velluto, D., Demurtas, D. & Hubbell, J.A. PEG-b-PPS diblock copolymer aggregates for hydrophobic drug solubilization and release: Cyclosporin A as an example. Mol Pharm 5, 632-642 (2008).

O’Neil, C.P., Suzuki, T., Demurtas, D., Finka, A. & Hubbell, J.A. A Novel Method for the Encapsulation of Biomolecules into Polymersomes via Direct Hydration. Langmuir 25, 9025-9029 (2009). Martino, M.M., Mochizuki, M., Rothenfluh, D.A., Rempel, S.A., Hubbell, J.A. & Barker, T.H. Controlling integrin specificity and stem cell differentiation in 2D and 3D environments through regulation of fibronectin domain stability. Biomaterials 30, 1089-1097 (2009). Kraehenbuehl, T.P., Ferreira, L.S., Zammaretti, P., Hubbell, J.A. & Langer, R. Cell-responsive hydrogel for encapsulation of vascular cells. Biomaterials 30, 4318-4324 (2009). Jo, Y.S., van der Vlies, A.J., Gantz, J., Thacher, T.N., Antonijevic, S., Cavadini, S., Demurtas, D., Stergiopulos, N. & Hubbell, J.A. Micelles for Delivery of Nitric Oxide. J. Am. Chem. Soc. 131, 14413-14418 (2009).

Arrighi, I., Mark, S., Alvisi, M., von Rechenberg, B., Hubbell, J.A. & Schense, J.C. Bone healing induced by local delivery of an engineered parathyroid hormone prodrug. Biomaterials 30, 1763-1771 (2009).

Rothenfluh, D.A., Bermudez, H., O’Neil, C.P. & Hubbell, J.A. Biofunctional polymer nanoparticles for intra-articular targeting and retention in cartilage. Nature Materials 7, 248-254 (2008). Rehor, A., Schmoekel, H.G., Tirelli, N. & Hubbell, J.A. Functionalization of polysulfide nanoparticles and their performance as circulating carriers. Biomaterials, 1958-1966 (2008). Kraehenbuehl, T.P., Zammaretti, P., Van der Vlies, A.J., Schoenmakers, R.G., Lutolf, M.P., Jaconi, M.E. & Hubbell, J.A. Three-dimensional extracellular matrix-directed cardioprogenitor differentiation: Systematic modulation of a synthetic cell-responsive PEG-hydrogel. Biomaterials 29, 2757-2766 (2008)

Block copolymer amphiphiles are used to condense plasmid DNA into 30 nm nanoparticles, which consist of a single copy of plasmid per particle. The particles, the smallest polymer-DNA complexes that have been reported, transfect cells very well in tumor killing models in mice. (Photo: Diana Velluto)

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51

IBI

EPFL School of Life Sciences - 2009 Annual Report


EPFL School of Life Sciences - 2009 Annual Report

IBI - Institute of Bioengineering

Lutolf Lab Head of Lab (PI) - http://lscb.epfl.ch/

Team Members

Matthias Lutolf

Steffen Cosson, PhD student Samy Gobaa, Postdoctoral fellow Sylke Hoehnel, PhD student Stefan Kobel, PhD student Raphaelle Luisier, PhD student Katarzyna Mosiewicz, PhD student Andrea Negro, PhD student Yuya Okawa, PhD student Adrian Ranga, PhD student Marta Roccio, Postdoctoral fellow Aline Roch, PhD student Nicola Vannini, Postdoctoral fellow

Tenure Track Assistant Professor

Introduction

Tissue homeostasis and regeneration are critically dependent on a limited number of adult stem cells, their self-renewal capability and their commitment to become specialized cells. Due to these unique properties, stem cells hold enormous potential for the treatment of many diseases. However, despite extensive research on elucidating molecular stem cell regulation, significant hurdles need to be overcome before stem cells can be used for therapy. Arguably one of the greatest challenges is controlling stem cell behavior outside of the body, as this would, for example, allow expanding them to sufficient numbers. Adult stem cells reside in specialized niches, comprised of complex mixtures of extracellular cues delivered by support cells in close proximity. Niches protect stem cells from rapid differentiation and regulate the delicate balance between self-renewal and differentiation. The underlying mechanisms remain poorly defined in mammals, mainly because of the difficulties in manipulating these intricate microenvironments in vivo. To address how micro-environmental signals control the behaviour of adult stem cells, we develop innovative bioengineering strategies that allow us to biochemically and structurally deconstruct in vivo adult stem cell niches, and reconstruct them in vitro. These well-defined artificial stem cell niches are applied in the lab to decipher adult stem cell regulation. We expect that some of these technologies have the potential to be translated into clinical settings, for example to expand rare hematopoietic stem cells to treat blood cancers.

Results Obtained in 2009

We have developed novel stem cell culture technologies that allow fate changes of individual stem cells to be monitored in vitro, under near-physiologic conditions and in real time. These artificial niches were fabricated from ‘smart’ poly(ethylene glycol) (PEG)

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hydrogels that allow key biochemical characteristics of adult stem cell niches to be mimicked and the physiological niche complexity deconstructed into a smaller, experimentally amenable number of distinct signalling interactions. Moreover, because many adult stem cell populations are inherently heterogeneous and current state-of-the-art culture techniques do not permit efficient dynamic analyses of fates of large numbers of single cells, 2D and 3D hydrogel patterning techniques were developed that allow to confine and array single stem cells for highthroughput experimentation. In order to mimic cellcell interactions typical of niches without the complexity of co-culture, we have for example invented protein micropatterning methods for hydrogels allowing to expose confined stem cells to tethered protein cues, singly or in combination, or to overlapping protein gradients. These artificial niches are being utilized to explore the fate of individual mouse hematopoietic stem cells (HSC) as well as neural stem/progenitor cells. For example, time-lapse microscopy of several thousand single HSC cultured in microwell arrays over several days, combined with subsequent image analyses allowed growth kinetics of selected populations to be statistically analyzed. Retrospective transplantation experiments in mice were performed in order to correlate proliferation kinetics with self-renewal function. A pronounced difference in cell division kinetics, that is predictive of their in vivo blood reconstitution potential, was observed when we compared the behavior of standard multipotent progenitors (LKS phenotype) with long-term repopulating HSC (LKS-CD150+). Testing of ca. 20 putative soluble HSC regulatory proteins, including Wnt and Notch ligands, as well as surface-tethered cell-cell adhesion proteins such as Cadherins, allowed identification of factors that dictate distinct HSC proliferation kinetics and in vivo functions. Ongoing experiments are geared towards the identification of the role of these niche factors in directing the symmetry of stem cell divisions. To address this question, we have developed microfluidic

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EPFL School of Life Sciences - 2009 Annual Report

Selected publications

Kobel, S., Limacher, M., Gobaa, S. Lutolf, M.P. Soft embossing of hydrogels, Langmuir, 25 (15), 8774–8779 (2009) Lutolf, M.P. Artificial ECM: Expanding the cell biology toolbox in 3D, Integrative Biology, 1, 235 – 241 (2009) Jo Y.S., Gantz, J., Hubbell, J.A., Lutolf, M.P. Tailoring hydrogel degradation and drug release via neighboring amino acid-controlled ester hydrolysis, Soft Matter, 5, 440 – 446 (2009)

Lutolf, M.P.*, Gilbert, P.M., Blau, H.M.* Designing materials to direct stem cell fate, Nature, 462, 433-441 (2009)

Lutolf, M.P.* and Blau, H.M.* Artificial stem cell niches, Advanced Materials, 21 (32), 3255-3268 (2009)

Charnley, M., Textor, M., Khademhosseini, A. and Lutolf, M.P. Integration column: microwell arrays for mammalian cell culture, Integrative Biololy, 1, 625 – 634 (2009)

Lutolf, M.P.*, Doyonnas, R., Havenstrite, K., Koleckar, K., Blau, H.M.* Pertubation of Single Hematopoietic Stem Cell Fate in Artificial Niches, Integrative Biology, 1, 59 (2009) (on Cover of 1st edition of Integrative Biology)

Cosson, S., Kobel, S., Lutolf, M.P. Biomolecule gradients on synthetic hydrogels, Advanced Functional Materials, 19 (21), 3411-3419 (2009) (on Inside Cover of AFM) Lutolf, M.P. Spotlight on hydrogels, Nature Materials, 8, 451 - 453 (2009)

Cordey, M., Limacher, M., Kobel, S., Taylor, V., Lutolf, M.P. (2008) Enhancing the Reliability and Throughput of Neurosphere Culture on Hydrogel Microwell Arrays, Stem Cells, 26:2586-2594

Microfluidic chip to study the symmetry of division of hematopoietic stem cells (Kobel et al., Lab on a Chip, 2010). The chip architecture is based on an array of hydrodynamic, self-regulating traps which allows to sequentially capture and analyze single progeny generated from a single ‘mother’ stem cell.

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53

IBI

chips that allow the automated separation of dividing stem cell progeny (see figure), i.e. the reliable tracking of the genealogical relationship between developing progeny over multiple generations. Our efforts to systematically ‘deconstruct’ stem cell niches may serve as a broadly applicable paradigm for defining and reconstructing artificial niches to accelerate the transition of stem cell biology to the clinic.


EPFL School of Life Sciences - 2009 Annual Report

IBI - Institute of Bioengineering

Naef Lab Head of Lab (PI) - http://naef-lab.epfl.ch/

Team Members

Arnaud Amzallag, Postdoctoral fellow (until Sept 2010) Mirko Bischofberger, PhD student Thomas d’Eysmond, PhD student Teresa Ferraro, Postdoctoral fellow Enrico Guarnera, Postdoctoral fellow (until July 2009) Bernhard Sonderegger, Postdoctoral fellow (until April 2009) Guillaume Rey, PhD student Laura Symul, trainee student (Sept-Dec 2009) Sophie Aquilar, Administrative Assistant

Felix Naef

Tenure Track Assistant Professor

Introduction

Life on earth is organized in temporal rhythms of 24 hours. These rhythms are manifestations of organism’s internal metronome called the circadian clock or oscillator, reflecting adaptation to the earth rotation. The clock drives periodic behavior and physiology through a complex network of feedback mechanisms acting at the transcriptional and post-transcriptional levels. In our group, we tackle two main problems in circadian biology: (i) the study of transcription regulatory networks that drive phase- and tissue-specific expression patterns, using a combination of bioinformatics and experimental methods; (ii) we investigate the factors that contribute to oscillator precision using mathematical modeling and analysis of time series data. With these approaches we hope to understand mechanisms and consequences of temporal organization at the cellular and organism level.

Keywords

Circadian biology, bioinformatics, gene regulation, modeling, stochastic processes, systems biology

Results Obtained in 2009

Circadian gene regulation. Our group recently identified using comparative genomics a highly conserved transcriptional regulatory element (termed the E1-E2 motif) bound by the main circadian transcription activator hetero-dimer CLOCK/BMAL1. This sequence element was found to regulate circadian genes in mammals, birds, frogs, fish, flies, mosquito and honey bee. Beyond these species, notably in the nematodes, no orthologous sequences could be found. The E1-E2 element was found both near core clock genes, and also in the vicinity or within genes involved in mediating clock output functions such as detoxification in the liver. We are pursuing this work along two main directions. The first consists in experimental validation of E1-E2 enhancers using both in vitro binding (EMSA) and

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chromatin immuno-precipitation (ChIP) in mouse liver. We have made significant progress on the in vitro analyses, showing that two instances of the CLOCK/ BMAL1 heterodimer bind the E1E2 element with high cooperativity. In addition, our ChIP experiments done at 4 hour intervals around the clock have refined mechanisms for BMAL1/CLOCK dependent transcription, and are revealing the dynamic nature of mammalian circadian protein-DNA interactomes on a genome-wide scale. In parallel, we are extending the bioinformatics studies to include a much broader class of binding elements to explain phase specific circadian gene expression profile in liver (Figure 1). Preliminary analysis shows that our methodologies are able to recover most of the described active circadian transcription factors, while also finding a set of novel putative circadian regulators. Modeling period length and precision of circadian oscillators. In collaboration with the Schibler Lab (U Geneva), we studied circadian oscillators under reduced transcription rates experimentally and theoretically. One of the key and counter-intuitive finding was that period length is shortened under reduced transcription, a property that was consistent with simulation studies of delayed feedback oscillators, such as Goodwin models. Oscillator precision, or the rate at which population of circadian oscillators desynchronize due to noise, is another key and physiologically relevant property of circadian clocks. To study this problem, we developed novel analytical and computational approaches to assess precision in common circadian oscillator models. The current is to identify molecular processes that consistently affect oscillator precision across a library of common circadian oscillator models, and eventually design perturbation experiments to validate these predictions.

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EPFL School of Life Sciences - 2009 Annual Report

Selected publications

Parisi F., Sonderegger B., Wirapati P., Delorenzi M., Naef F., “Relationship between estrogen receptor alpha location and gene induction reveals the importance of downstream sites

and cofactors”, BMC Genomics 10, 381 (2009). Parisi F., Koeppl H., Naef F., “Network inference by combining biologically motivated regulatory constraints with penalized regression”, Ann N Y Acad Sci 1158, 114 (2009). Hazen S., Naef F., Quisel T., Gendron J., Chen H., Ecker J., Borevitz J., Kay S., “Exploring the transcriptional landscape of plant circadian rhythms using genome tiling arrays”, Genome Biol 10, R17 (2009). Dibner C., Sage D., Unser M., Bauer C., d’Eysmond T., Naef F., Schibler U., “Circadian gene expression is resilient to large fluctuations in overall transcription rates”, The EMBO Journal 28, 123 (2009). Bénazet J. D., Bischofberger M., Tiecke E., Gonçalves A., Martin J. F., Zuniga A., Naef F., Zeller R., “A self-regulatory system of interlinked signaling feedback loops controls mouse limb patterning”, Science 323, 1050 (2009). Sauvain M-O, Dorr A.P., Stevenson B., Quazzola A, Naef F, Wiznerowicz M., Schütz F., Jongeneel V., Duboule D, Spitz F, Trono D., “Genotypic features of lentivirus transgenic mice”, J Virol 82, 7111 (2008). Rougemont J., Amzallag A., Iseli C., Farinelli F., Xenarios I, Naef F., “Probabilistic base calling of Solexa sequencing data”, BMC Bioinformatics 9, 431 (2008). Paquet E., Rey G., Naef F., “Modeling an Evolutionary Conserved Circadian Cis-Element”, PLoS Comput Biol 4, e38 (2008).

Phase (x-axis) enrichment profiles of binding sites for transcription regulators in TRANSFAC (y-axis) are inferred from mRNA times series data in mouse liver. Most phases are represented (left). Clusters of regulators are separated according to peak time (phase). E.g. E-box motifs peak around ZT10, as expected from CLOCK/BMAL1 activity.

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55

IBI

A 3D model of the developing Drosophila embryo. Segmentation of the Drosophila melanogaster embryo results from the dynamic establishment of spatial mRNA and protein patterns. Current mathematical models typically consider a simplified 1D geometry along a portion of the anterior-posterior (A-P) axis. We have modeled the gap gene network on the actual 3D surface of the blastoderm, including the head and pole regions. The reaction-diffusion model was calibrated using recent 3D spatio-temporal mRNA and protein expression atlases. We modeled the early dynamics of both mRNA and protein of the gap genes hunchback, Kruppel, giant and knirps, taking as regulatory inputs the maternal Bicoid and Caudal gradients, plus the zygotic Tailless and Huckebein proteins. The inferred network captures the expression patterns reliably, and predicts gap gene knock-outs and bicoid dosage mutants. Sensitivity analysis showed that the posterior domain of giant is among the most fragile features of an otherwise fairly robust network. Such 3D models offer clear advantages for studying morphogenesis in realistic geometries, accounting for cross-regulation between the A-P and dorso-ventral (D-V) systems to capture D-V asymmetries in the patterns of gap genes.


EPFL School of Life Sciences - 2009 Annual Report

IBI - Institute of Bioengineering - ISREC

Swartz Lab Head of Lab (PI) - http://lmbm.epfl.ch/ Team Members

Melody Swartz Associate Professor

Marie Ballester, PhD student Véroniqe Borel, Technician Carmen Bonvin, EPFL MS student (9/2008 – 6/2009) Dr. Brandon Dixon, Postdoctoral fellow (left the lab 8/2009) Didier Foretay, Technician Esra Guç, PhD student (started 4/2009) Ulrike Haessler, PhD student Patricia Corthésy Henrioud, technician (started 7/2009) Dr. Sachiko Hirosue, Scientist (joint with Hubbell lab) Laura Jeanbart, PhD student (joint with Hubbell lab, started 9/2009) Alba Jimenez, Erasmus MS student from Univ Barcelona (10/2008 – 4/2009) Dr. Tine Karlsen, visiting scientist (from 9/2008 – 6/2009) Dr. Witold Kilarski, Postdoctoral fellow Iraklis Kourtis, PhD student (joint with Hubbell lab) Dr. Amanda Lund, Postdoctoral fellow (started 7/2009) Ingrid Margot, Administrative Assistant Dimana Miteva, PhD student Carine Rosa Tsamo Nintedem, EPFL MS student (5/2008 – 3/2009) Miriella Pasquier, Technician Marco Pisano, PhD student (started 9/2009) Sandeep Raghunathan, PhD student (joint with Hubbell lab) Dr. Evan Scott, Postdoctoral fellow (joint with Hubbell lab) Dr. Adrian Shieh, Postdoctoral fellow Dr. Jacqueline Shields, Postdoctoral fellow Dr. Susan Thomas, Postdoctoral fellow Valentina Triacca, PhD student (started 9/2009)

Introduction

Immune cell transport and cancer metastasis depend on lymphatic function and are tied to interstitial fluid transport and lymph formation. Lymphatic vessels and their biomechanical drainage functions play important roles in inflammation, immunity, and the tumor micro-environment. Lymphatic drainage is known to greatly increase upon injury or certain types of acute inflammation, and dendritic cells enter lymphatic vessels when activated by pathogenic danger signals or certain inflammatory cytokines. However, how lymphatic vessels respond to, and modulate, their surroundings is poorly understood. We are interested in learning how lymphatic endothelium responds to such complex cues to modulate its function, and how these functional changes affect immunity and the tumor micro-environment. Furthermore, lymphatic vessels and the lymph nodes they drain to are emerging targets for immunomodulation. Our lab aims to elucidate and exploit the integrated physiology and biology of interstitial and lymphatic transport, as well as its affects on tumor progression, using interdisciplinary in vivo, in vitro, and in silico approaches. In doing so, we aim to better understand how lymphatic function is regulated and the specific roles it plays in cancer, and to develop new design principles for lymphatic tissue engineering and lymph node-targeted immunomodulatory therapeutics.

Keywords:

Lymphatic biology, cancer metastasis, tumor microenvironment, biological transport, interstitial flow, tissue engineering, lymphangiogenesis, lymph node, immunomodulation, mechanobiology, lymphedema

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Results Obtained in 2009

In 2009 we continued to contribute in several areas of lymphatic biology, specifically related to immunology and tumor micro-environment. In tumor-lymphatic interactions, we described a novel relationship between lymphatics and invasive tumor cells, showed that tumor expression of the lymphatic growth factor VEGF-C and the lymphoid homing receptor CCR7 act synergistically to promote their invasion towards lymphatics. First, VEGF-C increased lymphatic secretion of the lymphoid chemokine CCL21, driving CCR7-dependent tumor chemoinvasion towards lymphatics. Second, VEGF-C acted in an autocrine fashion to increase tumor invasiveness by increasing the proteolytic activity and motility of tumor cells in a 3D matrix. These findings bridge the pro-metastatic functions of CCR7 and VEGF-C in tumors, and demonstrate that beyond lymphangiogenesis, VEGF-C promotes tumor invasion towards lymphatics by both autocrine and CCR7-dependent paracrine signaling mechanisms (Issa et al, Cancer Res. 2009). We found that CCL21, critical for leukocyte and tumor cell homing to lymphatics, is also strongly regulated by flow, both in the lymph node stroma (Tomei et al, J. Immunol., 2009) and in lymphatic endothelium (Miteva et al, Circ Res (in press)). In addition to CCL21, we showed that leukocyte adhesion molecules and ultimately dendritic cell transmigration were regulated by transmural flow. Since increased fluid flow is the most immediate response to tissue injury or some inflammatory events, these findings suggest that lymphatics use fluid flow as an important inflammatory cue.

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EPFL School of Life Sciences - 2009 Annual Report

Finally, together with the Laboratory for Regenerative Medicine and Pharmacobiology, we expanded our work in exploiting the phenomenon of interstitial flow to target immunotherapeutics to the lymphatics. We have built up our team to focus on a number of applications with relevant collaborators, including vaccines against cancer (Merck-Serono), Lassa virus (Stefan Kunz), and tuberculosis (John McKinney), and are currently evaluating several different strategies.

Selected publications

JD Shields, IC Kourtis, AA Tomei, J. Roberts, and MA Swartz. Induction of lymphoid-like stroma and immune escape by tumors that express the chemokine CCL21. Science. (in press) DO Miteva, JB Dixon, W Kilarski, JM Rutkowski, JD Shields, and MA Swartz. Transmural flow modulates cell and fluid transport functions of lymphatic endothelium: A potential early cue for inflammation. Circ. Res. (in press). JA Pedersen, S. Lichter, and MA Swartz. Cells in 3D matrices under interstitial flow: Effects of pericellular matrix alignment on cell shear stress and drag forces. J. Biomech. (in press). C Bonvin, J Overney, AC Shieh, JB Dixon, and MA Swartz. A multichamber fluidic device for long-term 3D cultures under interstitial flow with live imaging. Biotechnol. Bioeng.

(in press). JM Rutkowski, CE Markhus, C.C. Gyenge, K. Alitalo, H. Wiig, MA Swartz. Dermal collagen and fat accumulation correlate with tissue swelling and hydraulic conductivity in murine lymphedema. Am. J. Pathol. (in press). JA Hubbell, SN Thomas, MA Swartz (2009). Materials engineering for immunomodulation. Nature 462(7272):449460. AA Tomei, S Siegert, MR Britschgi, SA Luther, MA Swartz (2009). Fluid flow regulates stromal cell organization and CCL21 expression in a tissue-engineered lymph node model. J Immunol 183(7):4273-83 (featured article) HY Lim, JM Rutkowski, J Helft, ST Reddy, MA Swartz, GJ Randolph, and V Angeli (2009). Hypercholesterolemic mice exhibit lymphatic vessel dysfunction and degeneration. Am J Pathol 175(3):1328-1337. JB Dixon, S Rangunathan, MA Swartz (2009). A tissue engineered model of the intestinal microenvironment for evaluating lipid uptake into lacteals. Biotech Bioeng 103(6):1224-35. (Cover photo) U Haessler, Y Kalinin, MA Swartz, M Wu (2009). An agarosebased microfluidic platform with a gradient buffer for 3D chemotaxis studies. Biomed. Microdev. 11(4):827-35. AA Tomei, F Boschetti, F. Gervaso, MA Swartz (2009). Culturing 3D collagen cultures under well-defined dynamic strain: a novel strain device with a porous elastomeric support. Biotech Bioeng 103: 217-225 (featured “Spotlight”). A Issa, TX Le, AN Shoushtari, JD Shields, and MA Swartz (2009). VEGF-C and CCL21 in tumor cell-lymphatic crosstalk promote invasive phenotype. Cancer Res. 69:34957. MA Swartz, JA Hubbell, and ST Reddy (2008). Lymphatic drainage function and its immunological implications: From dendritic cell homing to vaccine design. Semin Immunol 20(2):147-56. AA Tomei, MM Choe, and MA Swartz (2008). Effects of dynamic compression on Lentiviral transduction in an in vitro airway wall model. Am J Physiol Lung Cell Mol Physiol 294:L79-L86. (Faculty of 1000 featured article)

Lymphatic capillaries (green) attract activated dendritic cells (magenta), which transmigrate into the vessel at the terminal end. Blood capillaries are shown in red.”

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IBI

Lymphoid chemokines are not only important for chemoattraction to lymphatics. In 2009, we discovered that invasive tumor cells can also secrete CCL21 to start a chain of events that drive lymphoidlike changes in the tumor micro-environment, ultimately leading to immunological tolerance (Shields et al, Science, in press). We hypothesize that CCL21 attracts lymphoid tissue inducer cells that lead to fibroblast transformation towards lymphoid-like stroma, which are rich in regulatory functions and critical for regulatory T (Treg) cell activation. CCL21 also attracts Tregs and naïve T cells to the tumor stroma, where the highly tolerogenic micro-environment leads to a shift in host immune response to the tumor from immunogenic to tolerogenic. Thus, we propose that lymphoid chemokine expression by tumors can provide a mechanism for promoting immunological tolerance.


EPFL School of Life Sciences - 2009 Annual Report

IBI - Institute of Bioengineering

Wurm Lab Head of Lab (PI) - http://lbtc.epfl.ch/ Team Members

Florian M. Wurm Full Professor

Bachmann Virginie, Technical Assistant Baldi Unser Lucia, Senior Scientist Baras Jérémy, Trainee Hesso Benavides Tatiana, Master Student Broccard Gilles, Master Student Davami Fatameh, Visiting Phd Student Delegrange Fanny, Phd Student Divorne Marie-France, Administrative Assistant Engelhardt Eva Maria, Phd Student Favre Daniel, Visiting Scientist Hacker David, Senior Scientist Hadadi Noushin, Internship (15.09.2009 - 31.12.2009) Kiseljak Divor, Phd Student Manoli Sagar, Master Student (01.03.2009 - 31.12.2009 Matasci Mattia, Scientist Nallet Sophie, Phd Student Noll Sébastien, Trainee Hesso Oberbek Agata, Phd Student Rajendra Yashas, Phd Student Rudin Fabienne, Administrative Assistant Sinhadri Balaji, Master Student (15.03.2009 - 15.09.2009) Thomas Amelie, Trainee Hesso Tissot Stéphanie, Phd Student Wulhfard Sarah, Phd Student Zagari Francesca, Phd Student Zhang Xiaowei, Phd Student Xie Qiuling, Visiting Scientist (15.06.2009 - 31.12.2009)

Introduction

The major goal of the Laboratory of Cellular Biotechnology is the development of novel and/or improved tools for gene (DNA) transfer to cultured mammalian cells and subsequent high-level expression of recombinant proteins from such cells in scalable production systems (bioreactors). This work has become important since mammalian cells are now considered the most versatile and productive system for the manufacture of recombinant proteins for pharmaceutical applications. In fact this part of the pharma business is the fastest growing sector of global industrial activities and the value of sales of a small number of highly beneficial protein products through this technology exceeds 50 billion US$ annually

Keywords:

Recombinant protein expression – Mammalian cell culture – Bioreactor – Bioprocess control – Gene transfer – DNA integration – Microinjection - Stable cell line development – Tissue engineering – Orbital shaking

Results Obtained in 2009

Research at the LBTC is situated on the crossroads between biology and engineering, and it addresses the expression of recombinant proteins from suspension cultures of mammalian cells, which is the major approach to therapeutic protein production in modern biotechnology industry. We are investigating two major thematic areas: gene delivery and transient gene expression in mammalian cells and their respective impacts on the host cells physiology and

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genetics, orbital shaking technology and novel bioreactor systems. Gene delivery and transient gene expression. We have studied the cellular uptake and disassembly of PEI-DNA complexes in mammalian cells, and by combining our knowledge of cellular metabolism in suspension cultures with our most recent results we were able to increase recombinant protein productivity from transiently transfected cells. The highest titers ever reported for a transiently expressed recombinant antibody (1 g/L) in HEK-293 cells were obtained in an orbitally shaken, non-instrumented cultivation system. Currently the system is being up scaled to 100 and 1’000 Litres (see below). For recombinant genes that are stably integrated into the genome of the host cell lines we have focused on one cell line, CHO, which is the most widely used cell line in the biotech industry. We have investigated the cytogenetics of CHO-derived stable cell lines generated using different DNA delivery techniques. We are interested whether the widely observed phenomenon of gene silencing can be prevented. We have also investigated micro-injection as a tool for DNA delivery for cell line generation, which allows quantitative delivery of plasmid DNA inside a single cell. Lentiviral vectors and transposon (integrase) mediated DNA delivery is hoped to improve the integration of DNA into the active chromatin of the host cells. The orbitally shaken bioreactor technology for mammalian cell cultivation, designed in our lab, has been scaled-up to 1’000 L with a custom-made re-

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search bioreactor. This and other orbitally shaken cylindrical vessels (with nominal volumes from 50 mL to 250 L) are being extensively studied in order to characterize the hydrodynamics of this type of agitated systems. For these studies we have formed collaborations with the Professor Alfio Quarteroni (Chair of Modelling and Scientific Computing and Dr. Mohamed Farhat of the Laboratory of Hydraulic Machines to study the fluid dynamics in orbitally shaken bioreactors. In these studies, a good understanding of the interplay of Chemistry, Biology and Engineering is very important, therefore part of the work is also addressing question of cell physiology and metabolism.

Engelhardt EM, Houis S, Gries T, Hilborn J, Adam M, Wurm FM. Suspension-adapted Chinese hamster ovary-derived cells expressing green fluorescent protein as a screening tool for biomaterials. Biotechnol Lett. 2009 Aug;31(8):11439. Epub 2009 Apr 10.

Overall, our research provided useful insights for understanding cell cultivation in suspension, gene integration and protein expression. These studies are of general interest in biotechnology (gene therapy, protein production, industrial biotechnology). Through our research we also provide innovative tools for industrial applications and development.

Chenuet, S., Martinet, D., Besuchet-Schmutz, N., Wicht, M., Jaccard, N., Bon, A. C., Derouazi, M., Hacker, D. L., Beckmann, J. S., and Wurm, F. M. (2008) Calcium phosphate transfection generates mammalian recombinant cell lines with higher specific productivity than polyfection, Biotechnol Bioeng 101, 937-945.

Selected publications

Zhang X, Stettler M, De Sanctis D, Perrone M, Parolini N, Discacciati M, De Jesus M, Hacker D, Quarteroni A, Wurm F. Use of orbital shaken disposable bioreactors for Mammalian cell cultures from the milliliter-scale to the 1,000-liter scale. Adv Biochem Eng Biotechnol. 2010;115:33-53. Hacker DL, De Jesus M, Wurm FM. 25 years of recombinant proteins from reactor-grown cells - where do we go from here? Biotechnol Adv. 2009 Nov-Dec;27(6):1023-7. Epub 2009 May 20. Review. Nallet S, Amacker M, Westerfeld N, Baldi L, König I, Hacker DL, Zaborosch C, Zurbriggen R, Wurm FM. Respiratory syncytial virus subunit vaccine based on a recombinant fusion protein expressed transiently in mammalian cells. Vaccine. 2009 Oct 30;27(46):6415-9. Engelhardt EM, Stegberg E, Brown RA, Hubbell JA, Wurm FM, Adam M, Frey P. Compressed collagen gel: a novel scaffold for human bladder cells. J Tissue Eng Regen Med. 2009 Oct 19.

Chenuet S, Derouazi M, Hacker D, Wurm F. DNA delivery by microinjection for the generation of recombinant mammalian cell lines. Methods Mol Biol. 2009;518:99-112. Zhang, X., Stettler, M., Reif, O., Kocourek, A., Dejesus, M., Hacker, D. L., and Wurm, F. M. (2008) Shaken helical track bioreactors: Providing oxygen to high-density cultures of mammalian cells at volumes up to 1000 L by surface aeration with air, N Biotechnol 25, 68-75.

Bertschinger, M., Schertenleib, A., Cevey, J., Hacker, D. L., and Wurm, F. M. (2008) The kinetics of polyethyleniminemediated transfection in suspension cultures of Chinese hamster ovary cells, Molecular biotechnology 40, 136143. Backliwal, G., Hildinger, M., Kuettel, I., Delegrange, F., Hacker, D. L., and Wurm, F. M. (2008) Valproic acid: a viable alternative to sodium butyrate for enhancing protein expression in mammalian cell cultures, Biotechnol Bioeng 101, 182-189. Backliwal, G., Hildinger, M., Hasija, V., and Wurm, F. M. (2008) High-density transfection with HEK-293 cells allows doubling of transient titers and removes need for a priori DNA complex formation with PEI. Biotechnol Bioeng 99, 721-727. Backliwal, G., Hildinger, M., Chenuet, S., Wulhfard, S., De Jesus, M., and Wurm, F. M. (2008) Rational vector design and multi-pathway modulation of HEK 293E cells yield recombinant antibody titers exceeding 1 g/l by transient transfection under serum-free conditions. Nucleic Acids Res 36, e96.

Orbital shaking at the multi-hundred litres scale. The shaking bioreactor system was developed and tested for cell cultivation inside a disposable bag for volumes up to 200 L (manufactured by the Swiss company Kühner AG).

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IBI

EPFL School of Life Sciences - 2009 Annual Report


EPFL School of Life Sciences - 2009 Annual Report

IBI - Co-affiliated Research Groups

IBI - Co-affiliated Research Group - School of Engineering (STI)

Aminian Lab Head of Lab (PI) - http://lmam.epfl.ch/

Team Members

Kamiar Aminian Professor Titulaire STI

Rachid Aissaoui, Visiting Professor Danielle Alvarez, Administration Assistant Kamiar Aminian, Adjunct Professor Arash Arami, PhD student Arnaud Barré, PhD student Julien Chardonnens, PhD student Hooman Dejnabadi, Scientist Cyntia Duc, PhD student Julien Favre, lecturer Anthony Fleury, Postdoctoral Fellow Raluca Lidia Ganea, PhD student Jean Gramiger, Technicien Anisoara Ionescu, Scientist Benoît Mariani, PhD student Pascal Morel, Technicien Hossein Rouhani, PhD student

Research Interests

The multidisciplinary research of the Laboratory of Movement Analysis and Measurement aims to transfer bioengineering findings into clinical applications. We are particularly interested to characterize sport performances and pathologies affecting motor function such as osteoarthritis, frailty, pain or movement disorder by studying the movement ability. Our research involves biomechanical instrumentation for measuring and modelling human biodynamics in daily conditions, during spontaneous activity or physical exercises. Based on body worn sensors, we design wearable systems and algorithms for longterm monitoring of physical activity and gait analysis, for the estimation of the 3D joint kinematics and kinetics, and for the sport performance evaluation. This involves advanced signal processing, multiparametric approach, sensors’ fusion and functional calibration methods to devise new methods for activity recognition and to extract relevant disease/health related features hidden in human biomechanical signals. Based on these features and instruments new metrics are defined and validated to provide early diagnosis and objective clinimetry for outcome evaluation in orthopaedics and aging, to assess the change of motor function with disease and rehabilitation, to characterise improved performances in sport, and to classify movement disorders.

Selected publications

Paraschiv-Ionescu, K. Aminian, Nonlinear analysis of physiological time series, in Advanced Biosignal Processing, Springer, 307-334, Chapter 15, 2009 Najafi, J. Helbostad , R. Moe-Nilssen, W. Zijlstra, K. Aminian Does walking strategy in older people change as a function of walking distance? Gait & Posture, 261-266, 2009 Coley, BM. Jolles, A. Farron, K. Aminian, (2008), Arm position during daily activity, Gait & Posture, 581–587 S. Rochat, MD,E. Martin, C. Piot-Ziegler, B. Najafi, K. Aminian,C.J Büla, (2008), Falls Self-Efficacy and Gait Performance after Gait and Balance Training in Older People, J Am Geriatr Soc 56 (6): 1154-1156 J. Favre, B.M. Jolles, R. Aissaoui, K. Aminian, (2008), Ambulatory measurement of 3D knee joint angle, Journal of Biomechanics, 41, 1029-1035 Paraschiv-Ionescu, E. Buchser, B. Rutschmann, K. Aminian, (2008), Nonlinear analysis of the human physical activity patterns in health and disease, Phys. Rev. E 77, 021913 Coley, BM. Jolles, A. Farron, C. Pichonnaz, JP Bassin, K. Aminian, (2008), Estimating the upper-limb dominant segment during daily activity, Gait & Posture, 368-375 H. Dejnabadi, BM Jolles, K. Aminian, (2008), A New Approach for Quantitative Analysis of Inter-Joint Coordination during Gait, IEEE Transactions on Biomedical Engineering, 755-764

J. Favre, R. Aissaoui, BM. Jolles, JA de Guise, K. Aminian, Functional calibration procedure for 3D knee joint angle description using inertial sensors, Journal of Biomechanics, 42, 2330-2335, 2009 Coley, C, Jolles, BM, Farron, A, Aminian, K, Detection of the movement of the humerus during daily activity, Med. Biol. Eng. Comp, 47, 467–474, 2009

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EPFL School of Life Sciences - 2009 Annual Report

IBI - Co-affiliated Research Group - School of Engineering (STI)

Guiducci Lab

IBI

Head of Lab (PI) - http://clse.epfl.ch/

Team Members

Accastelli Enrico, Assistant-doctorant Bianchi Elena, Assistante-doctorante Cappi Giulia, Stagiaire Ferretti Anna, Assistante-doctorante Salimi Homeira, Secrétaire Temiz Yuksel, PhD student (in co-direction with Prof. Leblebici), Trippa Maria, Stagiaire

Carlotta Guiducci

Tenure Track Assistant Professor STI Swiss Up Engineering Chair

Research Interests

CLSE is a newly established laboratory in EPFL, affiliated to the Institute of Bioengineering (IBI) and to the Institute of Electrical Engineering (IEL). CLSE has interdisciplinary personnel and owns strong biological and electrical characterization competences. It is equipped with wet lab facilities, electrical characterization instrumentation and a room dedicated to cell culture. The research team’s activity focuses on the design and application of electronic biosensors, which are at the forefront of electronic engineering and bioengineering. The sensors address a wide range of applications, from nucleic acid, protein and drug detection to the measurements of bacterial and cell metabolism and they will be based on detection principles supporting electronic transduction, in order to couple directly and integrate the sensors themselves with electronic circuitry for data acquisition. Professor Carlotta Guiducci’s research effort has been devoted to the development and characterization of novel electronic sensing techniques on chip for life science applications. She developed in collaboration with Infineon technologies two CMOS test chips for DNA detection by capacitance measurements, which successfully demonstrated the feasibility of the technique. She has been working on electrical, electrochemical and optical techniques.

Selected publications

Overview of Electrochemical DNA Biosensors: New Approaches to Detect the Expression of Life. Stefano Cagnin, Marcelo Caraballo, Carlotta Guiducci, Paolo Martini, Marty Ross, Mark SantaAna, David Danley, Todd West, and Gerolamo Lanfranchi In Sensors, volume 9, number 4, page 3122 – 3148, 2009. High Parallelism, Portability, and Broad Accessibility: Tech-

nologies for Genomics . C Guiducci And C Nardini In Acm Journal On Emerging Technologies In Computing Systems, volume 4, number 1, pages 3:1-3:39, 2008. Novel front-end circuit architectures for integrated bioelectronic interfaces. C. Guiducci, A. Schmid, F. K. Guerkaynak, and Y. Leblebici In 2008 Design, Automation and Test in Europe, Vols 1-3, pages 1170-1175, 2008. A fully electronic label-free DNA sensor chip. C. Stagni, C. Guiducci, L. Benini, B. Ricco, S. Carrara, C. Paulus, M. Schienle, and R. Thewes In Ieee Sensors Journal, volume 7, number 3-4, pages 577-585, 2007. Electronic detection of DNA hybridization: Toward CMOS microarrays. L. Benini, C. Paulus, and C. Guiducci In Ieee Design & Test of Computers, volume 24, number 1, pages 38-48, 2007. CMOS DNA sensor array with integratedA/D conversion based on label-free capacitance measurement. C. Stagni, C. Guiducci, L. Benini, B. Ricco, S. Carrara, B. Samori, C. Paulus, M. Schienle, M. Augustyniak, and R. Thewes In Ieee Journal of Solid-State Circuits, volume 41, number 12, pages 2956-2964, 2006. Hydrogenated amorphous silicon ultraviolet sensor for deoxyribonucleic acid analysis. G. de Cesare, D. Caputo, A. Nascetti, C. Guiducci, and B. Ricco In Applied Physics Letters, volume 88, number 8, 2006. Fully Electronic CMOS DNA Detection Array Based on Capacitance Measurement with On-Chip Analog-to-Digital Conversion. C Stagni Degli Esposti, C Guiducci, C Paulus, M Schienle, M Augustyniak, G Zuccheri, B Samori, L. Benini, B Ricco, and R Thewes In Solid-State Circuits Conference, 2006. ISSCC 2006. Digest of Technical Papers. IEEE International, pages 69-78, San Francisco, CA, Feb 6-9, 2006. DNA detection by integrable electronics. C. Guiducci, C. Stagni, G. Zuccheri, A. Bogliolo, L. Benini, B. Samori, and B. Ricco In Biosensors & Bioelectronics, volume 19, number 8, pages 781-787, 2004.

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EPFL School of Life Sciences - 2009 Annual Report

IBI - Co-affiliated Research Group - School of Basic Sciences (BS)

Johnsson Lab Lab Hatzimanikatis Head of Lab (PI) - http://isic.epfl.ch/lcsb

Team Members

Vassily Hatzimanikatis

Angelino Paolo, Scientist Brunk Elizabeth Claire, Doctoral Assistant Brusnjak Alen, Bachelor/Master Student Fung Ho Ki, Scientist Hadadi Noushin, Bachelor/Master Student Kupper Christine, Secretary/Administrator Mettraux Yannick, Bachelor/Master Student Miskovic Ljubisa, Scientist Racle Julien, Doctoral Assistant Radivojevic Andrijana. Doctoral Assistant Seijo Marianne, Scientist Soh Keng Cher, Doctoral Assistant Stefaniuk Adam, Bachelor/Master Student Özdemir Muhittin Emre, Doctoral Assistant

Associate Professor SB

Research Interests

Computational biotechnology focuses on the development of mathematical models and systems engineering frameworks for accelerating the design and purposeful manipulation of complex cellular processes. The Laboratory of Computational Systems Biotechnology (LCSB) develops expertise in the formulation of mathematical models of cellular processes and in the development of process systems engineering methods for the integration and analysis of experimental information from different levels. However, most of this information is partial and it is subject to uncertainty. Researchers in LCSB develop methods that can account quantitatively for the uncertainty in the available information and can provide guidance on solving problems in biotechnology and medicine. LCSB is one of the leading laboratories in the study of energetics and thermodynamics of complex cellular processes. Research in LCSB has also pioneered the development of computational methods for the discovery of novel metabolic pathways for metabolic engineering and synthetic biology. The applications areas of research in LCSB are: metabolic engineering and metabolic diseases, bioenergetics, protein synthesis, and drug discovery.

Selected publications

“Network thermodynamics in the post-genomic era”, Keng Cher Soh and Vassily Hatzimanikatis, Curr. Opin. Microbiol., Apr 6. [Epub ahead of print] DOI 10.1016/j. mib.2010.03.001 (2010). “In silico feasibility of novel biodegradation pathways for 1,2,4-trichlorobenzene”, Stacey D. Finley, Linda J. Broadbelt, Vassily Hatzimanikatis, BMC Systems Biology, 4:7, doi:10.1186/1752-0509-4-7 (2010).

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“The Origins of Time-Delay in Template Biopolymerization Processes”, Luis Mier-y-Teran, Mary Silber, Vassily Hatzimanikatis, PLoS Computational Biology, Apr 1;6(4):e1000726 (2010). “Discovery and analysis of novel metabolic pathways for the biosynthesis of industrial chemicals: 3-hydroxypropanoate”, Christopher S. Henry, Linda J. Broadbelt, Vassily Hatzimanikatis, Biotechnology and Bioengineering, 106(3), 462-473 (2010). “Computational framework for predictive biodegradation”, Stacey D. Finley, Linda J. Broadbelt, and Vassily Hatzimanikatis, Biotechnology and Bioengineering, 104 (6), 1086-1097 (2009). “Thermodynamic analysis of biodegradation pathways”, Stacey D. Finley, Linda J. Broadbelt, and Vassily Hatzimanikatis, Biotechnology and Bioengineering, 103 (3), 532-541 (2009). “Group contribution method for thermodynamic analysis of complex metabolic networks”, Matthew D. Jankowski, Christopher S. Henry, Linda J. Broadbelt, and Vassily Hatzimanikatis, Biophysical Journal, 95 (3), 1487-1499 (2008). “Effects of codon distributions and tRNA competition on protein translation”, Hermioni D. Zouridis and Vassily Hatzimanikatis, Biophysical Journal, 95 (3), 1018-1033 (2008). “A genome-scale metabolic reconstruction for Escherichia coli K-12 MG1655 that accounts for 1261 ORFs and thermodynamic information”, Adam M. Feist, Chris S. Henry, Jennifer L. Reed, Markus Krummenacker, A. R. Joyce, Peter D. Karp, Linda J. Broadbelt, Vassily Hatzimanikatis, and Bernhard O. Palsson, Molecular Systems Biology, 3: Art. No. 121 (2007). “Thermodynamics-based Metabolic Flux Analysis”, C.S. Henry, L.J. Broadbelt, V. Hatzimanikatis, Biophysical Journal, 92 (5), 1792-1805 (2007).

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EPFL School of Life Sciences - 2009 Annual Report

IBI - Co-affiliated Research Group - School of Engineering (STI)

JohnssonLab Ijspeert Lab Head of Lab (PI) - http://biorob.epfl.ch/

Auke Ijspeert

Associate Professor STI

IBI

Team Members

Ajallooeian Mostafa, PhD student Bicanski Andrej, PhD student Bonardi Stéphane, PhD student Crespi Alessandro, Postdoctoral Researcher Dégallier Sarah, PhD student Fiaux Sylvie, Secretary Gay Sébastien, PhD student Karakasiliotis Konstantinos, PhD student Knüsel Jérémie, PhD student Möckel Rico, Postdoctoral Researcher Porez Mathieu, Postdoctoral Researcher Pouya Soha,PhD student Ronsse Renaud, Postdoctoral Researcher Spröwitz Alexander, PhD student Tuleu Alexandre, Research Assistant van den Kieboom Jesse, PhD student

Research Interests

Our research is at the intersection of robotics and computational neuroscience. It addresses the topics of movement control, sensorimotor coordination, and learning in autonomous robots with multiple degrees of freedom (from snake robots to quadruped robots to humanoid robots). Our ambition is twofold: (1) to program and design robots that exhibit motor skills with the same efficiency, adaptivity, and robustness as animals, and (2) to get a better understanding of the functioning of animals using numerical simulation and robots as scientific tools. Together with neurobiologists (Jean-Marie Cabelguen and Sten Grillner), we have developed mathematical models of the neural circuits controlling locomotion in lower vertebrates. We have demonstrated how a primitive neural circuit for swimming like the one found in the lamprey can be extended by phylogenetically more recent limb oscillatory centers to explain the ability of salamanders to switch between swimming and walking. These models have been tested in an innovative salamander-like robot capable of swimming and walking.

Ijspeert A.J., Central pattern generators for locomotion control in animals and robots: a review. Neural Networks, 21(4):642-653, 2008 Buchli J. and Ijspeert A.J.. Self-organized adaptive legged locomotion in a compliant quadruped robot. Autonomous Robots, 25(4):331-347, 2008. Buchli J., Righetti L., and Ijspeert A.J.. Frequency Analysis with a Nonlinear Dynamical System, Physica D, 237: 1705–1718, 2008. Sproewitz A., Moeckel R., Maye J., Ijspeert A.J., Learning to move in modular robots using central pattern generators and online optimization. International Journal of Robotics Research. 27(3-4):423-443, 2008 Crespi A. and Ijspeert A.J.. Online optimization of swimming and crawling in an amphibious snake robot. IEEE Transactions on Robotics, 24(1), 2008 pp 75-87. Chevallier S., Ijspeert A.J., Ryczko D., Nagy F. and Cabelguen J.-M., Organisation of the spinal central pattern generators for locomotion in the salamander: biology and modelling. Brain Research Reviews. 57(1), 2008, pp 147-161.

We also develop a dynamical systems approach for controlling movements in robots. For instance, we designed the concept of dynamical movement primitives: nonlinear dynamical systems with well-defined attractor properties that can learn demonstrated discrete or rhythmic movements. Our methods are applied to various robots (quadruped, humanoid and reconfigurable modular robots) and more recently to lower limb exoskeletons for patients with locomotor deficiencies.

Tsakarakis N.G., Metta G., Sandini G., Vernon D., Beira R., Becchi F., Righetti L., Santos-Victor J., Ijspeert A.J., Carrozza M.C., and Caldwell D.G.. iCub - The Design and Realization of an Open Humanoid Platform for Cognitive and Neuroscience Research. Journal of Advanced Robotics, 21(10), 2007, pp 1151-1175.

Selected publications

Righetti L., Buchli, J. and Ijspeert A.J.: Dynamic Hebbian learning in adaptive frequency oscillators, Physica D, 216(2), 2006 pp 269-281

A. Spröwitz, S. Pouya, S. Bonardi, J. van den Kieboom, R. Möckel, A. Billard, P. Dillenbourg, A.J. Ijspeert. Roombots: Reconfigurable Robots for Adaptive Furniture, IEEE Computational Intelligence Magazine, 5(3): 20-32, 2010

Ijspeert A.J., Crespi A., Ryczko D., and Cabelguen J.M.. From swimming to walking with a salamander robot driven by a spinal cord model. Science, 315(5817):1416-1420, 2007.

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EPFL School of Life Sciences - 2009 Annual Report

IBI - Co-affiliated Research Group - School of Basic Sciences (BS)

Johnsson Lab Head of Lab (PI) - http://isic.epfl.ch/lip Team Members

Kai Johnnson Full Professor SB

Avello Simoes Pires Claudia, Postdoctoral Researcher Bojkowska Karolina, Doctoral Assistant Brun Matthias, Doctoral Assistant Chidley Christopher, Doctoral Assistant Fellay Cindy, Assistant Fujishima Shohei, Doctoral Assistant Gasparini Marie-Claude, Secretary Haruki Hirohito, Scientist Hinner Marlon, Postdoctoral Researcher Lukinavicius Grazvydas, Postdoctoral Researcher Masharina Anastasiya, Doctoral Assistant Maurel Damien, Scientist Mollwitz Birgit, Doctoral Assistant Moser Simone, Scientist Pedersen Miriam Grønlund, Doctoral Assistant Peters Ruud, Doctoral Assistant Rengifo Gonzalez Monica, Scientific Assistant Reymond Luc, Scientist Schena Alberto, Doctoral Assistant Tan Kui Thong, Scientist Trefzer Claudia, Doctoral Assistant Umezawa Keitaro, Postdoctoral Researcher

Research Interests

The visualization and characterization of biologically relevant molecules and activities inside living cells continues to transform cell biology into a truly quantitative science. However, despite the spectacular achievements in some areas of cell biology, the majority of cellular processes still operate invisibly. Further progress will therefore depend increasingly on the development of new (fluorescent) sensors and chemical probes to target and characterize these activities. Our research addresses this need by developing and applying chemical approaches to observe and manipulate protein function in living cells. For example, we have introduced general methods for the covalent and specific labeling of fusion proteins with chemically diverse compounds that open up new ways of studying proteins (i.e. SNAP-tag, CLIP-tag and ACP-tag). We are pursuing the further development of such approaches and their application to biological problems that cannot be resolved by traditional approaches.

Selected publications

Arnaud Gautier, Eiji Nakata, Grazvydas Lukinavičius, KuiThong Tan, Kai Johnsson,”Selective crosslinking of interacting proteins using self-labeling tags”, J. Am. Chem. Soc. 131, 17954-62 (2009)

Kai Johnsson,“Visualizing biochemical activities in living cells” Nature Chemical Biology, 5, 63-5 (2009) Helen M. O’Hare, Rosario Durán, Carlos Cerveñansky, Marco Bellinzoni, Anne Marie Wehenkel, Otto Pritsch, Gonzalo Obal, Jens Baumgartner, Jérome Vialaret, Kai Johnsson, Pedro M. Alzari,“Regulation of glutamate metabolism by protein kinases in mycobacteria”Molecular Microbiology, 70, 1408-23 (2008) Christopher Chidley, Katarzyna Mosiewicz, Kai Johnsson,“A designed protein for the specific and covalent hetero-conjugation of biomolecules” Bioconjugate Chemistry, 19, 1753-6 (2008) Arnaud Gautier, Alexandre Juillerat, Christian Heinis, Ivan Reis Correa Jr., Maik Kindermann, Florent Beaufils, Kai Johnsson,“An engineered protein tag for multi-protein labeling in living cells” Chemistry&Biology, 15, 128-63 (2008) Helen O’Hare, Alexandre Juillerat, Petra Dianiskova, Kai Johnsson,“A split-protein sensor for studying protein-protein interactions in mycobacteria”,Journal of Microbiological Methods, 73, 79-84 (2008) Sambashiva Banala, Anke Arnold, Kai Johnsson, “Caged substrates for protein labeling and immobilization”, ChemBioChem, 9 38-41 (2008)

Matthias A. Brun, Kui-Thong Tan, Eiji Nakata, Marlon J. Hinner, Kai Johnsson,“Semisynthetic fluorescent sensor proteins based on self-labeling protein tags” J. Am. Chem. Soc., 131, 5873-84 (2009) Michael Bannwarth, Ivan R. Corrêa Jr, Monika Sztretye, Sandrine Pouvreau, Cindy Fellay, Annina Aebischer, Leandro Royer, Eduardo Ríos, Kai Johnsson,“Indo-1 derivatives for local calcium sensing”ACS Chemical Biology, 4, 179-190 (2009)

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EPFL School of Life Sciences - 2009 Annual Report

IBI - Co-affiliated Research Group - School of Engineering (STI)

Jolles-Haeberli Lab Head of Lab (PI) - http://cbt.epfl.ch Team Members

Brigitte Jolles-Haeberli Adjunct Professor STI Director of CBT

Research Interests

The mission of the Interinstitutional Center of Translational Biomechanics (CBT) is to promote and support the transfer of findings from the basic science laboratory to clinical application, and, vice versa, the transfer of clinical observations back to the lab, with a clear aim of improving patient care. The key point of this collaboration is its double-headed management, with a strong relationship between clinicians and engineers for each specific project. We develop medical devices and wearable systems to characterize human mobility and locomotion in daily conditions. Based on these instruments, we provide objective clinical metrics for diagnosis and outcome evaluation of treatments as well as useful parameters to increase sport performances. We also carry out work in tissue engineering of musculoskeletal tissues, implant and joints biomechanics, drug delivery systems and mechanobiology. A combination of biomechanical and biological approaches is used to describe and understand different clinical problems of interest such as bone loss following total joint arthroplasty, arthritis or intervertebral disc degeneration. Based on these analyses, original solutions are developed such as fetal cell therapy, scaffolds with high mechanical properties or orthopaedic implants used as drug delivery systems.

Selected publications

Favre J, Aissaoui R, Jolles BM, de Guise JA, Aminian K. Functional calibration procedure for 3D knee joint angle description using inertial sensors. J Biomech. 2009; 42(14):2330-5.

IBI

Aminian Kamiar, Adjunct Professor Arami Arash, PhD student Barré Arnaud, PhD student Chardonnens Julien, PhD student Crevoisier Xavier, Medical advisor Duc Cyntia, PhD student Farron Alain, Medical advisor Gortchacow Miguel, PhD student Gramiger Jean, Technician Jaccoud Sandra, Laboratory technician Krattinger Nathalie, Post-doc Laurent-Applegate Lee Ann, Group leader Morel Pascal, Technician Pioletti Dominique, Tenure Track Assistant Professor Quintin Aurélie, PhD student Ramondetti Silvio, Scientific collaborator Rouhani Hossein, PhD student Scaletta Corinne, Laboratory technician Schizas Constantin, Medical advisor Stadelmann Vincent, PhD student Terrier Alexandre, Group leader Vogel Arne, PhD student

Coley B, Jolles BM, Farron A, Aminian K. Detection of the movement of the humerus during daily activity. Med Biol Eng Comput. 2009; 47(5):467-74. Gremion G, Gaillard D, Leyvraz PF, Jolles BM. Effect of biomagnetic therapy versus physiotherapy for treatment of knee osteoarthritis: a randomized controlled trial. J Rehabil Med. 2009; 41(13):1090-5. Dejnabadi H, Jolles BM, Aminian K. A new approach for quantitative analysis of inter-joint coordination during gait. IEEE Trans Biomed Eng. 2008; 55(2 Pt 1):755-64. Favre J, Jolles BM, Aissaoui R, Aminian K. Ambulatory measurement of 3D knee joint angle. J Biomech. 2008; 41(5):1029-35. Jolles BM, Bogoch ER. Quality of life after TKA for patients with juvenile rheumatoid arthritis. Clin Orthop Relat Res. 2008; 466(1):167-78. Terrier A, Merlini F, Pioletti DP, Farron A. Comparison of polyethylene wear in anatomical and reversed shoulder prostheses. J Bone Joint Surg Br. 2009; 91(7):977-82. Quintin A, Schizas C, Scaletta C, Jaccoud S, Gerber S, Osterheld MC, Juillerat L, Applegate LA, Pioletti DP. Isolation and in vitro chondrogenic potential of human foetal spine cells. J Cell Mol Med. 2009; 13(8B):2559-69. Blecha LD, Rakotomanana L, Razafimahery F, Terrier A, Pioletti DP. Targeted mechanical properties for optimal fluid motion inside artificial bone substitutes. J Orthop Res. 2009; 27(8):1082-7. Terrier A, Sedighi-Gilani M, Roshan Ghias A, Aschwanden L, Pioletti DP. Biomechanical evaluation of porous biodegradable scaffolds for revision knee arthroplasty. Comput Methods Biomech Biomed Engin. 2009; 12(3):333-9.

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EPFL School of Life Sciences - 2009 Annual Report

IBI - Co-affiliated Research Group - School of Engineering (STI)

Maerkl Lab Head of Lab (PI) - http://lbnc.epfl.ch

Team Members

Sebastian Maerkl

Matthew Blackburn, PhD Student Nicolas Denervaud, PhD Student Luis Miguel Fidalgo, Postdoctoral fellow Jose Garcia-Cordero, Postdoctoral Fellow Marcel Geertz, Postdoctoral Fellow Bin He, PhD Student (Co-advised, Kreitman Lab, U. Chicago) Henrike Niederholtmeyer, PhD Student Jean-Bernard Nobs, PhD Student Tatjana Petrov, PhD Student Arun Rajkumar, PhD Student Sylvie Rockel, PhD Student Helen Chong, Administrative Assistant Viktoria Stepanova, Masters Student

Tenure Track Assistant Professor STI

Research Interests

In January of 2008 Prof. Maerkl established the Laboratory of Biological Network Characterization (LBNC) at the EPFL. The LBNC is principally interested in developing highly integrated microfluidic devices and applying these to pertinent problems in biology. Of particular interest to the lab at the moment is systems biology, which will benefit tremendously from the development of novel, high-throughput technologies. We are actively developing high-throughput methods for single cell analysis in S.cerevisiae and S.pombe, as well as M.smegmatis in collaboration with the McKinney Lab (SV/GHI). Using these methods we are interested in characterizing global protein expression dynamics on the single cell level (S.cerevisiae), understand how genotypic variants affect fitness (S.pombe), and discover leads towards understanding and possibly counteracting bacterial persistence (M.smegmatis). The lab is also interested in understanding transcriptional regulatory networks by developing and characterizing promoter variants in vivo, as well as through the biophysical characterization of transcription factors in vitro. We are additionally interested in synthetic biology, for which we are developing novel microfluidic methods. Prof. Maerkl also co-advises the annual EPFL iGEM team/ project course, which participates at a synthetic biology competition at MIT.

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Selected publications

Maerkl, S.J. and S.R. Quake (2009). “Experimental Determination of the evolvability of a transcription factor.” PNAS 106(44): 18650-5. Maerkl, S. (2009). “Integration column: Microfluidic highthroughput screening.” Integrative Biology 1(1): 19-29. Huang, L., S. J. Maerkl and O. J. F. Martin (2009). “Integration of plasmonic trapping in a microfluidic environment.” Optics Express 17(8): 6018-6024. Gerber, D., S. J. Maerkl and S. R. Quake (2009). “An in vitro microfluidic approach to generating protein-interaction networks.” Nature Methods 6(1): 71-4. Einav, S., D. Gerber, P. D. Bryson, E. H. Sklan, M. Elazar, S. J. Maerkl, J. S. Glenn and S. R. Quake (2008). “Discovery of a hepatitis C target and its pharmacological inhibitors by microfluidic affinity analysis.” Nature Biotechnology 26(9): 1019-1027. Maerkl, S. J. and S. R. Quake (2007). “A systems approach to measuring the binding energy landscapes of transcription factors.” Science 315(5809): 233-237. Thorsen, T., S. J. Maerkl and S. R. Quake (2002). “Microfluidic large-scale integration.” Science 298(5593): 580584.

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EPFL School of Life Sciences - 2009 Annual Report

IBI - Co-affiliated Research Group - School of Engineering (STI)

Millán Lab

IBI

Head of Lab (PI) - http://cnbi.epfl.ch

Team Members Bayati Hamidreza , PhD Student

José del Rocio Millán Assistant Professor Defitech Professor STI

Biasiucci Andrea, PhD Student Bourdaud Nicolas, PhD Student Carlson Tom Edward, Scientist Chavarriaga Ricardo, Senior Scientist Garipelli Gangadhar, PhD Student Gasser Maria-Eva, Assistant Kumar Mohit, PhD Student Leeb Robert, Scientist Lew Eileen Yi Lee, PhD Student Miller Heather, PhD Student Monnard Guillaume, Project Assistant Perdikis Serafeim, PhD Student Sagha Hesam, PhD Student Tavella Michele, PhD Student Tonin Luca, PhD Student Uscumlic Marija, PhD Student

Research Interests

The Defitech Foundation Chair in Non-Invasive BrainMachine Interaction carries out research on the direct use of human brain signals to control devices and interact with our environment. In thismultidisciplinary research, we are bringing together our pioneering work on the two fields of brain-machine interfaces and adaptive intelligent robotics. A brain-machine interface (BMI) monitors the user’s brain activity, extracts specific features from the brain signals that reflect the intent of the subject, and translates these features into actions —such as moving a wheelchair or selecting a letter from a virtual keyboard without using activity of any muscle or peripheral nerve. The central tenet of a BMI is the capability to distinguish different patterns of brain activity, each being associated to a particular intention or mental task. Hence mutual adaptation is a key component, as (i) the brain-controlled device must learn to respond to the individual mental commands of the user and (ii) users must learn to modulate their brainwaves voluntary to facilitate the recognition of their intent by the BMI. Non-invasive BMIs mainly use electroencephalographic (EEG) activity recorded from electrodes placed on the scalp.

Selected publications

Millán, J. d. R., Ferrez, P. W., and Seidl, T. (2009). Validation of Brain-Machine Interfaces during Parabolic Flight. International Review of Neurobiology, 86:189-197.

Menon, C., de Negueruela, C., and Millán, J. d. R. (2009). Prospects on Brain-Machine Interfaces for Space System Control. Acta Astronautica, 64(4):448-456. Garipelli, G., Chavarriaga, R., and Millán, J. d. R. (2009). Fast Recognition of Anticipation Related Potentials. IEEE Transactions on Biomedical Engineering, 56(4):125760. In press. Bourdaud, N., Chavarriaga, R., Galán, F., and Millán, J. d. R. (2008). Characterizing the EEG Correlates of Exploratory Behavior. IEEE Transactions on Neural Systems and Rehabilitation Engineering, 16(6):549-556. IDIAP-RR 08-28. Millán, J. d. R., Ferrez, P. W., Galán, F., Lew, E., and Chavarriaga, R. (2008). Non-Invasive Brain-Machine Interaction. International Journal of Pattern Recognition and Artificial Intelligence, 22(5):959-972. Millán, J. d. R. (2008). Brain-Controlled Robots. IEEE Intelligent Systems, 23(3):74-76. Galán, F., Nuttin, M., Lew, E., Ferrez, P. W., Vanacker, G., Philips, J., and Millán, J. d. R. (2008). A Brain-Actuated Wheelchair: Asynchronous and Non-Invasive Brain-Computer Interfaces for Continuous Control of Robots. Clinical Neurophysiology, 119(9):2159-2169. Ferrez, P. W. and Millán, J. d. R. (2008). Error-Related EEG Potentials Generated during Simulated Brain-Computer Interaction. IEEE Transactions on Biomedical Engineering, 55(3):923-929.

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EPFL School of Life Sciences - 2009 Annual Report

IBI - Co-affiliated Research Group - School of Engineering (STI)

Pioletti Lab Head of Lab (PI) - http://lbo.epfl.ch Team Members

Dominique P. Pioletti

Tenure Track Assistant Professor STI

Abdel-Sayed Philippe, Doctoral Assistant Beuchat Nicolas, Bachelor/Master Student Borges de Couraça Ana, Doctoral Assistant Darwiche Salim Elias, Doctoral Assistant Delabarde Claire, Doctoral Assistant Devaud Yannick, Bachelor/Master Student Farron Alain, Scientist Gortchacow Miguel, Doctoral Assistant Ingram David, Assistant Jaccoud Sandra, Laboratory Assistant Jolles-Haeberli Brigitte, Adjunct Professor Kokocinski Virginie, Secretary Krattinger Nathalie, Scientist Krause Alexandra, External Student Larrea Xabier, Scientist Laurent-Applegate Lee Ann, Group Leader Majd Hicham, Scientist Pennese Rafael, Bachelor/Master Student Ramondetti Silvio, Guest Roshan Ghias Alireza, Doctoral Assistant Röthlisberger Marion, Doctoral Assistant Scaletta Corinne, Laboratory Assistant Scheuber Patricia, Scientific Assistant Terrier Alexandre, Group Leader Vogel Arne, Doctoral Assistant Wettstein Michael, Scientist Zambelli Pierre-Yves, Scientist

Research Interests

The projects developed at the LBO are at different levels from basic to applied researches with anyway an overall strategy to bring the developed research to clinical application, the so-called translational research. The core aspect is to use and develop biomechanical descriptions to understand or develop new strategies in the field of musculo-skeletal system. In particular, our projects are organized in four categories: mechano-transduction, tissue engineering, biomechanics of joints and implants, and drug delivery system. A particularity of the LBO is to involve surgeons in most of the developed projects, allowing us to obtain a more effective way for the translational aspect of our research. Translation also means valorization, so we still continued our long lasting collaborations with several industries such Tornier, Stryker or Symbios. In particular, we also obtained financial support from the KTI to develop new solutions in orthopedic implant with some of these companies.

Selected publications

Stadelmann V.A., Terrier A., Gauthier O., Bouler J.-M., Pioletti D.P. Prediction of bone density around orthopedic implants delivering bisphosphonate. J Biomechanics, 42, 1206–1211, 2009. Terrier A., Merlini F., Pioletti D.P., Farron A. Comparison of polyethylene wear in anatomic and reversed shoulder prostheses. J Bone Joint Surg-B, 91-B, 977-982, 2009. Blecha L.D., Rakotomanana L., Razafimahery F., Terrier A., Pioletti D.P. Targeted mechanical properties for optimal fluid motion inside artificial bone constructs. J Orthop Res, 27, 1082-1089, 2009.

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Terrier A., Sedighi-Gilani M., Roshan Ghias A., Aschwanden L., Pioletti D.P. Biomechanical evaluation of porous biodegradable scaffolds for revision knee arthroplasty. Comp Meth Biomech Biomed Eng, 12, 333-339, 2009. Quintin A., Schizas C., Scaletta C., Jaccoud S., ChapuisBernasconi C., Gerber S., Juillerat L., Osterheld M.C., Applegate L.A., Pioletti D.P. Human foetal spine as a source of cells for intervertebral disc regeneration. J Cell Mol Med, 13, 2559-2569, 2009. Montjovent M.O, Bocelli-Tyndall C., Scaletta C., Scherberich A., Martin I., Laurent-Applegate L., Pioletti D.P. In vitro characterization of immune-related properties of human fetal bone cells for potential tissue engineering applications. Tissue Engineering, 15, 1523-1532, 2009. Terrier A., Merlini F., Pioletti D.P., Farron A. Total shoulder arthroplasty: downward inclination of the glenoid component to balance supraspinatus deficiency. J Shoulder Elbow Surg, 18, 360-365, 2009. Stadelmann V.A., Hocké J., Forster V., Merlini F., Terrier A., Pioletti D.P. 3D strain map of axially loaded a mouse tibia: a numerical analysis validated by experimental measurements. Comp Meth Biomech Biomed Eng, 12, 95-100, 2009. Applegate L.A., Scaletta C,. Hirt-Burri N., Raffoul W., Pioletti D.P. Whole-cell bioprocessing of human fetal cells for tissue engineering of skin. Skin Pharmacol Physio, 22, 63-73, 2009. Ramelet A.A, Hirt-Burri N, Raffoul W., Scaletta C., Pioletti D.P., Offord E., Mansourian R., Applegate L.A. Fetal cell therapy for chronic wound treatment and differential gene profiling compared to old skin cells. Exp Gerontology, 44, 208–218, 2009.

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EPFL School of Life Sciences - 2009 Annual Report

IBI - Co-affiliated Research Group - School of Engineering (STI)

Psaltis Lab

IBI

Head of Lab (PI) - http://lo.epfl.ch/

Team Members

Demetri Psaltis Full Professor Dean of the School of Engineering

Choi Jae-Woo, Doctoral Assistant Cuennet Julien, Doctoral Assistant Goy Alexandre, Doctoral Assistant Grange Roduit Rachel, Scientist Hsieh Chia-Lung, Doctoral Assistant Lanvin Thomas, Project Student Laporte Grégoire, External Student Loeffen Berthet Carole, Secretary Makris Konstantinos, Scientific Assistant Papadopoulos Ioannis, Doctoral Assistant Pu Ye, Postdoctoral Researcher Song Wuzhou, Doctoral Assistant Vasdekis Andreas, Scientist Yanisse Daniel, Project Student

Research Interests

The activities of the Optics Laboratory primarily focus onto two research areas. As our first line of research, we work within the field of Optofludics, where we integrate optics with microfluidics for lab-on-a-chip applications. We employ nanofabrication methods to create platforms, where -in a non-invasive mannerlight interrogates bioentities that are manipulated by microfluidic circuits. This work aims at chips capable of performing absorption and fluorescent spectroscopy, DNA and single molecule analysis, portable diagnostics and sensors. In our second major research focus, we explore how light interacts nonlinearly with matter and how such principles can be adapted in imaging and bio-imaging. We use nanodots (dimensions in the range of 10’s of nm) capable of emitting second harmonic radiation (SHG). These act as imaging probes when injected in cells and animals, while permitting their observation for significantly long periods. To the same end, we investigate how images can be transported by very intense light pulses with minimal resolution and information loss.

Selected publications

Luciano De Sio, Julien G. Cuennet, Andreas E. Vasdekis, and Demetri Psaltis. «All-optical switching in an optofluidic PDMS-Liquid Crystal grating defined by cast-molding» Appl. Phys. Lett, Vol. 96, 131112 (2010). Andreas E. Vasdekis, Conlin P. O’Neil, Jeffrey A. Hubbell and Demetri Psaltis. «Microfluidic assays for DNA manipulation based on a block-copolymer immobilization strategy» Biomacromolecules Vol. 11, 827 (2010).

Jae-Woo Choi, Samuel Rosset, Muhmed Niklaus, James R. Adleman, Herbert Shea and Demetri Psaltis, «3-dimensional electrode patterning within microfluidic channel using ion metal implantation» Lab on Chip Vol 10, 783 (2010). Wuzhou Song and Demetri Psaltis, «Pneumatically tunable optofluidic dye laser» Appl. Phys. Lett. Vol 96, 081101 (2010). James R. Adleman, David A. Boyd, David G. Goodwin, and Demetri Psaltis. “Heterogenous catalysis mediated by Plasmon heating”. Nanoletters, Vol. 9, 4417 (2009). Chia-Lung Hsieh, Rachel Grange, Ye Pu, and Demetri Psaltis, “Bioconjugation of barium titanate nanocrystals with immunoglobulin G antibody for second harmonic radiation imaging probes,” Biomaterials, Vol. 31, pp. 2272-2277 (2010). Ye Pu, Rachel Grange, Chia-Lung Hsieh, and Demetri Psaltis, “Nonlinear optical properties of core-shell nanocavities for enhanced second-harmonic generation,” accepted by Physical Review Letters (2010). Rachel Grange, Jae-Woo Choi, Chia-Lung Hsieh, Ye Pu, Arnaud Magrez, Rita, Smajda, Laszlo Forro and Demetri Psaltis, «Lithium niobate nanowires: synthesis, optical properties and manipulation», Appl. Phys. Lett., Vol. 95, 143105 (2009). Chia-Lung Hsieh, Rachel Grange, Ye Pu, Demetri Psaltis, “Three-dimensional harmonic holographic microscopy using nanoparticles as probes for cell imaging,” Optics Express, Vol. 17, Issue 4, pp. 2880-2891.

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EPFL School of Life Sciences - 2009 Annual Report

IBI - Co-affiliated Research Group - School of Engineering (STI)

Radenovic Lab Head of Lab (PI) - http://lben.epfl.ch/

Team Members

Annibale Paolo, PhD student Chong Helen, Administrative Assistant Dutto Fabrizia, PhD student Raillon Camille, PhD student Scarselli Marco, Postdoctoral fellow Traversi Floriano, Postdoctoral fellow

Aleksandra Radenovic

Tenure Track Assistant Professor STI

Research Interests

The research of the Laboratory of Nanoscale Biology focuses on developing tools and probes for single-molecule biophysics. The group uses optical tweezers, AFM, single-molecule fluorescence, Photo-Activated Light microscopy PALM and nanofabricated structures to study biomolecular systems and advance new nanotechnology. Current experimental work in our lab focuses on two interconnecting areas: Nanofabricated probes and platforms for single-molecule biophysics experiments Including nanofabricated SHG nanocylinders, solidstate nanopores, local nanolectrodes for molecular sensing and sequencing

Selected publications

P. Annibale, M. Scarselli, A. Kodiyan and A. Radenovic,. Photoactivatable Fluorescent Protein mEos2 Displays Repeated Photoactivation after a Long-Lived Dark State in the Red Photoconverted Form, The Journal of Physical Chemistry Letters, 2010, 1, pp 1506–1510 J. Miklossy, H.Qing, A. Radenovic, A.Kis, B. Villeno, F. Laszlo,L. Miller, R. Martins, G. Waeberf V.Mooser, F. Bosman, K Khalilii, N Darbinian-Sarkissian P. L. McGeer, Beta amyloid and hyperphosphorylated tau deposits in the pancreas in type 2 diabetes, Neurobiology of Aging, 2009 in press A.Radenovic, E.Trepagnier, R. Csencsits, K. Downing and J. Liphardt, Fabrication of 10 nm diameter hydrocarbon nanopore, Applied Physics Letters 93, 183101 (2008)

DNA nanotechnology Our main focus is to implement DNA origami structures into nanoelectronics. We use grapheme nanoribbon templates onto which different DNA origami structures can self-assemble and would enable us to register individual molecular nanostructures, to electronically address them, and to integrate them into functional devices. Local probe studies of single biomolecules For example RNA polymerase, DNA binding proteins, membrane proteins such G protein–coupled receptors (GPCRs )

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EPFL School of Life Sciences - 2009 Annual Report

IBI - Co-affiliated Research Group-School of Engineering

Stergiopulos Lab

IBI

Head of Lab (PI) - http://lhtc.epfl.ch/

Team Members

Aristotelis Agianniotis, PhD student Luca Augsburger, PhD student Michel Bachmann, Engineer Stéphane Bigler, Engineer Rafaela Fernandes da Silva, Postdoctoral fellow Philippe Reymond, PhD student Rana Rezakhaniha, PhD student Sylvain Roy, Scientific collaborator Tyler Thatcher, PhD student Alkiviadis Tsamis, PhD student Tamina Sissoko, administrative assistant Adan Villamarin, PhD Student

Nikos Stergiopulos Full Professor STI

Research Interests

The Laboratory of Hemodynamics and Cardiovascular Technology (LHTC) focuses is on the relation between blood flow and the development, progression and regression of cardiovascular disease. Development of vascular implants and non-invasive or miniinvasive technologies for the diagnosis and treatment of disease is also a major objective.

Keywords

Cardiovascular mechanics, Hemodynamics, Arterial remodelling, Cerebrovascular disease, Vascular prostheses, Active implants.

Selected publications

Tsamis, A. and N. Stergiopulos (2009). “Arterial remodeling in response to increased blood flow using a constituentbased model.” J Biomech 42(4): 531-6.

Thacher, T., V. Gambillara, et al. (2009). “Reduced cyclic stretch, endothelial dysfunction, and oxidative stress: an ex vivo model.” Cardiovasc Pathol. Thacher, T., R. F. da Silva, et al. (2009). “Differential effects of reduced cyclic stretch and perturbed shear stress within the arterial wall and on smooth muscle function.” Am J Hypertens 22(12): 1250-7. Roy, S., T. Thacher, et al. (2009). “Arterial biomechanics after destruction of cytoskeleton by Cytochalasin D.” J Biomech 42(15): 2562-8. Reymond, P., F. Merenda, et al. (2009). “Validation of a one-dimensional model of the systemic arterial tree.” Am J Physiol Heart Circ Physiol 297(1): H208-22. Ioannou, C. V., D. R. Morel, et al. (2009). “Left ventricular hypertrophy induced by reduced aortic compliance.” J Vasc Res 46(5): 417-25.

Thacher, T. N., P. Silacci, et al. (2009). “Autonomous Effects of Shear Stress and Cyclic Circumferential Stretch regarding Endothelial Dysfunction and Oxidative Stress: An ex vivo Arterial Model.” J Vasc Res 47(4): 336-345.

Fonck, E., G. G. Feigl, et al. (2009). “Effect of aging on elastin functionality in human cerebral arteries.” Stroke 40(7): 2552-6.

Thacher, T. N., V. Gambillara, et al. (2009). “Regulation of arginase pathway in response to wall shear stress.” Atherosclerosis.

Gambillara, V., T. Thacher, et al. (2008). “Effects of reduced cyclic stretch on vascular smooth muscle cell function of pig carotids perfused ex vivo.” Am J Hypertens 21(4): 425-31.

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GHI

EPFL School of Life Sciences - 2009 Annual Report

GHI - Global Health Institute The Global Health Institute (GHI) is contributing to the understanding, diagnosis, prevention and treatment of infectious diseases, which still claim 18 million human lives each year and account for half of the deaths in the developing world. The GHI comprises 8 groups, all engaged in different facets of research linked to human health but with strong emphasis on diseases of truly global importance such as HIV/AIDS, tuberculosis and malaria. Basic mechanisms of host-pathogen interactions and innate and acquired immunity against pathogens are being studied using multidisciplinary approaches. A unique feature of the GHI is its ability to tackle crucial world health issues by harnessing cutting edge technologies developed at EPFL and elsewhere. Among these the nanotechnologies, microengineering and informatics are proving particularly powerful at underpinning drug discovery and vaccinology research. Intensification of current research themes by applying novel technological innovations will continue and elucidating the basis of genetic predisposition to infectious diseases is also a priority as this will open new avenues for diagnostics and personal medicine. http://ghi.epfl.ch

Š Co opyright 2004-2010 0 EPFL for or a ma ater eria ia al pu publ blis ishe he ed iin n this rep port rt Copyright alllll m material published report info f .sv@ v@e epfl.c ..ch ch info.sv@epfl

71


EPFL School of Life Sciences - 2009 Annual Report

GHI - Global Health Institute

Blokesch Lab Head of Lab (PI) - http://blokesch-lab.epfl.ch/

Team Members

Olga de Souza Silva, Technician (from July 09) Caroline Guinchard, Administrative Assistant Rasmus Lykke Marvig, summer student (July / Aug 09) Mirella Lo Scrudato, PhD student (from November 09) Gaia Suckow, PhD student (from October 09

Melanie Blokesch

Tenure Track Assistant Professor

Introduction

There is increasing concern about the emergence of new infectious agents and the threat this poses to global health. Knowledge of how pathogens emerge, why it happens repeatedly and how it can be predicted or controlled is of fundamental importance for human health. The focus of our research is on how environmental bacteria evolve outside the human host to become pathogens. This is exemplified on Vibrio cholerae, the causative agent of the disease Cholera, as a model organism.

Keywords

Evolution of pathogens, horizontal gene transfer, environmental reservoirs, bacterial signal transduction pathways, single cell expression analysis

Results Obtained in 2009

The Laboratory of Molecular Microbiology was established in May 2009. After the initial setup we started our experiments with a primary focus on the developmental program Natural Competence of Vibrio cholerae. This program enables the bacterium to take up free DNA from the environment in order to incorporate it into its own genome. Such a change of genotype might lead to a different phenotype and therefore to bacterial variants that are better adapted to their environmental niche and / or more pathogenic to man. The knowledge on how this “behavior� is regulated is very limited and we aim at elucidating the major factors that are involved in the regulatory network. Using natural competence to genetically manipulate Vibrio species Learning how pathogens survive in their natural reservoir not only provides us with major insight into their evolution but also allows us to better understand their environmental lifestyle. In the laboratory we are making use of this knowledge, e.g. that Vibrio takes up free DNA under certain conditions,

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in order to develop it as a genetic tool. As the natural competence program is preserved in most if not all Vibrio species such a tool will facilitate the work with less genetically amenable Vibrios like Vibrio vulnificus. This opportunistic pathogen causes severe wound infection and gastroenteritis, the later being mostly caused by eating contaminated seafood. Our first results are with respect to the DNA uptake and integration capacity of V. cholerae. Once in the competence state the bacteria take up DNA and recombine it into the chromosome thereby getting naturally transformed. This recombination is only possible if homologous regions are present at both ends and a minimum length of 500 base pairs is required to allow this process to occur at detectable levels. As extracellular DNA gets easily degraded by nucleases an increase in the amount of free DNA is reflected by higher transformation frequencies. In agreement with this finding nuclease minus strains on the other hand are hypertransformable. The source of the DNA seems less important; genomic DNA, PCR amplified DNA fragments and plasmid DNA are all taken up though at different levels. Plasmids are not contained as such within the cell and maintenance of the plasmid DNA is only guaranteed if homologous recombination into the genome takes place. Keeping these results in mind, we established a method to knock-out genes in Vibrio cholerae applying natural competence in conjunction with the yeast Flp recombinase system. Genes were disrupted by insertion of antibiotic cassettes flanked by FLP recombination target sites (FRT). Antibiotic-resistant insertional mutants were subsequently cured by excision of the antibiotic cassette mediated by the Flp recombinase encoded on a plasmid. This method represents a simplified and fast alternative to standard gene deletion techniques, the latter requiring bacterial conjugation / mating as well as suicide vectors. Using a similar approach, we were able to insert

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EPFL School of Life Sciences - 2009 Annual Report

Future perspectives As the lab has finally reached a critical mass after our two graduate students joined the lab at the end of the year 2009, we are very ambitious for the future. The main questions we want to address are with respect to “what triggers the natural competence program of V. cholerae”, “how is horizontal gene transfer linked to this specialized environmental niche (the chitin surface)” and “what are the benefits of virulence factors in the environment”.

Selected publications

Blokesch, M. and G.K. Schoolnik (2008). The extracellular nuclease Dns and its role in natural transformation of Vibrio cholerae. J. Bacteriol. 190:7232-40. Blokesch, M. and G.K. Schoolnik (2007). Serogroup Conversion of Vibrio cholerae in Aquatic Reservoirs. PLoS Pathog. 3(6):e81.

GHI

DNA sequences encoding affinity tags (e.g. Histag) or commonly used reporter genes (e.g. mCherry encoding a red fluorescent protein). We hope this procedure will not only facilitate work in our own laboratory but will also be adapted by other researchers working on V. cholerae and other Vibrio species.

The human pathogen Vibrio cholerae often lives in association with zooplankton and their molts in its environmental reservoir, the ocean. The chitinous exoskeleton can serve as a nutrient source but also fosters the exchange of genes via horizontal gene transfer (HGT). The colonization of an artificial chitin surface is visualized by phase contrast microscopy (middle) and gene expression relevant to HGT is indicated by red (left) and green (right) fluorescence using reporter fusions.

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EPFL School of Life Sciences - 2009 Annual Report

GHI - Global Health Institute

Cole Lab Head of Lab (PI) - http://cole-lab.epfl.ch

Team Members

Stewart T. Cole Full Professor Director of GHI

Benjamin Blasco, PhD student Philippe Busso, Technician Jeffrey Chen, Postdoctoral fellow Elizabeth Fullam, Postdoctoral fellow Gabriela Gago, visiting scientist (June-September) Ruben Hartkoorn, Postdoctoral fellow Adamandia Kapopoulou, Scientific Assistant Suzanne Lamy, Administrative Assistant Jocelyne Lew, Scientific Assistant Sophie Magnet, Postdoctoral fellow Joao Neres, Postdoctoral fellow Iain Old, Administrative Project Manager Florence Pojer, Postdoctoral fellow Claudia Sala, Postdoctoral fellow Patricia Schneider, Technician Pushpendra Singh, Postdoctoral fellow Swapna Uplekar, PhD Student Ming Zhang, PhD Student

Introduction

We take a multidisciplinary approach to tackle global health problems such as tuberculosis (TB) and leprosy and are concentrating on the discovery of new TB drugs.

Keywords

Microbial Pathogenesis, TB drug discovery, signal transduction, protein secretion, phylogeography of leprosy.

Results Obtained in 2009

TB Drug Discovery We are leading a major effort to discover new drugs for the treatment of TB as part of the New Medicines for Tuberculosis Project, NM4TB, funded by the European Commission. In 2009, we described the identification and characterization of the 1,3-benzothiazin4-ones (BTZ), a new class of antimycobacterial agents that kill Mycobacterium tuberculosis in vitro, ex vivo and in murine models of TB. Using genetics and biochemistry, the enzyme decaprenylphosphoryl-βD-ribose 2’-epimerase was identified as a major BTZ target. Inhibition of this enzymatic activity abolishes formation of decaprenylphosphoryl arabinose, a key precursor required for the synthesis of the cell-wall arabinans, thus provoking cell lysis and bacterial death. The most advanced compound, BTZ043 is a candidate for inclusion in combination therapies for both drug-sensitive and extensively-drug resistant TB. Signal transduction, phosphorelays and cell wall biosynthesis in M. tuberculosis There is mounting evidence that cell growth and elongation is controlled by a phosphorelay involving serine-threonine protein kinases (STPK) and phosphatases, and forkhead-associated proteins that recognize phospho-threonine residues. We are intensively investigating the biological function of PknB, and trying to identify the ligand for this essential

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receptor kinase, a potential therapeutic target for new TB drugs. PknB localizes to the poles of tubercle bacilli. Protein secretion and pathogenicity The ESX-1 protein secretion system is the major virulence determinant operating in M. tuberculosis and has been lost by the vaccine strains M. bovis BCG and M. microti. ESX-1 is required for the export of small helical-hairpin proteins belonging to the ESAT6 family as well as other effector proteins of unknown function. ESX-1 mediates host cell entry of tubercle bacilli and triggers intercell spread. We are using an integrated approach involving biochemistry, genetics, X-ray crystallography and electron microscopy to establish the organization, architecture, structure and function of this ATP-driven secretory apparatus. The figure shows crystals of key proteins from the ESX-1 system, which represents an attractive target for chemical biology and drug discovery. A regulatory map of the M. tuberculosis genome We have adopted an integrated approach to studying gene regulation by using chromatin-immunoprecipitation of DNA-binding proteins in conjunction with high density oligonucleotide-based microarrays or high-throughput sequencing to map the genome. Using this approach we demonstrated that the BlaI repressor controls expression of five separate genomic loci that respond to beta-lactam antibiotics and mapped all the RNA polymerase binding sites in the genome. Regulatory information is being incorporated into TubercuList, the genome server dedicated to M. tuberculosis http://tuberculist.epfl.ch/, for which we are the official curators. Phylogeography of leprosy Despite the massive and highly successful implementation of mulitidrug therapy by the World Health Organisation, leprosy remains a serious public health

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EPFL School of Life Sciences - 2009 Annual Report

Selected publications

Alvey, L., S. Prado, B. Saint-Joanis, S. Michel, M. Koch, S. T. Cole, F. Tillequin & Y. L. Janin, (2009) Diversity-oriented synthesis of furo[3,2-f]chromanes with antimycobacterial activity. Eur J Med Chem 44: 2497-2505. Bitter, W., E. N. Houben, D. Bottai, P. Brodin, E. J. Brown, J. S. Cox, K. Derbyshire, S. M. Fortune, L. Y. Gao, J. Liu, N. C. Gey van Pittius, A. S. Pym, E. J. Rubin, D. R. Sherman, S. T. Cole & R. Brosch, (2009) Systematic genetic nomenclature for type VII secretion systems. PLoS Pathog 5: e1000507. Christophe, T., M. Jackson, H. K. Jeon, D. Fenistein, M. Contreras-Dominguez, J. Kim, A. Genovesio, J. P. Carralot, F. Ewann, E. H. Kim, S. Y. Lee, S. Kang, M. J. Seo, E. J. Park, H. Skovierova, H. Pham, G. Riccardi, J. Y. Nam, L. Marsollier, M. Kempf, M. L. Joly-Guillou, T. Oh, W. K. Shin, Z. No, U. Nehrbass, R. Brosch, S. T. Cole & P. Brodin, (2009) High content screening identifies decaprenyl-phosphoribose 2’ epimerase as a target for intracellular antimycobacterial inhibitors. PLoS Pathog 5: e1000645.

bovis strains and M. bovis BCG strains. Infect Immun 77: 2230-2238. Kaser, M., O. Gutmann, J. Hauser, T. Stinear, S. Cole, D. Yeboah-Manu, G. Dernick, U. Certa & G. Pluschke, (2009) Lack of insertional-deletional polymorphism in a collection of Mycobacterium ulcerans isolates from Ghanaian Buruli ulcer patients. J Clin Microbiol 47: 3640-3646. Makarov, V., G. Manina, K. Mikusova, U. Mollmann, O. Ryabova, B. Saint-Joanis, N. Dhar, M. R. Pasca, S. Buroni, A. P. Lucarelli, A. Milano, E. De Rossi, M. Belanova, A. Bobovska, P. Dianiskova, J. Kordulakova, C. Sala, E. Fullam, P. Schneider, J. D. McKinney, P. Brodin, T. Christophe, S. Waddell, P. Butcher, J. Albrethsen, I. Rosenkrands, R. Brosch, V. Nandi, S. Bharath, S. Gaonkar, R. K. Shandil, V. Balasubramanian, T. Balganesh, S. Tyagi, J. Grosset, G. Riccardi & S. T. Cole, (2009) Benzothiazinones kill Mycobacterium tuberculosis by blocking arabinan synthesis. Science 324: 801-804. Monot, M., N. Honore, T. Garnier, N. Zidane, D. Sherafi, A. Paniz-Mondolfi, M. Matsuoka, G. M. Taylor, H. D. Donoghue, A. Bouwman, S. Mays, C. Watson, D. Lockwood, A. Khamispour, Y. Dowlati, S. Jianping, T. H. Rea, L. Vera-Cabrera, M. M. Stefani, S. Banu, M. Macdonald, B. R. Sapkota, J. S. Spencer, J. Thomas, K. Harshman, P. Singh, P. Busso, A. Gattiker, J. Rougemont, P. J. Brennan & S. T. Cole, (2009) Comparative genomic and phylogeographic analysis of Mycobacterium leprae. Nat Genet 41: 1282-1289. Sala, C., D. C. Grainger & S. T. Cole, (2009) Dissecting regulatory networks in host-pathogen interaction using chIPon-chip technology. Cell Host Microbe 5: 430-437.

Demangel, C., T. P. Stinear & S. T. Cole, (2009) Buruli ulcer: reductive evolution enhances pathogenicity of Mycobacterium ulcerans. Nat Rev Microbiol 7: 50-60.

Sala, C., A. Haouz, F. A. Saul, I. Miras, I. Rosenkrands, P. M. Alzari & S. T. Cole, (2009) Genome-wide regulon and crystal structure of BlaI (Rv1846c) from Mycobacterium tuberculosis. Mol Microbiol 71: 1102-1116.

Garcia Pelayo, M. C., S. Uplekar, A. Keniry, P. Mendoza Lopez, T. Garnier, J. Nunez Garcia, L. Boschiroli, X. Zhou, J. Parkhill, N. Smith, R. G. Hewinson, S. T. Cole & S. V. Gordon, (2009) A comprehensive survey of single nucleotide polymorphisms (SNPs) across Mycobacterium bovis strains and M. bovis BCG vaccine strains refines the genealogy and defines a minimal set of SNPs that separate virulent M.

Taylor, G. M., S. Blau, S. Mays, M. Monot, O. Lee, D. E. Minnikin, S. T. Cole & P. Rutland, (2009) Genotyping of Mycobacterium leprae Amplified From an Archaeological Case of Lepromatous Leprosy from Central Asia. J Archaeol Science 36: 2408–2414.

Crystals of key proteins from the ESX-1 system

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problem in several countries probably due to our inability to identify infectious cases early enough. One of our goals is the development of an epidemiological tool to monitor transmission of the disease. This uses comparative genomics, particularly SNP (single nucleotide polymorphism) analysis of patient isolates, to monitor the phylogeography of leprosy. In collaboration with the WHO, we are also coordinating a worldwide effort to monitor the emergence of drug resistance.


EPFL School of Life Sciences - 2009 Annual Report

GHI - Global Health Institute

Harris Lab Head of Lab (PI) - http://harris-lab.epfl.ch

Team Members

Herbst, Tina, PhD student Kulagin, Manuel, Lab Assistant Massacand, Joanna, Postdoctoral fellow Mosconi, Ilaria, PhD Student Schär, Corinne, PhD student

Nicola Harris

Tenure-track Assistant Professor

Introduction

The intestinal mucosa represents an interface between the body and the external environment and is constantly exposed to environmental micro-organisms. Amongst these commensal bacteria and helminth pathogens are present almost ubiquitously within mammals. Our work aims to i) understand the impact commensal bacteria have on our bodies and ii) determine the means by which we can protect ourselves from heavy burdens of helminths that take a terrible toll on the health of children living in developing countries.

Keywords

Immunology, intestine, soil-transmitted helminths, commensal bacteria, antibodies (IgG, IgA), Th2 immune responses, cytokines, allergy, vaccination

Results Obtained in 2009

Protective immunity against soil-transmitted helminths Most children in developing countries are infected with intestinal helminths that cause malnutrition and learning difficulties. No effective vaccines are available. Our work has aimed at finding the mechanisms of protective immunity against intestinal helminths and we recently published a study that provides grounds for optimism in the search for effective vaccines. This work revealed that parasite-specific antibodies control intestinal worm infection. However we also demonstrated that helminths have evolved the ability to divert the host immune response away from production of ‘protective’ antibodies and towards ‘non-specific’ antibodies. Taken together these findings indicate that we need to choose antigens used for vaccination very carefully. We are now focused on finding protective antigens and understanding the immune mechanisms by which antibodies can kill intestinal helminths.

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Impact of intestinal micro-environment on antibody production Immunoglobulin A represents the predominant antibody isotype produced at the intestinal mucosa, where it plays an important role limiting the penetration of commensal intestinal bacteria and opportunistic pathogens. IgA is well recognized to exert protective function within the intestine, whilst preserving tissue integrity by damping down inflammation. The ability of the intestinal immune response to choose IgA production over inflammatory antibody isotypes is therefore crucial to the maintenance of crucial intestinal functions such as the absorption of nutrients and water. Our work has aimed to determine how the body makes the choice to produce IgA. We could show that a subset of immune cells, called dendritic cells, is educated by the intestinal microenvironment to promote IgA production. The ability of intestinal DC to promote IgA production was imparted by both intestinal commensal bacteria and close proximity to intestinal epithelial cells. This work highlights the importance that the local tissue microenvironment plays in ‘educating’ specialized immune cells such as DC. It also indicates that constant exposure of our gut to harmless commensal bacteria is important in maintaining tissue integrity. Role of Thymic stromal lymphopoietin (TSLP) in promoting Th2 immune responses Th2 immune responses evolved to provide us with protection against helminths, but unfortunately are also capable of causing extensive tissue damage during allergic reactions. TSLP is a cytokine produced by epithelial cells in the lung, skin and intestine that has been proposed to be crucial for generation of Th2 immune responses. Our goal was to investigate the role of TSLP in promoting protective Th2 immune responses against intestinal helminths. We found that TSLP was not required for Th2-mediated immunity against two distinct rodent helminth parasites Heligmosomoides polygyrus (H. polygyrus)

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EPFL School of Life Sciences - 2009 Annual Report

Selected publications

J.C. Massacand, R.C. Stettler, R.Meier, B.J. Marsland, R.K. Grencis, N.L. Harris. (2009) Parasite products bypass the need for TSLP in Th2 immune responses by directly

modulating dendritic cell function. Proc. Natl. Acad. Sci. USA18;106(33):13968-73 Heer AK, Harris NL, Kopf M, Marsland BJ. (2008)CD4+ and CD8+ T cells exhibit differential requirements for CCR7mediated antigen transport during influenza infection. J. Immunology 15;181(10):6984-94 McCoy, KD., Stoel, M., Stettler, R., Merky, P., Fink, K., Senn, B., Schaer, C., Massacand, J., Odermatt, B., Oettgen, H.C., Zinkernagel, RM., Bos, NS., Hengartner, H., Macpherson, AJ., Harris, NL. (2008). Polyclonal and specific antibodies mediate protective immunity against enteric helminth infection. Cell Host and Microbe 4:4 p362-373. Massacand, J., Kaiser, P, Ernst, B, Burki, K, Schneider P, Harris, NL. (2008) Intestinal Bacteria and Epithelial Cells Condition Dendritic Cells to promote IgA production. Plos One 2:e2588. Marsland, B.J., Kurrer, M., Reissman, R., Harris, N.L., Kopf, M. (2008) Nippostrongylus brasiliensis infection leads to the development of emphysema and long-term induction of alternatively activated macrophages. European Journal of Immunology 38(2):479-88.

Intestinal helminths represent a major cause of morbidity and mortality in developing countries. Given that many intestinal helminths reproduce outside the human host, parasite burdens increase to detrimental levels only through re-infection—a process that could be halted by adequate vaccination. We have reported an essential role for antibodies in mediating immunity against enteric helminth infection. Initial helminth infection elicits a robust polyclonal antibody response that allows parasite persistence while limiting parasite fecundity and spread. Later development of affinity-matured antibodies provides protection against reinfection and likely functions to limit total parasite burden and provides immunity to offspring in the form of maternal antibodies. The cover image depicts an intestinal mouse helminth Heligmosomoides polygyrus coated with host IgG antibodies (green

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GHI

and Nippostrongylus brasiliensis (N. brasiliensis). Yet paradoxically TSLP was needed to generate Th2 responses against the helminth Trichuris muris (T. muris). More detailed studies demonstrated that the main role of TSLP during T. muris infection was to limit production of the Th2 inhibitory cytokine IL-12. We further demonstrated that products from H. polygyrus and N. brasiliensis, but not T. muris, could directly suppress IL-12 production by mammalian immune cells. These findings indicate that the primary function of TSLP is not to promote Th2 immune responses directly, but rather indirectly through the suppression of IL-12. This information that will be important for the development of future therapeutics aimed at blocking TSLP in allergic patients. Our work also alerted us to the potent ability of helminths to directly modulate mammalian immune function, a finding that has formed the basis of a new project within our laboratory.


EPFL School of Life Sciences - 2009 Annual Report

GHI - Global Health Institute

Lemaître Lab Head of Lab (PI) - http://lemaitrelab.epfl.ch

Team Members

Olivier Binggeli, PhD Student (joined in April 2009) Jean-Philippe Boquete, Technician Nichole Broderick, Postdoctoral fellow Nicolas Buchon, Postdoctoral fellow Véronique Bulliard, Administrative Assistant Sveta Chakrabarti, PhD Student Jeremy Herren, PhD Student Takayuki Kuraishi, Postdoctoral fellow (joined in April 2009) Lionel Mury, Technician (joined in January) Claudine Neyen, Postdoctoral fellow (joined in July 2009) Onya Opota, Postdoctoral fellow David Welchman, Postdoctoral fellow

Bruno Lemaître Full Professor

Introduction

regulator of the Imd pathway (Scherfer et al., 2006 and 2008, Lhocine et al., 2008, Tang et al., 2006, 2008, Buchon et al., 2009a). We have also analyzed the contribution of hemocytes to the Drosophila host defense and uncovered a new immune defense mechanism in the male reproductive tract (Defaye et al 2008, Gendrin et al 2009).

Keywords

Drosophila intestinal response to bacterial infection The gut is the major interface between microbes and their animal hosts and constitutes the main entry route for pathogens. As a consequence gut cells must be armed with efficient immune defenses to combat invasion and colonization by pathogens. However, the gut also harbors a microbiota, with potentially beneficial effects for the host, which must be tolerated without a chronic, and harmful, immune response. However, in spite of growing interest in gut mucosal immunity, very little is known about the immune response of the Drosophila gut. Recently, we reported a comprehensive analysis of the gene expression changes that occur in the Drosophila gut following infection. This study revealed that immune responses in the gut are regulated by the Imd and JAK-STAT pathways, but not the Toll pathway. We were also the first to report that bacterial ingestion strongly stimulates epithelium renewal through intestinal stem cell division to repair gut damage (Buchon et al. 2009b). We further showed that a cytokine, Upd3, is produced by stressed enterocytes and stimulates stem cell proliferation through the JAK-STAT pathway, providing a homeostatic regulatory loop. We also reported that gut microbiota affect stem cell activation and epithelium renewal, concepts previously proposed in mammalian systems, but never fully demonstrated. Finally, we provide evidence that intestinal epithelium renewal is a central determinant of the infection process, by demonstrating how a pathogenic bacterium, Pseudomonas entomophila disrupts gut homeostasis by blocking renewal (Buchon et al 2009c). Future studies will investigate how immune and developmental signaling pathways shape the gut immune response to bacteria.

Our group studies the molecular basis of host defense responses to microbial infection using Drosophila as a model system. Analysing how insects combat microbial infection may improve our understanding of infectious processes and innate host defense in vertebrates.

Innate immunity, host-pathogen interactions, insect, genetic, gut, stem cells

Results Obtained in 2009

Microbial infections are characterized by a continual, dramatic interplay between pathogen and host: pathogens exploit an array of host cell functions during infection and hosts respond with efficient immune responses. Drosophila provides a powerful model system for dissecting innate host defense mechanisms. In response to an immune challenge, the fat body of this insect produces a battery of small peptides with antifungal and antibacterial activities. Two signaling pathways, Imd and Toll, control the expression of antimicrobial peptide encoding genes in Drosophila. These pathways exhibit striking similarities with the Toll-Like Receptors and Tumor Necrosis Factor, α-cascades that regulate NF-κB transcription factor activity in vertebrates (Leulier and Lemaitre 2008). The systemic immune response Our group employs genetic screens to identify novel factors regulating the immune response of Drosophila. These studies extend our understanding of how the Toll and Imd pathways activate immune responses, as well as how the host recognizes and distinguishes between different microbial pathogens. This approach led to our recent characterization of two immune-responsive serpins, several serine proteases with important roles in melanization (an arthropod specific immune defense) or Toll pathway activation, the identification of the novel clotting factor Fondue, and the description of Pims, a negative

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© Copyright 2004-2010 EPFL for all material published in this report info.sv@epfl.ch


Selected publications

Gendrin M., Welchman D., Poidevin M., Hervé M., Lemaitre B. Long-range activation of systemic immunity through peptidoglycan diffusion in Drosophila. PLoS Pathog. 2009 Dec;5(12):e1000694

Quevillon-Cheruel S, Leulliot N, Acosta-Muniz C, Vincent M, Gallay J, Boccard F, Lemaitre B & van Tilbeurgh H. (2009) Evf, a virulence factor produced by the Drosophila pathogen Erwinia carotovora is a S-palmitoylated protein with a new fold that binds to lipid vesicles. J. Biol. Chem 284(6):3552-62.

Vallet-Gely I, Novikov A, Augusto L, Liehl P, Bolbach G, Péchy-Tarr M, Cosson P, Keel C, Caroff M, Lemaitre B. Hemolytic activity of Pseudomonas entomophila, a versatile soil bacterium, is linked to cyclic lipopeptide production. Appl Environ Microbiol. 2009 Dec 18. [Epub ahead of print] PMID: 20023108

Defaye, A.; Evans, I.; Crozatier, M.; Wood, W.; Lemaitre, B.; Leulier, F. (2009) Genetic Ablation of Drosophila Phagocytes Reveals Their Contribution to Both Development and Resistance to Bacterial Infection. J Innate Immun 2009;1:322-334

Buchon, N., Broderick, N.A., Chakrabarti, S., and Lemaitre, B. (2009) Invasive and indigenous microbiota impact intestinal stem cell activity through multiple pathways in Drosophila. Genes and Dev. 23: 2333-2344.

Scherfer C, Tang H, Kambris Z, Lhocine N, Hashimoto C, Lemaitre B (2008). Drosophila Serpin-28D regulates hemolymph phenoloxidase activity and adult pigmentation. Dev Biol. 323(2):189-96.

Buchon, N., Broderick N.A., Poidevin M., Pradervand S. and Lemaitre B (2009). Host defense and stem cell proliferation in the gut of bacteria-infected Drosophila Cell Host & Microbes, 5, 200-211.

Tang, H., Kambris Z. Lemaitre B. and Hashimoto C. (2008) A serpin that regulates immune melanization in the respiratory system of Drosophila. Dev Cell 15, 617–626.

Buchon N, Poidevin M, Kwon HM, Guillou A, Sottas V, Lee BL, Lemaitre B. (2009) A single modular serine protease integrates signals from pattern-recognition receptors upstream of the Drosophila Toll pathway. Proc Natl Acad Sci U S A. Jul 28;106(30):12442-7.

Leulier F, and Lemaitre B. (2008) Toll-like receptors - taking an evolutionary approach. Nature Review Genetics. 9(3):165-78.

Injection of bacteria into fly provides an easy method to trigger the systemic immune response.

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EPFL School of Life Sciences - 2009 Annual Report


EPFL School of Life Sciences - 2009 Annual Report

GHI - Global Health Institute

McKinney Lab Head of Lab (PI) - http://mckinney-lab.epfl.ch/

Team Members

John McKinney Full Professor

Guillaume Castillon, Postdoctoral fellow (until November) Tarun Chopra, Postdoctoral fellow (joined in July) Cyntia De Piano, PhD Student Neeraj Dhar, Senior Staff Scientist Meltem Elitas, PhD Student (co-advised Maerkl and Renaud labs) Angelica Ferguson, PhD Student (joined in August) Sonia Garcia, Technician (until June) Ekaterina Gelman, PhD Student (joined in January) Suzanne Lamy, Administrative Assistant Manisha Lotlikar, PhD Student (co-advised Hatzimanikatis lab) Zeljka Maglica, Postdoctoral fellow (joined in August) Giulia Manina, Postdoctoral fellow (joined in August) Laetitia Martin, Technician (joined in August) Jean-Bernard Nobs, Master student (until April) Fred Ross, PhD Student (until November) Isabella Santi, Postdoctoral fellow Anna Tischler, Postdoctoral fellow Emre Özdemir, PhD Student (co-advised Hatzimanikatis lab)

Introduction

Most bacterial infections come and go in a matter of days or weeks, but tuberculosis resists elimination by the immune system and persists for the lifetime of the host. Although the live-attenuated Bacille Calmette Guérin (BCG) vaccine has been widely administered since the 1920s, the protective efficacy of BCG in clinical trials has been highly variable and generally poor. Although effective anti-uberculosis therapy has been available since the 1950s, successful treatment requires administration of multiple antibiotics for six months or longer, a regimen that most patients fail to complete unless closely supervised. In the Laboratory of Bacteriology (LBAC), we study the mechanisms of tuberculosis persistence in the face of host immunity and antibiotics. This work has generated new insights into the biology of bacterial persistence and new tools for developing and evaluating antimicrobial agents

Keywords

Microbiology, bacteriology, Mycobacterium tuberculosis, infectious disease, persistence, counter-immunity, carbon metabolism, antibiotic tolerance, growth control, biosafety Level 3 (BSL3); time-lapse fluorescence microscopy; microfluidics; micro-electromechanical systems (MEMS); computational modeling; mass spectrometry

Results Obtained in 2009

The Laboratory of Bacteriology (LBAC) studies the mechanisms responsible for recalcitrance of Mycobacterium tuberculosis to host immunity and antimicrobial therapy. Our research is focused in four areas: Counter-Immunity The host immune response is often sufficient to restrain M. tuberculosis growth, but not to eradicate

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infection. Our goal is to identify the strategies that M. tuberculosis deploys to counter host immunity in order to establish and maintain a lifelong infection. We identified a signal transduction pathway that is essential for bacterial resistance to the mammalian acquired immune response, and we showed that this pathway mediates bacterial resistance to immunerelated stressors. Now we are exploring the mechanistic basis of this pathway’s role in stress resistance and immune evasion. In Vivo Metabolism Central metabolism has emerged as an attractive target for antimicrobial drug discovery. Our goal is to identify the essential metabolic pathways and adaptations for M. tuberculosis growth and persistence during infection. In collaboration with the Hatzimanikatis lab (School of Basic Sciences), we constructed and characterized the first thermodynamically constrained genome-scale computational model of M. tuberculosis metabolism. The model has provided some surprising new insights into the metabolic capabilities and vulnerabilities of M. tuberculosis. We proved the model’s prediction that antibiotic-mediated killing is dependent on the carbon substrate used for bacterial growth. Now we are doing experiments to test other predictions generated by the model. Antibiotic Tolerance Tuberculosis is notoriously difficult to treat with antibiotics due to the recalcitrance of a subpopulation of bacteria. Our goal is to elucidate the epigenetic basis of cell-to-cell variation in antibiotic tolerance and persistence. Using microfluidics and timelapse microscopy, we tested and disproved the dogma that persistence is due to a subpopulation of dormant bacteria. Instead, we found that bacterial persistence to isoniazid, a frontline anti-tuberculosis drug, is due to stochastic variation in the expression of

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EPFL School of Life Sciences - 2009 Annual Report

Growth Control M. tuberculosis is also notorious for its slow intrinsic growth rate. Our goals are to understand why M. tuberculosis grows so slowly, to identify physiologic adaptations required for slow growth, and to analyze the scaling of physiological processes with growth kinetics. Unexpectedly, we found that mycobacteria display extreme cell-to-cell variation in cell cycle parameters (rate of biomass growth, time between cell divisions, size at cell birth and division, symmetry of division). These findings challenge the assumption that an individual cell’s phenotype is a predictable outcome of its genotype and its environment. Now we are building reporter systems to analyze bacterial chromosome and gene expression dynamics at the single-cell level. As part of an SNF Sinergia consortium involving collaborators at U. Geneva and U. Zurich, we are developing new microfluidic platforms to analyze the behavior of individual bacteria within phagocytic cells. We will use these new systems to

study the mechanistic basis of microbial non-genetic phenotypic heterogeneity.

Selected publications

Dhar, N. and McKinney, J. (2010). Mycobacterium tuberculosis persistence mutants identified by screening in isoniazid-treated mice. Proc. Natl. Acad. Sci. USA (in press). Tischler, A.D. and McKinney, J.D. (2010). Contrasting persistence strategies in Salmonella and Mycobacterium. Curr. Opin. Microbiol. 13(1): 93-99. Makarov, V., Manina, G., Mikusova, K., Möllmann, U., Ryabova, O., Saint-Joanis, B., Dhar, N., Pasca, M.R., Buroni, S., Lucarelli, A.P., Milano, A., De Rossi, E., Belanova, M., Bobovska, A., Dianiskova, P., Kordulakova, J., Sala, C., Fullam, E., Schneider, P., McKinney, J.D., Brodin P., Christophe, T., Waddell, S., Butcher, P., Albrethsen, J., Rosenkrands, I., Brosch, R., Nandi, V., Bharath, S., Gaonkar, S., Shandil, R.K., Balasubramanian, V., Balganesh, T., Tyagi, S., Grosset, J., Riccardi, G., Cole, S.T. (2009). Benzothiazinones kill Mycobacterium tuberculosis by blocking arabinan synthesis. Science 324(5928): 801-804. Savvi, S., Warner, D.F., Kana, B.D., McKinney, J.D., Mizrahi, V. and Dawes, S.S. (2008). Functional characterization of a vitamin B12-dependent methylmalonyl pathway in Mycobacterium tuberculosis: implications for propionate metabolism during growth on fatty acids. J. Bacteriol. 190(11): 3886-3895.

Stochastic expression of mycobacterial catalase determines persistence during antibiotic treatment. The anti-tuberculosis drug isoniazid is a prodrug that must be activated by the bacterial catalase in order to kill. The dynamics of catalase expression were tracked at the single-cell level using microfluidics and timelapse fluorescence microscopy by fusing the katG gene encoding catalase to the rfp gene encoding red fluorescent protein. Bacteria expressing the KatG-RFP fusion protein showed a pulsatile and apparently random pattern of KatG-RFP expression. KatG-RFP pulsing was positively correlated with cell death (P < 0.00001). After infusion of isoniazid images were recorded at t=0 min (A), t=90 min (B), and t=180 min (C).

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an antibiotic resistance determinant. This work also disproved the dogma that antibiotics kill by triggering unbalanced cell growth, because we found that cell growth arrests rapidly after antibiotic exposure, hours before the onset of cell death. Now we are doing experiments to explore the broader relevance of these new concepts of antibiotic-mediated killing and bacterial persistence.


EPFL School of Life Sciences - 2009 Annual Report

GHI - Global Health Institute

Trono Lab Head of Lab (PI) - http://tronolab.epfl.ch Team Members

Didier Trono

Professor Dean of the School of Life Sciences

Isabelle Barde, Postdoctoral fellow Karolina Bojkowska, PhD student Yannick Bulliard, PhD student Natali Castro Diaz, PhD student (from September 2009) Andrea Corsinotti, PhD student Marc Friedli, Postdoctoral fellow (from August 2009) Anna Groner, PhD student Hillary Layard-Liesching, PhD student (from May 2009) Pierre Maillard, PhD student Julien Marquis, Postdoctoral fellow Flavia Marzetta, PhD student (from November 2009) Thomas Meier, Visiting professor (from November 2009) Sylvain Meylan, PhD student Sujana Nylakonda, Technician (until June 2009) Sandra Offner, Technician Simon Quenneville, Postdoctoral fellow Shyam Sunder Reddy Peddy, visiting scientist (until September 2009) Charlène Raclot, Technician (from October 2009) Séverine Reynard, Administrative Assistant Helen Mary Rowe, Postdoctoral fellow Francesca Santoni de Sio, Postdoctoral fellow Priscilla Turelli, Senior Scientist Sonia Verp, Technician Nadine Zangger, Bioinformatician (from August 2009)

Introduction

Retroelements constitute important evolutionary forces for the genome of higher organisms, yet their uncontrolled spread, whether from endogenous loci or within the context of viral infections, can cause diseases such as cancer, hepatitis and AIDS. For many years, our work has aimed at characterizing the relationship between retroelements and their hosts. This has led us to investigate more broadly how epigenetic mechanisms shape the expression of mammalian genomes. This question has now become central to our research effort.

Keywords:

Molecular virology, HIV, hepatitis B virus, endogenous retroelements, innate immunity, lentiviral vectors, mammalian genetics, epigenetics, KRAB zinc finger proteins, KAP1, transcriptional repression.

Results Obtained in 2009

While we have pursued our structure-function analyses of APOBEC- and TRIM5α-mediated restriction of retroelements, an increasingly prominent part of our activity has focused on understanding the impact of epigenetics on the control of mammalian gene expression. The mouse and human genomes contain around four hundred genes encoding KRAB-containing zinc finger proteins (KRAB-ZFPs), a family of tetrapod-restricted sequence-specific DNA-binding transcriptional repressors. As such, these KRAB-ZFPs represent the single largest group of transcriptional regulators encoded by higher vertebrates, yet their functions remain largely unknown. Nevertheless, it has been established that they share an essential

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cofactor, the histone methyltransferase- and histone deacetylase-recruiting KAP1, and act by triggering the formation of heterochromatin. KAP1 is ubiquitous, and KRAB-ZFPs appear to be present in most if not all cells, albeit along patterns that are distinctly cell type-, stage- and state-specific. It suggests that KRAB/KAP1 gene regulation influences a very large number of physiological events. A few years ago, we started investigating this question through a combination of genetic, functional and molecular studies aimed at i) deciphering the genomic modalities of KRAB/KAP1-mediated transcriptional regulation, and ii) exploring its role in health and disease. On the mechanistic front, we first determined that KRAB/KAP1-mediated repression can be exerted on both chromosomal and episomal DNA, which has implications for the design of externally controllable non-integrating gene transfer systems (e.g. adenoassociated virus-derived vectors) and for the mechanisms by which some viruses (e.g. of the herpes family) establish latency via the persistence of episomal DNA. We then used a combination of lentivector-mediated gene trapping and a drug-controllable KRAB-containing synthetic repressor to examine the genomic modalities of KRAB/KAP1-induced repression. We could demonstrate that KRAB and its corepressor KAP1 can silence promoters located several tens of kilobases away from their DNA binding sites through heterochromatin spreading and blockade of transcriptional initiation. We confirmed the biological relevance of this phenomenon by documenting KAP1-dependent transcriptional repression at an endogenous KRAB-ZFP gene cluster, where KAP1 binds to the 3’end of genes and mediates propagation of

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EPFL School of Life Sciences - 2009 Annual Report

On the functional front, the conditional knockout of KAP1, the essential cofactor of all KRAB-ZFPs, logically turned out to constitute a formidable experimental tool, and as such rapidly became a cornerstone of our strategy. It allowed us to report last year that KAP1 expression in the forebrain controls behavioral vulnerability to stress. We had also observed that KRAB/KAP1 can induce permanent gene silencing during the early embryonic period via promoter DNA methylation. We explored the functional significance of this phenomenon by generating conditional KAP1 knockout embryonic stem cell (ESC) lines, which revealed that KAP1 is essential for the pluripotent selfrenewal of ESC. Furthermore, we found that KAP1 deletion results in a marked upregulation of a range of endogenous retroviruses (ERVs), in particular IAPs (intracisternal A type particles), in mouse ESC and early embryos. We could further demonstrate that KAP1 acts synergistically with DNA methylation to silence IAPs and that it is enriched at the 5’ untranslated region (5’UTR) of IAP genomes, where KAP1 deletion leads to loss of H3K9 trimethylation, a hallmark of KAP1-mediated repression. Correspondingly, we demonstrated that IAP-5’UTR sequences can impose in cis KAP1-dependent repression on a heterologous promoter in ES cells. Our results thus establish that KAP1 controls endogenous retroelements during early embryonic development, and strongly suggest that these rapidly mutating genetic invaders contributed to the strong positive selection undergone by KRAB-ZFP genes during evolution.

Selected publications

P.V. Maillard, G. Ecco, M. Ortiz and D. Trono (2010). The Specificity of TRIM5α-medited restriction is influenced by its coiled-coil domain. J. Virol., e-publ. March 10.

A.C. Groner, S. Meylan, A. Ciuffi, N. Zangger, G. Ambrosini, N. Dénervaud, Philipp Bucher and D. Trono (2010). KRABzinc finger proteins and KAP1 can mediate long-range transcriptional repression through heterochromatin spreading. PLoS Genetics, 6: e1000869. H. Rowe, J. Jakobsson, D. Mesnard, J. Rougemont, S. Reynard, T. Aktas, P.V. Maillard, H. Layard-Liesching, S. Verp, J. Marquis. F. Spitz, D.B. Constam and D. Trono (2010). KAP1 controls endogenous retroviruses in embryonic stem cells. Nature, 463: 237-240. S. Meylan and D. Trono (2009). Immunity against retroviral pathogens: from an ambiguous genetic self to novel therapeutic approaches. Swiss Med. Wkly, 139: 706-711. Y. Bulliard, P.Turelli, U.G. Röhrig, V. Zoete, B. Mangeat, O. Michielin and D. Trono (2009). Functional analysis and structural modeling of human APOBEC3G reveals the role of evolutionarily conserved elements in HIV1 and Alu inhibition. J. Virol. 83: 12611-12621. F. R. Santoni de Sio and D. Trono (2009). APOBEC3G-depleted resting CD4+ cells remain refractory to HIV1 infection. PLoS One 4: e6571. I. Barde, E. Laurenti, S. Verp, A. Groner, C. Towne, V. Padrun, P. Aebischer, A. Trumpp and D. Trono (2009). Regulation of episomal gene expression by KRAB/KAP1-mediated histone modifications. J. Virol. 83: 5574-5580. J. Jakobsson, M.I. Cordero, R. Bisaz, A. Groner, V. Busskamp, J.-C- Bensadoun, F. Cammas, R. Losson, I.M. Mansuy, C. Sandi and D. Trono (2008). KAP1-mediated epigenetic repression in the forebrain modulates behavioral vulnerability to stress. Neuron 60: 818-831. M.-O-. Sauvain, A. Dorr, B. Stevenson, A. Quazzola, F. Naef, M. Wiznerowicz, F. Schütz, V. Jongeneel, D. Duboule. F. Spitz and D. Trono (2008). Genotypic features of lentiviral transgenic mice. J. Virol. 82: 7111-7119. P. Turelli, A. Quazzola, S. Verp, S. Jost and D. Trono (2008). APOBEC3-independent IFN-induced viral clearance in Hepatitis B virus transgenic mice. J. Virol. 82: 6585-6590.

IAP-specific in situ hybridization of E 5.5 KAP1+/- intercross embryos, with KAP1-positive and KAP1-knockout embryos on top and bottom rows, respectively. In the absence of KAP1, embryos are smaller, fail to gastrulate, and exhibit a massive upregulation of IAPs. Size bar: 100 μm

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H3K9me3 and HP1β towards their 5’ end. These results not only suggest auto-regulatory mechanisms in the control of KRAB-ZFP gene clusters, but also provide important cues for interpreting future genome-wide DNA binding data of KRAB-ZFPs and KAP1.


EPFL School of Life Sciences - 2009 Annual Report

GHI - Global Health Institute

Van Der Goot Lab Head of Lab (PI) - http://vdg.epfl.ch

Team Members

F. Gisou van der Goot

Laurence Abrami, Scientific collaborator Sanja Blaskovic, PhD student (Start June 2009) Valérie Burger, Administrative Assistant Julie Deuquet, PhD Student Michal Feldman, Post doctoral fellow Barbara Freche, Post doctoral fellow (end April 2009) Manuel Gonzalez, PhD Student Romain Groux, Technician Ioan Iacovache, PhD Student Béatrice Kunz, Technician Asvin Lakkaraju (Start May 2009) Nuria Reig, Post doctoral fellow (end February 2009) Geneviève Rossier, Administrative assistant for LipidX project Suzanne Salvi, Technician Nadine Zangger, Bioinformatician

Full Professor

Introduction

The laboratory’s effort is focused on understanding the molecular and cellular mode of action of bacterial protein toxins, such as pore-forming toxins or anthrax toxin, which are major determinants of human infectious diseases. Our studies are also aimed at understanding the physiological role and the cell biology of the anthrax toxin receptors and their partner proteins. Our work lies at the frontier of cell biology and cellular microbiology - bacterial toxins provide us with a powerful tool to study basic cellular processes, in addition to their role as virulence factors.

Keywords:

Bacterial virulence factors, toxins, anthrax, poreforming toxins, cellular microbiology, cell biology, structure of membrane proteins, Systemic Hyalinosis, Hyaline Fibromatosis

Results Obtained in 2009

Pore-forming toxin aerolysin Pore-forming toxins (PFTs) are molecules that are produced as soluble proteins but finally form pores in the target cell membrane. Our aim is to understand the molecular mechanisms that underlie this metamorphosis. Two question of interest are: during the initial folding of PFTs, what drives them towards the soluble rather than the transmembrane conformation? What prevents pore-formation in the membrane of the producing cell? We have shown for the prototypical PFTs aerolysin, the protein is produced with a C-terminal extension that serves as an intramolecular chaperone, which possibly via conformational selection drive the folding of the toxin. In a second step, the extension prevents premature formation of the oligomeric pore-forming complex. Biosynthesis of the anthrax toxin receptor and effects of Hyaline Fibromatosis mutations The anthrax toxin is composed of three polypeptide chains: the protective antigen (PA), the lethal factor (LF) and the edema factor (EF). LF is a zinc dependent

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metalloprotease that cleaves all MAP kinase kinases; EF is a calmodulin dependent adenylate cyclase that is responsible for the edema observed in anthrax patients ; PA has no enzymatic activity and is involved in escorting EF and LF to the cytoplasm. Toxicity is strictly dependent upon the delivery of the enzymatic subunits to the cytoplasm, an event that strictly depends on the anthrax toxin receptors (ATRs), of which there are two: capillary morphogenesis gene 2 (CMG2) and tumor endothelial marker 8 (TEM8), two poorly characterized membrane proteins. We have analyzed the post-translational modifications undergone by the ectodomain in the ER and how these impact on protein folding and targeting to the plasma membrane. We found that both CMG2 and TEM8 are glycosylated at multiple sites, modifications that are not required for ER exit, but affect the plasma membrane localization. We also found that the CMG2 ectodomain contains 3 disulfide bonds, formation of which is essential for ER folding and exit. Interestingly, in collaboration with Dr. A. Superti-Furga (Univ. Freiburg), we found three novel Hyaline Fibromatosis mutations that affect disulfide bond formation, leading to ER retention and thus loss of function. Endocytosis of the anthrax toxin We have recently focused on the initial events at the cell surface that promote toxin endocytosis. We found that the toxin through the binding to its receptors triggers multiple intracellular signaling events. Binding triggers the dissociation of TEM8 from the actin cytoskeleton, leading to an increase in surface mobility. Heptamerization of PA triggers activation of the tyrosine kinase src, which phosphorylates the cytosolic tail of the receptor. This leads to the sequential recruitment of the adaptor protein ß-arrestin and the E3 ubiquitin ligase Cbl, which in turn ubiquitinates a cytosolic lysine on the receptors. These events finally initiate clathrin-mediated endocytosis of the receptor in an adaptor AP-1 dependent

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EPFL School of Life Sciences - 2009 Annual Report

Selected publications

N. Reig, A. Jiang, R. Couture, F. S. Sutterwala, Y. Ogura, R. A. Flavell, I. Mellman, and F. G. van der Goot (2008) Maturation Modulates Caspase-1 Independent Responses of Dendritic Cells to Anthrax Lethal Toxin. Cellular Microbiology 10(5):1190-207 Abrami, L., Kunz, B., Iacovache, I., & van der Goot, F. G. (2008) Palmitoylation and ubiquitination regulate exit of the Wnt signaling protein LRP6 from the endoplasmic reticulum. Proc Natl Acad Sci U S A 105(14):5384-9. Abrami, L., Kunz, B., J. Deuquet, Bafico, A. and van der Goot, F. G. (2008) Functional interactions between the anthrax toxin receptors and the Wnt signaling protein LRP6. Cellular Microbiology 10:2509-2519 Deuquet J., Abrami L., Ramirez M.C.M., DiFeo A., Martignetti J. and van der Goot F.G. (2009) Systemic hyalinosis mutations in the CMG2 ectodomain leading to loss of function through retention in the endoplasmic reticulum. Human Mutations 30 : 583-589 Charollais, J., and van der Goot, F. G. (2009). Palmitoyla-

tion of membrane proteins (Review). Mol Membr Biol 26: 55-66 Bischofberger M., Gonzalez M.R. and van der Goot, F.G. (2009) Membrane injury by pore-forming proteins (2009) Current Opinion in Cell Biology 21:589-95Feldman M. and van der Goot, F.G. Novel ubiquitin-dependent quality control in the endoplasmic reticulum (2009) Trends in Cell Biology 19:357-63 van der Goot, F.G. and J.A. Young (2009) Receptors of anthrax toxin and cell entry. Mol Aspects Med. 6: 406-412. Iacovache, I, Bischofberger M. and van der Goot, F.G. (2010) Structure and assembly of pore-forming proteins. Current Opinion in Structural Biology (in press) Abrami, L., Kunz, B. and van der Goot, F.G. (2010) Anthrax toxin triggers the activation of src like kinases to mediates its own uptake. Proc Natl Acad Sci U S A 107:14201424 Iacovache, I. Biasini, M., Kowal, J., Kukulski, W., Chami, M. van der Goot, F.G., Engel, A. and Rémigy, R.W. (2010) The 2DX robot: a membrane protein 2D crystallization Swiss Army kniffe. J. Struct. Biol. 169:370-378 Abrami, L., Bischofberger M, Kunz, B., Groux, R. and van der Goot, F.G. (2010) Endocytosis of the anthrax toxin is mediated by clathrin, actin and unconventional adaptors PLoS Pathogen 6(3): e1000792.

Schematic representation of the endocytosis of the anthrax toxin. TEM8-1 is preorganized at the cell surface by the cortical actin cytoskeleton while CMG2 is not. Upon PA binding, processing and oligomerization, the toxin receptor complex moves to lipid rafts. There, ß-arrestin mediates the recruitment of the E3 ligase Cbl to the cytoplasmic tail of the receptor. The ubiquinated receptor subsequently recruits the heterotetrameric adaptor AP-1 and finally clathrin. Completion of the endocytic process and pinching off of the toxin containing clathrin-coated vesicle requires both actin and dynamin. (Abrami, L., M. Bischofberger, B. Kunz, R. Groux, and F.G. van der Goot. 2010. Endocytosis of the anthrax toxin is mediated by clathrin, actin and unconventional adaptors. PLoS Pathog. 6:e1000792.)

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manner. In contrast to endocytosis of the diphtheria toxin, we found that update of the anthrax toxin was strongly dependent on actin. These studies identify specific molecular players involved in anthrax toxin endocytosis and highlight the highly modular nature of clathrin-mediated endocytosis.


ISREC - Swiss Institute for Experimental Cancer Research ISREC has now been integrated into the School of Life Sciences at EPFL, where it shares a new building (Batiment SV) with the Global Health Institute. Its faculty continue to investigate a spectrum of fundamental biological systems that are variously co-opted or disrupted during the development of cancer. Prominent amongst the research topics are signalling pathways that normally regulate aspects of embryogenesis and organogenesis, and mechanisms orchestrating the cell division cycle and the maintenance of genomic integrity during cell proliferation. Increasingly, genetically engineered mouse models of human cancer are being employed to elucidate the roles of such signalling circuits and regulatory mechanisms in tumors, as well as the complex interplay of cancer cells with ostensibly normal cells in their collective “tumor micro-environment”; such heterotypic cell-cell interactions are proving instrumental for malignant progression of tumors to lethal disease. A new director has been appointed – Douglas Hanahan, from the University of California at San Francisco – and with his arrival a new strategic plan for ISREC is developing, involving increasing focus via new faculty recruitments on directly studying mechanisms of cancer in model systems and in human tumors, notably involving outreach to the medical oncology community in the region. Toward that end, ISREC will establish a translational oncology branch at the University of Lausanne’s medical campus (CHUV) intended to foster by its proximity and emphasis catalytic interactions and cooperative research with clinical cancer researchers. Both branches of ISREC will seek to expedite progress toward a deeper understanding of the biology and genetics of cancer, in turn leveraging new knowledge forthcoming to develop and test innovative drug targeting strategies aimed to disrupt critical mechanisms of the disease and thereby improve the benefits of therapy. For further information please see http://isrec.epfl.ch.

© Co opyright 2004-2010 0 EPFL for or a ma ater eria ia al pu publ blis ishe he ed iin n this rep port rt Copyright alllll m material published report info f .sv@ v@e epfl.c ..ch ch info.sv@epfl

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ISREC

EPFL School of Life Sciences - 2009 Annual Report


EPFL School of Life Sciences - 2009 Annual Report

ISREC - Swiss Institute for Experimental Cancer Research

Aguet Lab Head of Lab (PI) - http://aguet-lab.epfl.ch

Team Members

Pascale Anderle, Postdoctoral fellow (50%) Sylvie André, Technician Frédérique Baruthio, Postdoctoral fellow Jennyfer Bultinck, Postdoctoral fellow Juergen Deka, Staff scientist (50%), Scientific manager NCCR «Molecular Oncology» (50%) Vania Sergy-Schmidt, Administrative assistant NCCR «Molecular Oncology» (50%) Norbert Wiedemann, PhD student

Michel Aguet

Full Professor Director of NCCR «Molecular Oncology»

Introduction

The Wnt pathway regulates critical processes during embryonic development and adult tissue renewal, and aberrant activation of this pathway is associated with colorectal and other cancers. Our group is focusing on the phenotypic characterization of mouse Wnt pathway mutants in which the transcriptional co-activators Bcl9 and Bcl9l were conditionally ablated. These Wnt pathway components were discovered in Drosophila where they are essential for canonical Wnt signaling. Bcl9 proteins act as linkers to tether Pygo to the ß-catenin-Tcf activation complex (Kramps et al., Cell 109, 47-60, 2002).

Keywords

Mouse Wnt signaling mutants, Tumor cell differentiation

Results Obtained in 2009

Phenotypic characterization of mice lacking Bcl9/9l To elucidate the role of Bcl9 proteins during embryonic development and adult tissue homeostasis in the mouse, we generated loxP-flanked alleles of Bcl9/9l and derived several conditionally mutant strains. Our recent work has focused on the following two projects: Role of Bcl9/9l in homeostasis and regeneration of adult intestinal epithelium In the intestinal epithelium, locally confined Wnt signals regulate and compartmentalize cell proliferation and differentiation along the crypt villus axis. In contrast to other Wnt pathway mutants, ablation of Bcl9/9l in the adult intestinal epithelium did not result in any detectable anomaly and had no effect

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on cell proliferation and lineage determination. However, mutant mice proved highly susceptible to the irritant dextrane sulfate sodium (DSS) and developed a markedly more severe colitis as compared to wild-type mice (see Figure), likely due to impaired epithelial regeneration during wound healing. Genes with enriched expression in intestinal stem cells were expressed at markedly lower levels in Bcl9/Bcl9l mutant colon epithelium. While these findings imply that in the mouse Bcl9/9l are by and large not obligatory for Wnt-mediated gene regulation, differences in gene expression indicate that they may selectively regulate a subset of Wnt target genes that is involved in stem cell control. Role of Bcl9/9l in tumor development Constitutive activation of Wnt/β-catenin signaling is an initiating event in the development of colorectal carcinomas in humans. To assess the role of Bcl9 proteins in colorectal tumorigenesis, colon tumors were induced in mice through exposure to the mutagen N,N’-dimethylhydrazine (DMH) and subsequent challenge with DSS. Unexpectedly, wild-type and Bcl9/9l mutants developed tumors to the same extent and tumors had comparable morphology. Transcriptional profiles of micro-dissected tumor tissue, however, revealed major differences, pointing again to a likely role of Bcl9/9l in regulating a selective set of Wnt target genes that may control cell differentiation. Interestingly, some transcriptional signatures observed in our colon cancer model that are largely abrogated by the loss of Bcl9/9l function overlap with gene expression patterns that correlate with poor outcome in human cancers.

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EPFL School of Life Sciences - 2009 Annual Report

Selected publications

Brack, A.S., Murphy-Seiler, F., Hanifi, J., Deka, J., Eyckerman, S., Keller, C., Aguet, M., and Rando, T.A. (2009). BCL9 is an essential component of canonical Wnt signaling that mediates the differentiation of myogenic progenitors during muscle regeneration. Dev. Biol. 335, 93-105. Farmer, P., Bonnefoi, H., Anderle, P., Cameron, D., Wirapati, P., Becette, V., Andre, S., Piccart, M., Campone, M., Brain, E., et al. (2009). A stroma-related gene signature predicts resistance to neoadjuvant chemotherapy in breast cancer. Nat. Med. 15, 68-74.

ISREC

Outlook In view of these recent findings our efforts are now largely focusing on characterizing the subset of Bcl9/9l-regulated genes both in the epithelial regeneration and in the tumor model, and on investigating the functional implications notably in tumor progression and dissemination. In vivo models are being adapted to assess the role of Bcl9/9l-regulated genes for tumor engraftment, progression and dissemination. In vitro assays are being developed that recapitulate the observed transcriptional profile changes, as a basis for addressing the mechanisms of Bcl9/9l-mediated gene selective transcriptional regulation.

Immunohistochemistry using a Bcl9 specific antibody on A) wild-type and B) Bcl9/9l deleted mouse colon adenocarcinomas confirming complete loss of Bcl9 expression in the mutant tumor epithelium.

Gene expression changes between wild-type (WT) and Bcl9/9l mutant (KO) colon adenocarcinomas revealing substantial transcriptional differences and pointing to a role of Bcl9/9l in a subprogram of Wnt regulated cell functions.

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EPFL School of Life Sciences - 2009 Annual Report

ISREC - Swiss Institute for Experimental Cancer Research

Beard Lab Head of Lab (PI) - http://beard-lab.epfl.ch/

Team Members

Anika Ekrut, PhD student Michail Fragkos, Postdoctoral fellow Laurence Goulet, Master student Leila Laredj, Postdoctoral fellow Florence Magnin, PhD student Geneviève Massy, Administrative assistant Nicole Paduwat, Senior laboratory assistant

Peter Beard

Professor Titulaire

Introduction

The cellular response to DNA damage is a crucial factor in tumour formation. Our work uses a virus (adeno-associated virus, AAV) as a biological probe to study DNA damage signalling pathways in cells. We found that AAV infection triggers a damage response that can lead to death of p53-defective tumour cells. AAV therefore provides a unique opportunity to study this response without actually damaging the cellular DNA. Understanding the tumour-suppressive activity of AAV may lead to novel approaches to cancer therapy

Keywords

Adeno-associated virus, cancer, DNA damage response, cell cycle, cell death

Aims

The goal of our work is to understand what is needed for a successful infection of different cell types by AAV and how the cells react to this infection.

Results Obtained in 2009

The DNA damage response to non-replicating adeno-associated virus: centriole overduplication and mitotic catastrophe independent of the spindle checkpoint. Adeno-associated virus (AAV) type 2 or UV-inactivated AAV (UV-AAV2) infection provokes a DNA damage response that leads to cell cycle arrest at the G2/M border. p53-deficient cells cannot sustain the G2 arrest, enter prolonged impaired mitosis, and die. We studied how non-replicating AAV2 kills p53-deficient osteosarcoma cells. We found that the virus uncouples centriole duplication from the cell cycle, inducing centrosome overamplification that is dependent on Chk1, ATR and CDK kinases, and on G2 arrest. Interference with spindle checkpoint components Mad2 and BubR1 revealed unexpectedly that mitotic catastrophe occurs independently of spindle checkpoint function. We conclude that, in the p53-deficient cells, UV-AAV2 triggers mitotic catastrophe associat-

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ed with a dramatic Chk1-dependent over-duplication of centrioles and the consequent formation of multiple spindle poles in mitosis (see figure). As AAV2 acts through cellular damage response pathways, the results provide information on the role of Chk1 in mitotic catastrophe after DNA damage signaling in general. How adeno-associated virus Rep78 relocalises Cdc25B to arrest the cell cycle: the multiple interactions of Rep78 with cellular regulatory, replication and recombination proteins. The AAV Rep78 protein has been found to block the cell cycle at several stages. Our work focuses on the question of whether Rep78 exerts its effect through interaction with the cell division cycle 25 (Cdc25) phosphatases. Rep78 interacts with Cdc25A and inactivates it. As Cdc25B has an equally important role in cell cycle regulation, we investigated the interaction between Rep78 and Cdc25B. Expression of Rep78, which is nuclear, leads to the relocalisation of Cdc25B to the nucleus. Our model is that Rep78 by interacting with Cdc25B, retains it in the nucleus. This work establishes a novel mechanism in which sequestration of Cdc25B in the nucleus prevents it from activating the proteins in the cytoplasm needed to give the entry signal into mitosis. Mitotic catastrophe can occur in the absence of apoptosis in p53-null cells with a defective G1 checkpoint. Cell death occurring during mitosis, or mitotic catastrophe, often takes place in conjunction with apoptosis, but the conditions under which mitotic catastrophe may exhibit features of programmed cell death are still unclear. In the work presented here, we studied mitotic cell death by making use of a UV-inactivated parvovirus (adeno-associated virus; AAV) that has been shown to induce a DNA damage response and subsequent death in mitosis, without affecting the integrity of the host genome. p53-deficient osteosarcoma cells (U2OSp53DD) lack the G1

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EPFL School of Life Sciences - 2009 Annual Report

Selected Publications

Fragkos M, Jurvansuu J, Beard P. (2009). H2AX is required for cell cycle arrest via the p53/p21 pathway. Mol Cell Biol. May;29(10):2828-40. Fragkos M, Breuleux M, ClĂŠment N, Beard P. (2008). Recombinant adeno-associated viral vectors are deficient in provoking a DNA damage response. J Virol. Aug;82(15):737987. Garner E, Martinon F, Tschopp J, Beard P, Raj K. (2007). Cells with defective p53-p21-pRb pathway are susceptible to apoptosis induced by p84N5 via caspase-6. Cancer Res. Aug 15;67(16):7631-7. Jurvansuu J, Fragkos M, Ingemarsdotter C, Beard P. (2007). Chk1 instability is coupled to mitotic cell death of p53-deficient cells in response to virus-induced DNA damage signaling. J Mol Biol. Sep 14;372(2):397-406.

Ingemarsdotter C, Keller D, Beard P. (2010). The DNA damage response to non-replicating adeno-associated virus: Centriole overduplication and mitotic catastrophe independent of the spindle checkpoint. Virology. Mar 1. Epub ahead of print.

AAV2-infected mitotic osteosarcoma cells display multiple spindle poles which lead to mitotic catastrophe. Top panel: uninfected cells show normal mitosis; lower two panels: UVinactivated AAV2-infected cells 4 days post infection enter catastrophic mitotis. Staining: DAPI, blue; centrin-3, green; alphatubulin, red.

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cell cycle checkpoint and respond to AAV infection through a transient G2 arrest. Infected U2OSp53DD cells died by mitotic catastrophe with no signs of chromosome condensation or DNA fragmentation. Moreover, cell death was independent of caspases, apoptosis-inducing factor (AIF), autophagy and necroptosis. On the other hand, p53-defective cells that retained a functional G1 checkpoint responded differently to the virus and died by apoptosis. These data, together with the results of time-lapse microscopy, suggest that in the absence of a functional G1 checkpoint, mitotic catastrophe can occur in these p53-null cells as a result of mechanical damage induced by centrosome overduplication, and not as a consequence of a suicide signal.


EPFL School of Life Sciences - 2009 Annual Report

ISREC - Swiss Institute for Experimental Cancer Research

Brisken Lab Head of Lab (PI) http://brisken-lab.epfl.ch

Team Members

Ayyanan Ayyakkannu, Senior technician Caikovski Marian, Postdoctoral fellow Gutierrez Najera Maria,Technician Jimenez Rojo Lucia, Postdoctoral fellow Ouahiba Laribi, Postdoctoral fellow Rajaram Renuga Devi, PhD studentostdoctoral fellow Rodel Anne-Marie, Administrative Assistant Schrick Christina, Technician (end September 2009) Tamara Tanos, Postdoctoral fellow Yalçin Özden, PhD student

Cathrin Brisken

Tenure Track Assistant Professor

Introduction

Breast cancer strikes one out of eight women in Switzerland. A woman’s risk to get breast cancer is linked to her life time exposure to endogenous and exogenous hormones. While early pregnancies have a protective effect, breast cancer risk increases with the number of menstrual cycles a woman experiences prior to her first pregnancy. Hormones also influence the course of the disease. We study how hormones control the breast in vivo, in particular the mechanisms by which they elicit cell proliferation and changes in structure of the breast tissue, to gain insights into the genesis of the disease and to develop new preventive and therapeutic strategies. The use of mutant mice to study breast development The mouse mammary gland provides a unique experimental system to study in vivo how systemic hormones impinge on molecular determinants of development in the breast and how deregulation of these pathways leads to tumorigenesis. The mammary gland is the only organ to undergo most of its development after birth and can be readily manipulated. In prepubertal female mice, the part of the inguinal mammary glands that contains the milk ducts can be surgically removed creating a “cleared fat pad”. When epithelial tissue or primary cells are engrafted into a cleared fat pads, they repopulate it and form a well-organized ductal tree, which responds to hormonal stimuli (see scheme 1). We have used this model and hormone receptor deficient mouse strains to define the role of estrogens,

progesterone and prolactin in mammary gland development and to analyze the mechanism by which the hormones control local developmental signaling pathways in the breast (summarized in scheme 2).

Scheme 2: Schematic representation of mammary gland development (black) and our current working model of how various factors control different morphogenetic steps (color) based on our previous work.

Hormones exert control in the breast tissue by regulating interactions between different cells. We have shown that a subset of cells in the mammary gland, which have the receptors for estrogens and progesterone act as « sensor cells » and translate the hormonal stimulus into local signals which they pass on to different neighboring cells. As a result, cells in close vicinity to the « sensor cells » proliferate. The Progesterone RANKL connections While estrogens are still widely believed to be the major culprit of hormonally-induced breast cancer, our data indicate that progesterone has an equally important if not prevalent role. In fact, we have

Scheme 1: Mammary gland reconstitution: In the 3-weekold female mouse the ductal tree growing out from the nipple has only partially penetrated the mammary fat pad (left panel). It can be surgically removed leaving behind a cleared fat pad. Primary mammary epithelial cells injected into this fat pad can form new ducts that grow out to populate the fat pad (right panels).

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EPFL School of Life Sciences - 2009 Annual Report

We demonstrated that progesterone triggers cell proliferation in the milk ducts by two distinct mechanisms. Progesterone triggers proliferation of hormone receptor positive mammary epithelial cells by a cylin D1-dependent mechanism and proliferation of hormone receptor negative mammary epithelial cells by a paracrine mechanism. The latter requires the tumor necrosis factor a family member RANKL. These findings provided the first evidence that hormone receptor positive and hormone receptor negative mammary epithelial cells divide by distinct molecular mechanism.

Selected publications

Beleut, M., Rajaram, R., Caikovski, M., Ayyanan, A., Germano, D., Choi, Y., Schneider, P., Brisken, C. (2010). Two distinct mechanisms underlie progesterone-induced proliferation in the mammary gland. Proc Natl Acad Sci U S A. 2010 Feb 16;107(7):2989-94. Booth BW, Boulanger CA, Anderson LH, Jimenez-Rojo L, Brisken C, Smith GH. (2009) Amphiregulin mediates self-renewal in an immortal mammary epithelial cell line with stem cell characteristics. Exp Cell Res. 2010 Feb 1;316(3):422-32. Cicalese, A*.; Bonizzi, G*.; Pasi, C.E., Faretta, M., Ronzoni, S., Giulini, B., Brisken, C., Minucci, S., Di Fiore, P.P., Pelicci, P.G. (2009). The tumor suppressor p53 regulates polarity of self-renewing divisions in mammary stem cells. Cell 18; 138(6): 1083-95 .

Barbaroux, J.B., Beleut, M., Brisken, C., Mueller, C.G., Groves, R.W. (2008) Epidermal Receptor Activator of NFkB Ligand Controls Langerhans Cells Numbers and Proliferation, Langerhans cells numbers and proliferation. J Immunol. 181(2): 1103-8. Mani, S. A., Guo, W., Liao, M.J., Ng. Eaton, E., Ayyanan, A., Zhou, A., Brooks, M., Reinhard, F., Zhang, C.C., Shipitsin, M., Campbell, L. l., Polyak, K., Brisken, C., Yang, J., Weinberg, R. A. (2008). The epithelial-mesenchymal transition generates cells with properties of stem cells. Cell 133(4): 704-15. Boulay, A., Breuleux, M., Stephan, C., Fux, C., Brisken, C., Fiche, M., Wartmann, M., Stumm, M., Lane, H. A., Hynes, N.E. (2008). The Ret Receptor Tyrosine Kinase Pathway Functionally Interacts with the ERα Pathway in Breast Cancer. Cancer Res. 68(10): 3743-51.

Reviews :

Dietrich, C., Weiss, C., Bockamp, E., Brisken, C., Roskams, T., Morris, R., Oesch-Bartlomowicz, B., Oesch, F. (2009). Stem cells in chemical carcinogenesis Arch Toxicol. E pub ahead of print. Yalçın-Özuysal, O., Brisken, C. (2009) From normal cell types to malignant phenotypes. Breast Cancer Research, 11(6): 306. Tanos, T., Brisken, C. (2008) What signals operate in the mammary niche? Breast Disease 29: 69-82 Brisken, C. (2008) Endocrine Disruptors and Breast Cancer. Chimia 62(5): 1-4.

Scheme 3: Model of estrogen-induced proliferation : estrogens bind to the estrogen receptor in the sensor cell. In response, the sensor cell secretes the growth factor amphiregulin that acts on stromal cells. In response to an unknown signal from the stroma, cells next to the sensor cells, divide.

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found that progesterone is the strongest growth stimulus in the adult mammary gland with estrogens having only a permissive role.


EPFL School of Life Sciences - 2009 Annual Report

ISREC - Swiss Institute for Experimental Cancer Research

Constam Lab Head of Lab (PI) - http://constam-lab.epfl.ch

Team Members

Stéphane Baflast, Technician Anja Dietze, PhD student Bruno Filippi, Postdoctoral fellow Christophe Fuerer, Postdoctoral fellow Susanna Kallioinen, PhD student Virginie Kokocinski, Administrative Assistant Charlotte Maisonneuve, PhD student Daniel Mesnard, Postdoctoral fellow Nathalie Piazzon, Postdoctoral fellow Alberto Toso, Postdoctoral fellow Séverine Urfer-Beck, Technician

Daniel Constam Associate Professor

Introduction

We use genetic and biochemical approaches to determine how pluripotent cells in the mouse embryo communicate with their microenvironment to coordinately differentiate into tissues and organs, and how tumor cells evade such regulation. We also investigate mechanisms of RNA silencing that suppress cystic growth in the kidney and pancreas.

Keywords

Development and cancer; stem cell fate; polycystic kidney disease; cilia and planar polarity signaling; protein processing and trafficking; microRNA.

Results Obtained in 2009

Cripto binds the Nodal precursor and its convertases to spatially couple processing and signaling. The allocation of pluripotent cells to distinct germ layers and the establishement of antero-posterior and left-right asymmetries are orchestrated by the TGFβ-related precursor protein Nodal and its proprotein convertases Furin and Pace4. Nodal signaling sustains trophoblast stem cells in the extra-embryonic ectoderm and prevents precocious neural differentiation of pluripotent progenitors in the inner cell mass of the implanted blastocyst. By contrast, during gastrulation, Nodal induces endoderm and mesoderm formation, and ectopic pathway activation in human melanoma has been linked to metastasis. How such disparate activities are regulated is poorly understood. Our previous work established that inhibition of Nodal precursor processing does not prevent binding to ActRII and Acvr1 signaling receptors and only blocks a subset of Nodal activities in mouse embryos. However, Nodal cleavage is necessary to potentiate activation of the transcription factors Smad2 and-3 by a complex with the GPI-anchored coreceptor Cripto. To elucidate the role of Cripto, we asked whether it is necessary for Nodal trafficking. Biochemical analysis in transfected cells revealed that Cripto captures Nodal already during exocytosis, in part by tethering the prosegment, leading to an increase of Nodal precursor in lipid rafts marked by Flotillin. This interac-

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tion is important since mutation of a Cripto-binding region in the Nodal prosegement inhibited signaling. Furthermore, Cripto was found to also bind Furin and Pace4, suggesting a new role in localizing Nodal processing to specific membrane microdomains (Blanchet et al., 2008a). To investigate how the localization of Nodal by Cripto might potentiate Smad signaling, we tested whether Cripto controls access of Nodal to endosomal signaling platforms harboring the Smad anchor for receptor activation (SARA). Indeed, Cripto attenuated the sequestration of Nodal from the endosome-limiting membrane to intralumenal vesicles. Mutant forms of Cripto that failed to inhibit intra-lumenal sorting were unable to stimulate Nodal signaling, even though binding of Nodal and Alk4 at the plasma membrane and subsequent endocytosis were unaffected (Blanchet et al., 2008b). These observations suggest that Cripto guides Nodal trafficking to couple proteolytic maturation and signaling in target cells to ensure that internalized processed Nodal can efficiently signal to Smad2,3 in the cytoplasm (Constam, 2009). The RNA-binding protein Bicaudal-C governs left-right axis formation and renal morphogenesis After gastrulation, Nodal induces its own expression on only one side of the embryo to establish asymmetric patterning of the visceral organ primordia. We recently found that this asymmetry is randomized in embryos where we inactivated the RNA-binding protein Bicaudal-C by a targeted deletion. Video microscopy and scanning electron microscopy analysis revealed that loss of Bicaudal-C disrupts the planar alignment of node cilia and a cilia-propelled fluid flow that is needed to bias Nodal signaling to the left side. Furthermore, disruption of Bicaudal-C triggers cystic growth in kidneys and pancreas, leading to renal failure and early postnatal lethality. These phenotypes were reminiscent of inv/inv mutant lacking the ankyrin repeat protein Inversin, a known inhibitor of Dishevelled in the Wnt/β-catenin signaling pathway. Reporters of canonical Wnt signaling were also inhibited by Bicaudal-C. Inhibition occurred at the level of

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EPFL School of Life Sciences - 2009 Annual Report

Selected Publications

Maisonneuve, C.M., Guilleret, I., Vick, B., Weber, T., Andre, P., Beyer, T., Blum, M., and Constam, D.B. (2009). BicaudalC, a novel regulator of Dvl signaling abutting RNA-processing bodies, controls cilia orientation and nodal flow. Development 136 (17):3019-3030. Constam, D.B. (2009a) Riding shotgun: A dual role for the EGF-CFC protein Cripto in Nodal trafficking. Traffic 10(7):783-791. Constam, D.B. (2009b). Intracellular trafficking and signaling in development. F1000 Biol. Reports doi: 10.3410/ B1-59. Constam, D.B. (2009c) Running the gauntlet: An overview of the modalities of travel employed by the putative mor-

phogen Nodal. Curr. Opin. Genet. Dev. 19 (4):302-307. Blanchet M-H, Le Good JA, Oorschot V, Baflast S, Minchiotti G, Klumperman J, and Constam DB (2008) Cripto localizes Nodal at the limiting membrane of early endosomes. Sci. Signal. 1(45); ra13. Blanchet, M.-H., Le Good, J.A., Mesnard, D., Baflast, S., Minchiotti, G., Klumperman, J., and Constam, D.B. (2008) Cripto recruits Furin and PACE4 and controls Nodal trafficking during proteolytic maturation. EMBO J. 27 :25802591. Szumska, D., Pieles, G., Essalmani, R., Bilski, M., Mesnard, D., Kaur, K., Franklyn, A., El Omari, K., Jefferis, J., Bentham, J., Taylor, J., Schneider, J., Arnold, S., Johnson, P., Tymowska-Lalanne, Z., Stammers, D., Clarke, K., Neubauer, S., Morris, A., Brown, S., Shaw-Smith, C., Cama, A., Capra, V., Ragoussis-J., Constam, D., Seidah, N.G., Prat, A., Bhattacharya, S. (2008). VACTERL / Caudal Rregression / Currarino Syndrome-like malformations in mice with mutation in the proprotein convertase Pcsk5. Genes Dev. 22 :1465-1477. Dâ&#x20AC;&#x2122;Andrea, D., Liguori, G.L., Le Good, J.A., Lonardo, E., Andersson, O., Constam, D.B., Persico, M.G., Minchiotti, G. (2008). Cripto promotes A-P axis specification independently of its stimulatory effect on Nodal autoinduction. J. Cell Biol. 180:597-605.

Targeted inactivation of Bicaudal-C perturbs the planar orientation of node cilia and induces polycystic kidney disease. (A) Left-right asymmetry is specified by motile cilia (green) that generate a leftward fluid flow in the ventral node (VN) at E8.0. Due to this flow, Nodal produced by the VN signals preferentially to the left side to upregulate its own expression in lateral plate mesoderm (black arrows), whereas the right side is shielded by Nodal antagonists (blue). ec: ectoderm ; m: mesoderm ; en: endoderm. (B) The VN expresses Bicc1 mRNA. Bicaudal-C comprises 3 RNA-binding Khomology (KH) domains and a sterile alpha motif (SAM). (C) Nodal mRNA expression in control and Bicc1 knockout embryos. Loss of Bicc1 leads to absent, bilateral or inverted Nodal signaling (85%, n=60). (D) Analysis of motile node cilia and directional flow. Top left: The trajectories (colored lines) of cilia tips are offset towards the posterior (p) relative to the base (red dots) in the wild-type. By contrast, a majority of Bicc1 KO cilia tilt their rotational axes in a random orientation (blue dots), suggesting their effective strokes are not coordinated. The frequencies of cilia beating (white numbers) were unaffected. Bottom: Gradient time track analysis revealed leftward flow of fluorescent beads in control nodes, but not in the VN of Bicc1 mutants. Right: Scanning electron microscopy analysis of a Bicc1 mutant. Misoriented cilia are indicated (blue arrows). (E) Cilia harbor the calcium channel polycystin-2 (PC2) and Inversin (Invs), which is upregulated by flow to target cytoplasmic Dishevelled (Dvlc) for proteasomal degradation. Both loss of cilia and mutations in PC2 or Invs give rise to left-right asymmetry defects and cystic kidneys, possibly due to hyperactivation of Dvl. (F) Multiple cysts also develop in Bicc1 KO kidneys, leading to renal failure within days after birth. (G, H) In transfected cells, Bicaudal-C inhibits Dvl2 activity in cytoplasmic scaffolds that accommodate P-bodies marked by Dcp1a (G). Inhibition of Dvl involves the SAM domain (pink), which concentrates BicC around P-bodies (H). Thus, mutation of BicC stimulates Wnt/b-catenin signaling. Hyperactivation of b-catenin is sufficient to induce renal cysts and is expected to counteract Wnt/PCP signals that govern cilia positioning.

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Dishevelled and was enhanced by the sterile alpha motif (SAM) of Bicaudal-C that mediated an interaction with RNA processing bodies. Thus, Bicaudal-C links cilia positioning and renal and pancreatic tubule morphogenesis to an organelle that degrades targets of microRNAs to mediate post-transcriptional gene silencing. Future identification of functionally relevant Bicaudal-C target RNAs should provide important insights into the mechanisms that disrupt tubular morphogenesis in polycystic kidney diseases.


EPFL School of Life Sciences - 2009 Annual Report

ISREC - Swiss Institute for Experimental Cancer Research

Duboule Lab Head of Lab (PI) - http://duboule-lab.epfl.ch/

Team Members

Guillaume Andrey, PhD student Saskia Delpretti, PhD student Caroline Guinchard, Administrative Assistant István Gyurján, Postdoctoral fellow Elisabeth Joye, Technician Marion Leleu, Bioinformatician Nicolas Lonfat, PhD student Thomas Montavon, Postdoctoral fellow Daan Noordermeer, Postdoctoral fellow Patrick Schorderet, PhD student Bernhard Sonderegger, Bioinformatician Maxence Vieux-Rochas, Postdoctoral fellow

Denis Duboule Full Professor EPFL & University of Geneva

Introduction

The aim of this research is to understand how genes are regulated during mammalian embryonic development and in embryonic stem cells. We are particularly interested to study the relationships that exist between genomic organization (e.g. gene topology) and the control of transcriptional activity, both at the genetic and epigenetic levels, by using one of the Hox gene loci as a paradigm.

nated activation of Hoxd genes during development, is associated with a progressive and linear modification of the epigenetic landscape covering this gene locus, and is established early on in ES cells. Our focus is to understand what mechanism underlies this progressive chromatin modifications as well as what determines the extent of the repressive landscape, which is organized early on in ES cells to prevent these genes to be activated too early.

Keywords

Selected publications

Embryos, development, evolution, transcription, epigenetic regulation, Hox gene clusters, enhancers

Results Obtained in 2009

SystemsHox.ch; an in vivo System Approach to Hox Genes Regulation in Vertebrates. We would like to understand the relationship between genomic topology and the control of transcription, using the HoxD locus as a paradigm. We use a systems approach in the mouse, combining genetic tools, evolutionary genomics and biochemistry to try and model various modes of large-scale gene regulations occurring during development and in embryonic stem cells. Our approach makes use of a unique collection of mutants at this locus along with both transcript profiling and quantitation, ChIP of several proteins (modifications thereof-) indicative of chromatin states, chromosome conformation capture (4C) and transgenesis after phylogenetic footprint. We focus on a 2 Mb large DNA interval centered around the HoxD locus, which contains a range of conserved non-coding DNA sequences and includes a gene-rich island, flanked by two large gene-deserts bordered again by gene-rich regions. The aim is to conduct a systematic analyses of these multiple parameters in various embryonic tissues, at different times to understand where and when Hox genes are required. Recently, we have shown that the coordi-

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Soshnikova N, Duboule D. (2009) Epigenetic regulation of vertebrate Hox genes ; a dynamic equilibrium. Epigenetics, 4, 537-540. Soshnikova N, Duboule D. (2009) Epigenetic temporal control of mouse Hox genes in vivo. Science, 324, 13201323. Tschopp P, Tarchini B, Spitz F, Zakany J, Duboule D. (2009) Uncoupling time and space in the collinear regulation of Hox genes. PLoS Genet. ,5, e1000398. Di-Poï N, Montoya-Burgos JI, Duboule D. (2009) Atypical relaxation of structural constraints in Hox gene clusters of the green anole lizard. Genome Res. 19, 602-610. Woltering J.M. and Duboule D (2009) Conserved elements within open Reading frames of mammalian Hox genes. J. Biol., 8, 17 Minoux M, Antonarakis GS, Kmita M, Duboule D, Rijli FM. (2009) Rostral and caudal pharyngeal arches share a common neural crest ground pattern. Development, 136, 637-645. Montavon T, Le Garrec JF, Kerszberg M, Duboule D. Modeling Hox gene regulation in digits: reverse collinearity and the molecular origin of thumbness. Genes Dev. 2008 Feb 1;22(3):346-59.

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EPFL School of Life Sciences - 2009 Annual Report

Morey C, Da Silva NR, Kmita M, Duboule D, Bickmore WA. Ectopic nuclear reorganisation driven by a Hoxb1 transgene transposed into Hoxd. J Cell Sci. 2008 Mar 1;121(Pt 5):571-7.

Soshnikova N, Duboule D. Epigenetic regulation of Hox gene activation: the waltz of methyls. BioEssays. 2008 Mar;30(3):199-202.

ISREC

Spitz F, Duboule D. Global control regions and regulatory

landscapes in vertebrate development and evolution. Adv Genet. 2008;61:175-205.

Forebrains of E13.5 embryos are dissected, fixed and incubated with antibodies against histone H3K27 trimethyl. The immunoprecipitated material is then hybridized on a 2Mb customized tiling array and normalized to total input. Enrichments are plotted on a log2 scale according to their genomic position. The upper profile shows the normal situation, whereas the two profiles below shows two mutant strains, with an internal deletion. In these latter cases, the recruitment of PRC2 does not seem to be severely affected (figure: Patrick Schorderet)

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EPFL School of Life Sciences - 2009 Annual Report

ISREC - Swiss Institute for Experimental Cancer Research

Gönczy Lab Head of Lab (PI) - http://gonczy-lab.epfl.ch/

Team Members

Pierre Gönczy Full Professor

Katayoun Afshar, Postdoctoral fellow Alexandra Bezler, PhD student Simon Blanchoud, PhD student Yemima Budirahardja Suryani, Postdoctoral fellow Coralie Busso, Technician Nicole de Montmollin, Administrative assistant Isabelle Fluckiger, Technician Virginie Hachet, Postdoctoral fellow Debora Keller, PhD student Daiju Kitagawa, Postdoctoral fellow Gregor Kohlmaier, PhD student Sachin Kotak, Postdoctoral fellow Tamara Mikeladze-Dvali Petr Strnad, Postdoctoral fellow Kalyani Thyagarajan, PhD student

Introduction

We aim to better understand fundamental cell division processes, with a particular focus on those that are critical for genome integrity. We utilize a unique combination of genetic, functional genomic, cell biological and live imaging approaches in the embryo of the nematode Caenorhabiditis elegans as well as in human cells in culture.

Keywords

Cell biology, developmental biology, centrosome duplication, asymmetric cell division

Results Obtained in 2009

Our research focuses principally on two cell division processes that are critical for genome integrity: centrosome duplication and asymmetric cell division. Centrosome duplication. The centrosome is the principal microtubule-organizing center of animal cells and comprises two microtubule-based centrioles surrounded by pericentriolar material. Just like the genetic material, the centrosome must duplicate once per cell cycle to ensure genome integrity. Formation of a single procentriole next to each centriole is key for initiating the entire centrosome duplication cycle. Together with other laboratories, we identified five proteins required for procentriole formation in the nematode C. elegans: the kinase ZYG-1, as well as the coiled-coil proteins SAS-4, SAS-5, SAS-6 and SPD-2. We established recently that ZYG-1 exerts its function in part by phosphorylating SAS-6 at Serine 123, and that this phosphorylation event is critical for the maintenance of SAS-6 at the emerging procentriole. The proteins identified as being essential for procentriole formation in C. elegans turn out to have relatives in other metazoans. For instance, we discovered that a protein related to SAS-6 is present in every species with centrioles, and also that HsSAS-6 is essential for procentriole formation in human cells. Recently,

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we reported that the SAS-4-related protein CPAP is similarly required for centrosome duplication in human cells. Interestingly in addition, we found that overexpression of CPAP results in the formation of overly long centrioles, together indicating that CPAP levels are critical for proper procentriole formation and elongation. Overall, such observations indicate that lessons learned in C. elegans are applicable to other species and thus will have a significant impact on our understanding of genome integrity in human cells. Asymmetric cell division. Asymmetric division is crucial for generating cell diversity, both during development and in stem cell lineages. Accurate spindle positioning is critical for proper asymmetric cell division. Together with the work of other laboratories, our findings support a model for spindle positioning in C. elegans whereby two Gα proteins recruit the GoLoco protein GPR-1/2 and the coiled-coil protein LIN-5 to the cell cortex to generate force on the plus-end of astral microtubules. The LIN-5/GPR-1/2/Gα complex serves in turn to recruit the minus-end directed microtubule motor dynein to the cell cortex. Together with microtubule depolymerization, dynein activity then allows pulling forces to be exerted along astral microtubules, which ensures proper spindle positioning. More recently, we found that the protein phosphatase PPH-6 is an important modulator of pulling forces during spindle positioning in C. elegans embryos, by virtue of ensuring the presence of GPR-1/2 and LIN-5 at the cell cortex. Moreover, our results to date suggest that PPH-6 acts by modulating the subcellular distribution of the evolutionarily conserved protein kinase Aurora-A (called AIR-1 in C. elegans). Given that Gα proteins and their associated partners, including Aurora-A, have been implicated in asymmetric cell division across metazoan evolution, we expect the mechanisms characterized in C. elegans to be of broad significance.

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EPFL School of Life Sciences - 2009 Annual Report

Kitagawa D., Busso C., Flückiger I. and Gönczy P (2009). Phosphorylation of SAS-6 by ZYG-1 is critical for centriole formation in C. elegans embryos. Dev Cell. 17:900-907.

Strnad P. and Gönczy P. Mechanisms of procentriole formation. Trends Cell Biol. 18: 389-396 (2008).

Kohlmaier G., Lončarek J., Meng X., McEwen B.F., Mogensen M., Spektor A., Dynlacht B.D., Khodjakov A. and Gönczy P. (2009). Overly long centrioles and defective cell division upon excess of the SAS-4-related protein CPAP. Curr. Biol. 19: 1012-1018.

Johnston C.A., Afshar K., Snyder J.T., Tall G.G., Gönczy P, Siderovski D.P. and Willard F.S (2008). Structural determinants underlying the temperature-sensitive nature of a Ga mutant in C. elegans asymmetric cell division. J. Biol. Chem. 283: 21550-21558.

Budirahardja Y. and Gönczy P. Coupling the cell cycle to development. Development. 136:2861-2872 (2009).

Gönczy P. Mechanisms of asymmetric cell division: flies and worms pave the way. Nat Rev Mol Cell Biol. 9: 355366 (2008).

van der Voet M., Berends C.W., Perreault A., Nguyen-Ngoc T., Gönczy P., Vidal M., Boxem M. and van den Heuvel S (2009). NuMA-related LIN-5, ASPM-1, calmodulin and dynein promote meiotic spindle rotation independently of cortical LIN-5/GPR/Ga. Nat. Cell Biol. 11: 269- 277.

Budirahardja Y. and Gönczy P (2008). PLK-1 asymmetry contributes to asynchronous cell division of C. elegans embryos. Development 135: 1303-1313.

(Top panel) Schematic representation of nuclear and cytoplasmic regions detected in human HeLa cells by a custom-written algorithm expressing the centriolar marker GFP-Centrin1 (see bottom panel); cells are pseudo-colored for ease of identification. The rectangle is ~200 μm-long. (Bottom panel) Automatic detection of centrioles (two brightest regions expressing GFP-Centrin1) by a custom-written algorithm within the cell denoted by the rectangle in the top panel.

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Selected publications:


EPFL School of Life Sciences - 2009 Annual Report

ISREC - Swiss Institute for Experimental Cancer Research

Grapin-Botton Lab Head of Lab (UPGRA) http://grapinbotton-lab.epfl.ch/

Team Members

Cédric Cortijo, PhD student Filippo De Franceschi, Postdoctoral fellow Emilie Gésina, Postdoctoral fellow Joan Goulley, Postdoctoral fellow Mathieu Gouzi, PhD student Chiara Greggio, PhD student Yung Hae Kim, Postdoctoral fellow Yvan Pfister, Technician Marine Rentler Courdier, PhD student Anne-Marie Rodel, Secretary Nancy Thompson, Research associate

Anne Grapin-Botton Tenure Track Assistant Professor

Introduction

Our research addresses how the pancreas normally develops during embryogenesis, how this can be used to generate replacement cells for diabetes therapy and how these mechanisms are reactivated during pancreas cancer progression.

Keywords

Development, embryo, gut, pancreas, diabetes, endoderm, Wnt, patterning, beta-cell, chick, mouse, architecture

Results Obtained in 2009

Our work was focused on several stages of pancreas development in vertebrates. Organizing endoderm organs along the body axis We had previously shown that the pancreas is induced by signals from the mesoderm in contact with endoderm and that graded levels of Fgf4 are necessary to form posterior foregut, mid- and hindgut and accurately maintain the boundaries between different digestive tract organs. We recently showed that increasing levels of retinoic acid along the anteroposterior axis are also necessary to form these regions (Bayha et al., 2009). We are pursuing similar experiments with the Wnt pathway. Regulation of pancreas organogenesis: role of the bHLH transcription factor Ptf1a in pancreas progenitor maintenance Signaling activity from the mesoderm induces pancreatic genes at a given position along the anteroposterior axis. The transcription factors Pdx1 and Ptf1a maintain pancreas progenitors. At a later stage Ptf1a becomes restricted to exocrine cells. We compared the transcriptome of early pancreas progenitors lacking Ptf1a to that of wild type progenitors. Our experiments identified the molecular mechanisms by which Ptf1a maintains pancreas progeni-

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tors. Ptf1a blocks the expression of intestinal genes while activating a network of transcription factors of known importance in pancreas progenitors. In addition, we identified new targets of Ptf1a that we are studying functionally. Regulation of pancreas organogenesis: role of the bHLH transcription factor Neurogenin 3 (Ngn3) in endocrine cell differentiation and migration In addition to exocrine cells, the pancreas gives rise to multiple endocrine cells whose main function is the regulation of glucose homeostasis. The transcription factor Ngn3 is absolutely necessary to generate endocrine cell and to promote their migration from the epithelium. All pancreatic endocrine cells, producing glucagon, insulin, somatostatin or PP, differentiate from Pdx1+ progenitors that transiently express Neurogenin3. We recently identified targets of Ngn3 which mediate its ability to trigger migration and differentiation. In particular we found that Ngn3 induces planar cell polarity genes and are currently using mouse mutants for this pathway to study its function during development. Significance of this work for diabetes and pancreatic cancer Although the function of the pancreas in controlling glucose homeostasis is compensated by insulin injection in diabetic patients, the physiological effects are inexact and too variable. Among approaches that are currently being explored to find a cure for diabetes are the isolation and propagation of embryonic or adult stem cells that can be engineered to produce endocrine hormones and then transplanted to patients. Our experiments are aimed at identifying the critical cellular transcription factors and signaling molecules that are sufficient to transform cells into pancreas and β cells. Collaborations within the context of the European 6th framework project BetaCellTherapy allow us to test these ideas by introducing transcription factors in stem cells or exposing

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EPFL School of Life Sciences - 2009 Annual Report

these cells to signalling molecules in vivo or in vitro to force their differentiation into β cells. To assist diabetes therapy, we developed optical coherence microscopy in collaboration with the Lasser laboratory (EPFL) to image islets of Langerhans live in mice (Villiger et al., 2009).

Bayha, E., Jørgensen, M., Serup, P. and Grapin-Botton , A. (2009) Retinoic acid signaling organizes endodermal organ specification along the entire antero-posterior axis. PLoS one, 4:e5845. Villiger, M., Goulley, J., Friedrich, M., Grapin-Botton, A., Meda, P., Pralong, W., Lasser, T. and Leitgeb, R.A. (2009) In vivo imaging of endocrine islets with extended focus Optical Coherence Microscopy. Diabetologia, 52(8):1599-607 Grapin-Botton, A. (2008) Endoderm specification- In StemBook. http://www.stembook.org/

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In addition, pre-cancerous and cancerous cells often reactivate the expression of developmental genes. In pancreatic carcinoma many developmental genes are reactivated. Ongoing work on developmental genes may give a better understanding of pancreas cancer development and may point to new therapeutic targets.

Selected publications:

Optical imaging allows for visualization of the pancreas 3D architecture. The scheme on the left shows the pancreas with ducts, exocrine acini and islets of Langerhans. The right panel shows these structures in a developing embryo, as visualized on a confocal slice extracted from a 3D stack. The progenitors lining the ducts are shown in blue (anti Pdx1 antibody). From these progenitors, islet cell progenitors shown in dark green (anti Ngn3 antibody) are generated and start to exit the epithelium to subsequently aggregate into islets of Langerhans. The epithelial structure with membranes in red is shown (anti E-cadherin) as well as the forming duct lumen in light green (apical side anti aPKC). Optical coherence microscopy allows for the tomographic visualization of islets of Langerhans as shown in the panels in the middle. Without labeling, islets can be monitored in normal live mice (top). Reduced numbers and contrast can be detected in disease models where islets are destroyed (bottom).

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EPFL School of Life Sciences - 2009 Annual Report

ISREC - Swiss Institute for Experimental Cancer Research

Hanahan Lab Head of Lab (PI)

Team Members

Jennifer Brady, Executive Assistant Elena Gasparotto, Technician Anguraj Sadanandam, Postdoctoral Fellow Ksenya Shchors, Staff Scientist Leane Yuan, PhD student

Douglas Hanahan

Merck-Serono Professor of Molecular Oncology Director of ISREC

Introduction

The Hanahan laboratory uses engineered mouse models of human cancer types to investigate the mechanisms by which tumors develop and progress to lethal states of malignancy. Then, leveraging knowledge of such mechanisms of disease, the lab performs pre-clinical trials with drugs aimed to disrupt key capabilities, seeking to develop therapeutic strategies that could be translated into clinical trials, with the prospect for expediting innovative treatments that will greatly benefit human cancer patients.

Keywords

Angiogenesis, invasion, and metastasis; tumorpromoting inflammation; pancreas, breast, cervical, and brain cancer

Results Obtained in 2009

The Hanahan laboratory began its transition from the University of California, San Francisco, to the EPFL in mid 2009, and the principal effort at EPFL has involved hiring staff, ordering equipment, and setting up the laboratory. The groundwork was laid for new research directions at the EPFL in breast and pancreatic cancer, and glioblastoma. The lab will continue to focus on the functional contributions of the tumor micro-enviromnment, composed of overtly transformed cancer cells and a constellation of recruited and subverted normal cells, including inflammatory cells, endothelial cells and pericytes of the angiogenic tumor vasculature, and cancer-associated fibroblasts. In parallel, as Director of ISREC, the Swiss Institute of Experimental Cancer Research, a division of the

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School of Life Sciences, Douglas Hanahan has been engaged in leadership initiatives relating to the strategic goals to enhance ISREC’s pre-eminence in basic and translational cancer research via new faculty recruitments, and to foster the formation of an innovative, multi-institutional cancer center that integrates the diverse talent and expertise in biomedical science & engineering and clinical research in Lausanne and south-western Switzerland.

Selected publications:

Olson P, Lu J, Zhang H, Shai A, Chun MG, Wang Y, Libutti SK, Nakakura EK, Golub TR, Hanahan D. MicroRNA dynamics in the stages of tumorigenesis correlate with hallmark capabilities of cancer. Genes Dev. 200,; 2:2152-65. Olson P, Hanahan D. Cancer. Breaching the cancer fortress. Science. 2009, 324:1400-1. Pàez-Ribes M, Allen E, Hudock J, Takeda T, Okuyama H, Viñals F, Inoue M, Bergers G, Hanahan D, Casanovas O. Antiangiogenic therapy elicits malignant progression of tumors to increased local invasion and distant metastasis. Cancer Cell. 2009, 15:220-31. Hager JH, Ulanet DB, Hennighausen L, Hanahan D. Genetic ablation of Bcl-x attenuates invasiveness without affecting apoptosis or tumor growth in a mouse model of pancreatic neuroendocrine cancer. PLoS One. 2009, 4:e4455. Nolan-Stevaux O, Lau J, Truitt ML, Chu GC, Hebrok M, Fernández-Zapico ME, Hanahan D. GLI1 is regulated through Smoothened-independent mechanisms in neoplastic pancreatic ducts and mediates PDAC cell survival and transformation. Genes Dev. 2009, 23:24-36. Bergers G, Hanahan D. Modes of resistance to anti-angiogenic therapy. Nat Rev Cancer. 2008, 8:592-603.

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EPFL School of Life Sciences - 2009 Annual Report

Pietras K, Pahler J, Bergers G, Hanahan D. Functions of paracrine PDGF signaling in the proangiogenic tumor stroma revealed by pharmacological targeting. PLoS Med. 2008, 5:e19.

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Pahler JC, Tazzyman S, Erez N, Chen YY, Murdoch C, Nozawa H, Lewis CE, Hanahan D. Plasticity in tumor-promoting inflammation: impairment of macrophage recruitment evokes a compensatory neutrophil response. Neoplasia. 2008, 10:329-40.

Evasive resistance to an anti-angiognic drug via heightened invasiveness of cancer cells. Preclinical trials in a mouse model of pancreatic cancer with a potent inhibitor of angiogenesis have revealed an unexpected form of drug resistance, whereby tumors adapt to their inability to grow tumor blood vessels (the process of angiogenesis) in the face of a potent inhibitory drug. After a period of tumor shrinkage due to the collapse of tumor vasculature, cancer cells switch to a highly invasive mode, percolating throughout the pancreas, evidently co-opting normal tissue vasculature to survive and grow. Enduring therapies will therefore likely require concordant inhibition of angiogenesis and invasion, a topic of current investigation by the Hanahan laboratory. In this photomicrograph of a cancerous pancreas tissue section, the cancer cells are visualized in red, and the blood vessels in green. For details see: Pàez-Ribes, M., Allen. E., Hudock J.,Takeda, T., Okuyama, H., Viñals, F., Inoue. M., Bergers, G., Hanahan, D. & Casanovas, O. (2009). Anti-angiogenic therapy elicits malignant progression of tumors to increased local invasion and distant metastasis. Cancer Cell 15: 220–231

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EPFL School of Life Sciences - 2009 Annual Report

ISREC - Swiss Institute for Experimental Cancer Research

Huelsken Lab Head of Lab (PI) http://huelsken-lab.epfl.ch/

Team Members

Ilaria Malanchi, Postdoctoral fellow Anja Irmisch, Postdoctoral fellow (from September 2009) Paloma Ordóñez Morán, Postdoctoral fellow (from December 2009) Ablert Santamaria Martínez, Postdoctoral fellow (from September 2009) Fabien Kuttler, Postdoctoral fellow (until October 2009) Evelyn Susanto, PhD student Wang-Hun Fan, PhD student Caroline Urech, PhD student (from August 2009) Jean-Paul Abbuehl, PhD student (from August 2009) Marzia Armaro, PhD student (until August 2009) Ursula Winter, Administrative Assistant

Joerg Huelsken

Tenure Track Assistant Professor Debiopharm Chair in Signal Transduction in Oncogenesis

Introduction

The last years of cancer research have established the concept of cancer stem cells (CSC) as sub-population of cells within a tumor entirely responsible for long-term tumor growth. This has aroused expectations that targeting specifically these cancer stem cells would allow effective tumor eradication. We now provide evidence that these cells are also essential for metastatic disease and identify the interaction between stem cells and their environment as a promising target for intervention to block the spreading of cancer to secondary sites.

Keywords

Stem cells, Cancer stem cells, Stem cell niches, Wnt signaling, Metastatic colonization

Results Obtained in 2009

Key for a potential targeting of cancer stem cells for therapy is the identification of signals which are essential for these transformed stem cells, however, not for their normal tissue-specific counterpart which are required for organ homeostasis and repair. We have shown recently that in the skin, the prospect of targeting Wnt signaling to affect tumor formation appears favorable and without major side effects. We found cutaneous squamous cell carcinomas to contain CSCs which depend on β-catenin signaling in order to sustain their stemness. Deletion of β-catenin in established tumors was sufficient to provoke complete tumor regression which was initiated by a rapid loss of CSCs. Furthermore, we provided direct evidence that normal stem cells are the target for oncogenic mutations and can very efficiently produce tumors. We have now set out to understand the basis of this effect and to identify the downstream events which mediate β-catenin control of stem cell function. Based on the identification of cancer stem cells (CSC) in various tumors, we predicted that niche signals, i.e. signaling molecules and extracellular matrix components produced by surrounding stromal

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cells, may participate in the control of cancer stem cell function and may play a role in expanding these stem cells. We furthermore suggest that such niche derived signals may be of particular importance during the early phases of metastatic colonization, when cancer cells are suddenly exposed to a new environment where they may critically depend on a “known” set of signaling molecules which are present at the primary site. With these ideas in mind, we performed several screens for niche signals using a combination of expression profiling on micro-dissected material and in situ hybridization. In the following, we decided to concentrate on one of the identified genes which encodes for an extracellular matrix molecule (ECM) with sequence similarity to an insect cell adhesion molecule. We observed this niche ECM molecule to be expressed in the mammary gland in the stem cell niche as well as in the stroma of breast cancer. Interestingly, it is not expressed in the normal lung, however, is induced when tumor cells seed to the lung. Importantly, we were able to show that genetic ablation of this protein blocks expansion of cancer stem cells in vitro and in vivo. Consequently, mice lacking this gene are resistant to metastasis formation in the lungs. This clearly demonstrates that molecular signals that participate in tumor– host cross-talk could emerge as a viable approach to metastasis prevention and intervention if they target the stem cell niche communication. It is well described that the formation of metastasis is a very inefficient process which is successful only for a very few cells which leave the primary tumor whereas the majority of cells is either lost rapidly or switches into a dormant state which does not lead to metastatic growth. Moreover, many cancers have preferred target sites of metastatic spread which cannot be explained by simple passive seeding via the circulation. We think that we can now explain part of these restrictions by the apparent need for the incoming cancer cell to “educate” or re-program the secondary environment for its support. This can be anticipated to be more or less complete depending on the target tissue. It is in particular this early metastatic colonization phase that can be expected to be sensitive

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EPFL School of Life Sciences - 2009 Annual Report

Selected Publications:

Hussenet, T., J. Exinger, S. Dali, B. Jost, D. Dembelé, C. Thibault, J. Huelsken, E. Brambilla and S. du Manoir (2010). SOX-2 is a new oncogene activated by recurrent 3q26.3 amplifications in lung Squamous Cell Carcinomas. PLOS One, in press. Buchert, M., D. Athineos, H.E. Abud, Z.D. Burke, M.C. Faux, M.S. Samuel, A.G. Jarnicki, C.E. Winbanks, I.P. Newton, V.S. Meniel, H.Suzuki, S.A. Stacker, I.S. Näthke, D. Tosh, J. Huelsken, A.R. Clarke, J.K. Heath, O.J. Sansom and M. Ernst (2009). Genetic dissection of differential signaling threshold requirements for the Wnt/β-catenin pathway in vivo. PLoS Genetics, in press

foe? Cell Res. 19:279-81. Malanchi, I., & J. Huelsken (2009). Cancer stem cells: never Wnt away from the niche. Curr. Opin. Oncol., 21, 41-6. Xu, Y.H., R. Reboulet, B. Quinn, J. Huelsken, D. Witte, & G.A. Grabowski (2008). Dependence of reversibility and progression of mouse neuronopathic Gaucher disease on acid beta-glucosidase residual activity levels. Mol. Genet. Metab, 94, 190-203. Cain, S., G. Martinez, K. Turner, R.J. Richardson, M.I. Kokkinos, H.E. Abud, J. Huelsken, M.L. Robinson, & R.U. de Iongh (2008). Differential requirement for beta-catenin in epithelial and fiber cells during lens development. Dev. Biol., 321, 420-33. Malanchi, I., H. Peinado, D. Kassen, T. Hussenet, D. Metzger, P. Chambon, M. Huber, D. Hohl, A. Cano, W. Birchmeier and J. Huelsken (2008). Cutaneous cancer stem cell maintenance is dependent on β-catenin signaling. Nature, 452, 650-3.

Zhang, Y., P. Tomann, T. Andl, N.M. Gallant, J. Huelsken, B. Jerchow, W. Birchmeier, R. Paus, S. Piccolo, M.L. Mikkola, E.E. Morrisey, P.A. Overbeek, C. Scheidereit, S.E. Millar & R. Schmidt-Ullrich (2009). Reciprocal requirements for Eda/Edar/NF-κB and Wnt/β-catenin signaling pathways in hair follicle induction. Dev. Cell, 17, 49-61.

Making the paper: Joerg Huelsken. Genetic labelling reveals a role for hair follicle stem cells in skin cancer. Nature, 452, xiii

Huelsken, J. & W. Held (2009). Letter to the editor. Eur J Immunol. 39, 3582-83.

Braun, K.M. (2008). Cutaneous cancer stem cells: betacatenin strikes again. Cell Stem Cell. 2(5), 406-08.

Braeuning, A., R. Sanna, J. Huelsken, & M. Schwarz (2009). Inducibility of Drug-Metabolizing Enzymes by Xenobiotics in Mice with Liver-Specific Knockout of Ctnnb1. Drug Metab Dispos 37, 1138-45.

Jeannet, G., M. Scheller, L. Scarpellino, S. Duboux, N. Gardiol, J.Back, F. Kuttler, I. Malanchi, W. Birchmeier, A. Leutz, J. Huelsken* & W. Held* (2008). Long-term, multilineage hematopoiesis occurs in the combined absence of β-catenin and γ-catenin. Blood, 111, 142-9. *these two authors contributed equally

Huelsken, J. (2009). Tissue-specific stem cells: friend or

This article has been highlighted in:

The establishment of a metastasis in a distant organ is a very inefficient process. Most often, incoming tumor cells can either not survive at all or enter a state of dormancy without growth or expansion (right). Only few tumor cells manage to induce an appropriate stromal reaction (left) which provides essential factors the tumor needs for its growth. These requirements depend on the origin of the tumor cell as well as the stage of the metastatic colonization process and are therefore variable over time. This fine-tuned interplay between tumor cells and surrounding tissue determines the outcome of tumor progression.

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to therapeutic intervention as the dependence of the cancer cell for niche signals is highest. We suggest that targeting this stroma-produced stem cell niche holds the promise to be less prone to rapid genetic changes in cancer cells and therefore could turn out to show better resistance against therapy escape.


EPFL School of Life Sciences - 2009 Annual Report

ISREC - Swiss Institute for Experimental Cancer Research

Kühn Lab Head of Lab (PI) http://kuhn-lab.epfl.ch

Team Members

Liviu Vanoaica, PhD student Ramona Batschulat, PhD student (from December 2008) Barbara Grisoni-Neupert, Scientific collaborator Larry Richman, Specialist technician Geneviève Massy, Administrative assistant

Lukas Kühn

Professor Titulaire

Introduction

We study mice with a conditional gene deletion of ferritin H affecting iron storage. In the liver, heart and B lymphocytes, the deletion provokes oxidative tissue damage, while in the intestine we have evidence for a role of ferritin in the control of iron absorption. The mice are important to study oxidative stress in vivo and contribute to the understanding of hereditary diseases of iron overload and anemia.

Keywords

Conditional knock-out mice for ferritin H, oxidative cell damage, iron physiology, mRNA degradation, RNA-protein interactions

Results Obtained in 2009

Analysis of ferritin H knock-out mice Iron is an essential metal for life and at the same time a hazard since, in its free form, it catalyzes the formation of hydroxyl radicals, which are a cause of cell damage and mutations in DNA. Therefore, body iron absorption from nutrients and free iron in cells are delicately controlled in order to avoid the excess or deprivation of iron. In the human population, an excess of iron is observed in the hereditary disease of hemochromatosis, which is accompanied by tissue damage in liver, heart and pancreas. It can be the cause of liver cancer. Anemia due to insufficient body iron is on the other hand a frequent problem in developing countries and can affect the immune system. We study the role of ferritin in the regulation of iron physiology. Ferritin, a complex composed of ferritin H and L chains, stores excess free iron and thereby protects cells against radical formation. We have generated mouse strains with a conditional deletion of the ferritin H gene in adult animals using the Cre lox strategy. Over the last year we have completed a study with mice having an intestine-specific deletion of ferritin H. These mice accumulate too much iron in the liver and spleen, similar to the condition of hemochroma-

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tosis. Accordingly, the mice show a high liver hepcidin 1 mRNA expression, a reduced expression of the iron export protein ferroportin, and reduced mRNA levels for iron transport proteins DMT1 and Dcytb. In spite of these feedback regulatory mechanisms, we newly found, in collaboration with K. Schümann, Technische Universität München, that 59Fe-absorption is increased. We conclude that hepcidin 1 is not sufficient to regulate iron absorption in absence of ferritin H. We propose that ferritin H is needed to capture excess iron on its transit through intestinal absorptive cells between the apical to basolateral membrane. Deletion of ferritin H in the heart provoked fibrosis indicating damage of heart muscle cells. We attribute this to increased oxidative stress, as suggested by the induction of the marker gene heme oxygenase 1 and other stress-induced genes. In collaboration with T. Pedrazzini, CHUV Lausanne, we assessed parameters of heart physiology by echocardiography. Five days after ferritin H deletion mice were strongly affected in heart muscle contraction with an ejection fraction of 23% instead of 55% in control animals (Figure). The mice recovered rapidly and showed no further signs of illness up to 10 months. However, when mice received a single subcutaneous injection of iron dextran prior to the ferritin H deletion, the signs of tissue damage and oxidative stress were strongly increased and mice did not fully recover. A deletion of ferritin H by CD19-Cre provoked a diminished number of mature B lymphocytes. The evidence gathered show that a subpopulation of cells has an increased intracellular free iron pool which is directly causative of an increase in reactive oxygen species. Upon differentiation and maturation a high proportion of these cells end up missing. Mechanisms of rapid mRNA degradation In parallel, we study rapid mRNA degradation as it occurs in a large number of mRNAs that harbor instability elements, such as AU-rich sequences, in their

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EPFL School of Life Sciences - 2009 Annual Report

Selected Publications:

Darshan D., Vanoaica, L., Richman L., Beermann, F., and Kühn L.C. (2009). Conditional deletion of ferritin H in mice induces loss of iron storage and liver damage. Hepatology, 50, 852-860. Kühn L.C. (2009) How iron controls iron. Cell Metabolism, 10, 439-441.

Vanoaica, L. (2009) Analysis of mice with conditional ferritin H deletion. PhD thesis EPFL. Surdej P., Richman L. and Kühn L.C. (2008) Differential translational regulation of IRE-containing mRNAs in Drosophila melanogaster by endogenous IRP and a constitutive human IRP1 mutant. Insect Biochem. Mol. Biol. 38, 891894. Paschoud, S., Dogar, A.M., Kuntz, C., Grisoni-Neupert, B., Richman, L. and Kühn, L.C. (2006). Destabilization of interleukin-6 mRNA requires a putative RNA stem-loop structure, an AU-rich element and the RNA-binding protein AUF1. Mol. Cell. Biol. 26: 8228-8241.

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3’ untranslated regions. We have devised methods to enrich RNA-protein complexes from cells or tissues by immunoprecipitation. These are combined with microarray analysis, high-throughput sequencing and identification of target genes under various physiological conditions.

M1-mode echocardiography of mice before and after ferritin H gene deletion. The inner diameter of the left ventricle is measured at the diastolic and systolic phase of the heartbeat. In A, before ferritin H deletion, it was 3.91 mm and 2.92 mm, respectively. In B, 5 days after ferritin H deletion, the two values were 4.22 mm and 3.94 mm showing a much weaker contraction of the heart. Measurements like these carried out on many deleted and control mice showed that these differences were significant and that the fractional shortening and ejection fraction were severely affected in mutant mice. However, in absence of additional stress by increased iron, the cardiac performance returned to normal after 10 days.

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EPFL School of Life Sciences - 2009 Annual Report

ISREC - Swiss Institute for Experimental Cancer Research

Lingner Lab Head of Lab (UPLIN) http://lingner-lab.epfl.ch/ Team Members

Joachim Lingner Full Professor

Larissa Angehrn, PhD student Elena Aritonovska, PhD student Chen Liuh-Yow, Postdoctoral fellow (from June 2009) Alix Christen, PhD student (from October 2009) Jérôme Crittin, PhD student (from April 2009) Sascha Feuerhahn, Postdoctoral fellow Nahid Iglesias, Postdoctoral fellow Brian Luke, Postdoctoral fellow (to September 2009) Vered Machluf, PhD student Nicole de Montmollin, Secretary-Administrator Andrea Panza, PhD student Antonio Porro, Postdoctoral fellow Sophie Redon, Scientific collaborator Patrick Reichenbach, Senior technician Katarzyna Sikora, PhD student Susan Smith, Visiting professor (from November 2009)

Introduction

Telomeres are the physical ends of eukaryotic chromosomes. They function as guardians of genome stability, cellular clocks and tumor suppressors. Our laboratory studies telomere structure and maintenance in human cells and in the yeast Saccharomyces cerevisiae in order to gain a detailed understanding of the function of these fascinating structures. This may allow manipulation of telomere function in tumors in the future.

Keywords

Telomeres, telomerase, TERRA, genome stability

Results Obtained in 2009

Telomerase is regulated at individual chromosome ends to mediate telomere length homeostasis. In humans, telomere length is set in most tissues early in embryogenesis as telomerase is repressed later in life. Short telomeres that accumulate with increasing numbers of cell division cycles induce cellular senescence and this is thought to counteract growth of pre-malignant lesions in our body. Most cancers reexpress telomerase to overcome this growth barrier. Our work during 2009 concentrated on the question of how the telomerase enzyme is regulated at chromosome ends. In addition, we extended our studies on telomeric repeat containing RNA (TERRA), a large non-coding (nc) RNA at telomeres whose functions may include heterochromatin assembly and the regulation of telomerase Regulation of telomerase at chromosome ends Telomerase is a cellular reverse that counteracts telomere shortening that occurs due to incomplete DNA end replication and nucleolytic processing. Telomerase extends chromosome 3’ ends by iterative reverse transcription of a small region of its tightly associated telomerase RNA moiety. Telomerase activity is regulated at individual chromosome ends by telomere binding factors. Using single telomere extension analysis, we demonstrated in the past in

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yeast that telomerase exhibits an increasing preference for telomeres as their lengths decline. We also identified that the DNA checkpoint kinase Tel1 (ortholog of human ATM) and Tbf1p, a protein that binds subtelomeric sequence, activate telomerase at short telomeres. Recruitment and activation of telomerase at chromosome ends is not well understood in complex eukaryotes including humans. Therefore, we developed assays to measure association of human telomerase with chromosome ends by chromatin immunoprecipitation, and our collaborators from the Terns-lab (University of Georgia) could for the first time detect human telomerase at chromosome ends by fluorescence in situ hybridization. Through our analyses, we identified that Cajal bodies, subnuclear structures implicated in ribonucleoprotein maturation are critical for telomerase activation at telomeres. Furthermore, through downregulation of telomere binding proteins by RNA interference, we identified proteins that mediate the recruitment of human telomerase to chromosome ends. Telomeric repeat containing RNA (TERRA) Telomeres consist of simple repetitive DNA repeats and a large set of specialized proteins that are crucial for telomere function. We recently discovered in eutherian mammals and subsequently in the yeast S. cerevisiae that telomeres are transcribed into large nc RNAs termed TERRA. TERRA forms an integral part of telomeric heterochromatin (Figure 1). The identification of TERRA is paradigm-shifting because telomeric heterochromatin had been accepted as being a transcription silencer. The role of nc RNAs previously thought to be limited to special cases such as Xist RNA in X-chromosome inactivation is now extended to telomeres. Current challenges are to decipher the biogenesis (Figure 1) and biological functions of TERRA. So-far, we could identify several factors that control TERRA turnover and telomere association (see Figure 1). This includes RNA surveillance factors, which in human cells not only regulate

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EPFL School of Life Sciences - 2009 Annual Report

Selected Publications

Chang, M., Luke, B., Kraft, C., Li, Z., Peter, M., Lingner, J., and Rothstein, R. (2009). Telomerase is essential to alleviate pif1-induced replication stress at telomeres. Genetics 183:779-791. Luke, B. and Lingner, J. (2009). TERRA: telomeric repeatcontaining RNA, EMBO J. 2:2503-2510. Iglesias, N. and Lingner, J. (2009). Related mechanisms for end processing at telomeres and DNA double-strand breaks. Mol. Cell 35:137-138.

Lingner, J. (2009). Telomere und Telomerase - was bedeuten sie für die Medizin? Radio DRS 2: Kontext, Wednesday, 07.10.2009, from 09:06-09:35. To listen: http://pod.drs. ch/mp3/kontext/kontext_200910071000_10101924.mp3 Luke, B., Panza, A., Redon, S., Iglesias, N., Li, Z. and Lingner, J. (2008). The Rat1p 5’ to 3’ exonuclease degrades telomeric repeat-containing RNA and promotes telomere elongation in Saccharomyces cerevisiae. Mol. Cell 32:465477. Chang, M. and Lingner, J. (2008) Cell signaling: Tel2 finally tells one story. Science 320:60-61. Azzalin, C. M., and Lingner, J. (2008). Telomeres: the silence is broken. Cell Cycle 7:1161-1165.

TERRA Biogenesis, telomere association and displacement from telomeres. TERRA is a RNAPII transcript whose transcription initiates within the sub-telomeric sequences and proceeds into the telomeric tract. A fraction of TERRA is polyadenylated via the canonical poly (A) polymerase, Pap1. The 5’ to 3’ exonuclease, Rat1, and the noncanonical poly (A) polymerase, Trf4, mediate TERRA turnover. The NMD factors UPF1, SMG1 and EST1A/SMG6 all counteract TERRA abundance at telomeres. EST1A also physically interacts with telomerase.

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TERRA abundance at telomeres but they also physically interact with telomerase. In addition, we could provide genetic evidence in yeast that TERRA regulates telomere length and telomerase.


EPFL School of Life Sciences - 2009 Annual Report

ISREC - Swiss Institute for Experimental Cancer Research

Radtke Lab Head of Lab (PI) http://radtke-lab.epfl.ch/

Team Members

Freddy Radtke

Associate Professor

April Bezdek, PhD student Christelle Dubey, Technician Nicolas Fasnacht, Postdoctoral Fellow Fabian, Junker PhD student (from December 2009) Ute Koch, PhD Scientist Michela Marani Postdoctoral Fellow Craig Nowell, Postdoctoral Fellow (from April 2009) Catherine Pache, Administrative Assistant Matteo di Piazza, Postdoctoral Fellow Caroline Poisson, PhD student Rajwinder Lehal, PhD student Luca Pellegrinet, PhD student Olivier Randin, Technician Emma Smith, Postdoctoral fellow Agnieszka Wendorff, PhD student Silvia Wirth, PhD student (from September 2009)

Introduction

Our group is interested in the molecular mechanisms controlling stem cell maintenance, lineage commitment and differentiation in self-renewing systems such as the hematopoietic system, the skin and the gut. The basic principle of self-renewing tissues is that they constantly produce cells from a stem cell reservoir that gives rise to proliferating transient amplifying cells, which subsequently differentiate and migrate to the correct compartment. These processes have to be under stringent control to ensure life-long homeostasis. In recent years a substantial body of evidence has accumulated to support the notion that signaling pathways known to be important during embryonic development (such as e.g. Shh, Wnt and Notch) play important roles in regulating self-renewing tissues. Moreover, the same pathways are often deregulated during tumorigenesis due to mutations of key elements of these pathways. The general concept is that a better understanding of the mechanisms controlling stem maintenance versus differentiation may lead to the identification of novel therapeutic targets, as well as improving strategies for influencing these players during tumorigenesis. Currently, attention is being focused on the evolutionarily conserved Notch signaling pathway, which plays pleiotropic roles in different self-renewing tissues and cancer.

Keywords

Notch, stem and progenitor cells, self-renewing tissues, differentiation, cancer, genetic mouse models

Results Obtained in 2009

Notch: lineage specifier, oncogene, tumor suppressor and progenitor gate-keeper Notch signaling in T cell development and leukemia. Using conditional gene targeting strategies we have established an essential role for Notch1 in specifying the T cell lineage. More recently, we explored the ability of the Notch ligand Delta4 (DL4) to induce T cell lineage commitment and/or maturation

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in vivo from BM progenitors by generating mice in which DL4 could be specifically inactivated in thymic epithelial cells (TECs). Loss of DL4 in TECs led to a complete block in T cell development coupled with the ectopic appearance of immature B cells in the thymus. These immature B cells were phenotypically indistinguishable from those developing in the thymus of conditional Notch1 mutant mice. Collectively, our results demonstrate that DL4 is the essential and non-redundant Notch1 ligand responsible for T cell lineage commitment within the thymus. Another aspect of our work focuses on the role of Notch in T cell leukemia. Aberrant Notch1 signaling within the hematopoietic system results in the development of acute lymphoblastic T cell leukemia in mice and humans. Thus Notch1 functions as a oncogene in the hematopoietic system. We are currently investigating the molecular mechanisms by which uncontrolled Notch1 signaling exerts its oncogenic functions. Notch signaling in skin and hematopoietic disorders In contrast to the previously established role of Notch1 as an oncogene and lineage specifier in the hematopoietic system, we unexpectedly identified novel roles for Notch signaling in the skin. Inducible ablation of Notch1 in the skin results in hair loss and epidermal hyperplasia within four weeks of deletion. By one year the same mice develop basal cell carcinoma-like tumors suggesting that Notch1 functions as tumor suppressor in the skin. Since the skin expresses multiple Notch receptors we completely abrogated Notch signaling in the skin to investigate whether these mice now develop skin tumors faster, or whether the tumor type changes. Skin specific loss of Notch signaling in the embryo results in skin barrier defects accompanied by a B-lymphoproliferative disease. Surprisingly, postnatal epidermis-specific inactivation of Notch signaling induces massive inflammation resulting in the development of a severe form of atopic dermatitis (AD, also known as eczema)

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EPFL School of Life Sciences - 2009 Annual Report

Our studies demonstrated a critical role for Notch in repressing TSLP production in keratinocytes thereby maintaining integrity of the skin and the hematopoietic system.

Selected Publications

Koch, U.; Fiorini, E.; Benedito, R.; Besseyrias, V.; SchusterGossler, K.; Pierres, M.; Manley, NR; Duarte, A.; MacDonald, HR; and Radtke F. (2008). Delta-like 4 is the essential, non-redundant ligand for Notch1 during thymic T cell lineage commitment. J.Ex.Med. Oct 27;205(11):2515-23. Epub 2008 Sep 29.

Siveke JT, Lubeseder-Martellato C, Lee M, Mazur PK, Nakhai H, Radtke F, and Schmid RM., Notch signaling is required for exocrine regeneration after acute pancreatitis. (2008) Gastroenterology. Feb;134(2):544-55. Epub 2007 Nov 4. Maillard, I., Koch, U., Dumortier, A., Shestova, O., Xu, L., Sai, H., Pross, S.E., Aster, J.C., Bhandoola, A., Radtke, F., and Pear, WS. (2008). Canonical Notch signaling is dispensable for the self-renewal of adult hematopoietic stem cells. Cell Stem cell, Apr 10;2(4):356-66. Riccio, O., van Gijn, M.E., Bezdek, A.C., Pellegrinet, L., van ES, J.H., Zimber-Strobl, U., Strobl, L., Honjo, T., Clevers, H. and Radtke, F. (2008). Loss of intestinal crypt progenitor cells owing to inactivation of both Notch1 and Notch2 is accompanied by derepression of CDK inhibitors p27Kip1 and p57Kip2. EMBOreports Apr;9(4):377-83, Epub 2008 Feb 15. Radtke F. and MacDonald HR., Is there molecular cross talk between Notch and the pre-TCR during thymocyte development?, Nature Immunology 2009 April Vol 10, No4, Online Focus, Brief Insights, Immune signaling Cross talk Gao J, Graves S, Koch U, Liu S, Jankovic V, Buonamici S, El Andaloussi A, Nimer SD, Kee BL, Taichman R, Radtke F, Aifantis I. (2009). Hedgehog signaling is dispensable for adult hematopoietic stem cell function. Cell Stem Cell. Jun 5;4(6):548-58. Roeder I and Radtke F., Stem cell biology meets systems biology. Development. 2009 Nov;136(21):3525-30.

Moriyama, M., Durham, AD., Moriyama, H., Hasegawa, K., Nishikawa, SI., Radtke, F. and Osawa, M.Multiple Roles of Notch signaling in the regulation of epidermal development (2008) Dev. Cell 14, 1-11.

Model for the role of Notch signaling in adult skin and how its loss results in skin and hematological disorders. Notch receptors are expressed in the suprabasal cell layer of the skin. Skin specific loss of Notch signaling leads to pronounced secretion of TSLP by epithelial cells. High TSLP serum levels in the embryo or neonates cause a cell non-autonomous B-lymphoproliferative disease (B-LPD). In contrast, the presence of TSLP in adult mice results in the recruitment of mast cells and eosinophiles within the dermis of Notch mutant mice, thereby contributing to massive inflammation and the development of an atopic dermatitis-like disease. At very high TSLP serum levels, a currently unknown cell type produces G-CSF, causing the cell non-autonomous development of a myeloproliferative disorder (MPD) and osteopenia.

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in mice. Likewise, patients suffering from AD, but not psoriasis or lichen planus (two other common skin disorders), have a marked reduction of Notch receptor expression in the skin. Loss of Notch in keratinocytes induces the production of thymic stromal lymphopoietin (TSLP), a cytokine deeply implicated in the pathogenesis of AD. The AD-like associated inflammation is accompanied by a myeloproliferative disorder (MPD) characterized by an increase in immature myeloid populations in the bone marrow and spleen. Transplantation studies revealed that the MPD is a cell non autonomous disease, caused by dramatic alterations within micro-environment. Genetic studies demonstrated that G CSF mediates the MPD as well as changes in the bone marrow microenvironment leading to osteopenia.


EPFL School of Life Sciences - 2009 Annual Report

ISREC - Swiss Institute for Experimental Cancer Research

Simanis Lab Head of Lab (PI) http://simanis-lab.epfl.ch/

Team Members

CANO Del ROSARIO Elena, senior laboratory assistant GOYAL Anupama, Doctoral assistant KRAPP Andrea, Scientist LATTMANN Evelyn, Doctoral assistant PACHE Catherine, Secretary

Viesturs Simanis Associate Professor

Introduction

All cells arise by division. Failure to coordinate cell cycle events properly and execute them with high fidelity can lead to death of the progeny of a division event, or alterations of the genome which can contribute to the genesis of tumours. We use a simple model system to study how cell division (cytokinesis) is coordinated with chromosome segregation (mitosis). As these are mechanistically conserved processes, our findings should be applicable to understanding how division is controlled in human cells.

Keywords

Yeast, cytokinesis, mitosis, cell division, protein kinase, meiosis, mitosis

Results Obtained in 2009

Overview In the yeast S. pombe, cytokinesis is coordinated with mitosis through cyclin-dependent kinase activity and a signal transduction network called the septation initiation network or SIN (see Lattmann et al., 2009 and Krapp and Simanis (2008) for reviews). The SIN is required for cytokinesis during the mitotic cycle, and for spore formation in meiosis. We have used a combination of classical and reverse genetics to identify and study regulators of mitosis and cytokinesis in the mitotic cycle and of cellularisation (spore formation) in the meiotic cycle. We also collaborated with the Hauser lab in the Institute of Microbiology at the CHUV to study Tbf1p. Analysis of the role of dma1p in meiosis (Andrea Krapp, Elena Cano del Rosario); submitted for publication. Meiosis is a specialised form of the cell cycle that gives rise to haploid gametes. In S. pombe, the products of meiosis are four spores. They are formed by encapsulation of the four meiosis II nuclei by membrane vesicles that are recruited to the spindle poles in meiosis II to form the forespore membrane, followed by deposition of spore wall material. We investigated the role of the conserved SIN regulator

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dma1p in meiosis; we found that though both meiotic divisions occur in the absence of dma1p, asci frequently contain fewer than four spores. Our data suggest that dma1p contributes to the temporal regulation of the abundance of the meiosis-specific SIN component mug27p. Analysis of S. pombe monopolin function (Collaboration with the McCollum lab, Boston, USA); see Choi et al., 2009 It is unknown how oscillations in Cdk1 activity drive the dramatic changes in chromosome and spindle dynamics that occur at the metaphase/anaphase transition. This study presented data that the Schizosaccharomyces pombe monopolin complex has distinct functions in metaphase and anaphase, which are determined by the phosphorylation state of its Mde4 subunit. When Cdk1 activity is high in metaphase, Mde4 is hyperphosphorylated on Cdk1 phosphorylation sites and localizes to kinetochores. A nonphosphorylatable mutant of Mde4 does not localize to kinetochores, appears prematurely on the metaphase spindle, and interferes with spindle dynamics and chromosome segregation. When Cdk1 activity drops in anaphase, dephosphorylation of Mde4 triggers monopolin localization to the mitotic spindle, where it promotes spindle elongation and integrity, coupling the late mitotic loss of Cdk1 activity to anaphase spindle dynamics. Taken together, the data obtained illustrate how the sequential phosphorylation and dephosphorylation of monopolin helps ensure the orderly execution of discrete steps in mitosis. Functional differentiation of tbf1 orthologues in fission and budding yeasts (Collaboration with the Hauser lab, Institute of Microbiology, CHUV, Lausanne) In Saccharomyces cerevisiae, TBF1, an essential gene, influences telomere function but also has other roles in the global regulation of transcription. We identified a new member of the tbf1 gene family in the mammalian pathogen Pneumocystis carinii. We demonstrate by transspecies complementation that

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EPFL School of Life Sciences - 2009 Annual Report

Selected Publications

Cockell M.M., Lo Presti, L., Cerutti, L., Cano Del Rosario, E., Hauser P.M. and Simanis, V., Functional differentiation of tbf1 orthologues in fission and budding yeasts (2009) Eukaryotic Cell 8, 207 – 216. Choi, S. H., Peli-Gulli, M. P., McLeod, I., Sarkeshik, A., Yates, J. R., 3rd, Simanis, V. and McCollum, D., Phosphorylation state defines discrete roles for monopolin in chromosome attachment and spindle elongation. (2009) Current Biology 19, 985-95. Lattmann, A., Krapp, A., and Simanis,Cytokinesis: Closure resets your SIN (2009) V. REVIEW, Current Biology, 19, r1040 – 1042.

inge, C., Simanis, V., Magistretti, P. J. and Marquet, P.,Noninvasive characterization of the fission yeast cell cycle by monitoring dry mass with digital holographic microscopy (2009),Journal of Biomedical Optics 14, 034049. Hachet, O. and Simanis, V., Mid1p/anillin and the Septation Initiation Network orchestrate contractile ring assembly for cytokinesis (2008),Genes and Development 22, 3205 - 3216. Dischinger, S, Krapp, A., Xie, L., Paulson J.R., and Simanis, V., Chemical genetic analysis of the role of cdc2p in regulating the S.pombe septation initiation network. (2008), Journal of Cell Science 121, 843 853 Krapp, A, Collin, P, Cano del Rosario, E and Simanis, V., Homoeostasis between the GTPase spg1p and its GAP in the regulation of cytokinesis in S. pombe. (2008) Journal of Cell Science 121, 601 – 608. Krapp, A., and Simanis, V. (REVIEW) An overview of the SIN (2008),Biochemical Society Transactions 36, 411 – 415.

Rappaz, B., Cano, E., Colomb, T., Kuhn, J., Depeurs-

Aberrant spore formation during meiosis in the absence of the conserved SIN regulator dma1-D. Matings between h- and h+ wild-type or h- and h+ dma1-D cells expressing psy1p-GFP were filmed during forespore membrane (FSM) development in meiosis II. The time interval between frames is 2 mins in the wild-type and 4 mins in the dma1-D cells. Note the aberrant FSM formation in the dma1-D cells.

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its ectopic expression can provide the essential functions of Schizosaccharomyces pombe tbf1 but that there is no rescue between fission and budding yeast orthologues. Our findings indicated that an essential function of this family of proteins has diverged in the budding and fission yeasts and suggest that effects on telomere length or structure are not the primary cause of inviability in S. pombe tbf1 null strains.


EPFL School of Life Sciences - 2009 Annual Report

ISREC - Swiss Institute for Experimental Cancer Research

Beermann Group Group Leader http://isrec.epfl.ch/page27137.html

Team Members

Iraz Topak Aydin, PhD student Sandra Brunschwiler, Technician (January to October 2008) Irina Pshenichnaya, PhD student Karine Schouwey, PhD student

Friedrich Beermann

Group Leader Maître d’Enseignement et de Recherche (MER)

Introduction

Pigment cells are responsible for hair graying, albinism, vitiligo, and melanoma. We use these cells to study gene regulation, development and interplay between stem and differentiating cells, and tumorigenesis. The transgenic service (of the CPG) provides support for producing genetically modified mice for research.

Keywords

Melanocyte, melanoma, pigmentation, transgenic, knockout, mouse

Results Obtained in 2009

Melanocyte development Melanocytes differentiate from pluripotent neural crest cells in early embryogenesis in the mouse, migrate along the dorsolateral pathway and subsequently proliferate through the dermis horizontally to the ventral region. By mid-gestation, melanocytes exit from the dermis and invade into the epidermis to finally be located in the skin and the hair follicles. Many genes are implicated in specific aspects of melanocyte/melanoblast differentiation and more than 300 loci are currently known to affect pigmentation in the mouse. We have addressed the role of the Notch signaling pathway and of the c-Myc oncogene in melanocyte development and homeostasis, and have demonstrated that both genes / pathways lead to precocious hair graying if affected. Disruption of the Notch pathway by inactivating Notch1 and Notch2, or RbpJk in the melanocyte lineage resulted in a dosage-dependent precocious hair graying, due to an elimination of melanocytes and melanocyte stem cells. Expression from a NotchIC-expressing transgene rescued this deficiency, providing further evidence that Notch signaling acts via RbpJk. In contrast to Notch signaling, melanocyte-specific removal of c-Myc did not lead to

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a progressive hair graying phenotype, but, instead, the degree of graying remained constant over life. Further analyses pointed to a major defect in midgestation embryos, with less cells in epidermis probably due to a proliferation defect. Our results illustrate how different pathways can affect melanocyte homeostasis and thus hair graying, either by acting more specifically on presumptive melanocyte stem cells or by severely affecting the overall population of melanocytes without specific effects on stem cells. We are currently extending these studies to the role of Notch signaling in the retinal pigment epithelium, and plan to investigate these pathways in melanoma formation, taking advantage of a transgenic melanoma model established in the laboratory. Mart1/mlana gene expression and function Mart-1 (encoded by the Mlana gene in the mouse) is an important melanoma-associated antigen which is widely studied as a target for immunotherapy. We had performed in vitro analyses (transfections) as well as in vivo analyses (transgenics) to unveil the important 5’ regulatory sequences. Whereas 0.6 kb of mouse Mlana promoter was sufficient for in vitro expression in melanocyte and RPE cell lines transgenic expression obtained with 6 kb of the promoter was variable and did not fully reproduce the anticipated endogenous Mlana expression. We then generated Mlana::lacZ BAC transgenic mice which closely reproduced the endogenous expression pattern of Mlana/Mart-1 (Figure), highlighting the potential use of this promoter for targeted gene expression in mice. Transgenic service The transgenic service is integrated into the Center of Phenogenomics of the EPFL, and we have performed pronuclear microinjection of DNA constructs either derived from plasmid vectors or from BAC DNA. Furthermore, the service was involved in the rederivation of mouse lines.

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EPFL School of Life Sciences - 2009 Annual Report

Aydin, I.T., Beermann, F. (2009) Melanocyte and RPEspecific expression in transgenic mice by mouse MART-1/ Melan-A/mlana regulatory sequences. Pigment Cell Mel. Res. 22: 854-856. Levin, M.D., Lu, M.M., Petrenko, N.B., Hawkins, B.J., Gupta, T.H., Lang, D., Buckley, P.T., Jochems, J., Liu, F., Spurney, C.F., Yuan, L.J., Jacobson, J.T., Brown, C.B., Huang, L., Beermann, F., Margulies, K.B., Muniswamy, M., Eberwine, J.H., Epstein, J.A., Patel, V.V. (2009) Melanocyte-like cells in the heart and pulmonary veins contribute to atrial arrhythmia triggers. J. Clin. Invest. 119: 3420-3436. Mérillat, A.M., Charles, R.P., Porret, A., Maillard, M., Rossier, B., Beermann, F., Hummler, E. (2009) Conditional gene targeting of the ENaC subunit genes Scnn1b and Scnn1g. Am. J. Physiol.- Renal Physiol. 296: F249-F256. Walker, G.J., Kimlin, M.G., Hacker, E., Ravishankar, S., Muller, H.K., Beermann, F., Hayward, N.K. (2009) Murine neonatal melanocytes exhibit a heightened proliferative response to ultraviolet radiation and migrate to the epidermal basal layer. J. Invest. Dermatol. 129: 184-193.

Rubera, I., Hummler, E., Beermann, F. (2009) Transgenic mice and their impact on kidney research. Pflügers Arch. - Eur. J. Physiol. 458: 211-222. Inoue-Narita, T., Hamada, K., Sasaki, T., Hatakeyama, S., Fujita, S., Kawahara, K., Sasaki, M., Kishimoto, H., Eguchi, S., Kojima, I., Beermann, F., Kimura, T., Osawa, M., Itami, S., Mak, T.W., Nakano, T., Manabe, M., and Suzuki, A. (2008) Pten deficiency in melanocytes results in resistance to hair graying and susceptibility to carcinogen-induced melanomagenesis. Cancer Res. 68: 5760-5768. Schouwey, K. and Beermann, F. (2008) The Notch pathway: hair graying and pigment cell homeostasis. Histol. Histopathol. 23: 609-619. Murisier, F., Aydin, I.T., Guichard, S., Brunschwiler, S. and Beermann, F. (2008) Expression from a bacterial artificial chromosome containing the Dct gene locus. Pigment Cell Mel. Res. 21: 212-215.

Expression from a Mlana::lacZ BAC in pigment cells of skin and eye.

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Selected Publications


EPFL School of Life Sciences - 2009 Annual Report

ISREC - Swiss Institute for Experimental Cancer Research

Bucher Group Group Leader- http://isrec.epfl.ch/bucher/

Team Members

Giovanna Ambrosini, Scientist Slavica Dimitrieva, PhD student (from July 2009) Helah Yasrebi, PhD student

Philipp Bucher Group Leader

Introduction

New high-throughput assays allow for comprehensive and detailed characterization of the molecular processes that cause a healthy cell to become cancerous. We develop computational methods that will help to extract insight from the ever increasing amounts data produced by such technologies.

Keywords

Computational genomics, sequence analysis methods, ChIP-Seq data analysis, survival prediction from molecular profiles, human biographic data analysis

Results Obtained in 2009

Computers have become indispensable tools for accessing, visualizing, analyzing, managing, and publishing biological data. Over the last decade, highthroughput sequencing and functional genomics projects have generated unprecedented quantities of biological data which can neither manually be processed, nor comprehensively inspected by eye. The emergence of bioinformatics as a research field in its own right, is a response to this development. Cancer can be considered a gene regulatory disease. Normal regulation of genes permits the development and maintenance of a healthy human being. Abnormal regulation leads to various diseases. Cancer cells are maintained in a specific pathological state by gene regulatory circuits. Transcription factors are key elements of such circuits in that they control the expression of other genes, while themselves are being regulated by the products of genes. Our research aims at an understanding of transcriptional regulatory mechanisms, in particular those which are affected by genetic lesions that cause cancer. Studying transcriptional regulation with chromatin-IP data Transcriptional control mechanisms in higher organisms are poorly understood. We are, for instance, still not able to predict the effects of genetic variations and somatic mutations in gene regulatory regions. The recently introduced genome-wide chromatin im-

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munoprecipitation assays (ChIP-chip and ChIP-seq) open new perspectives for studying gene regulation. For the first time, we are able to observe molecular interactions at gene regulatory regions at near-basepair resolution. The comparison of histone modification profiles from different cell types constitutes a completely new way of analyzing differential gene activity, centering on the activity status of cis-regulatory regions rather than mRNA abundances. We have swiftly responded to this new trend by devoting major research efforts to the development of new computational methods for the analysis of ChIP-Seq and other â&#x20AC;&#x153;mass genome annotationâ&#x20AC;? data produced with massively parallel sequencing technology. These new methods have already turned out to be useful in a number of research collaborations with experimentalists. Analysis of breast cancer gene expression data sets A novel breast cancer survival gene, CYB5D1, potentially useful for diagnosis and personalized treatment, was discovered in the course of a methodological research project aimed at improving prognostic machine learning algorithms by integrating expression data from different clinical studies (Figure 1). The data used for this project were selected, extracted and reorganized with the aid of the CleanEx database, a resource initially developed by our group and now independently maintained by Viviane Praz at the Center of Integrative Genomics in Lausanne. CYB5D1 is a member of the Cytochrome b5 family, which presumably escaped earlier discovery as a survival gene because it was not present on the gene expression profiling chips used in the classical breast cancer gene expression studies. Our results also called into question the current popular paradigm that composite gene signatures are more reliable disease outcome predictors than single-molecule markers. Collaborative projects with experimentalists With Nicolas Mermod (University of Lausanne) we have analyzed ChIP-Seq data for transcription factor CTF/NFI in mouse embryonic fibroblasts. With

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EPFL School of Life Sciences - 2009 Annual Report

Bioinformatics meets sociology: For the first time, we published bioinformaticsinspired interdisciplinary research in a leading sociology journal (Gauthier et al. 2009). The fruitful collaboration leading to this achievement involved researchers from the PaVie lab (Laboratoire d’étude des Parcours de Vie) at Lausanne and Cédric Notredame from CRG (Barcelona), a leading scientist in multiple sequence alignment. In this work, we incorporated techniques from molecular sequence alignment into algorithms for comparing human biographic data, in particular professional and personal career trajectories. It is not uncommon that computational methods developed in one field turn out be useful in a completely different context. For instance, the current state-of-the-art methods for identifying new members of protein families rely on techniques originally developed for speech recognition. Regarding molecular sequence alignment algorithms, we anticipate further applications in the analysis of clinical time series data such as patients’ health histories or disease follow-up records.

Development of bioinformatics resources Our group has developed a number of public databases and software resources over the last two decades including the Eukaryotic Promoter Database EPD, CleanEx (Praz and Bucher, 2009), the Signal Search Analysis (SSA) server for DNA motif discovery, and the ChIP-Seq tools for analyzing data from chromatin-immunoprecipitation experiments and other high-throughput functional genomics assays. These resources are publicly available at ccg.vital-it. ch/chipseq.

Selected Publications

Gauthier, J.-A., Widmer, E., Bucher, P. and Notredame, C. (2009). How much does it cost? Sociological Methods & Research 38(1):197-231. Yasrebi, H., Sperisen, P., Praz, V. and Bucher, P. (2009). Can survival prediction be improved by merging gene expression data sets? PLoS One 4(10):e7431. Praz, V. and Bucher, P. (2009). CleanEx: new data extraction and merging tools based on MeSH term annotation. Nucleic Acids Res. 37:D880-D884. Leimgruber, E., Seguín-Estévez, Q., Dunand-Sauthier, I., Rybtsova, N., Schmid, C.D., Ambrosini, G., Bucher, P. and Reith W. (2009). Nucleosome eviction from MHC class II promoters controls positioning of the transcription start site. Nucleic Acids Res. 37(8):2514-2528. Krawczyk, M., Seguín-Estévez, Q., Leimgruber, E., Sperisen, P., Schmid, C., Bucher, P. and Reith W. (2008). Identification of CIITA regulated genetic module dedicated for antigen presentation. PLoS Genet. 4(4):e1000058

Kaplan-Meier plot showing the correlation of CYB5D1 expression with survival in a cohort of breast cancer patients participating in clinical tests (based on data from Sorlie et al., Proc. Natl. Acad. Sci. USA 100, 8418–8423, 2003). Note that this gene is not part of the 70-gene signature derived from expression data which is already in use for breast cancer diagnosis. CYB5D1 apparently escaped the attention of other researchers because if was not included in the Affymetrix gene chips used in earlier studies.

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Walter Reith (University of Geneva) we characterized the complete set of target loci of the regulatory protein CIITA using ChIP-chip data (Krawczyk et al. 2008). With Didier Trono (EPFL) we analyzed a large collection of retroviral integration sites generated with a gene trap system to gain insights into KAP1mediated silencing. With Fritz Müller (University of Fribourg) we are currently analyzing ChIP-Seq data for the nucleosome-remodeling protein Let-418 in the nematode.


EPFL School of Life Sciences - 2009 Annual Report

External Adjunct Professor

Molinari Group Head of Lab (PI) - http://www.irb.ch/

Team Members

Riccardo Bernasconi, PhD student Siro Bianchi, Technician Verena Calanca, Technician Carmela Galli, Scientific collaborator Danilo Presotto, PhD student Tatiana SoldĂ , Scientific collaborator

Maurizio Molinari

External Adjunct Professor Institute for Research in Biomedicine Bellinzona

Introduction

Cell and organism homeostasis relies on a balanced activity of the protein folding, quality control and degradation machineries operating in the lumen of the endoplasmic reticulum (ER). The capacity to manipulate protein folding, quality control and degradation will be instrumental to delay progression or even to cure diseases caused by inefficient functioning of the cellular protein factory such as cystic fibrosis and several types of tumor and of neurodegenerative diseases. It will also increase productivity of recombinant proteins to be employed in the clinics and in the industry (reviewed in Hebert, D.N. and Molinari, M. (2007) Physiol. Rev. 87, 1377-1408).Results obtained in 2009 Recognition and dislocation across the ER membrane of terminally misfolded polypeptides. The endoplasmic reticulum of eukaryotic cells is the site of maturation for proteins destined to intracellular organels of the endocytic and secretory pathway, to the plasma membrane and to the extracellular space. Part of our work aims at the characterization of mechanisms that regulate clearance from the ER of terminally misfolded polypeptides (ER-associated degradation, ERAD). Most polypeptides entering the ER lumen are covalently modified at asparagine side chains with glucose3-mannose9-N-acetylglucosamine2- oligosaccharides. Their maturation is assisted by a dedicated folding machinery comprising the oligosaccharide-binding chaperones calnexin and calreticulin and the oxidoreductase ERp57. Processing of oligosaccharides displayed on misfolded conformers by ER-resident Îą1,2-mannosidases irreversibly extracts folding-defective polypeptides from the lectin-operated folding machinery. We have characterized the function in ER quality control of few proteins derived from alternative splicing of the OS9 and the XTP3-B genes. These proteins are ubiquitously expressed in human tissues, are amplified in tumors and are transcriptionally induced upon activation of

the Ire1/Xbp1 ER-stress pathway. They do not associate with folding-competent proteins. Rather, they selectively bind folding-defective ones thereby inhibiting transport of non-native conformers through the secretory pathway. Their intralumenal level inversely correlates with the fraction of folding-defective glycoproteins that escapes retention-based ER quality control. OS9 and XTP3-B play a dual role in mammalian ER quality control: firstly as crucial retention factors for misfolded conformers, and secondly as promoters of protein disposal from the ER lumen. We have defined OS-9 and XTP3-B as ERAD shuttles because they transport soluble, extensively demannosylated terminally misfolded glycopolypeptides (ERAD-Ls substrates) from the ER lumen to the site of dislocation across the ER membrane. The ERAD shuttles deliver ERAD-Ls substrates to a multiprotein complex comprising the membrane receptor SEL1L, the associated E3 ubiquitin ligase HRD1 and an elusive dislocation (retro-translocation) channel. Extensively de-mannosylated membrane-tethered polypeptides with the same luminal defects (ERADLm substrates) are cleared from the ER lumen even upon complete inactivation of the ERAD-Ls pathway. ERAD tuning Another focus of our research is the characterization of mechanisms that regulate the content in the ER lumen of molecular chaperones assisting rate-limiting steps in the processes of clearance of misfolded polypeptides from the ER lumen. This regulation is crucial for maintenance of cell and organism homeostasis because hyper-activation of the ER disposal machinery causes premature interruption of ongoing folding programs (folding-intermediates are improperly recognized as misfolded end-products and are cleared from the folding environment before they have a chance to attain the native structure). EDEM1 is a crucial ERAD regulator that extracts non-native glycopolypeptides from the folding ma-


EPFL School of Life Sciences - 2009 Annual Report

chinery that has been characterized in our lab (Science 299, 1397). Under normal growth conditions, the intralumenal level of EDEM1 must be low to prevent premature interruption of ongoing folding programs. Our studies showed that in unstressed cells, EDEM1 is segregated from the bulk ER into LC3-Icoated vesicles and is rapidly degraded. The rapid turnover of EDEM1 (and of other ERAD regulators) has been named ERAD tuning. It is regulated by a novel mechanism that shows similarities but is clearly distinct from macroautophagy. Cells with defective EDEM1 turnover contain unphysiologically high levels of EDEM1, show enhanced ERAD activity and are characterized by impaired capacity to efficiently complete maturation of model glycopolypeptides.

Selected publications

Hebert, D.N. Bernasconi, R. and Molinari, M. (2010) ERAD Substrates: Which Way Out? Semin. Cell Dev. Biol. IN PRESS Aebi, M., Bernasconi, R., Clerc, S. and Molinari, M. (2010) N-Glycan Structures: Recognition and Processing in the ER. TIBS 35, 74-82. Bernasconi, R., Galli, C., Calanca, V., Nakajima, T. and Molinari, M. (2010) Stringent requirement for HRD1, SEL1L and

OS-9/XTP3-B for disposal of ERAD-Ls substrates. J. Cell Biol. 188, 223-235.- Highlights in J. Cell Biol. Calì, T., Galli, C., Olivari, S. and Molinari, M. (2008) Segregation and Rapid Turnover of EDEM1 Modulates Standard ERAD and Folding Activities. Biochem. Biophys. Res. Commun. 371, 405-410. Bernasconi, R., Pertel, T., Luban, J. and Molinari M. (2008) A Dual Task for the Xbp1-Responsive OS-9 Variants in the Mammalian ER: Inhibiting Secretion of Misfolded Protein Conformers and Enhancing Their Disposal. J. Biol. Chem. 283, 16446-16454. Vanoni, O., Paganetti, P. and Molinari, M. (2008) Consequences of Individual N-Glycan Deletions and of Proteasomal Inhibition on Secretion of Active BACE. Mol. Biol. Cell 19, 4086-4098. Bernasconi, R. and Molinari M. (2008) ER-Associated Folding and Degradation: Learning From Yeast? In Protein misfolding: New Research O’Doherty, C.B. and Byrne, A.C. Eds, Nova Science Publishers, Inc., Hauppauge, NY, 113-123. Calì, T., Vanoni, O. and Molinari, M. (2008) The Endoplasmic Reticulum: Crossroads for Newly Synthesized Polypeptide Chains. Progress in Mol. Biol. Transl. Sci. 83, 135-179.

The ERAD mechanism as depicted in this figure, can be divided in four steps: First, the folding-incompetent glycoprotein is extracted from the calnexin cycle through the concerted activity of several mannose-trimming enzymes (GH47); Second, the lectin ERAD shuttles OS-9 and XTP3-B transport the misfolded glycopolypeptide from the ER lumen to the membrane-anchored adaptor protein SEL1L, which is associated with the E3 ubiquitin ligase HRD1; Third, the misfolded glycopolypeptide has to be unfolded (e.g., reduction of aberrant disulfide bonds and cis/trans isomerization of peptidyl-prolyl bonds) in order to; Fourth, allow dislocation across the ER membrane through an elusive channel, poly-ubiquitylation and degradation by the 26S proteasome. The different mannose composition of the N-glycan is depicted in color code: gray, oligosaccharide with nine mannose (Man9); red, five to seven mannose residues (Man5-7); and white, oligosaccharide with an undefined number of mannose residues.


EPFL School of Life Sciences - 2009 Annual Report

External Adjunct Professor

Rainer Group Head of Lab (PI) - http://www.unifr.ch/inph/vclab

Team Members

Xiaozhe Zhang, Postdoctoral fellow Laetitia Fouillen, Postdoctoral fellow Anwesha Bhattacharyya, PhD student Sara Falasca, PhD student Julia Veit, PhD student Abbas Khani, PhD student Filomena Petruzziello, PhD student Pierre Joris, PhD student (joint supervision with Prof. Philippe Renaud)

Gregor Rainer External Adjunct Professor University of Fribourg

Introduction

The primate visual system is comprised of numerous brain regions, connected to each other in a highly recurrent fashion. Our research aims at understanding how visual information is processed and stored, enabling us to recognize objects and interact with our environment. Understanding the neural code of the visual system is a fundamental requirement for advancing sensory brain-machine interfaces. We combine sophisticated behavioural psychophysical methods with multi-channel electrophysiological recordings, electrical microstimulation and pharmacological manipulations. We are particularly interested in studying how cholinergic neuromodulation and associated neuropeptides affect visual processing mechanisms and behavioural performance. Laminar recordings in the visual cortex We are examining neural activity at the level of single neurons and local field potentials in the primary visual cortex using tetrodes as well laminar probes that are designed at EPFL in a collaboration funded by the Stoicescu program in Neurotechnology. We are developing and refining these probes for different applications including laminar cortical recordings, deep brain stimulation and epidural recording. We employ a variety of visual stimuli, designed to probe particular computational functions of the visual cortex, allowing us to describe layer-specific relationships between cortical oscillatory activity at the mesoscopic level and information transmission by single neurons. We apply cholinergic receptor agonists and antagonists as well as electrical microstimulation in the basal forebrain to up- and downregulate cholinergic neuromodulation, and study how these manipulations affect behaviour as well as neural activations in the visual system. Cholinergic

neuromodulation has linked to a number of neurological disorders including Alzheimerâ&#x20AC;&#x2122;s disease and Schizophrenia. Neurochemical Dynamics We are developing methods based on Liquid Chromatography and Mass Spectrometry (LC-MS) as well as electrochemical detection (ECD) to track the concentration of amino acids, small molecule neurotransmitters such as Acetylcholine, Acetylcarnitine, Dopamine or Serotonin and their metabolites and neuropeptides with high temporal resolution. Our work includes the development of direct brain-mass spectrometer interface for fast and automated quantitation of neurochemicals, as well as characterization of brain-region specific neuropeptides that are linked to visual cognition and cholinergic neuromodulation. Recent advances in neurochemical monitoring have shown that the cholinergic system, as well as other neuromodulators, can act at very fast time scales. These systems are thus significantly more specific than has previously been assumed. Exploring this fast action is thus likely to provide important novel insights into their mechanisms of action during cognition.

Selected publications

Nielsen, K., N. K. Logothetis and G. Rainer: Object features used by humans and monkeys to identify rotated shapes. Journal of Vision 8(2): 9, 1-15 (2008) Zhang, X., A. Rauch, H. Xiao, G. Rainer and N. K. Logothetis: Mass spectrometry-based neurochemical analysis: perspectives for primate research. Expert Review of Proteomics 5(5), 641-652 (2008) Rainer, G.: Localizing cortical computations during visual search. Neuron 57, 480-81 (2008)


EPFL School of Life Sciences - 2009 Annual Report

Liebe, S., Fischer, E., Logothetis, N. K., Rainer, G. Color and shape interactions in the recognition of natural scenes by human and monkey observers. Journal of Vision, 9(5):14, 1-16 (2009)

Bießmann F., Meinecke F.C., Gretton A., Rauch A., Rainer G., Logothetis N.K., Müller K.R. Temporal Kernel CCA and its Application in Multimodal Neuronal Data Analysis. Machine Learning (2009)

On the left, a polyimide laminar neural recording probe is shown with contact spacing of 100μm and three different contact sizes. An example single trial local field potential recording in the primary visual cortex is shown in the center panel; the vertical line represents the onset of the visual stimulus. On the left, action potentials recorded on one of the probe channels are shown.


EPFL School of Life Sciences - 2009 Annual Report

External Adjunct Professor

Schorderet Group Head of Lab (PI) - http://irovision.ch/

Team Members

Daniel Schorderet External Adjunct Professor Director IRO Institute for Research in Ophthalmology, Sion

Nathalie Allaman-Pillet, senior postdoctoral fellow Raphaël Roduit, senior postdoctoral fellow Hana Abouzeid, senior postdoctoral fellow Bozena Polok, postdoctoral fellow Nathalie Produit-Zengaffinen, postdoctoral fellow Anne Oberson, biologist Leila Tiab, biologist Gaëlle Boisset, postdoctoral fellow Sylvain Bolay, ingénieur HES Fabienne Marcelli, PhD student Séverine Hamann, PhD student Désirée von Alpen, PhD student Tatiana Favez, technician Céline Agosti, technician Sylviane Métrailler, technician Martine Emery, technician Carole Herkenne, technician Isabelle Favre, technician Etienne Bagnoud, informaticien CFC Cédric Schöpfer, technician ES Pascale Evéquoz, administrative assistant Sandra Théodoloz, administrative assistant

Introduction

The goal of my laboratory is to understand the genetics of vision, from the development of the eye until late in life, in order to develop better diagnostic tools and new therapies.

Keywords:

Gene identification, cell signaling, animal models, mouse, zebrafish, vision, eye, development, gene therapy, degenerative disorders, cataract, retinitis pigmentosa, glaucoma, age-related macular degeneration, apoptosis, autophagy

Results obtained in 2009

In order to understand the genetics of vision, we use various approaches to identify new genes and to analyze their role in the development or the death of ocular cells. Eye development follows a complicated program during which activating and inhibiting signals cohabit. For example, an excess of retinal cells is produced, then apoptosis is turned on in order to fine tune the final number of photoreceptors and eliminate those which did not make the proper connections. Our goal is to understand the details of this program and to develop new therapies by modulating the pathways involved using small cell-penetrating peptides or gene therapy. Human diseases involving eye malformations are observed in clinical settings. They may be due to spontaneous or inherited mutations. We have collected DNA from members of numerous families with blinding disorders and have embarked on identifying all the genes involved. This is done by linkage analysis using SNP microarray and large-scale resequenc-

ing. Recently, we identified CNNM4 and TRPM1 as responsible for the Rod-cone dystrophy/amelogenesis imperfecta syndrome and a form of congenital stationary night blindness, respectively (Polok et al., 2009; Audo et al., 2009) and elucidate how mutations in NR2E3, a retinal-specific nuclear receptor, were responsible for three different blinding disorders (Roduit et al., 2009; Schorderet et al., 2009; Escher et al., 2009). We also identified a new autosomal dominant syndrome called CCMMCA characterized by Congenital Cataract, Microcornea, Microphthalmia, atypical Coloboma in a family living in Alexandria (Egypt) (Abouzeid et al., 2009) and mapped it to human chromosome 2. For many blinding diseases, accession to affected tissues is crucial for the characterization of the physiopathology involved. However, obtaining human retina and other ocular tissues is difficult. We therefore have to rely on animal models. In my lab, we are using mouse and zebrafish for the study of eye development and the generation of disease models (see picture of a mouse with non-functional Nkx5-3, a gene responsible for the human Schorderet-Munier oculo-auricular syndrome). Zebrafish have a number of advantages over other animal models, including external fertilization and development, transparency of the eggs and embryo, and easy access. Zebrafish eggs can be used in many studies, from investigating the role of specific genes to mass screening of active compounds.

Selected publications

Polok B, Escher P, Ambresin A, Chouery E, Bolay S, Meunier I, Nan F, Hamel C, Munier FL, Thilo B, Mégarbané A, Schorderet DF. Mutations in CNNM4 cause recessive cone-


EPFL School of Life Sciences - 2009 Annual Report

rod dystrophy with amelogenesis imperfect. Am J Hum Genet. 84(2):259-265,2009 Abouzeid H, Meire FM, Osman I, Elshakankiri N, Bolay S, Munier FL, Schorderet DF. A New Locus for Congenital Cataract, Microcornea, Microphthalmia, and Atypical Iris Coloboma Maps to Chromosome 2. Ophthalmology. 116(1):154-162,2009 Audo I, Kohl S, Leroy BP, Munier FL, Guillonneau X, Mohand-Saïd S, Bujakowska K, Nandrot EF, Lorenz B, Preising M, Kellner U, Renner AB, Bernd A, Antonio A, Moskova-Doumanova V, Lancelot ME, Poloschek CM, Drumare I, Defoort-Dhellemmes S, Wissinger B, Léveillard T, Hamel CP, Schorderet DF, De Baere E, Berger W, Jacobson SG, Zrenner E, Sahel JA, Bhattacharya SS, Zeitz C. TRPM1 Is Mutated in Patients with Autosomal-Recessive Complete Congenital Stationary Night Blindness. Am J Hum Genet. 85(5):720-729,2009 Escher P, Gouras P, Roduit R, Tiab L, Bolay S, Delarive T, Chen S, Tsai CC, Hayashi M, Zernant J, Merriam JE, Mermod N, Allikmets R, Munier FL, Schorderet DF. Mutations in NR2E3 can cause dominant or recessive retinal degenerations in the same family. Human Mutation 30(3):342351,2009

Roduit R, Escher P, Schorderet DF. Mutations in the DNABinding Domain of NR2E3 Affect In Vivo Dimerization and Interaction with CRX. PLoS One. 4(10):e7379,2009 Schorderet DF, Escher P. NR2E3 mutations in enhanced Scone sensitivity syndrome (ESCS), Goldmann-Favre syndrome (GFS), clumped pigmentary retinal degeneration (CPRD), and retinitis pigmentosa (RP). Human Mutation 30(11):1475-1485,2009. Hamann S, Schorderet DF, Cottet S. Bax-induced apoptosis in Leber’s congenital amaurosis: a dual role in rod and cone degeneration. PLoS One. 4(8):e6616,2009 Cottet S, Schorderet DF. Mechanisms of apoptosis in retinitis pigmentosa. Curr Mol Med. 9(3):375-83,2009 Produit-Zengaffinen N, Pournaras CJ, Schorderet DF. Retinal ischemia-induced apoptosis is associated with alteration in Bax and Bcl-x(L) expression rather than modifications in Bak and Bcl-2. Molec Vision 15:2101-2110,2009 Abouzeid H, Youssef MA, ElShakankiri N, Hauser P, Munier FL, Schorderet DF. PAX6 aniridia and interhemispheric brain anomalies. Molec Vision 15:2074-2083,2009.

The Dumbo mouse harbors a non-sense mutation in the Nkx5-3 gene and is an animal model of the human Schorderet-Munier oculoauricular syndrome, characterized by small eyes and abnormal ears as shown in the photo.


EPFL School of Life Sciences - 2009 Annual Report

External Adjunct Professor

Marcel Tanner http://www.swisstph.ch

Research Keywords Epidemiology, public health, vaccines and drug resistance

Marcel Tanner

External Adjunct Professor Director Swiss TPH Institute Basel

Swiss TPH (Tropical and Public Health Institute) and the EPFL School of Life Sciences have started a joint research project on Mycobacterium ulceransdisease (Buruli ulcer) with a Swiss TPH PostDoc working part time at EPFL. In addition, joint parasitological research projects have been identified and are in an advanced planning phase. These include use of Caenorhabditis elegans as model for helminth drug discovery, complementation of in vivo and in vitro screening for antimalarials with new approaches for in silco modeling and the analysis of protein-protein interactions to elucidate complex functions and host-pathogen interactions in malaria. Overall goal is to bring together complementary expertise of the two institutions in research on infectious diseases and the development of new diagnostics, drugs and vaccines.


EPFL School of Life Sciences - 2009 Annual Report

Welcome To Our New Collaborators! School of Life Sciences

Former Home Institution

Head of Global Research, Roche Ltd.

EPFL School of Life Sciences since December 2009

Keywords

Translation Research, Cancer Therapy

Jonathan Knowles Full Professor

Former Home Institution

Novocell, Inc. San Diego, California EPFL School of Life Sciences since December 2009

Research Keywords

Stem Cells, Diabetes, Bioengineering, Biotechnology

Emmanuel Baetge

External Adjunct Professor

GHI - Global Health Institute Former Home Institution

Inserm U609, Wellcome Trust Centre for Molecular Parasitology, University of Glasgow, Scotland, UK EPFL School of Life Sciences since November 2009

Research Keywords Molecular parasitology, Phosphosignalling and functional kinomics of the human malaria parasite Plasmodium falciparum Christian Doerig

Directeur de researche, Inserm EPFL - INSERM Joint Laboratory


EPFL School of Life Sciences - 2009 Annual Report

6th edition 2009/2010 Produced and edited by the EPFL School of Life Sciences Printed at the EPFL â&#x20AC;&#x2DC;Atelier de Reprographieâ&#x20AC;&#x2122; Editor: Alice Emery-Goodman With many thanks to Roland Chabloz, Harald Hirling and Bruno Liardon for their help and support!

2009 Annual Report  

the annual report of the EPFL School of Life Sciences

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