Research ALS Today - Fall 2015

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Association Expands Support Post-Doctoral Fellowships Whole Genome Sequencing Call for Grants Journal News

FALL 2015

Association Expands its Support for Research with ALS Ice Bucket Funds

The pursuit of the causes of and treatments for ALS took a dramatic step forward with the global support for ALS research known as the ALS Ice Bucket Challenge. Those new funds, combined with strong ongoing contributions from many other sources, has allowed The ALS Association to fund more than $11 million in new research directed at the discovery of treatments for the disease in these key areas: Genes, Models and Mechanisms The Association is providing significant support for major international collaborations to identify new genes that may account for more than 80 percent of ALS cases for which the cause is unknown. Whole genome sequencing efforts, combined with detailed clinical phenotyping, are designed to uncover genotype-phenotype correlations to better understand the basis of different clinical forms of ALS and potentially design therapeutic approaches to subtypes that may have better chances of success in clinical trials. These efforts as part of The Association’s strategic focus on Precision Medicine, or Personalized Medicine, include collaborative efforts together with Biogen-Idec at the New York Genome Center, Project MinE and the CReATe Consortium. Other researchers will pursue understanding of genes for motor neuron development, and genetic modifiers of disease severity. New genes must be developed into disease models, and recently models of ALS using the C9orf72 mutation have emerged and are beginning to yield new insights. ALS Association-funded researchers will be examining the consequences of the mutation in human cell and animal models, seeking to understand how the pathogenic consequences of the mutation, including effects on gene expression and RNA metabolism, among other processes, are

involved in disease. Other groups will investigate the effects of mutations in FUS and TDP-43, both involved in RNA processing. Studies of neuroinflammation, neuronal hyperexcitability and environmental influences on disease are also being supported. Biomarkers The ALS Association has identified biomarker development as a central focus of research, in order to speed clinical trials and make the results of them more informative. Funded work includes a study of intermuscular coherence, a painless and noninvasive clinical measure, with the potential to track disease progression; development of imaging biomarkers for detecting survival and movements of transplanted cells; and study of C9orf72-related changes in blood, to monitor disease progression and response to therapy. Therapy Development and Clinical Trials Significant investment will be provided for The Association’s Drug Development Contract Program encouraging public-private partnerships between academia and industry. To ensure that new treatment approaches reach the clinic, The Association is investing in translational research efforts to derisk ALS programs and encourage industry partners to further the development once the target or Continued on page 7

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Milton Safenowitz Post-Doctoral Fellowships Promising Advances Provide New Avenues for Treatment Development This has been an extremely active year for ALS research. Since the announcement of our four major initiatives as a result of the ALS Ice Bucket Challenge, The ALS Association has funded more than 58 new projects ranging from early discovery to small Phase II clinical trials. While numerous new grants began this year, ALS Association-funded investigators continue to publish advances in the field. In the last few months, highlighted in our Journal News, studies are beginning to reveal potential mechanisms for the C9orf72 hexanucelotide repeat expansions, the most common gene mutation in ALS. While significant progress is being made to use antisense technology as a therapeutic for the C9orf72 expansion, two new studies are being funded using alternative therapeutic approaches. One study will test therapeutic efficacy of humanderived antibodies targeting dipeptide-repeat proteins, and a second will be evaluating small molecules targeting the C9orf72 repeat expansion. Through The Association’s Drug Development Contract program, several new companies are partnering to advance therapies for ALS. Lucie Bruijn, Ph.D., M.B.A. Chief Scientist The ALS Association

In parallel, programs in The Association and with partner organizations are expanding to address the heterogeneity of ALS. Through the New York Genome effort and Project MinE, featured in this edition, we will learn more about the genetics underlying both sporadic and familial ALS. Precision medicine, or personalized medicine, has significantly impacted therapy development in the cancer field. Borrowing from these successes, we hope that efforts aimed at characterizing each person with ALS at the phenotypic, genotypic, proteomic and metabolomic level will provide a better understanding of which treatment approaches are likely to be beneficial in the different sub-populations. In doing so, we will also develop better biomarkers, signatures for diagnosis, disease progression and treatment validation. We congratulate this year’s Milton Safenowitz fellows. The program has grown over the years and has seen talented young fellows establish their own laboratories and become mentors to the new fellows. More than 90 percent of Milton Safenowitz fellows remain in ALS research and contribute significantly to the advances made in the field. We encourage researchers to contact The ALS Association with their research ideas and submit to our grant programs. –Lucie Bruijn, Ph.D., M.B.A.

The ALS Association offers the Milton Safenowitz Post-Doctoral Fellowship for ALS Research Award, designed to encourage and facilitate promising young scientists to enter the ALS field. The award program was founded by the Safenowitz family, through the Greater New York Chapter of The ALS Association, and awards are given in memory of Mr. Safenowitz. Fellows work with a senior mentor and receive extensive exposure to the ALS research community through meetings and presentations. This year’s fellowship award recipients are: Determine the mechanisms of ALS-associated poly (GR) toxicity and screening for therapeutic drugs Dejun Yang, M.D., Ph.D. University of Massachusetts Medical School Worcester, Massachusetts

The potential pathogenic role of dipeptide repeat (DPR) proteins will be investigated by Dr. Yang, under the guidance of Fen-Biao Gao, Ph.D. Dr. Yang has shown that an important signaling pathway called Notch in both fruit flies and C9orf72 patient-derived neurons is disrupted by multiple DPRs in a length-dependent manner. He will now go on to further dissect the mechanisms of toxicity and to screen for FDA-approved therapeutic drugs that mitigate this effect. “I feel extremely honored and grateful to have been chosen as a recipient of the Milton Safenowitz Post-Doctoral Fellowship for ALS Research,” said Dr. Yang. “This two-year fellowship will strongly support our efforts to further investigate the mechanisms of toxicity for C9orf72 repeat expansion, following up my recent exciting findings, and more importantly, screen for therapeutic drugs. The knowledge from our studies will shed new light on the pathogenic mechanisms of ALS and may help future drug development.”

Deciphering the pathogenic mechanisms of C9orf72 dipeptide proteins Marian Hruska-Plochan, Ph.D. Institute of Molecular Life Sciences University of Zurich

An unexpected discovery following identification of the C9orf72 repeat expansion was that the expanded RNA is used for non-canonical peptide synthesis, using the GGGGCC repeat, and its opposite strand, in every possible reading frame, to generate a set of different dipeptide repeat (DPR) proteins. Some evidence indicates that some of these DPRs may be toxic. Dr. Hruska-Plochan, under the mentorship of Magdalini Polymenidou, Ph.D., will investigate toxicity of the DPRs, using ex-vivo mouse organotypic brain and spinal cord slice cultures. He will also determine the most common DPRs in patient brain and spinal cord. “I am thrilled and deeply honored to have been awarded the Milton Safenowitz PostDoctoral Fellowship for ALS Research,” Dr. Hruska-Plochan said. “Thanks to this generous support, I will be able to characterize the role of dipeptide protein aggregation in the pathogenesis of C9orf72 ALS and FTD. Results of my research will hopefully expand our knowledge about the pathological mechanisms triggered by C9orf72 mutation, potentially identifying new therapeutic targets for future treatment strategies.” Continued on page 3


Safenowitz Post-Doctoral Fellowships Continued from page 2

Functional analysis of C9orf72 mutations in ALS via targeted genome engineering Antonia Dominguez, Ph.D. Stanford University Palo Alto, California

The discovery of the C9orf72 mutation, the most common cause of inherited ALS, set off an urgent search to understand the mechanism by which the GGGGCC repeat expansion causes disease. Dr. Dominguez, under the guidance of Lei Stanley Qi, Ph.D., and Steven Finkbeiner, M.D., Ph.D., will use genome editing technology to pursue this important question. She will work with patient and control induced pluripotent stem cells to understand the effects of the mutation, as well as probe the downstream effects of the repeat RNA, specifically testing the consequences of sequestration of important RNA-binding proteins by the accumulated RNA of the repeat. “By understanding the molecular mechanism and regulatory landscape of C9orf72, we hope to define new RNA or proteins as potential druggable targets for treating ALS,” Dr. Dominguez says. “I am honored and grateful to have received The ALS Association Milton Safenowitz PostDoctoral Fellowship,” said Dr. Dominguez. “With this generous support, I will apply emerging CRISPR genome engineering tools to conduct a functional analysis of C9orf72 mutations in the survival of motor neurons derived from induced pluripotent stem cells. The results from these studies have potential to define new targets for future ALS therapeutics.”

Modulating retinoid signaling as a therapeutic approach for ALS David Medina, Ph.D. Barrow Neurological Institute Phoenix, Arizona

New drug targets in ALS are desperately needed, based on an understanding of disease mechanisms. The retinoic acid (RA) signaling pathway represents an intriguing option. RA is a derivative of vitamin A and has been shown to have many important roles in the nervous system, such as neuronal development and neuroregeneration. Furthermore, recent evidence has shown that changes in proteins of the RA signaling pathway are correlated with ALS pathology. Dr. Medina, under the leadership of Robert Bowser, Ph.D., and Rachael Sirianni, Ph.D., will explore the potential of targeting this pathway for therapy development. He will analyze the role of RA signaling in ALS development and progression, and test a novel therapeutic approach using targeted polymeric nanoparticles to deliver adapalene, which increases retinoid signaling, to motor neurons in the spinal cord. Adapalene is an FDA-approved drug used for treating acne and cervical cancers. It has been shown to be neuroprotective in a cell culture system. “I am incredibly honored and grateful to be awarded the Milton Safenowitz PostDoctoral Fellowship for ALS Research. With the support from The ALS Association, I will be able to investigate the role of members of the retinoid signaling family in ALS progression. Furthermore, I will assess the therapeutic potential of modulating retinoid signaling while utilizing novel targeted drug delivery systems.”

Which potassium channel drives ALS motor neuron hyperexcitability? Kasper Roet, Ph.D. Boston Children’s Hospital Boston, Massachusetts

Recently it has been discovered that motor neurons derived from people with ALS who carry one of several disease-causing mutations are hyperexcitable relative to those from control subjects, with higher spontaneous action potential discharge. This hyperexcitability is due to a reduction in activity of delayed-rectifier potassium channels, which may in turn be due to calcium overload and endoplasmic reticulum stress. There are multiple channel subtypes, and Dr. Roet’s work, under the guidance of Clifford Woolf, M.D., Ph.D., will investigate the question of which subtype or -types are responsible. Dr. Roet has developed a new high-throughput thallium-flux assay that very precisely quantifies potassium ion flow across the membrane, which he will combine with channel RNAi knockdown to pursue this research. “I plan to find out which potassium channel drives hyperexcitability of motor neurons that causes increased cell death in ALS. This is important because the identification of this channel could lead to the development of new therapeutics. I will combine advanced single-cell and high-throughput imaging techniques with state-of-the-art human stem cell technology to reach this goal. I would like to thank The ALS Association, the Safenowitz family and the Greater New York Chapter of The ALS Association for their amazing support for this work!”

Genome wide identification of RNA editing dysregulation and upstream regulators in ALS Tao Sun, Ph.D. Stanford University Palo Alto, California

After transcription, some RNAs undergo editing to form a transcript with a sequence that differs from that predicted by genome sequencing. Some evidence indicates this process occurs in the mRNA for a glutamate receptor found in the spinal cord in people with ALS. “Given that millions of new RNA editing sites have been recently identified,” Dr. Sun says, “it is very intriguing to study whether dysregulation of additional editing events also contributes to ALS pathogenesis.” Under the mentorship of Jin Billy Li, Ph.D., and Aaron Gitler, Ph.D., Dr. Sun will perform next-generation sequencing to study the extent of RNA editing in spinal cord and brain tissues from both controls and ALS patients. Unusual RNA editing events will be further studied to test whether they may contribute to ALS pathogenesis. In addition, it is poorly understood how the RNA editing is dysregulated. “I am extremely honored and grateful to receive the Milton Safenowitz Post-Doctoral Fellowship. With this support, I will be able to systematically study the role of RNA editing deregulation in the pathogenesis of ALS, which I hope will be an important step in deciphering the molecular mechanisms of this devastating disease and contribute to the development of better diagnostics and therapeutics for ALS in the future.”




Whole Genome Sequencing of Well Characterized Clinical Samples Pave the Way for Personalized Medicine for ALS Project MinE USA

Discovery of new genes for ALS is the foundation of the search for new treatments. New genes offer new pathways of disease and open the way to finding new targets that can be hit with drugs or other treatments. The ALS Association has provided major funding for two large-scale gene sequencing initiatives: Project MinE and the Center for Genomics of Neurodegenerative Disease. Here, leaders of the two initiatives explain their efforts.

John Landers, Ph.D., Co-Director of Project MinE USA, Associate Professor of Neurology University of Massachusetts Medical School Worcester, Massachusetts

All human diseases have a genetic component; however, the mechanisms by which genes contribute to disease are very diverse. Simple or Mendelian (after the geneticist Gregor Mendel) diseases are caused by mutations in a single gene. Examples of this include cystic fibrosis and sickle-cell anemia. In contrast, the genetics of other diseases are categorized as complex or multi-factorial. These diseases typically involve multiple genes, each of which has variants that can increase the risk of developing the disease. The overall risk of developing a complex disease is the result of these numerous additive genetic factors, as well as any contributing environmental factors. A person harboring too many deleterious risk factors may be pushed beyond a threshold and become affected with the disease. Examples of complex diseases include coronary artery disease and type II diabetes. Interestingly, ALS fits into both of these categories. Approximately 10 percent of all ALS cases are defined as familial, in which more than one family member has been affected with the disease. Familial ALS is classified as a Mendelian disease due to the fact that it is usually caused Continued on page 5 1985: The ALS Association funds study of inherited motor neuron disease

TIMELINE 1869: French neurologist Jean-Martin Charcot identifies ALS

50s: DNA structure solved 50s: Nerve growth factor (NFG) identified–protective, growth promoting factor for nerve cells

1968: SOD1 enzyme identified

70s: Programmed cell death in motor neurons demonstrated





1986: Genes for muscular dystrophy identified

1990: Congress declares the 1990s the “Decade of the Brain”

1989: The ALS Association funds search for a common genetic link to ALS

1990: Growth factor CNTF is found to increase survival of motor neurons

1991: Researchers link familial ALS to Chromosome 21

The ALS Association begins workshops Glutamate transporter shown to be defective in ALS Growth factor BDNF found to increase survival of motor neurons






Whole Genome Sequencing: Project MinE USA

Dr. Jonathan Glass Co-Director of Project MinE Emory University

Continued from page 4

by a single gene mutation. To date, numerous causative genes for familial ALS have been identified, including C9orf72, SOD1 and TDP-43. In fact, we can actual determine the genetic cause in approximately two-thirds of all familial ALS cases. The remaining 90 percent of cases, called sporadic ALS, are thought to be due to multiple genetic risk factors and are thus categorized as a complex disease. Unfortunately, progress in the identification of such risk factors is far behind our success in deciphering familial ALS. Nevertheless, the identification of these risk factors is vital. Knowing which genes are risk factors may reveal new therapeutic targets for drug development, identify new mechanisms leading to disease or possibly help us identify environmental factors that contribute to ALS. The identification of such genetic risk factors is quite challenging. The human genome is three billion base pairs long, and it is filled with variants in its sequence. In fact, if you compare the entire genome of any two people, you will find approximately three million differences in their DNA. Even more remarkably, geneticists have identified more than 100 million DNA variants across the entire population. These variations can have a variety of effects. These variants are what make each of us unique; however, some of these variants are those that may increase our risk for human diseases. Due to the large variation in the human genome within the general population, how can scientists identify those variants that increase ALS risk from those that are benign variants? One strategy is to sequence the entire genomes of a large cohort of people with and without ALS. If we can compare a large enough group of genomes, we will start to identify those variants that are more frequent in ALS patients and somehow contribute to disease. Such a study is no small endeavor. In order to differentiate the risk variants from benign variants, thousands to tens of thousands of genomes are needed. Furthermore, given that the cost to sequence the human genome of a person is approximately $2,000 (a dramatic decrease from the $3 billion for the Human Genome Project due to advances in sequencing technology) and the large computational requirements, this project is too cost prohibitive and labor intensive for any one laboratory.

Recognizing these obstacles, the ALS scientific community has chosen a divide-and-conquer approach. Based on this premise, numerous laboratories across the globe have joined forces for an effort called Project MinE ( The goal of Project MinE as a whole is to sequence 15,000 genomes from ALS patients and 7,500 from healthy individuals. Efforts are currently underway in 16 countries spanning five continents, with teams in each country focuses on raising funds within their respective country to sequence as many genomes as possible. The Project MinE USA effort was kicked off at the end of 2014 through a $1 million donation from The ALS Association. I am working with Dr. Jonathan Glass at Emory University as Co-Directors of Project MinE USA. This has allowed us to sequence nearly 400 genomes so far, and we anticipate another 400 by the end of the year. We have also received help raising funds for Project MinE from volunteers. Most recently, these volunteers organized a charity event in which more than 350 people swam a mile in New York City’s Hudson River, raising more than $450,000 for Project MinE USA. Despite this success in fundraising, Project MinE USA needs more money to be completed. Our efforts are primarily limited by funding. As we receive donations, we immediately turn around and sequence additional genomes. The DNA is ready. We just need the funds. We recognize there are additional sequencing projects ongoing by other groups. However, while on the surface they may seem quite similar, they are all distinct in their approach, each with its own advantages and disadvantages. Most importantly, though, these approaches are complementary to each other. Whenever we identify a new genetic factor, our first course of action is to validate our findings in an independent study. Other ongoing efforts, such as that occurring at the New York Genome Center, are a prime source for such validation. Reciprocally, Project MinE can facilitate the validation of results from other studies. We all have the same goal: find the causes of ALS.

Continued on page 6

A transgenic rat is designed; efforts start on fly model

TIMELINE cont. SOD1 gene mutation (chromosome 21) discovered in familial ALS Trials using glutamate blocker riluzole begin

Animal studies combining CNTF and BDNF demonstrate decreased motor neuron loss GDNF rescues degenerating motor neurons during development in an in vitro experiment



FDA approves riluzole

Toxic properties of the SOD1 enzyme discovered and linked to familial ALS

RNAi discovered by Craig Mello and Andrew Fire

The ALS Association co-sponsors workshop on high-throughput drug screening with NINDS NINDS issues first ever RFA (request for applications) specifically for ALS research





The ALS Association holds scientific workshop on “Environmental Factors and Genetic Susceptibility”

Attention turns to support cells of nerve tissue to find role in ALS Inflammation and programmed cell death gather research interest ALS2 gene (alsin protein) linked to juvenile ALS The ALS Association/NINDS collaborative effort begins screening drugs

Aggressive search for new ALS genes funded by The ALS Association



Scientists complete map of mouse genome Agency of Toxic Substances and Disease Registries awards five grants focused on ALS Department of Defense approves funding for ALS-specific research




Whole Genome Sequencing: NYGC Continued from page 5


NYGC’s Research Program on the Genetics and Genomics of ALS

The ALS Association Research Investigator-Initiated Research Grant Program supports innovative research of high scientific merit and relevance to ALS, offering investigators awards in the following categories: Multi-year Grants: The ALS Association will support research that is projected for periods of up to three (3) years. Funding for multi-year grants is committed for one (1) year only, with noncompetitive renewals conditioned upon results. These applications require strong preliminary data. Awards will be in the amount of up to $100,000 per year.

Hemali Phatnani, Ph.D., Co-Director of CGND Center for Genomics of Neurodegenerative Disease, New York Genome Center New York, New York

Starter Grants: One-year awards for new investigators entering the field of ALS. Alternatively, they can be pilot studies by ALS investigators. These applications do not require strong preliminary data but must emphasize innovation, scientific merit, feasibility and relevance to ALS. The maximum amount awarded is $50,000.

Just about a year ago, The ALS Association announced its commitment to the Center for Genomics of Neurodegenerative Disease (CGND) at the New York Genome Center (NYGC). The CGND’s vision is to establish a framework to apply state-of-the-art genomics and bioinformatics to the study of disease mechanisms in ALS, by building partnerships with clinicians, basic scientists, geneticists and computational biologists.

The Milton Safenowitz Post-Doctoral Fellowship for ALS Research Awards: The maximum amount awarded is $50,000 per year for two (2) years. Eligibility is limited to those who have been a fellow for one year or less.

The CGND’s partners include not only NYGC’s founding members, such as Columbia and Rockefeller, but also ALS centers throughout the Northeast, such as the University of Pennsylvania, Massachusetts General Hospital and Hershey Medical Center at Penn State University, among others. Through the support of The ALS Association, our efforts also synergize with other Association-funded consortia, such as ALS ACT and the GTAC (Genomics Translation for ALS Clinical care) consortium.

Call for abstracts December 1, 2015. To be added to the mailing list for grant notifications email

Through a multidisciplinary collaborative effort that spans multiple ALS centers and combines the efforts of ALS clinicians and scientists, we are using whole genome sequencing to discover and study mutations and mechanisms underlying ALS. Broadly stated, the goals of this consortium are the following: Continued on page 7

TIMELINE cont. Study shows surrounding support cells play key role in ALS Study shows that human embryonic stem cells can be stimulated to produce motor neurons Gulf War study shows that vets deployed to Persian Gulf in 1991 developed ALS at twice the rate of those not deployed there IGF-1 gene therapy study proves beneficial in mice with ALS VEGF gene abnormalities shown to be potential factor in ALS The ALS Association collaborates with U.S. Department of Veterans Affairs to enroll all vets with ALS in registry Early tests of ceftriaxone appear to increase survival in mice with ALS Combination of creatine and minocycline prove more effective together in mouse model than either drug alone

Study implicates smoking as likely risk factor in sporadic ALS Study releases evidence that mitochondrial malfunction may play an important role in ALS Study funded by The ALS Association to find biomarkers in cerebrospinal fluid and blood

Ceftriaxone increases levels of the glutamate transporter GLT1 in a mouse model of ALS First international workshop on frontotemporal dementia discusses link to ALS Stem cells engineered to make GDNF survive when transplanted into rats modeling ALS Early data suggests that mutant SOD1 may be secreted by and may activate microglia Launch of TREAT ALS initiative (Translational Research Advancing Therapies for ALS) to accelerate clinical trials in ALS VEGF increases survival in a rat model of ALS while improving motor

ALS patient samples collected to NINDS ALS Repository Repository samples allow genome analysis for sporadic ALS First TREAT ALS clinical trials funded First TREAT ALS clinical trials begun TDP-43 discovered as a common link in FTD, ALS Chromosome 9 region intense focus for FTD

Stem cell study shows SOD1 mutant support cells can kill any motor neuron ALS U.S. registry efforts gaining ground in Congress Fish model of ALS: Progress reported SOD1 in altered form common to both sporadic and inherited ALS Engineered stem cells making GDNF help motor neurons survive in SOD1 mutant rats First genome screening data published based on NINDS ALS Repository







Whole Genome Sequencing: NYGC

Association Expands its Support Research

Continued from page 6

Continued from page 1

> Integrate whole genome sequencing with RNA sequencing to interrogate relationships between mutations, gene expression and disease mechanisms. RNA sequencing analyses combined with whole genome sequencing will help us to identify how changes in DNA are expressed in the brain and spinal cord, and how this affects the presentation and course of disease.

ciated with specific clinical outcomes. This may ultimately make truly “personalized” medicine possible.

> Design and create ALS models to test effects of mutations in stem cell-derived neurons and in mouse models using state-of-the-art genomic manipulation methods. To study the function of any sequence variants that we identify, and to understand how they affect disease mechanisms, we will collaborate with our research partners to make new models of disease such as iPS cells and mouse models. We can use these models to study, for example, how mutations affect the different cell types that are known to play a role in ALS, such as astrocytes, microglia and oligodendrocytes, all of which are known to affect motor neurons in ALS. Using these models, we will be able to examine regulatory mechanisms affecting the transcriptome, as well as mechanisms underlying intercellular interactions in disease. To further these analyses, we are developing tools to integrate the experimental and computational analysis of large-scale data that includes transcriptomes of specific cell types in the central nervous system, profiles of RNA-binding proteins implicated in disease, and high-resolution imaging. The combination of new deep sequencing methods, sample acquisition, data analysis pipelines and molecular and phenotypic characterizations using new mouse models will provide mechanistic insights that were previously not possible.

> Integrate genomic and clinical data to iden-

tify genetic modifiers of disease onset/ progression/presentation. Our partners’ clinical phenotyping efforts will enable us to sequence well-stratified patient cohorts, so that we can eventually identify mutations that are associated with different forms of the disease, or gene variants that can modify the presentation of the disease and could be further studied to identify pathways for the targeted development of therapies.

> Create and maintain a data warehouse for genomic

data that can be broadly accessed by the academic community. Our sequencing data will be made freely available to the research community. Resource and data sharing is an integral aspect of our efforts, because we want the data that we generate to be as useful as possible to as many researchers as possible. Broad sharing will only accelerate the pace of discovery and therapeutics, which are crucially needed in ALS. Such broad sharing and collaborative efforts are ultimately geared towards making the best use of sequencing data. For example, comparing clinical profiles to genomic profiles can enable us to determine whether specific mutations are asso-

“These large sequencing efforts are absolutely critical to our mission of finding new treatments for ALS,” according to ALS Association Chief Scientist Lucie Bruijn, Ph.D., M.B.A. “As Dr. Landers pointed out, the multiple efforts are different in focus, and therefore, each is likely to offer significant independent value. As well, each has the ability to validate the findings of the other, which is critical for establishing the most robust findings and moving forward on model development and treatment discovery. We are hugely grateful to both teams for their efforts in finding new therapeutic approaches in ALS.”

treatment approach has been well defined. The development of antisense for neurodegenereative diseases can be directly attributed to this program and The Association’s investments. This approach is now being translated for clinical trials for ALS, Spinal Muscular Atrophy (SMA) and Huntington’s Disease by BiogenIdec and Roche. Several new contracts have just been announced and include key areas believed to be important targets for drug development in ALS. Neuroinflammation is believed to contribute strongly to disease progression. Association-supported researchers are pursuing treatments to reduce neuroinflammation, including the use of decoy immune receptors, delivery of cytokines and reducing damage by peripheral macrophages. Other avenues being pursued include stabilizing disease proteins to prevent misfolding, and altering gut microbiota, based on observed changes in a model of the disease. The ALS Association is proud to continue its partnership with NEALS (Northeast ALS Consortium) and the Neurological Clinical Research Institute (NCRI), a nationwide group of clinical researchers and a clinical trials network dedicated to advancing discovery and testing of new therapies for ALS. During its long collaboration with The Association, NEALS and NCRI have grown to become the most extensive clinical trials network in the field of ALS. ALS Association-funded programs that are part of the network include investigator training to ensure standardization between clinical trial sites, pilot trial support and patient/family ambassador education through the Clinical Research Learning Institute (CRLI). “Each part of our research program is designed to dovetail with the rest,” said Lucie Bruijn, Ph.D., M.B.A., Chief Scientist for The ALS Association. “Gene discovery allows us to make new models of ALS, which can provide new insights into pathogenesis and new targets for therapy. Our support of translational research as a whole leads the field to develop new treatments that can be rapidly tested in clinical trials. With the growth of a new generation of highly promising biomarkers, we are poised to make those trials as informative as they can be as we search for treatments to slow the disease.”

Any models that we develop through our collaborative efforts will be made freely available to the research community––this is in fact a condition of any partnerships that we undertake. In addition, the conceptual framework and infrastructure should be widely applicable to other neurodegenerative diseases.

TIMELINE cont. Stem cells generated from ALS patients Discovery of DPP6 in two genomewide association studies in ALS Mutations in TDP-43 linked to familial and sporadic ALS Induced Pluripotent Stem Cell Technology opens up new avenues for ALS

Identification of new gene linked to familial ALS, Fused in Sarcoma (FUS) on Chromosome 16 FDA approval of SOD1 antisense and stem cell trials in U.S.

First patients enrolled for antisense and stem cell trials in U.S.




For more details about the recently funded projects:

Ubiquilin-2 discovery; C9orf72 discovery

March: The ALS Association hosts 2nd Drug Discovery Workshop for ALS September: Researchers find genetic region influencing age at which people develop ALS

February: Identification of C9RAN translated peptide March: First human antisense trial published–– approach safe in people with ALS November: Progress in understanding effects of C9orf72 gene in ALS

March: Mutations in Matrin 3 identified linked to ALS August: ALS Ice Bucket Challenge September: Mutations in mitochondrial gene CHCHD10 linked to ALS October: Mutations in microtubule associated gene TUB4A linked to ALS

July: New studies shed light on TDP-43 disease mechanism August: C9orf72 repeat expansion mutations––disease mechanisms begin to unfold










Ostrow LW, Matunis MJ, Wang J, Sattler R, Lloyd TE, Rothstein JD. The C9orf72 repeat expansion disrupts nucleocytoplasmic transport. Nature. 2015 Sep 3;525(7567):56-61

C9orf72 Mutation Causes Nuclear Transport Defects Three new papers Jovičić A, Mertens J, Boeynaems S, Bogaert E, Chai N, Yamada SB, Paul JW 3rd, Sun S, Herdy JR, show that the C9orf72 mutation causes defects in nuclear transport. The Bieri G, Kramer NJ, Gage FH, Van Den Bosch L, Robberecht W, Gitler AD. Modifiers of C9orf72 studies, by independent labs, all conclude that a major consequence of the dipeptide repeat toxicity connect nucleocytoplasmic transport defects to FTD/ALS. Nat Neuhexanucleotide repeat expansion in the gene is to disrupt transport through rosci. 2015 Aug 26;18(9):1226-9 the nuclear pore, a very large and very long-lived protein complex that regu- NEW FINDINGS IN C9ORF72 ALS lates passage of proteins and RNA across the nuclear membrane. First C9orf72 Mouse Model Recapitulates Human Pathology The GGGGCC repeat expansion in the C9orf72 gene is the most common genetic cause of ALS, accounting for up to 40 percent of familial ALS and 6 Using adeno-associated virus, ALS Association-supported authors expressed percent of sporadic ALS, as well as a large fraction of cases of frontotem- the C9orf72 gene containing 66 repeats throughout the mouse central nerporal dementia. RNA transcripts aggregate into juxtanuclear foci, which vous system. At six months, mice displayed several key features of human have been shown to bind multiple RNA-binding proteins. In addition, non- mutation-related pathology, including RNA foci, dipeptide repeat proteins standard translation of the repeat RNA leads to production of “dipeptide and TDP-43 aggregates, along with neuronal loss and behavioral abnormalirepeat” (DPR) proteins, from both the sense and antisense strands, and ties. The model represents the first murine model of C9orf72-related disease. in each possible reading frame. Some evidence suggests the DPRs may be Chew J, Gendron TF, Prudencio M, Sasaguri H, Zhang YJ, Castanedes-Casey M, Lee CW, Jansen-West K, Kurti A, Murray ME, Bieniek KF, Bauer PO, Whitelaw EC, Roustoxic, through unknown mechanisms. seau L, Stankowski JN, Stetler C, Daughrity LM, Perkerson EA, Desaro P, Johnston A,

Freibaum et al., performed an unbiased screen of about 80 percent of the Overstreet K, Edbauer D, Rademakers R, Boylan KB, Dickson DW, Fryer JD, Petrucelli fly genome and identified 18 modifiers of the degenerative eye phenotype L. Neurodegeneration. C9orf72 repeat expansions in mice cause TDP-43 patholcaused by the repeat expansion. The identified genes encoded components ogy, neuronal loss, and behavioral deficits. Science. 2015 Jun 5;348(6239):1151-4. of the nuclear pore, as well as other genes that regulate transport through the pore. They found an increase in the nuclear/cytoplasmic ratio of RNA in Widespread Transcriptome Changes in C9orf72 Brain fly, human cells in culture and patient tissue. In human patient neurons, the The C9orf72 mutation causes profound changes in the transcription of genes ratio was 35% greater than in controls. in multiple brain regions, according to a study for which two first authors are Zhang et al., following up their earlier work identifying multiple binding recipients of The ALS Association’s Milton Safenowitz Post-Doctoral Fellowpartners for the repeat expansion, showed that binding of RanGAP contrib- ship. Changes include thousands of alternative splicing events and alternauted to neurodegeneration in human induced pluripotent stem cells (iPS tive polyadenylation defects in both cerebellum and frontal cortex. Changes cells), an effect that could be mitigated by overexpression of RanGAP. Ran- were also seen in brain of sporadic ALS, with relatively little overlap in the GAP is an obligate cofactor for maintenance of the cross-membrane Ran affected genes. “Disruption of normal RNA processing is part of the pathoGTPase gradient, essential for nuclear transport. Mislocalization of RanGAP logical signature of C9orf72 ALS and sporadic ALS,” the authors conclude. was seen in iPS cells and in patient tissue. A compound that disrupts the tertiary structure of the RNA expansion, the G quadruplex, reduced the bind- Prudencio M, Belzil VV, Batra R, Ross CA, Gendron TF, Pregent LJ, Murray ME, Overstreet Piazza-Johnston AE, Desaro P, Bieniek KF, DeTure M, Lee WC, Biendarra SM, Davis MD, ing of RanGAP to the RNA and rescued nuclear import defects, suggesting KK, Baker MC, Perkerson RB, van Blitterswijk M, Stetler CT, Rademakers R, Link CD, Dickson DW, that the RNA expansion is directly toxic in C9orf72 disease. Boylan KB, Li H, Petrucelli L. Distinct brain transcriptome profiles in C9orf72-associated and Jovičić et al., working in yeast, also identified nuclear transport proteins as central to disease pathogenesis, but due to DPRs instead. Yeast were engineered to express the full variety of DPRs independent of the repeat expansion. Arginine-containing DPRs were the most toxic, including poly-(prolinearginine). A genomic screen turned up 27 suppressors of poly-(Pro-Arg) toxicity and 35 enhancers. Six of these were members of the karyopherins, nuclear import proteins. Ran GTPase regulators were also identified.

sporadic ALS. Nat Neurosci. 2015 Aug 18;(8):1175-82.

GENES AND MECHANISMS New ALS Gene TBK1 Identified, Linked to Optineurin

Two new studies identify TANK-binding kinase 1 (TBK1) as a causative gene for ALS and frontotemporal dementia. TBK1 binds to optineurin and p62, both of which have been previously linked to ALS. Freischmidt et al. identiTaken together, these studies point strongly to defective nuclear transport fied 8 loss-of-function variants in 13 familial ALS pedigrees. Mutation led to as a central mechanism in C9orf72 ALS. Because TDP-43 depends on Ran- loss of interaction with optineurin in-vitro. GAP for its normal localization in the nucleus, and because TDP-43 accumuA, Wieland T, Richter B, Ruf W, Schaeffer V, Müller K, Marroquin N, Nordin F, Hübers lates cytoplasmically in most forms of ALS, the results may also implicate Freischmidt A, Weydt P, Pinto S, Press R, Millecamps S, Molko N, Bernard E, Desnuelle C, Soriani MH, Dorst nuclear transport defects in non-C9orf72 ALS. J, Graf E, Nordström U, Feiler MS, Putz S, Boeckers TM, Meyer T, Winkler AS, Winkelman J, “These studies, which independently arrive at the same conclusion, show the potential importance of nuclear transport dysfunction in C9orf72 ALS,” said ALS Association Chief Scientist Lucie Bruijn, Ph.D., M.B.A. “They highlight the possibility that transport defects may play a role in other forms of the disease as well. Exploring that possibility will now become a major focus of new research.” Freibaum BD, Lu Y, Lopez-Gonzalez R, Kim NC, Almeida S, Lee KH, Badders N, Valentine M, Miller BL, Wong PC, Petrucelli L, Kim HJ, Gao FB, Taylor JP. GGGGCC repeat expansion in C9orf72 compromises nucleocytoplasmic transport. Nature. 2015 Sep 3;525(7567):129-33 Zhang K, Donnelly CJ, Haeusler AR, Grima JC, Machamer JB, Steinwald P, Daley EL, Miller SJ, Cunningham KM, Vidensky S, Gupta S, Thomas MA, Hong I, Chiu SL, Huganir RL,

de Carvalho M, Thal DR, Otto M, Brännström T, Volk AE, Kursula P, Danzer KM, Lichtner P, Dikic I, Meitinger T, Ludolph AC, Strom TM, Andersen PM, Weishaupt JH. Haploinsufficiency of TBK1 causes familial ALS and fronto-temporal dementia. Nat Neurosci. 2015 May 18;(5):631-6 Cirulli ET, Lasseigne BN, Petrovski S, Sapp PC, Dion PA, Leblond CS, Couthouis J, Lu YF, Wang Q, Krueger BJ, Ren Z, Keebler J, Han Y, Levy SE, Boone BE, Wimbish JR, Waite LL, Jones AL, Carulli JP, Day-Williams AG, Staropoli JF, Xin WW, Chesi A, Raphael AR, McKenna-Yasek D, Cady J, Vianney de Jong JM, Kenna KP, Smith BN, Topp S, Miller J, Gkazi A; FALS Sequencing Consortium, Al-Chalabi A, van den Berg LH, Veldink J, Silani V, Ticozzi N, Shaw CE, Baloh RH, Appel S, Simpson E, Lagier-Tourenne C, Pulst SM, Gibson S, Trojanowski JQ, Elman L, McCluskey L, Grossman M, Shneider NA, Chung WK, Ravits JM, Glass JD, Sims KB, Van Deerlin VM, Maniatis T, Hayes SD, Ordureau A, Swarup S, Landers J, Baas F, Allen AS, Bedlack RS, Harper JW, Gitler AD, Rouleau GA, Brown R, Harms MB, Cooper GM, Harris T, Myers RM, Goldstein DB. Exome

Acknowledgement: Richard Robinson, Science Writer sequencing in amyotrophic lateral sclerosis identifies risk genes and pathways. Science. 2015 Mar 27;347(6229):1436-41

TDP-43 Suppresses Cryptic Exons The normal function of TDP-43 includes suppression of cryptic exons, according to a new study supported by The ALS Association. Cryptic exons are intronic sequences not normally used to make protein, which introduce frameshifts or stop sequences when included in mRNA. Targeted deletion of TDP-43 in mouse embryonic stem cells led to inclusion of cryptic exons, causing nonsense-mediated decay and reducing cell viability. Suppression of cryptic exons was impaired in tissue from individuals with ALS-FTD, suggesting that loss of TDP-43 function contributes to RNA dysregulation in the disease. Ling JP, Pletnikova O, Troncoso JC, Wong PC. Neurodegeneration. TDP-43 repression of nonconserved cryptic exons is compromised in ALS-FTD. Science. 2015 Aug 7;349(6248):650-5

Nonsense-mediated Decay Implicated in FUS/TDP-43 ALS The harmful effects of FUS or TDP-43 mutations can be mitigated by increasing expression of human up-frameshift protein 1 (hUPF1), according to this ALS Association-funded study. hUPF1 is a master regulator of nonsensemediated decay, and inhibition of NMD prevented rescue by hUPF1, suggesting that NMD may be impaired in ALS caused by either FUS or TDP-43. Barmada SJ, Ju S, Arjun A, Batarse A, Archbold HC, Peisach D, Li X, Zhang Y, Tank EM, Qiu H, Huang EJ, Ringe D, Petsko GA, Finkbeiner S. Amelioration of toxicity in neuronal models of amyotrophic lateral sclerosis by hUPF1. Proc Natl Acad Sci U S A. 2015 Jun 23;112(25):7821-6

No Effect of Prior Head Injury on Pathology or Progression A history of head injury does not influence either pathology or disease progression in ALS, according to this study of 100 ALS cases, including 24 with a report of head injury prior to diagnosis. No effect was seen on rate of decline as measured by the ALSFRS score. In 47 autopsy cases, including 9 with a history of head injury, no difference was seen in frequency of tauopathy, brain TDP-43 pathology or Alzheimer’s disease pathology. The results contribute to the ongoing debate about the role of prior head injury in ALS pathogenesis. Fournier CN, Gearing M, Upadhyayula SR, Klein M, Glass JD. Head injury does not alter disease progression or neuropathologic outcomes in ALS. Neurology. 2015 Apr 28;84(17):1788-95

THERAPY DEVELOPMENT AND BIOMARKERS Half-life Determine of SOD1 Provides Foundation for Antisense Trial For the first time, researchers have determined the half-life of SOD1 protein in human cerebrospinal fluid, providing critical data for testing the efficacy of antisense therapy against the mutant SOD1 gene. In the study, supported by The ALS Association, the half-life of the normal protein was approximately 25 days; turnover of the mutant protein will be determined in future studies. Antisense therapy has been shown to be safe in people with ALS due to SOD1 mutation. “These data will be used to help decide when and how often we will need to measure remaining mutant SOD1, in order to judge the effectiveness of antisense treatment,” said Lucie Bruijn, Ph.D., M.B.A., Chief Scientist for The ALS Association. “In that light, this study represents true progress toward a trial.” Crisp MJ, Mawuenyega KG, Patterson BW, Reddy NC, Chott R, Self WK, Weihl CC, Jockel-Balsarotti J, Varadhachary AS, Bucelli RC, Yarasheski KE, Bateman RJ, Miller TM. In-vivo kinetic approach reveals slow SOD1 turnover in the CNS. J Clin Invest. 2015 Jul 1;125(7):2772-80 National Office

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