Research ALS Today Fall 2014

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Four Major Initiatives Diverse Portfolio Grant Call ALS Research–C9orf72 Safenowitz Fellows Journal News

FALL 2014

When the “Ice Bucket Challenge” went viral this past summer, it not only raised awareness worldwide about amyotrophic lateral sclerosis (ALS), it also raised more than $100 million in new funds for research to support the fight against the disease. That overwhelming response makes possible four major new initiatives being supported by The ALS Association with $22.6 million of grants. Even as these projects move forward, The Association is in the process of gathering stakeholder feedback to develop an expanded strategic plan, including additional funding for research towards a variety of initiatives including solicitation for proposals from the research community. “We recognize the sense of urgency felt by people living with the disease and their families, and our number one commitment is to make decisions that get treatments to patients in the fastest way possible,” said Barbara Newhouse, President and CEO of The ALS Association. “Our roadmap to treatments involves collaboration with other ALS organizations and with industry, university investigators, government agencies, pharmaceutical and biotech companies and other nonprofit organizations committed to the fight against ALS.”


ALS Accelerated Therapeutics (ALS ACT) In partnership with General Electric, a leading global research and technology company, and the Neurological Clinical Research Institute at Massachusetts General Hospital (MGH), ALS ACT will enact a multi-pronged approach to expediting clinical trials in ALS. Efforts will include (1) development of neuroimaging tools as potential biomarkers for ALS progression, a key unmet need in trials; (2) development of therapeutic approaches to decrease production of misfolded proteins within motor neurons and reverse neuroinflammation, two major contributors to the disease process; (3) a challenge grant program to overcome key roadblocks in the search for therapies; and (4) support for phase IIA pilot clinical trials using biomarkers. In addition, ALS ACT will strengthen ongoing collaborative efforts in support of clinical trials, including NeuroBANK, a central repository for clinical research data in ALS, and the NEALS (Northeast ALS Consortium) Biorepository. Initially established through the TREAT ALS NEALS Clinical Trials Network, NeuroBANK will host, curate and disseminate proteomic, genomic and clinical data. Cooperative synergies among ALS ACT, New York Genome Center, Continued on page 2

The ALS Association Research Institute


Centered around people living with ALS, the Institute builds on the TREAT ALSTM platform and provides open source access for the global ALS community through its new and planned cooperative alliances.

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Four Major Initiatives Great Ideas Accelerate Meaningful Therapies This is an extraordinary time for ALS Research. The Ice Bucket Challenge this past summer has brought global attention to a disease many had never heard of before. In addition, the significant outpouring of funds for research provides the field with a tremendous opportunity to make significant impact towards treatments and a cure. The Association’s initial investments focus on the urgent need to bring teams together to generate data from people living with the disease to better understand the heterogeneity, disease process and pathways to intervene with treatments. Three cooperative alliances have been formed, ALS ACT, Neurocollaborative and the New York Genome Center for ALS Research. In addition we are pleased to be an international partner to Project MinE.

Lucie Bruijn, Ph.D., M.B.A. Chief Scientist The ALS Association

Over the next year, several opportunities will be developed for the broader community to propose important research directions. We will continue to support new investigators, young scientists and clinicians through our fellowship programs. Featured in this issue are this year’s recipients of The Milton Safenowitz Post-Doctural Fellowship program. These investigators provide promise now and in the future to find the answers to ALS.

While we continue to support early stage research, we will also forge additional partnerships with the industry to ensure that great ideas move along the development path. Acceleration of this process promises to bring meaningful therapies to patients in the near future. It is encouraging to see the pace of research into C9orf72, a mutation that accounts for 10 percent of ALS and 40 percent of frontotemporal dementia. Antisense oligonucleotide therapy, proven to be safe in a subset of ALS patients and now in clinical trial for Spinal Muscular Atrophy, is in development through academic efforts and in partnership between ISIS Pharmaceuticals and Biogen. The year ahead promises to be an exciting one for clinicians and scientists dedicated to ALS research. The ALS Association looks forward to working in collaboration with teams globally to reach our common goal of a cure for ALS. –Lucie Bruijn, Ph.D., M.B.A.

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Neurocollaborative and Project MinE will increase the quantity, and most importantly, the value of data available for ALS research. Merit Cudkowicz, Co-Chair of NEALS and Chief of Neurology and the ALS Program at MGH, notes that these efforts will accelerate diagnosis, speed development of new treatments for people with ALS, and break down roadblocks to finding cures for people with ALS.” Investigators: Robert J. Brown, Jr. M.D., D.Phil., University of Massachusetts; Merit Cudkowicz, M.D., MSc, Massachusetts General Hospital; Stanley H. Appel, M.D., Houston Methodist Hospital System; and Clive Svendsen, Ph.D., Cedars-Sinai Medical Center; Nadeem Ishaque, Ph.D., GE Global Research; and Tom Gentile, Senior Vice President, General Electric

INITIATIVE 2: Consortium for Genomics

of Neurodegenerative Disease The mission of the Consortium for Genomics of Neurodegenerative Disease is to harness stateof-the-art genetic, genomic and bioinformatics tools to gain insights into motor neuron disease mechanisms and to use this knowledge to identify new diagnostic and therapeutic approaches. Funding by The ALS Association will further the work of the center as it combines the expertise of 16 world-class scientists and its state-of-the-art whole-genome sequencing facility to discover new genomic contributors to ALS, discoveries that can then serve as the starting point for development of novel therapies. As part of its mission to bring this type of “big data” approach to ALS discovery, the center will serve as a repository for all published and unpublished DNA and RNA sequencing data that pertains to ALS and provide tools to the larger ALS community to analyze, curate and query the data. Investigators: Robert B. Darnell, M.D., Ph.D., Rockefeller University; Hemali Phatnani, Ph.D.; Merit Cudkowicz, M.D., MSc, Massachusetts General Hospital; Robert J. Brown, Jr. M.D., D.Phil., University of Massachusetts Medical School; Virginia Lee, Ph.D., University of Pennsylvania; John Q. Trojanowski, M.D., Ph.D., University of Pennsylvania, Alex Sherman, Massachusetts General Hospital; Susan Solomon; and New York Stem Cell Foundation, Tom Jessell, Ph.D., Columbia University

INITIATIVE 3: Neurocollaborative

The Neurocollaborative will employ a threepronged approach, drawing on the unparalleled expertise of the three associated labs. Donald Cleveland, Ph.D., of University of California, San Diego, will spearhead the development of antisense therapy against the C9orf72 gene, the most common genetic cause of ALS. Steven Finkbeiner, Ph.D., of the Gladstone Center at University of California, San Francisco, will further develop robotic technology for screening drugs in motor neuron cell culture. Targets include reducing protein misfolding and increasing misfolded protein clearance mechanisms, both key problems in ALS. Clive Svendsen, Ph.D., of Cedars-Sinai Medical Center in Los Angeles, will develop the Stem Cell and Motor Neuron Core Facility to create clinical-grade induced pluripotent stem (iPS) cell lines, which will be openly shared with the ALS research community. iPS cells have emerged as a key research tool and potential source of therapeutic cells in ALS and iPS cells are a key source of motor neurons for drug discovery efforts, such as those at the Gladstone Center. Investigators: Don Cleveland, Ph.D., University of California, San Diego; Steve Finkbeiner, M.D., Ph.D., Gladstone Institute, University of California, San Francisco; Clive Svendsen, Ph.D., Cedars-Sinai Medical Center; and collaborators Martin Marsala, M.D., University of California, San Diego; and Brian Kaspar, Ph.D., Children’s Hospital and Ohio State University

INITIATIVE 4: Project MinE

Project MinE is a global collaboration with the goal to sequence the genomes of at least 15,000 people with ALS to discover new genes that affect ALS risk. Discovering these variants and understanding how they contribute to disease––or protect against it––is likely to lead to novel approaches to ALS therapies. Funding by The Association will bring Project MinE to the United States, with a goal of sequencing 1,000 Americans with ALS. U.S. Investigators: John Landers, Ph.D., University of Massachusetts, Worcester; Jonathan Glass, M.D., Emory University. International partners include the Netherlands, United Kingdom, Ireland, Spain, Portugal and Belgium.


Diverse Portfolio of ALS Research In addition to the major new initiatives made possible through Ice Bucket Challenge donations, The ALS Association will distribute more than $3.4 million to individual scientists in seven countries and across the United States for new research. These awards, which support 21 new projects, are part of The Association’s Translational Research Advancing Therapies for ALS (TREAT ALS) portfolio, a diverse portfolio of ALS research to find treatments and a cure for the disease. Grants are awarded in each of the major focus areas that form the basis of the TREAT ALS portfolio.

mutation may cause disease: toxicity of accumulated RNA made from the gene expansion; production and aggregation of unusual proteins (C9RAN proteins) encoded by the expansion; alteration of nuclear transport; alteration of epigenetic markers and thus gene expression; and reduced levels of normal C9orf72 protein. RAN (repeat-associated non-standard translation) proteins have emerged as novel and potentially important toxic entities in multiple neurodegenerative diseases.

Models and Mechanisms Disease models are critical for understanding how ALS begins and how it progresses. Funded scientists will create Understanding ALS Genes The discovery of a a series of genetic models of ALS using the gene for ALS is the beginning, not the end, of the search for new treatments. Researchers will be roundworm C. elegans, whose well-described investigating how ALS genes contribute to disease developmental genetics should help researchers to and using that fundamental information to develop group ALS disease genes into functional pathways. Other researchers will investigate the important therapies. Recently discovered genes include FUS, contributions to disease made by astrocytes and TDP-43, and C9orf72. The normal functions of oligodendrocytes to motor neuron dysfunction. both FUS and TDP-43 include RNA and DNA binding, while the pathogenic mutation of C9orf72 While genes are clearly implicated in some forms of ALS, and likely contribute to many more, the leads to the accumulation of toxic RNA, strongly suggesting that disruption of RNA metabolism is a large proportion of ALS cases have no known genetic cause, suggesting a role for environmental common mechanism across many forms of ALS. factors in the disease. Funded scientists will pursue FUS and TDP-43: Funded researchers will be the possibility that two aquatic toxins, betadeveloping and sharing new FUS mouse models methylamino-l-alanine, or BMAA, from an aquatic and studying the effects of the mutant protein on motor neurons. Scientists will be asking how muta- bacterium, and methyl mercury, a byproduct of aquatic ecosystems, may contribute to disease, tions in TDP-43 impair the cell’s ability to repair through exploration of geographic distribution its DNA when it breaks and examining TDP-43 of ALS cases in northern New England. aggregates, which occur in most forms of ALS

and may spread from cell to cell, propagating the disease. In addition, scientists will examine the role of TDP-43 in another genetic form of ALS, caused by mutations in the Profilin1 gene. The team will investigate whether mutant Profilin 1 toxicity depends on alteration in TDP-43 functions in motor neurons.

C9orf72: Mutation in the C9orf72 gene is the most common genetic cause of ALS. Funded researchers will explore the contributions of five different potential mechanisms by which the

Biomarkers A biomarker is any measurable

substance that can be used to diagnose disease, follow its progression, or determine its response to therapy. Finding biomarkers for ALS is critical for speeding clinical trials and finding treatments that can alter the progression of disease. One group of scientists will further develop a promising urinary biomarker, by testing urine samples over time in a large number of people with ALS, including people with presymptomatic forms of confirmed genetic disease. Another group will investigate energy metabolism in fibroblasts from people with

NEW GRANT CALL! 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 $80,000 per year. 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 $40,000. 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. Request an abstract form for any of these categories from You will be notified within two-to-three weeks of the abstract submission due date whether you are eligible to submit a full application. See schedule below. Grant Schedule: Call for Abstracts December 1, 2014 Abstracts Due January 13, 2015 Request for Full Proposal January 30, 2015 Full Application Due March 2, 2015 Award Announcements July 2015 Funding Commences August 1, 2015

ALS, based on studies showing disease-related changes in this property. These studies aim at establishing fibroblast metabolism as a predictive factor for the evolution of ALS, and most importantly they could help predict the individual responsiveness to therapies. Stem Cells The ALS Association is a leading funder of stem cell-related ALS research.

Funded scientists will deliver stem cells producing growth factors to the muscles of the ALS rat, to determine whether these cells can improve upon established methods for delivering growth factors to muscle. The team will also generate muscle cells using stem cells derived from people with ALS and use them for disease modeling and drug screening. Therapy Development The ultimate goal of all research funded by The ALS Association is to develop therapies to halt the disease. While we still have much to learn about the basic mechanisms of ALS, many important leads are currently being explored to bring new therapies to trial. Scientists funded this year will screen for drugs to stabilize neurofilaments, based on work showing disruption of these cytoskeletal proteins in the disease. The work will be done in motor neurons derived from induced pluripotent stem cells (iPS cells) from people with ALS. Development of ALS iPS cell lines has been significantly supported by The ALS Association since the discovery of this technology.

Other treatment approaches being funded this year include delivery of glial cell-derived neurotrophic factor (GDNF) growth factor using a proprietary delivery system to overcome previous problems with brain and spinal cord penetration of this therapeutic molecule; development of gamma-secretase modulators; and replacement of cerebrospinal fluid with new fluid enriched with neuroprotective molecules.




For ALS Research–


A pathological hallmark of the novel C9orf72 mutation are so-called RNA foci (visualized in red dots), shown here to be present in iPS motor neuron nuclei.

harles Dickens’ A Tale of Two Cities describes the political and economic situation in London and Paris leading up to the French Revolution. The themes of duality and revolution are somewhat emblematic of the current landscape in ALS research. “It was the best of times, it was the worst of times”––symbolizing the desperate need for new therapeutics for people with ALS at a time of surging enthusiasm and engagement in the scientific ALS community since the discovery in 2011 of the highly prevalent mutation in the C9orf72 gene, as well as the technological advancements of adult pluripotent stem cells as a unique tool to study ALS disease pathogenesis. Combining genome analyses data obtained worldwide, the hexanucleotide GGGGCC (G4C2) repeat expansion of C9orf72 has been identified as the cause of approximately 36 to 46 percent of familial and up to eight percent of sporadic ALS cases, making this the most common genetic cause of ALS to date. Additionally, this same mutation was also found to be highly prevalent in a type of dementia known as “frontotemporal dementia” (FTD). “Repeat expansion” describes the repeat of a nucleotide sequence (here G4C2) over hundreds or thousands of times in a patient’s genome, while a healthy individual only has between 20 to 30 repeats. Due to the high prevalence of mutant C9orf72, pre-clinical efforts in identifying therapeutic targets and developing novel therapeutics for this mutation are being aggressively pursued in the hope of providing a desperately needed disease-modifying treatment for ALS patients and

other populations affected by the C9orf72 mutation. In addition to the development of novel drugs, efforts are also underway to provide so-called biomarkers, which are designed to allow clinical researchers to quickly and non-invasively monitor the biological activity of a given drug in each patient during a clinical trial. In other words, these biomarkers will determine whether a patient shows the expected biological drug-to-target response before measuring any clinical efficacy, which in the case of ALS usually takes months to be noticable. Three major mechanisms have been proposed to explain mutant C9orf72-mediated ALS disease progression: (1) C9orf72 protein loss-of-function; (2) toxic G4C2 repeat RNA gain-offunction and (3) toxic repeat-associated non-ATG (RAN) translation peptides. Let’s see what kind of progress has been made over the last three years to better understand these possible disease mechanisms, and most importantly, let’s find out how those studies have enabled us to move a step closer towards the development of novel drugs and/or biomarkers for future ALS clinical trials. C9orf72 protein loss-of-function has been proposed as a potential disease mechanism based on the reduced levels of C9orf72 RNA found in patient tissue. This type of deficiency is also known as haploinsufficiency. So-called epigenetic changes are one of the mechanisms suggested to explain the loss of C9orf72 transcripts, similar to what is known for other Continued on page 5

1985: The ALS Association funds study of inherited motor neuron disease

TIMELINE 1869: French neurologist Jean-Martin Charcot identifies ALS

Rita Sattler, Ph.D. Department of Neurology and Brain Science Institute Johns Hopkins University School of Medicine Baltimore, Maryland

Green = neuronal marker MAP2 Blue = nuclear marker DAPI

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

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



C9orf72 Continued from page 4

neurological disorders characterized by RNA repeat expansions, such as FragileX syndrome. Epigenetic changes result in alterations in gene expression that do not involve changes in somebody’s DNA sequence. Those detected in C9orf72 patient tissue include altered methylation (addition of a methyl group to specific DNA nucleotides) of specific regions along the DNA strand (CpG islands) as well as on major protein complexes of the chromosome structure, known as histones. These altered methylation states interfere with normal gene expression and can therefore lead to reduced levels of the target protein, in this case C9orf72. T o test whether loss of C9orf72 protein is responsible for disease pathogenesis, C9orf72 expression was inhibited in zebrafish, mice and cultured human induced pluripotent stem cells (iPSCs). While the loss of C9orf72 in zebrafish and worms leads to motor neuron deficits, reducing the levels of C9orf72 in adult mice or cultured human cells did not support the protein loss-of-function mechanism as the major contributor to C9orf72 disease pathogenesis. Therefore, until an allelic knock down of C9orf72 is achieved for the whole lifespan of an animal, the role of C9orf72 protein and its loss during disease progression remains controversial. Consequently no efforts have been made yet to target C9orf72 haploinsufficiency for therapeutic intervention. The presence of a toxic G4C2 repeat RNA gain-of-function as a mechanism in C9orf72 pathogenesis is strongly supported by numerous studies performed on postmortem patient autopsy tissue as well as human patient-derived iPSCs. The G4C2 repeat expansion leads to the accumulation of repeat RNA (so-called RNA foci) in both the nucleus as well as the cytoplasm of brain cells (neurons and glial cells) in C9orf72 patient tissue and patient iPSCs. Due to its structural arrangement, these RNA foci sequester RNA-binding proteins, which under normal healthy conditions are responsible for fundamental RNA-processing events within cells. The binding and sequestration to repeat RNA foci leads to a loss-of-function of the sequestered RNA-binding proteins, which in turn causes dysfunctional RNA metabolism and alterations in essential molecular processes such as RNA editing, RNA splicing and micro RNA biogenesis. In simpler terms, the long repeats of G4C2 act like a spider web to which certain molecules get stuck; those molecules then cannot do their normal jobs in the cell, and as a result the cell is defected and more susceptible to cellular stressors. number of RNA-binding proteins have been identified to specifically bind to the G4C2 repeat A RNA, and it is only a matter of time before specific downstream pathways will be proposed to confirm a contributing role in C9orf72 pathogenesis. Interestingly, RNA foci are also formed due to the accumulation of the bidirectionally transcribed (generated from the opposite direction) antisense strand to G4C2, which is known as C4G2. No antisense-specific RNA-binding proteins have been identified yet, but one would expect to find similar sequestration events.

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

The formation of toxic repeat-associated non-ATG (RAN) translation peptides represents the third proposed mechanism leading to C9orf72 pathogenesis. Generally, RNA is translated into proteins initiated from an ATG start codon. These peptides, however, are formed due to translation of RNA in an unusual ATG-independent manner, leading to the generation of five different polypeptides composed of repeating di-amino acids, which form pathological inclusions in C9orf72 patient tissue and cultured human iPSCs. Recent studies in cultured cells and fly models strongly support the hypothesis that these RAN translation di-peptides are toxic and therefore play a significant role in C9orf72 disease pathogenesis. The question is, have any of these mechanistic studies helped with the development of novel ALS therapeutics? And the answer is: Yes! Significant efforts are underway to target the G4C2 (and the bidirectional C4G2) repeat expansion pharmacologically with the goal of developing a therapeutic intervention for C9orf72 patients. One approach is to block or eliminate the repeat expansion using antisense oligonucleotides (ASOs), similar to what is being developed for repeat expansions in Huntington’s Disease as well as Myotonic Dystrophy. Treating human C9orf72 fibroblasts and iPSCs with ASOs leads to a reduction in the number of RNA foci, as well as the loss of RNA-binding protein sequestration. In addition, other cellular and molecular disease phenotypes that were identified in human motor neurons differentiated from iPSCs were also rescued with ASO treatment, strongly suggesting that this therapeutic approach presents a promising novel drug development for ALS. In addition to ASO, small molecule compounds designed to bind to the repeat RNA have been shown to reduce RNA foci and RAN translation di-peptides in cultured human cells, offering an alternative therapeutic approach for C9orf72. Finally, RAN translation di-peptides have been shown to be present in human cerebral spinal fluid and are therefore a strong candidate for the development as a biomarker to monitor drug efficacy in clinical trials. In conclusion, one can indeed say that “it is the best of times” for ALS research, given the incredible progress that has been made in regards to the identification of disease mechanisms and the development of therapeutic interventions within only three years of the discovery of the C9orf72 mutation. Due to the high number of C9orf72-positive patients, these therapeutic interventions will be beneficial for a large ALS patient population. In addition, the use of biomarkers during clinical trials shows great improvement over past clinical trial designs. Most importantly, though, the efficiency of scientific investigation for this specific mutation could set the stage for future mechanistic studies for other mutations in ALS as well as sporadic ALS cases, thereby providing potential therapies to more subpopulations of ALS patients.

RNAi discovered by Craig Mello and Andrew Fire

A transgenic rat is designed; efforts start on fly model

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

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

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

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A central part of The ALS Association’s research strategy is to foster the early careers of promising young researchers who are New considering the ALS field. That effort is supported through offering The Milton Safenowitz Post-Doctoral Fellowship for ALS Research Awards. Founded by the Safenowitz family through the Greater New York of The ALS Association and in memory of Mr. Safenowitz, who died of Safenowitz Fellows Chapter ALS in 1998, these two-year fellowships provide support for young scientists under Focus on Gene-related Defects the mentorship of senior researchers in internationally recognized laboratories.

Pathomechanisms of C9orf72 in real time

Characterizing the contributions of epigenetic changes in c9FTD/ALS

Philip McGoldrick, Ph.D. University of Toronto Canada A repeat expansion in C9orf72 is the most common cause of ALS and frontotemporal dementia (FTD), which are now recognized as a spectrum of the same underlying disease mechanism. Currently three pathways have been suggested to contribute to disease in C9orf72 patients; (1) RNA toxicity, associated with the formation of nuclear RNA foci composed of the repeat expansion; (2) expression and aggregation of proteins generated from the repeat expansion; and (3) reduced C9orf72 protein levels. Dr. McGoldrick will be testing the hypothesis that each potential pathomechanism contributes to disease. Using cultured motor neurons and cortical neurons, the cell types affected in disease, he and his colleagues will test whether they act alone or in concert with one another. In a novel set of experiments, they plan to use live cell imaging to directly examine RNA foci and proteins formed from the repeat to examine their dynamics in neurons to determine how they may be toxic. From these experiments, they aim to separate the different toxicities associated with these mechanisms. Importantly, they plan to do this in

both motor neurons and cortical neurons to see whether differences in these cell types may reflect disease vulnerability. This knowledge will provide important information on how the repeat expansion causes disease, allowing development of therapeutic strategies to abrogate these effects.

“I am incredibly excited to have received The Milton Safenowitz Post-Doctoral Fellowship,” said Dr. McGoldrick. “This award will allow me to examine the mechanisms and dynamics of C9orf72-mediated neurodegeneration in real time, which will provide insight into how these processes affect neurons and contribute to disease.”

Veronique Belzil, Ph.D. Mayo Clinic Jacksonville, Florida ALS and frontotemporal dementia (FTD) are two devastating conditions seen together in up to 50 percent of individuals affected with either disease. ALS and FTD are believed to result from the same or overlapping defective biological processes, with recent findings demonstrating that a mutation in the C9orf72 gene is present in a significant number of ALS and FTD patients. Much effort is now being dedicated to identifying how the mutation impairs biological processes, leading to reduced gene expression levels and abnormal cell functioning. Dr. Belzil and colleagues recently showed that epigenetic changes, or modifications in gene expression that occur independently of the gene’s structure, are responsible for the abnormal reduction in C9orf72 gene expression. They also demonstrated these epigenetic changes, which are unique to patients carrying the mutation, are detectable in blood. In the current study, they aim to generate a full epigenetic and expression profile of C9orf72 mutants, evaluate whether epigenetic variation also contributes to mRNA-mediated toxicity, and assess whether these changes may be used as

biomarkers of disease. Considering that epigenetic variation, a common theme in neurodegeneration, can be targeted by therapeutics, this study may be very important to the future development of treatments for ALS and FTD.

“I am tremendously grateful to The ALS Association and The Milton and Marilyn Safenowitz Family Foundation for this award,” Dr. Belzil said. “Their generosity will support our efforts to establish a completely new paradigm for understanding the causes of ALS with the aim of providing biological evidence that epigenetic modifications induced by environmental factors may play a central role in disease. Such studies will guide the development of future ALS therapeutic strategies.” 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

2003 2004 2005 2006 2007


Safenowitz Fellows

The role of nuclear transport defects in the pathogenesis of ALS/FTD

Continued from page 6

Ke Zhang, Ph.D. Johns Hopkins University Baltimore, Maryland

Contribution of protein aggregation in the pathogenesis of ALS: seeding, spreading and toxicity Florent Laferriere, Ph.D. University of Zurich Switzerland ALS, like other neurodegenerative diseases such as Alzheimer’s or Parkinson’s disease, is associated with the accumulation of misfolded proteins in neuronal and glial cells in the central nervous system. The major misfolded proteins linked with ALS pathology are SOD1, FUS/TLS and TDP-43. The latter, a DNA/RNA-binding protein, is the main component of cytoplasmic inclusions in almost all sporadic cases of ALS (of all ALS), accompanied by its nuclear depletion. Moreover, mutations in this protein are associated with inherited ALS, and the affected neurons of those with the disease present abnormal localization and aggregation of TDP-43. This protein has an intrinsically high propensity to aggregate and has the ability to propagate its misfolding and aggregation, in a prion-like manner. Despite intensive research, the contribution of TDP-43 aggregation in the pathogenesis of ALS remains poorly understood. To shed light to this crucial issue, Dr. Laferriere and colleagues will separate the TDP-43 aggregates present in ALS patients’ brains according to their size. Then they will be able to determine the relationship between protein aggregation and its pathogenic properties.


“ I am extremely honored and grateful to receive a Milton Safenowitz Post-Doctoral Fellowship for ALS Research,” said Dr. Laferriere. “Support from this award will allow me to characterize the role of protein aggregation in the pathogenesis of ALS, which we hope will contribute to a better understanding of disease mechanisms, and possibly represent an asset to future therapy development.”

Nuclear transport is tightly controlled to regulate the entrance to and exit from the nucleus of biological molecules. The precise regulation of such processes is essential for physiological functions and is disrupted in many human diseases, including neurodegenerative disease. Dr. Zhang and colleagues’ recent data show that nuclear transport is severely disrupted in the most common inherited form of ALS, caused by mutations in the C9orf72 gene. Using a fruit fly model, they have found that the machinery that drives molecules to enter the nucleus in neurons is defective and appears to be due to abnormal function of a protein called RanGAP. This leads to redistribution of proteins that are very important for the nerve cells to survive. The team’s goal is to understand why and how

such problems occur in ALS and whether these abnormalities can be corrected by drug therapies. They will first address the key molecular mechanisms and then will test drugs that potentially keep these proteins inside the nucleus.

“I am extremely honored and grateful to be a recipient of The Milton Safenowitz Post-Doctoral Fellowship,” Dr. Zhang said. “This generous support will allow me to study fundamental protein transport processes, a potentially important target for new ALS treatments.” This study is generously funded by the Greater Philadelphia Chapter of The ALS Association.

Characterizing the pathogenic role of TDP-43 in PFN1-linked ALS Claudia Fallini, Ph.D. University of Massachusetts Medical School Worcester, Massachusetts Mutations in the gene encoding Profilin1 (PFN1) are the cause of ALS in one to three percent of familial cases. Mutant PFN1 protein aggregates in the cell body of motor neurons. Dr. Fallini and colleagues have found that PFN1 sequesters TDP43 into these aggregates. Mutations in the gene encoding TDP-43 itself are a cause of ALS, and the aggregation of the TDP-43 protein is a common phenomenon in degenerating motor neurons in both sporadic and familial ALS. Together, these observations suggest that TDP-43 may be responsible for motor neuron degeneration in PFN1-associated ALS. In this project the team will investigate the link between mutations in PFN1 and the

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.




aggregation of TDP-43 and whether mutant PFN1 toxicity depends on alteration in TDP-43 functions in motor neurons. The results from this project will help the understanding of the processes that are altered in ALS motor neurons, thus paving the way toward the identification of therapeutic targets for the treatment of this disease.

“I am very grateful and honored to be a recipient of The Milton Safenowitz Post-Doctoral Fellowship for ALS Research,” Dr. Fallini said. “Thanks to the support from The ALS Association, I will be able to investigate the pathogenic effects of two distinct ALS genes, identifying common pathways that are altered in ALS motor neurons. I am hopeful that results from my research will shed light on the pathogenesis of ALS, thus opening new avenues for the identification of therapeutic targets for the treatment of this disease.”

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 September: Mutations in mitochondrial gene CHCHD10 linked to ALS October: Mutations in microtubule associated gene TUB4A linked to ALS








JOURNAL NEWS C9orf72 RAN Translation Proteins: Toxic, Targetable, and Potentially a CSF Biomarker

and smaller, allowing us to test a therapy much more rapidly. The particularly exciting aspect of the C9RAN proteins as biomarkers is that they are a direct consequence of the disease-causing entity. Being able to measure a RAN translation proteins from the C9orf72 gene mutation may be toxic, can treatment’s effect on them should not only give us a potentially valuable be targeted with small molecules, and are present in the cerebrospinal fluid biomarker for a clinical trial, but also give us insight into the pathogenesis of people with ALS due to mutation in the gene, according to new studies. of the disease.” Expansion of a GGGGCC repeat in the C9orf72 gene is the most common Su Z, Zhang Y, Gendron TF, Bauer PO, Chew J, Yang WY, Fostvedt E, Jansen-West K, Belzil VV, Degenetic cause of ALS. In RNA form, the repeat, ranging up to thousands of saro P, Johnston A, Overstreet K, Boeve BF, Dickson D, Floeter MK, Traynor BJ, Morelli C, Ratti A, extra copies, forms aggregates, or “foci” in neurons, which may sequester Silani V, Rademakers R, Brown RH, Rothstein JD, Boylan KB, Petrucelli L, Disney MD. Discovery RNA-binding proteins (see below for further research on this aspect of of a Biomarker and Lead Small Molecules to Target r(GGGGCC)-Associated Defects in c9FTD/ C9orf72 toxicity). Recently, it was shown that the repeat expansion leads ALS. Neuron. Sept. 3, 2014; 83(5):1043-50. to the phenomenon of “repeat-associated non-standard” (RAN), in which Zhang YJ, Jansen-West K, Xu YF, Gendron TF, Bieniek KF, Lin WL, Sasaguri H, Caulfield T, Hubbard translation occurs in the absence of an AUG start signal. RAN translation has J, Daughrity L, Chew J, Belzil VV, Prudencio M, Stankowski JN, Castanedes-Casey M, Whitelaw also been demonstrated in myotonic dystrophy and spinocerebellar ataxia E, Ash PE, DeTure M, Rademakers R, Boylan KB, Dickson DW, Petrucelli L. Aggregation-prone c9FTD/ALS poly(GA) RAN-translated proteins cause neurotoxicity by inducing ER stress. Acta type 8. In C9orf72 ALS, the product of RAN translation is a set of repeated dipeptides, including poly-(glycine-alanine), poly-(glycine-proline) and poly-(proline-arginine), each produced from a different reading frame. Whether these so-called C9RAN proteins are implicated in ALS pathogenesis is unclear. Three new studies address that question. In neuronal cell culture, Zhang et al. showed that poly-(glycine-alanine) could induce endoplasmic reticulum-related stress, which could be mitigated by ER-stress inhibitors. Mizielinska et al. found that expression of poly-(glycine-arginine) and poly-(proline-arginine) led to neurodegeneration in a fly model. Kwon and colleagues showed that both poly-(glycine-arginine) and poly-(prolinearginine) bound to heterogeneous ribonucleoprotein A2, an RNA-binding protein implicated in a rare form of ALS. They showed that in cell culture, the C9RAN proteins bound to nucleoli, inhibiting RNA production and causing cell death. Whether these C9RAN proteins are also causative in ALS remains unclear, and will need to be further investigated before contemplating making them a therapeutic target. But the proof of principle that their levels could be reduced with small molecules was demonstrated by Su et al., who showed that, in cell culture, a molecule structurally designed to bind to the repeat reduced expression of both poly-(glycine-proline) and poly-(glycine-alanine) in a dose-dependent manner. Treatment also reduced the number of RNA foci, through an unknown mechanism. Treatment did not reduce production of poly-(proline-arginine), although whether that was due to actual lack of effect of the treatment, or too low a signal from the protein-detecting antibody used to measure its production, is unclear. Perhaps of most significance in the Su study was the demonstration that the C9RAN protein poly-(glycine-proline) could be detected in CSF from people with ALS due to C9orf72 mutation. This raises the possibility that this protein could serve as a pharmacodynamic biomarker to monitor the effectiveness of anti-C9 therapy. Currently, antisense molecules against the C9orf72 gene are being readied for clinical trials. “We are very excited to determine whether measuring these proteins could offer the first gene-specific and treatment-specific biomarker in ALS,” said principle investigator Leonard Petrucelli, Ph.D., Professor of Neuroscience at the Mayo Clinic in Jacksonville, Florida. Further work will be needed to determine the natural history of the proteins in the CSF, and how responsive they are to antisense treatment in cell and potentially animal models. “Biomarkers are one of our most critical unmet needs in ALS,” according to Lucie Bruijn, Ph.D., Chief Scientist for The ALS Association. “With the appropriate biomarker in hand, we can design clinical trials that are faster

Acknowledgement: Richard Robinson, Science Writer

that colocalization was seen in only a small proportion of foci, “suggesting dynamic sequestration rather than irreversible binding.” In addition, irrespective of the presence of foci, neurons were depleted of nuclear TDP-43, and displayed inclusions of the C9RAN protein poly-GA. They suggest these results indicate two non-exclusive pathogenic mechanisms: disruption of splicing due to depletion of RNA-processing proteins, and inappropriate export of expanded C9orf72 pre-messenger RNA, leading to production of C9RAN proteins. Cooper-Knock J, Walsh MJ, Higginbottom A, Robin Highley J, Dickman MJ, Edbauer D, Ince PG, Wharton SB, Wilson SA, Kirby J, Hautbergue GM, Shaw PJ. Sequestration of multiple RNA recognition motif-containing proteins by C9orf72 repeat expansions. Brain. July 2014; 137(Pt 7):2040-51.

Genome-wide Analysis Suggests ALS Heritability is Significantly Higher than Thought

Familial ALS, in which the disease affects more than one immediate family member, accounts for about 10% of cases, with the majority of those acKwon I, Xiang S, Kato M, Wu L, Theodoropoulos P, Wang T, Kim J, Yun J, Xie Y, McK- counted for by known genes. But it has also been expected that some fracnight SL. Poly-dipeptides encoded by the C9ORF72 repeats bind nucleoli, im- tion of sporadic cases are also due to genetic causes, a suspicion strengthpede RNA biogenesis, and kill cells. Science. Sept. 5, 2014; 345(6201):1139-45. ened with the discovery of the C9orf72 gene, which has been found to be the cause of about 6% of sporadic ALS. Genome-wide association studies are Mizielinska S, Grönke S, Niccoli T, Ridler CE, Clayton EL, Devoy A, Moens T, Norona FE, Wool- limited in their ability to discover new genetic risks for ALS, since they examlacott IO, Pietrzyk J, Cleverley K, Nicoll AJ, Pickering-Brown S, Dols J, Cabecinha M, Hendrich O, ine the significance of individual genetic variants only. In contrast, genomeFratta P, Fisher EM, Partridge L, Isaacs AM. C9orf72 repeat expansions cause neurodegeneration in Drosophila through arginine-rich proteins. Science. Sept. 5, 2014; 345(6201):1192-4. wide complex trait analysis (GCTA) examines all known variants simultaneously, so that variants with smaller but still important individual effects are found. “In essence,” say the authors of a new study, “a GWAS generally only Potential Cerebrospinal Fluid Biomarker Identified identifies large or moderate effect variants, whereas GCTA includes rarer Diagnostic and progression biomarkers are needed in ALS to confirm early effect variants in its calculations.” diagnosis and monitor response to treatment in trials of potentially disease- Using this approach, the authors estimate the heritability of ALS in 1,223 modifying therapies. One candidate that has emerged is phosphorylated tau cases and 1,591 controls in publically available databases. They found that (ptau) in the cerebrospinal fluid. In a new study, Grossman et al. show that the overall heritability of ALS was approximately 21%, “indicating that addithe ratio of ptau to total tau (ttau) was significantly lower in 51 individuals tional genetic variation influencing risk of ALS loci remains to be identified.” with ALS versus 23 individuals with a non-ALS 4-repeat tauopathy. In a vali- They also identified 17 regions of the genome that “display significantly high dation cohort, “the receiver operating characteristic area under the curve heritability estimates,” including 11 novel regions. for the ptau:ttau ratio was 0.916, and the comparison of ALS with 4-repeat Keller MF, Ferrucci L, Singleton AB, Tienari PJ, Laaksovirta H, Restagno G, Chiò A, Traynor BJ, tauopathy showed 92.0% sensitivity and 91.7% specificity.” In addition, the Nalls MA. Genome-wide analysis of the heritability of amyotrophic lateral sclerosis. JAMA ratio correlated with measures of disease severity, including the Mini-Men- Neurol. Sept. 1, 2014; 71(9):1123-34. tal State Examination and the ALS Functional Rating Scale-Revised. Finally, the ratio correlated with NMR evidence of disease in a subset of patients for New Risk Gene Implicates Microglia whom imaging was available. A variant in a gene that promotes inflammation in the central nervous sysGrossman M, Elman L, McCluskey L, McMillan CT, Boller A, Powers J, Rascovsky K, tem more than doubles the risk for ALS, according to new research. The Hu W, Shaw L, Irwin DJ, Lee VM, Trojanowski JQ. Phosphorylated tau as a candidate gene, called TREM2, activates microglia, and the variant, which is called biomarker for amyotrophic lateral sclerosis. JAMA Neurol. April 2014; 71(4):442-8. p.R47H and which causes a switch of one amino acid in the TREM2 protein, has been associated with an increased risk of Alzheimer’s disease. Here, the authors sequenced the gene in almost 1,000 people with ALS and 2,000 conC9orf72 Expansion Traps Multiple RNA-binding Proteins trols. They found that the variant was 2.4 times as common in those with How the GGGGCC repeat expansion in the C9orf72 gene causes ALS is un- ALS versus controls, and that there was more TREM2 protein than expected clear. Initial evidence suggests the repeat can sequester RNA-binding pro- in the spinal cord of those with ALS and in SOD1 mutant mice, suggesting teins, a process analogous to that shown to contribute to disease in myo- that dysregulation of the gene contributes to disease. tonic dystrophy type 1. Here, Cooper-Knock and colleagues performed pull-down assays in conjunction with mass spectrometry to identify candi- Cady J, Koval ED, Benitez BA, Zaidman C, Jockel-Balsarotti J, Allred P, Baloh RH, Ravits J, Simpson E, Appel SH, Pestronk A, Goate AM, Miller TM, Cruchaga C, Harms MB. TREM2 variant date binding partners of the GGGGCC repeat expansion. They report, “Pro- p.R47H as a risk factor for sporadic amyotrophic lateral sclerosis. JAMA Neurol. April 2014; teins containing RNA recognition motifs and involved in splicing, messenger 71(4):449-53. pmc/articles/PMC3989393/ RNA nuclear export and/or translation were significantly enriched.” Specifically, in CNS tissue from gene-positive ALS patients, RNA repeat foci colocalNational Office ized with SRSF2, hnRNP H1/F, ALYREF and hnRNP A1 in cerebellar granule 1275 K Street NW, Suite 250 cells and with SRSF2, hnRNP H1/F and ALYREF in motor neurons. They note Neuropathol. October 2014; 128(4):505-24.

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