Research ALS Today - Fall 2016

RESEARCH ALS TODAY IMAGING DISEASE MECHANISMS IN PEOPLE WITH ALS

By Nazem Atassi, M.D. Associate Director, Neurological Clinical Research Institute (NCRI) at Massachusetts General Hospital, Assistant Professor of Neurology at Harvard Medical School

Our ALS imaging research is focused on understanding the mechanisms of ALS and designing efficient clinical trials by building advanced human imaging platforms.

ALS drug development faces many challenges that result in very high failure rates of tested therapies. Unfortunately, most ALS therapies that show encouraging results in the lab do not translate into effective treatments for people with ALS. One of the biggest challenges in ALS drug development is the lack of a mechanistic readout of drug efficacy in patients at early phases of clinical drug development. Our team at Mass General Hospital (MGH) is laser-focused on

INSIDE Imaging Disease Mechanisms Message from Chief Scientist Postdoctoral Fellowships The “Mice” Bucket Challenge Journal News

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building these needed imaging mechanistic platforms.

We are currently developing multiple positron emission tomography (PET) imaging platforms to measure key mechanistic pathways in people with ALS including inflammation, glutamate toxicity and epigenetics.

Imaging Inflammation in People With ALS Our research shows increased PBR28 uptake in the motor cortices and brainstem in people with ALS. We also show that the anatomical distribution of increased PBR28 uptake corresponds to ALS clinical presentation; for example, people with limb-onset weakness had higher PBR28 uptake in the motor cortices and patients with bulbar-onset weakness had higher PBR28 uptake in the brainstem (Figure 1). Furthermore, higher PBR28 uptake was strongly correlated with worse functional status measured by ALSFRS-R and worse pathological reflexes.

[Atassi N et al. Neuroimage. 2215] We are now testing the same technology using a novel tracer called GE180 as part of the TRACK ALS project that is funded by The ALS Association and ALS Finding a Cure Foundation. GE180 has a longer half-life and is available in commercial cassettes.

This would allow us to scale up this imaging platform to multicenter studies and clinical trials.

In addition, we are now taking this technology to a critical new level by studying inflammation in people who carry an ALS gene but don’t have any of the clinical symptoms of ALS. This will allow us to measure the pre-clinical pathological changes in the brain in people who are at risk of developing ALS. (Continued on page 8)

Figure 1 A. [11C]PBR-28 SUVR 60-90 min for 10 individual ALS patients and 10 age- and rs6971 genotype-matched healthy controls. B. Mean [11C]PBR-28 SUVR 60-90 min images for the ALS and control groups, including comparisons between limb- and bulbar-onset patients C. Brain regions that exhibit significantly higher binding in ALS compared to the control group in the voxel-wise whole brain analysis, pFWE<0.05

RESEARCH ALS TODAY NEW MODEL SYSTEMS, EMERGING DISEASE PATHWAYS AND IMAGING TOOLS HELP ACCELERATE DRUG DISCOVERY AND DEVELOPMENT FOR ALS

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

This year marks the second anniversary of the ALS Ice Bucket Challenge, and with the resulting increased awareness and resources for ALS research, significant advances are being made, including the discovery of new genes such as NEK1 and C21orf2. As the discovery of additional genes associated with ALS continues to grow, disease pathways are emerging that can be targeted to develop new therapeutic approaches. These opportunities have escalated the number of

industry and academic partnerships in drug development for ALS. Alongside research progress, clinical trials for ALS are either about to start, in progress or completed and awaiting review of results.

Cytokinetics announced completion of enrollment this summer of their phase III trial testing a fast skeletal muscle troponin activator to increase muscle strength and respiratory function. Results from the Arimoclomol trial, targeting SOD1 misfolding, are anticipated later this year. Researchers in California received FDA approval for a combination cell gene-therapy approach, entering the clinic for safety testing in the New Year.

Several new mouse models are emerging including those expressing ALS-associated mutations in ubiquilin and profilin genes.

These models mimic many important aspects of the human disease including motor neuron loss, muscle weakness, accumulations of mutant protein and TDP43 (present in almost all cases of the human disease).

Finally, I would like to recognize this year’s recipients of the Milton Safenowitz Postdoctoral Fellowship for ALS Research. The award program was founded in memory of Mr. Safenowitz, who died of ALS in 1998 to encourage young researchers in the field of ALS. Many former recipients now lead their own laboratories and are mentors to a new generation of fellows.

Tiffany Todd, Ph.D.

Mayo Clinic Jacksonville; Jacksonville, Fla.

MILTON SAFENOWITZ POSTDOCTORAL FELLOWSHIPS

Modeling selective vulnerability and disease-specific functions in mice by the comparison of C9orf72 repeat models to a novel disease control

The ALS Association offers the Milton Safenowitz Postdoctoral Fellowship for ALS Research to encourage and facilitate promising young scientists to enter the ALS field. The Safenowitz family, through the Greater New York Chapter of The ALS Association, founded this award program in memory of Mr. Safenowitz, who died of ALS in 1998. Fellows work with a senior mentor and receive extensive exposure to the ALS research community by attending meetings and delivering presentations about their exciting work.

The 2016 fellowship award recipients include the following:

Disease-related repeat expansions of C9orf72 in ALS and another neurodegenerative disease, spinocerebellar ataxia type 36 (SCA36), are likely a source of toxicity in neurons. Dr. Todd, under the mentorship of Dr. Leonard Petrucelli, aims to compare these two repeats using mouse models in order to understand why certain neurons respond differently to each repeat paving the way to ALS treatments.

“I am excited to see what insights this comparison study will give into the mechanisms underlying ALS and how the knowledge gained from this study will influence future research and therapeutic development.”

Vicente Valenzuela, Ph.D.

University of Chile; Santiago, Chile Gene therapy to attenuate ER stress alterations in ALS The role of the endoplasmic reticulum stress in ALS disease will be explored by Dr. Valenzuela under the mentorship of

- Lucie Bruijn, Ph.D., M.B.A.

TIMELINE French neurologist Jean-Martin Charcot identifies ALS

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

SOD1 enzyme identified

Programmed cell death in motor neurons demonstrated

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The ALS Association funds study of inherited motor neuron disease Genes for muscular dystrophy identified

The ALS Association begins workshops

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

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

Growth factor CNTF is found to increase survival of motor neurons

Researchers link familial ALS to chromosome 21

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Glutamate transporter shown to be defective in ALS. Growth factor BDNF found to increase survival of motor neurons

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Claudio Hetz, Ph.D. He recently obtained results demonstrating that gene therapy based on triggering an artificial unfolded protein response protects ALS mouse models. In collaboration with other key laboratories, he will move this gene therapy approach forward.

“The fellowship allows me to project the exciting preliminary results that I currently have obtained, and enables me to develop cutting-edge science in Chile. I really appreciate The Association’s support.”

Bruno Miguel da Cruz Godinho, Ph.D. University of Massachusetts Medical School; Worcester, Mass.

Silencing mutant SOD1 and C9orf72 in vivo using innovative hydrophobic siRNA scaffolds: a novel therapeutic path for the treatment of ALS Therapeutic gene silencing holds great promise as a clinical strategy to treat ALS. Dr. Godinho, under the guidance of Robert Brown, M.D., Ph.D., synthesized a novel gene silencing strategy to silence

both SOD1 and C9orf72 that exhibits efficient uptake in neurons. He plans to validate and test the efficiency of this strategy in the brain and spinal cord of established ALS mouse models, leading the way to develop effective drugs to treat ALS.

“The generous support from the Milton Safenowitz Postdoctoral Fellowship will allow me to investigate the nuclear pore complex (NPC) in patient tissue and motor neurons derived from induced pluripotent stem cells.”

“I am deeply honored and truly grateful to have been selected as a fellowship recipient, and believe my work will contribute to the development of adequate therapeutic gene silencing platforms that may have profound impact on the treatment of ALS.”

Sergey Stavisky, Ph.D.

Amanda Gleixner, Ph.D.

University of Pittsburgh; Pittsburgh, Pa. Linking impaired nucleocytoplasmic trafficking in C9orf72 ALS to altered nuclear pore complex O-linked N-acetylglucosamine (O-GlcNAc) posttranslational modifications Deficits in nucleocytoplasmic trafficking have been observed in C9orf72-associated ALS, but why this occurs in disease is unknown.

Dr. Gleixner, under the mentorship of Christopher Donnelly, Ph.D., will explore the link between impaired nucleocytoplasmic trafficking in C9orf72 ALS and alterations in the nuclear pore complex, specifically by modulating O-GlcNAcylation of nucleoporins.

Stanford University; Stanford, Calif.

Clinically-useful brain-machine interface control of a robotic prosthetic arm by people with ALS Assistive technology offers people living with ALS prolonged independence in their daily activities. Dr. Stavisky, under the guidance of Jaimie Henderson, M.D., will develop and optimize a clinically useful brain-machine interface control of a robotic prosthetic arm and hand for people living with ALS.

Jeanne E. McKeon, Ph.D.

University of Massachusetts Medical School; Worcester, Mass.

Disruption of actin dynamics as a pathogenic mechanism in ALS Mutations in profilin 1 (PFN1) cause one type of familial ALS that is important in regulating actin dynamics.

Dr. McKeon, under the mentorship of Daryl Bosco, Ph.D., will study the alteration of actin dynamics and actin-dependent cellular processes as a pathogenic mechanism of ALS. “I am very grateful for research support from The ALS Association which will help me yield critical insight into pathogenic mechanisms and druggable targets in the more common form of the disease.”

“The focus of my work could restore patients’ ability to move their own muscles, improving the quality of life of people with ALS, while other ongoing research advances towards treating the disease itself.”

A transgenic rat is designed; efforts start on fly model

SOD1 gene mutation (chromosome 21) discovered in familial ALS

Animal studies combining CNTF and BDNF demonstrate decreased motor neuron loss

Trials using glutamate blocker riluzole begin

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

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The ALS Association cosponsors workshop on highthroughput drug screening with NINDS RNAi discovered by Craig Mello and Andrew Fire

NINDS issues first ever RFA (request for applications) specifically for ALS research

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Attention turns to support cells of nerve tissue to find role in ALS Inflammation on and programmed cell death gather research interest

The ALS Association holds scientific workshop on “Environmental Factors and Gene Susceptibility” Aggressive search for new ALS genes funded

by The ALS Association Scientists complete map of mouse genome

ALS2 gene (alsin protein) linked to juvenile ALS

Agency of Toxic Substances and Disease Registries awards five grants focused on ALS

The ALS Association/NINDS collaborative effort begins screening drugs

Department of Defense approves funding for ALS-specific research

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RESEARCH ALS TODAY THE “MICE” BUCKET CHALLENGE

By Cat Lutz, Ph.D., M.B.A. Director of Rare and Orphan Disease Center, Director of Mouse Repository & In Vivo Pharmacology, The Jackson Laboratory

The genetic conservation between humans and mice, ease of animal husbandry, and the technologies available to manipulate the mouse genome all have propelled the mouse as the premier mammalian model organism to study disease. Over the years, single gene, loss of function “knock-outs” in mice have contributed a wealth of information on gene function, and for some diseas-

es, have resulted in preclinical models for testing therapeutics. These diseases are typically Mendelian in fashion, where a single known gene is responsible for the disease in a patient population. However, generating mouse models for human diseases becomes challenging when the disease itself is complex, meaning that there is likely more than one causative gene or predisposing genes, that environmental triggers play a role, and a wide range of clinical presentations are present in the patient population. ALS is one such disease.

Approximately 10% of ALS cases have been classified as familial in nature, meaning simply that there is a heritable component that can be traced within a family pedigree. The identification of these genes and subsequent understanding of how they function can help researchers identify underlying mechanisms of disease that may be applicable to sporadic ALS as well. Thus, each gene represents an important piece of the puzzle in the development of therapeutics. For many years, causative genes for ALS were unknown and it was only in 1993 when dominant missense mutations in the superoxide dismutase 1 (SOD1) gene were found to be causative in a subset of ALS patients.

SOD1 mutations represented 20% of the familial ALS population, indeed a significant discovery. With a gene now in hand, the

mouse models expressing SOD1 mutations quickly followed. Numerous models were made harboring patient sequence variants and while they differed slightly, generally, the SOD1 overexpressing transgenic mice presented with significant motor neuron loss, axonal denervation, progressive paralysis and reduced lifespan. The mice were, and continue to be, used extensively to study the pathophysiology of the disease; contributing immensely to our understanding of glutamate toxicity, astroglia involvement, axonal transport defects, mitochondrial abnormalities, protein misfolding and other cellular abnormalities related to disease.

Thus, SOD1 mouse models represent the first significant splash in ALS mouse bucket.

Filling the Bucket Over the next decade, researchers slowly chipped away at the genetics of familial ALS. In 2008, the identification of TARDBP and FUS as causative genes for ALS marked a significant milestone in ALS genetics. Both genes function in RNA metabolism, and interestingly, TARDBP cytoplasmic inclusions represents a common neuropathology observed in both familial and sporadic ALS patients.

In parallel, genetic engineering advanced significantly. Beyond

traditional knock-outs and overexpressing transgenes, researchers were now employing the use of conditional and inducible systems to introduce both loss of function and gain of function mutations in a time dependent and tissue specific manner.

The number of mouse models in the ALS bucket was accumulating at a rapid pace. In 2011, excitement grew in the ALS research community when the elusive genetic mutation in ALS families was discovered to be a hexanucleotide repeat expansion in a gene called C9orf727. The genetic mutation represented an astounding 40% of all familial ALS cases. The C9orf72 mutation created a whole new challenge in ALS mouse models. The repeat was isolated from patient cells, but the engineering of the repeats in mice was challenging, as the repeat often contracted in the cloning process prior to microinjection. A number of labs have reported the creation of transgenic animals carrying the hexanucleotide (GGGGCC) repeat 1, 2 3 4. Each published the presence of repeat-associated nonATG (RAN) translation, RNA foci and RAN translation products in the brain-a similar pathology seen in people living with ALS. At

TIMELINE CONT. Ceftriaxone increases levels of the glutamate transporter GLT1 in a mouse model of ALS

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

First international workshop on frontotemporal dementia discusses link to ALS Study implicates smoking as likely risk factor in sporadic ALS Study releases evidence that mitochondrial malfunction may play an important role in ALS

Combination of creatine and minocycline prove more effective together in mouse model than either drug alone

Study funded by The ALS Association to find biomarkers in cerebrospinal fluid and blood

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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

Stem cell study shows SOD1 mutant support cells can kill any motor neuron 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

VEGF increases survival in a rat model of ALS while improving motor function

TDP-43 discovered as a common link in FTD, ALS Chromosome 9 region intense focus for FTD

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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

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least one lab has reported gross physiological abnormalities and paralysis in a subset of animals4. These C9orf72 models represent the first generation of C9orf72 and already hold great potential for evaluating therapeutics targeting the repeat expansion.

Another Faucet Genetic mutations in ALS have traditionally been identified through linkage analysis. With advances in genome sequencing, a new path forward has emerged.

Genome sequencing of large numbers of people with ALS is a strategy now employed by a number of groups. These “big data” sets provide us not only with new causative genes in ALS, but also a catalog of rare variants and allow us to explore the role of coding, noncoding and intergenic genetic variation in the pathogenesis of motor neuron degeneration. Already, new genes such as TBK1, TUBA4A, NEK1, and CHCHD10 have been identified using this approach and our knowledge of genetic variants in ALS patients is increasing exponentially! This sets the future stage for a personalized medicine approach to ALS, where therapies identified as being helpful would not be a treatment

Stem cells generated from ALS patients Discovery of DPP6 in two genome- wide association studies in ALS Mutations in TDP-43 linked to familial and sporadic ALS

Identification of new gene linked to familial ALS, Fused in Sarcoma (FUS) on Chromosome 16

for all ALS, but tailored to the specific mutation harbored by the patient.

Coupled with the identification of new causative genes in ALS are major advances in genome editing technologies allowing us to genetically engineer mice at an unprecedented rate. The CRISPR/ Cas9 system allows researchers to reproduce pathogenic mutations found in humans in animal models. The simplicity of technique even affords introduction of the same mutation in different genetic backgrounds (i.e. different mouse strains) at once. The reduced time and cost associated with the CRISPR/Cas9 system, along with the veritable ease of technology, streamlines production of these mouse models.

Sharing the Wealth The ALS genetic landscape is changing dramatically. Our knowledge of the genetic variants and the ability to quickly engineer the mouse models has the potential to rapidly increase our understanding of the pathophysiology and disease mechanisms of ALS. The Jackson Laboratory (JAX) is committed to making these emerging ALS

The ALS Association hosts 2nd Drug Discovery Workshop for ALS September

Induced Pluripotent Stem Cell Technology opens up new avenues for ALS

FDA approval of SOD1 antisense and stem cell trials in U.S.

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

Ubiquilin-2 discovery; C9orf72 discovery

Researchers find genetic region influencing age at which people develop ALS

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mouse models available to the scientific community. In 2010, The ALS Association, the ALS Therapy Alliance and the Tow Foundation provided the funding necessary to initiate the ALS Mouse Model Resource at JAX. The role of the repository was to import and distribute mouse models provided by ALS investigators. This ensures adequate commercial supply and equal accessibility of models to all investigators; an important factor in maximizing the potential of the models as agents of therapeutic discovery.

This is especially true for new and emerging models, where unlimited and equal access will lead to the most efficient characterization. Today, JAX is not only distributing mouse models of ALS but also collaborating with scientists and clinicians to leverage the genetic engineering, reproductive sciences and scaled breeding facilities to engineer these new ALS models at JAX. In doing so, they put the animals in the hands of ALS researchers more quickly and complete the second site validation of disease relevant phenotypes. With this full bucket of resources at the disposal, effective therapeutics can become a closer reality for ALS patients.

References 1. O’Rourke JG, et al Neuron 2015, 88:892-901 2. Hukema RK, et al R Acta Neuropathol Commun 2014, 2:166 3. Liu Y, et al Neuron 2016, 90:521-534 4. Jiang J, et al Neuron 2016, 90:535-550

Identification of C9RAN translated peptide First human antisense trial published— approach safe in people with ALS Progress in understanding effects of C9orf72 gene in ALS

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RESOURCES ALS Mutations Database http://alsod.iop.kcl.ac.uk/home.aspx ALS iPS Cell Lines http://cedars-sinai.edu/Research/ Research-Cores/InducedPluripotentStem-Cell-Core-/ALSiPSC-Lines.aspx ALS Epidemiology http://aces.stanford.edu/ForRes.html SOD1 Mutant Rats, Taconic http://www.taconic.com/rat-model/ sod1-rat Jackson Laboratories ALS Mouse Repository https://www.jax.org/ search?q=ALS+mouse+repository ALS Mouse Model Summary http://www.alzforum.org/researchmodels/als ALS Untangled http://www.wfnals.org/alsu.html ALS Association Research Webinars http://www.alsa.org/research/ research-webinars.html ALS Research Forum http://www.alsresearchforum.org/

Mutations in Matrin 3 identified, linked to ALS ALS Ice Bucket Challenge Mutations in mitochondrial gene CHCHD10 linked to ALS

New studies shed light on TDP-43 disease mechanism

Mutations in microtubule associated gene TUB4A linked to ALS

C9orf72 repeat expansion mutations—disease mechanisms begin to unfold

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Antisense clinical trial for SOD1 started Discovery of ALS genes NEK1, C21orf2

2016

RESEARCH ALS TODAY: Journal News GENES AND DISEASE MECHANISMS: C9ORF72 This year has seen major progress in understanding the consequences of the C9orf72 expansion mutation, the most common genetic cause of ALS and frontotemporal dementia (FTD). New work has also begun to reveal the normal function of the protein, which may be important for understanding the full effects of the gene mutation, and for predicting the effect of shutting down the gene through antisense therapy or other means.

New Mouse Models Display ALS-like and FTD-like Phenotypes Two groups of researchers, both funded by the ALS Association, developed mouse models of the expansion (carrying up to 500 GGGGCC units, versus fewer than 30 in the normal gene), using a bacterial artificial chromosome (BAC) as a vector for the gene. In addition to the entire gene, the BAC can carry the gene’s promoter and other regulatory regions, increasing the fidelity of the resulting mouse model. Both models developed the full range of pathology seen in people with the mutation, including accumulations of RNA known as “foci,” which form from the expansion, and dipeptide repeat proteins (DPRs), which are transcribed from the expanded RNA. Both models also developed behavioral symptoms and neurodegeneration; the model created by Lui et al. also displayed motor impairment. Jiang et al. also showed than antisense oligonucleotides directed against the mutant RNA reduced both foci and DPRs, and mitigated anxiety-like behaviors. The work strengthens the case that antisense treatment against

the mutation, which is currently in development, may be therapeutic in ALS patients.

Liu Y, Pattamatta A, Zu T, Reid T, Bardhi O, Borchelt DR, Yachnis AT, Ranum LP. C9orf72 BAC Mouse Model with Motor Deficits and Neurodegenerative Features of ALS/FTD. Neuron. 2016 May 4;90(3):521-34 https://www.ncbi.nlm.nih.gov/pubmed/27112499 Jiang J, Zhu Q, Gendron TF, Saberi S, McAlonis-Downes M, Seelman A, Stauffer JE, Jafar-Nejad P, Drenner K, Schulte D, Chun S, Sun S, Ling SC, Myers B, Engelhardt J, Katz M, Baughn M, Platoshyn O, Marsala M, Watt A, Heyser CJ, Ard MC, De Muynck L, Daughrity LM, Swing DA, Tessarollo L, Jung CJ, Delpoux A, Utzschneider DT, Hedrick SM, de Jong PJ, Edbauer D, Van Damme P, Petrucelli L, Shaw CE, Bennett CF, Da Cruz S, Ravits J, Rigo F, Cleveland DW, Lagier-Tourenne C. Gain of Toxicity from ALS/ FTD-Linked Repeat Expansions in C9ORF72 Is Alleviated by Antisense Oligonucleotides Targeting GGGGCC-Containing RNAs. Neuron. 2016 May 4;90(3):535-50 https://www.ncbi.nlm.nih.gov/pubmed/27112497

An Alternative to Antisense While antisense therapy against the repeat expansion looks promising, it may require targeting both the sense strand of DNA, and its complement, the antisense strand, since both strands are transcribed and both lead to RNA foci and DPR production. An alternative explored by Kramer et al. is to target the transcription elongation factor Spt4, a protein required for transcribing repetitive DNA. Knocking down the protein in cell culture suppressed production of mutant transcripts, reducing both foci and DPRs, suggesting it may be a valuable strategy for therapy development.

Kramer NJ, Carlomagno Y, Zhang YJ, Almeida S, Cook CN, Gendron TF, Prudencio M, Van Blitterswijk M, Belzil V, Couthouis J, Paul JW 3rd, Goodman LD, Daughrity L, Chew J, Garrett A, Pregent L, Jansen-West K, Tabassian LJ, Rademakers R, Boylan K, Graff-Radford NR, Josephs KA, Parisi JE, Knopman DS, Petersen RC, Boeve BF, Deng N, Feng Y, Cheng TH, Dickson DW, Cohen SN, Bonini NM, Link CD, Gao FB, Petrucelli L, Gitler AD. Spt4 selectively regulates the expression of C9orf72 sense and antisense mutant transcripts. Science. 2016 Aug 12;353(6300):708-12. https://www.ncbi.nlm.nih.gov/pubmed/27516603

Pathogenic Mechanisms of Mutant C9orf72 The expansion mutation in the C9orf72 gene produces both RNA foci and dipeptide repeat proteins. Evidence is accumulating that each may contribute to pathogenesis,

through different mechanisms. Zhang et al. show that one of the DPRs, poly(glycine-arginine), forms aggregates that sequesters both HR23 proteins, involved in proteasomal degradation, and proteins that mediate nuclear transport. Similar sequestration was seen in patient samples. Conlon et al. explored the consequences of higher-order folding of the expanded RNA transcribed from the gene.

Previous work has shown the RNA forms so-called G quadruplex structures, which can bind proteins. They found that these structures primarily bound the splicing factor hnRNP H, which led to dysregulation of splicing of multiple transcripts in brains of patients with the C9orf72 mutation. And Lopez-Gonzalez et al. showed that there was an increase in the level of DNA damage in cells derived from C9orf72 patients, and that DPRs caused DNA damage in normal cells, suggesting that the proteins were directly responsible for the damage seen in ALS cells.

The damage was associated with reduction in mitochondrial protein synthesis, leading to oxidative stress. Reduction in oxidative stress reduced the DNA damage.

Zhang YJ, Gendron TF, Grima JC, Sasaguri H, Jansen-West K, Xu YF, Katzman RB, Gass J, Murray ME, Shinohara M, Lin WL, Garrett A, Stankowski JN, Daughrity L, Tong J, Perkerson EA, Yue M, Chew J, Castanedes-Casey M, Kurti A, Wang ZS, Liesinger AM, Baker JD, Jiang J, Lagier-Tourenne C, Edbauer D, Cleveland DW, Rademakers R, Boylan KB, Bu G, Link CD, Dickey CA, Rothstein JD, Dickson DW, Fryer JD, Petrucelli L. C9ORF72 poly(GA) aggregates sequester and impair HR23 and nucleocytoplasmic transport proteins. Nat Neurosci. 2016 May;19(5):668-77 https://www.ncbi.nlm.nih.gov/pubmed/26998601 Conlon EG, Lu L, Sharma A, Yamazaki T, Tang T, Shneider NA, Manley JL. The C9ORF72 GGGGCC expansion forms RNA G-quadruplex inclusions and sequesters hnRNP H to disrupt splicing in ALS patient brains. Elife. 2016 Sep 13;5. https://www.ncbi.nlm.nih.gov/pubmed/27623008 Free: https://elifesciences.org/content/5/e17820 Lopez-Gonzalez R, Lu Y, Gendron TF, Karydas A, Tran H, Yang D, Petrucelli L, Miller BL, Almeida S, Gao FB. Poly(GR) in C9ORF72-Related ALS/ FTD Compromises Mitochondrial Function and Increases Oxidative Stress and DNA Damage in iPSC-Derived Motor Neurons. https://www.ncbi.nlm.nih.gov/pubmed/27720481

Wild-type C9orf72 Protein Regulates Autophagy Several new studies show that the normal C9orf72 protein helps regulate autophagy, a cellular proteostasis pathway important for degrading aggregates and organelles.

Webster, Smith and colleagues used RNA interference to reduce C9orf72 expression, leading to a reduction in the number of autophagosomes compared to control after treatment with an autophagy trigger. They showed that the protein interacts with the ULK1 complex, which is required for the initiation of autophagy, and that the protein binds directly to Rab1a, which is necessary for ULK1 translocation to the phagophore.

The results may explain the reduction in autophagy seen in C9orf72 ALS patients, whose levels of un-mutated C9orf72 protein are below normal.

Meanwhile, Yang and colleagues showed that the normal C9orf72 protein is part of a multiprotein complex that includes the autophagy regulator ATG101 and a protein of previously unknown function, Smith-Magenis syndrome chromosomal region candidate gene 8 (SMCR8). Knockout of either C9orf72 or Smrc8 led to a reduction in autophagy, indicating a codependence in their interaction with ULK1.

Yang M, Liang C, Swaminathan K, Herrlinger S, Lai F, Shiekhattar R, Chen JF. A C9ORF72/SMCR8-containing complex regulates ULK1 and plays a dual role in autophagy. Sci Adv. 2016 Sep 2;2(9):e1601167. https://www.ncbi.nlm.nih.gov/pubmed/27617292 Free: https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/27617292/ Webster CP, Smith EF, Bauer CS, Moller A, Hautbergue GM, Ferraiuolo L, Myszczynska MA, Higginbottom A, Walsh MJ, Whitworth AJ, Kaspar BK, Meyer K, Shaw PJ, Grierson AJ, De Vos KJ. The C9orf72 protein interacts with Rab1a and the ULK1 complex to regulate initiation of autophagy. EMBO J. 2016 Aug 1;35(15):1656-76. https://www.ncbi.nlm.nih.gov/pubmed/27334615 Free: http://emboj.embopress.org/content/35/15/1656.long

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GENES AND DISEASE MECHANISMS: OTHER ALS GENES Optineurin Suppresses Necroptosis Optineurin mutation is a rare cause of ALS, but a recent study suggests optineurin may be involved in ALS pathogenesis more widely. Previous work by the authors of the new paper had shown that loss of optineurin sensitizes cells to death by necroptosis, a form of programmed cell death distinct from apoptosis. In the new study, Ito et al. show that optineurin normally promotes the degradation of a pro-necroptosis protein called RIPK1. Loss of optineurin allows RIPK1 to trigger inflammation and necroptosis specifically in oligodendrocytes, leading to loss of myelin and neuronal dysfunction.

Because optineurin binds ubiquitin, which is found in protein aggregates in multiple neurodegenerative diseases, including ALS, suggesting ubiquitinated protein aggregates may induce a functional optineurin deficiency and may contribute to neurodegeneration far beyond cases of optineurin mutation.

Ito Y, Ofengeim D, Najafov A, Das S, Saberi S, Li Y, Hitomi J, Zhu H, Chen H, Mayo L, Geng J, Amin P, DeWitt JP, Mookhtiar AK, Florez M, Ouchida AT, Fan JB, Pasparakis M, Kelliher MA, Ravits J, Yuan J. RIPK1 mediates axonal degeneration by promoting inflammation and necroptosis in ALS. Science. 2016 Aug 5;353(6299):603-8. https://www.ncbi.nlm.nih.gov/pubmed/27493188

Profilin 1 Mouse Replicates Multiple Aspects of ALS To date, only the SOD1 mutant mouse has replicated the major features of human ALS: degeneration of motor neurons, progressive weakness, and early death.

In a new study, Yang et al. present a new model that also displays these features, as well as cellular pathology reminiscent of

the human disease. They show that mice expressing mutant profilin 1, a rare cause of ALS, develop weakness beginning at 350 days and become paralyzed at 421 days.

Expression in motor neurons led to motor neuron degeneration in the spinal cord, with presymptomatic axonal degeneration, cytoskeletal disorganization and relatively late development of ubiquitin-positive protein aggregates. In addition to its value in understanding profilin-linked ALS, this new model progressive neurodegeneration may allow identification of cellular-level commonalities between profilin- and SOD1-based ALS, which may provide clues to new targets for therapies.

Yang C, Danielson EW, Qiao T, Metterville J, Brown RH Jr, Landers JE, Xu Z. Mutant PFN1 causes ALS phenotypes and progressive motor neuron degeneration in mice by a gain of toxicity. Proc Natl Acad Sci U S A. 2016 Sep 28. https://www.ncbi.nlm.nih.gov/pubmed/27681617

Whole-genome Sequencing Identifies NEK1 and Other Risk Genes In recent years, whole-genome sequencing has emerged as a new standard in the search for risk genes. International consortia involving scores of laboratories in dozens of countries have brought the tools of “big data” to bear on the understanding of the genetic architecture of ALS, and the discovery of new genes. In a study of over 15,000 patients and over 25,000 matched controls, van Rheenen et al. established evidence that ALS is a complex trait, with multiple genes contributing to risk in individual cases. In a separate study that combined whole-genome analysis of more than 1,000 patients with analysis of variants in a small isolated population, Kenna et al. found that loss-offunction variants in the gene NEK1 occurred in almost 3% of ALS cases. NEK1 is involved in microtubule stability and related func-

tions, adding to the evidence that neuronal transport defects may contribute to ALS risk. van Rheenen W, Shatunov A, Dekker AM, McLaughlin RL, Diekstra FP, Pulit SL, van der Spek RA, Võsa U, de Jong S, Robinson MR, Yang J, Fogh I, van Doormaal PT, Tazelaar GH, Koppers M, Blokhuis AM, Sproviero W, Jones AR, Kenna KP, van Eijk KR, Harschnitz O, Schellevis RD, Brands WJ, Medic J, Menelaou A, Vajda A, Ticozzi N, Lin K, Rogelj B, Vrabec K, Ravnik-Glavač M, Koritnik B, Zidar J, Leonardis L, Grošelj LD, Millecamps S, Salachas F, Meininger V, de Carvalho M, Pinto S, Mora JS, Rojas-García R, Polak M, Chandran S, Colville S, Swingler R, Morrison KE, Shaw PJ, Hardy J, Orrell RW, Pittman A, Sidle K, Fratta P, Malaspina A, Topp S, Petri S, Abdulla S, Drepper C, Sendtner M, Meyer T, Ophoff RA, Staats KA, Wiedau-Pazos M, Lomen-Hoerth C, Van Deerlin VM, Trojanowski JQ, Elman L, McCluskey L, Basak AN, Tunca C, Hamzeiy H, Parman Y, Meitinger T, Lichtner P, Radivojkov-Blagojevic M, Andres CR, Maurel C, Bensimon G, Landwehrmeyer B, Brice A, Payan CA, Saker-Delye S, Dürr A, Wood NW, Tittmann L, Lieb W, Franke A, Rietschel M, Cichon S, Nöthen MM, Amouyel P, Tzourio C, Dartigues JF, Uitterlinden AG, Rivadeneira F, Estrada K, Hofman A, Curtis C, Blauw HM, van der Kooi AJ, de Visser M, Goris A, Weber M, Shaw CE, Smith BN, Pansarasa O, Cereda C, Del Bo R, Comi GP, D’Alfonso S, Bertolin C, Sorarù G, Mazzini L, Pensato V, Gellera C, Tiloca C, Ratti A, Calvo A, Moglia C, Brunetti M, Arcuti S, Capozzo R, Zecca C, Lunetta C, Penco S, Riva N, Padovani A, Filosto M, Muller B, Stuit RJ; PARALS Registry; SLALOM Group; SLAP Registry; FALS Sequencing Consortium; SLAGEN Consortium; NNIPPS Study Group, Blair I, Zhang K, McCann EP, Fifita JA, Nicholson GA, Rowe DB, Pamphlett R, Kiernan MC, Grosskreutz J, Witte OW, Ringer T, Prell T, Stubendorff B, Kurth I, Hübner CA, Leigh PN, Casale F, Chio A, Beghi E, Pupillo E, Tortelli R, Logroscino G, Powell J, Ludolph AC, Weishaupt JH, Robberecht W, Van Damme P, Franke L, Pers TH, Brown RH, Glass JD, Landers JE, Hardiman O, Andersen PM, Corcia P, Vourc’h P, Silani V, Wray NR, Visscher PM, de Bakker PI, van Es MA, Pasterkamp RJ, Lewis CM, Breen G, Al-Chalabi A, van den Berg LH, Veldink JH. Genome-wide association analyses identify new risk variants and the genetic architecture of amyotrophic lateral sclerosis.

Nat Genet. 2016 Sep;48(9):1043-8 https://www.ncbi.nlm.nih.gov/pubmed/27455348 Kenna KP, van Doormaal PT, Dekker AM, Ticozzi N, Kenna BJ, Diekstra FP, van Rheenen W, van Eijk KR, Jones AR, Keagle P, Shatunov A, Sproviero W, Smith BN, van Es MA, Topp SD, Kenna A, Miller JW, Fallini C, Tiloca C, McLaughlin RL, Vance C, Troakes C, Colombrita C, Mora G, Calvo A, Verde F, Al-Sarraj S, King A, Calini D, de Belleroche J, Baas F, van der Kooi AJ, de Visser M, Ten Asbroek AL, Sapp PC, McKenna-Yasek D, Polak M, Asress S, Muñoz-Blanco JL, Strom TM, Meitinger T, Morrison KE; SLAGEN Consortium, Lauria G, Williams KL, Leigh PN, Nicholson GA, Blair IP, Leblond CS, Dion PA, Rouleau GA, Pall H, Shaw PJ, Turner MR, Talbot K, Taroni F, Boylan KB, Van Blitterswijk M, Rademakers R, Esteban-Pérez J, García-Redondo A, Van Damme P, Robberecht W, Chio A, Gellera C, Drepper C, Sendtner M, Ratti A, Glass JD, Mora JS, Basak NA, Hardiman O, Ludolph AC, Andersen PM, Weishaupt JH, Brown RH Jr, Al-Chalabi A, Silani V, Shaw CE, van den Berg LH, Veldink JH, Landers JE. NEK1 variants confer susceptibility to amyotrophic lateral sclerosis. Nat Genet. 2016 Sep;48(9):1037-42. https://www.ncbi.nlm.nih.gov/pubmed/27455347

Epidemiology The national ALS Registry, maintained by the Centers for Disease Control, has provided an update on the prevalence of ALS in the United States, combining data from national

health databases and the online patient portal. The total number of cases in 2012 was 14,713 and in 2013 was 15,908, giving U.S. prevalence rates of 4.7 and 5.0 per 100,000, respectively. Using published incidence studies, and publically available data on population structures and expected growth, Arthur et al. calculate that the total number of ALS cases worldwide will grow by almost 70% in 25 years, largely due to aging of the world population, with the largest increases expected in developing nations. In the world as a whole, the number of ALS cases will increase from 222,801 in 2015 to 376,674 in 2040, they predict.

Mehta P, Kaye W, Bryan L, Larson T, Copeland T, Wu J, Muravov O, Horton K. Prevalence of Amyotrophic Lateral Sclerosis - United States, 2012-2013. MMWR Surveill Summ. 2016 Aug 5;65(8):1-12 https://www.ncbi.nlm.nih.gov/pubmed/27490513 Arthur KC, Calvo A, Price TR, Geiger JT, Chiò A, Traynor BJ. Projected increase in amyotrophic lateral sclerosis from 2015 to 2040. Nat Commun. 2016 Aug 11;7:12408 https://www.ncbi.nlm.nih.gov/pubmed/27510634 Free: https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/27510634/

RESEARCH ALS TODAY IMAGING DISEASE MECHANISMS IN PEOPLE WITH ALS (CONTINUED FROM PAGE 1)

By Nazem Atassi, M.D. This information will be extremely important to understand all forms of ALS (genetic and non-genetic).

Imaging Glutamate Toxicity in People With ALS Our team is now building a new imaging platform using GE-179 PET tracer to measure NMDA excitotoxicity in people with ALS. NMDA is activated by glutamate, which is the major excitatory neurotransmitter in the Central Nervous System (CNS).

While it normally plays important roles as a neurotransmitter, increased glutamate concentrations can be toxic to neurons (a phenomenon known as excitotoxicity).

Increased levels of glutamate have been found in the cerebrospinal fluid of people with ALS and glutamate excitotoxicity is considered one of the main mechanisms leading to motor neuron degeneration in ALS. Of relevance to ALS therapeutics, riluzole, the only FDA-approved medication for ALS, is thought to reduce glutamate levels, thus reducing glutamate signaling and excitotoxicity. This study will generate important preliminary data about the feasibility and value of imaging NMDA receptor activity in people with ALS, which would have major applications in measuring the biological activity of candidate ALS therapies that target the excitotoxicity pathway. Ultimately, such mechanism-based biomarkers will increase our understanding of disease mechanisms and accelerate the pace of ALS drug development. This project is funded by The ALS Association, ALS Finding a Cure and the ALS ONE foundation.

Imaging Epigenetics in People With ALS

Figure 2: [11C]Martinostat images of healthy subjects show high cortical binding and distinct gray-white matter differences. (A) [11C]Martinostat images averaged from 60 to 90 min after radiotracer injection (SUV60-90 min) from a representative subject overlaid on anatomical magnetic resonance (MR) image. (B) [11C]Martinostat SUVR60-90 min images of individual subjects.

The histone deacetylases (HDAC) are naturally occurring enzymes that regulate gene transcription and are implicated in ALS pathophysiology. ALS animal models and postmortem tissue show increased expression of certain subtypes of HDACs. Martinostat is a novel tracer which was developed at Mass General Hospital (MGH) and allows noninvasive quantification of HDAC expression in people (Figure 2) [Insights into epigenetics through human HDAC PET imaging. Science Translational Medicine.

2016]. We are now launching this exciting

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program and soon we will start enrolling in this study.

Mechanism-based imaging research has direct and tangible impact on the lives of ALS patients and their families in two main ways: I. Understand the Causes of ALS in Patients The study of ALS mechanisms used to be limited to pathological and animal studies in the lab. We are now at a point in history where we have advanced imaging technologies that allow us to study ALS causes in patients, and this is the core focus of our research. These advanced imaging technologies will open the window to study mechanisms such as inflammation, excitotoxicity and epigenetics in patients who suffer from ALS, and immediately translate these discoveries to effective treatments for ALS patients.

treatments that were approved in the past 10 years. Our goal is to replicate the MS successes in ALS by building imaging tools that are available for researchers and companies around the world in order to accelerate drug discoveries for ALS patients. This is already happening with the launch of three ALS clinical trials (Ibudilast, RNS60, AMX0035) that are all using our imaging technologies within less than a year of our initial publication in 2015.

Please visit The ALS Associationâ&#x20AC;&#x2122;s new research web pages at ALSA.org/research to learn more about our global research program to find treatments and a cure for the disease.

II. Accelerate the Pace of ALS Drug Discovery ALS drug development used to rely solely on clinical outcomes which require designing very large and inefficient clinical trials. Our imaging team is currently building an exciting and diverse pipeline of mechanistic imaging platforms to be used as specific readouts of drug efficacy in many efficient ALS clinical trials. Similar imaging pipelines allowed the multiple sclerosis (MS) field to develop many successful and effective

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