Aaron D. Gitler - US grants

08: Neuroscience Using Yeast Cells To Define Mechanisms of Human Neurodegenerative Diseases The United States and other countries around the world are experiencing a demographic sea change owing to the rapidly growing elderly and 'Baby Boomer' populations. As our population continues to age, neurodegenerative disease will increase in prevalence and thus pose a daunting challenge to public health worldwide. These truly disastrous disorders include Alzheimer's, Huntington's, Parkinson's, amyotrophic lateral sclerosis and the frontal temporal dementias. Interestingly, though disparate in their pathophysiology, many of these diseases share a common theme manifest in the accumulation of insoluble protein aggregates in the brain. The long-term goal of my laboratory is to elucidate the mechanisms causing these proteins to misfold and aggregate, identify the genes and cellular pathways affected by misfolded human disease proteins, and understand their function in normal biology. We are taking an innovative approach to attacking this exceedingly difficult problem: harnessing the baker's yeast, Saccharomyces cerevisiae, as a model system to study the mechanisms underpinning protein-misfolding diseases. Surviving cellular stresses caused by misfolded proteins is an ancient problem that all cells struggle with and many of the mechanisms employed to deal with protein misfolding are conserved from yeast to man. We propose to create yeast models of human neurodegenerative diseases and to perform high-throughput genome-wide screens to elucidate the basic cellular mechanisms of toxicity. These yeast models will provide us with a unique opportunity to observe and understand protein folding and misfolding in real time as it occurs in a living cell and then to ask big questions on a genome- wide scale about the cellular pathways affected by the aberrant accumulation and/or function of human disease proteins. We hypothesize that the mechanisms identified by our studies will have broad applicability to multiple neurodegenerative diseases. The innovative aspect of our approach is not just that we are working in yeast, but that we are willing and able to use this system as a discovery tool, which we will validate in more relevant animal models. We have done this successfully in the past (via collaboration and on our own) and this will allow us to proceed with future experiments from a knowledgeable point of view, knowing the relative strengths of various organisms and methods.

DESCRIPTION (provided by applicant): Amyotrophic lateral sclerosis (ALS) is a devastating adult-onset neurodegenerative disease that wreaks havoc on motor neurons. A progressive and fatal muscle paralysis ensues, causing death within 2 to 5 years. Recently, a central role for RNA binding proteins and RNA metabolism pathways has emerged. The protein TDP-43 was recently identified as the major disease protein in pathological inclusions in both ALS and frontal temporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U). Moreover, mutations in the TDP-43 gene have now been identified in sporadic and familial ALS patients. Pathology and genetics both converge on TDP-43 being central to the pathogenesis of these diseases. In the first five years of this research project, we have generated in vitro and in vivo TDP-43 proteinopathy models to explore TDP-43. We have harnessed the simple yeast model system to study TDP-43's properties and the effect of ALS-linked mutations. Our preliminary data demonstrate: 1) a critical role for the RNA recognition motif and carboxy-terminal region of TDP-43 in mediating aggregation and cellular toxicity, 2) increased aggregation and toxicity caused by a disease-linked TDP-43 mutation, and 3) genetic screens identified multiple RNA binding proteins as potent toxicity modifiers. One of these, Pbp1, is the yeast homolog of human ataxin 2 and we identified polyglutamine expansions in ataxin 2 as a major genetic risk factor for ALS in humans. We also discovered that deletion of the Dbr1 gene potently suppresses TDP-43 toxicity. The identification of a major genetic risk factor for ALS in humans and a novel and unexpected therapeutic target for ALS starting from the simple yeast model, illustrates the power of this approach. We have also pursued studies beyond TDP-43, focusing on additional RNA-binding proteins, such as FUS, TAF15, EWSR1, and several more. We have discovered a prion-like domain in TDP-43 and FUS and have used this domain to link additional RNA-binding proteins to a class of proteins with similar structural and functional properties. For the next fiv years of this project, with the goal to define TDP-43 disease mechanisms from multiple angles we propose three Specific Aims: 1) Continuing to characterize hits from our yeast TDP-43 toxicity modifier screens to elucidate additional mechanisms of TDP- 43 toxicity and to perform an additional yeast screen of ~1,000 essential genes; 2) Defining the mechanism by which Dbr1 inhibition suppresses TDP-43 toxicity and extending these studies to mammalian cells and animal models; 3) Testing the hypothesis that aggregation-prone RNA-binding proteins contribute broadly to ALS using next generation sequencing approaches.

DESCRIPTION (provided by applicant): Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease that wreaks havoc on motor neurons. Recently, a central role for RNA binding proteins and RNA metabolism pathways has emerged. TDP-43 is an RNA binding protein that has been identified as a component of pathological inclusions in neurons of most ALS patients. Further, pathogenic mutations in the gene encoding TDP-43 (TARDBP) are associated with familial and sporadic ALS cases. These data argue that pathological activities of TDP-43 are critical to ALS. Despite the central importance of TDP-43, however, little is understood about how it causes disease and protein interaction partners that lead to ALS. This Co-PI research proposal brings together two experts in yeast and fly genetics who have made fundamental discoveries into mechanisms of devastating human age-related diseases such as Parkinson's disease and ALS using these simple but powerful systems. Importantly, discoveries made in yeast are translatable to Drosophila disease models, and from yeast and flies to human patients. Using these systems, we have found that ataxin-2, a polyglutamine (polyQ) protein, whose polyQ repeat expansions cause spinocerebellar ataxia type 2 (SCA2), is a potent modifier of TDP-43 toxicity in yeast and fly. Launching from this finding, we tested and found that polyQ expansions of 27-33Qs in the human ataxin-2 gene, ATXN2, are significantly associated with ALS. These data argue that ATXN2 is a new and relatively common ALS susceptibility disease gene, and that the TDP-43/Atx2 interaction is a critical target in ALS and perhaps other diseases associated with TDP-43 pathology. With the goal to reveal mechanistic insight into this interaction, we propose three Specific Aims. In Aim 1, we will use mammalian cell culture and Drosophila to define the domains of the TDP-43 and ataxin-2 proteins that are critical for the synergistic interaction in degeneration and function. These studies will provide insight into the activity of the proteins and processes affected in the pathological situation. In Aim 2, we will use the power of fly genetics to define additional genes critical for the synergy between TDP-43 and ataxin-2. This will reveal understanding of the processes perturbed and new pathways for therapeutic targeting. Finally, in Aim 3, we will address the greater role of ataxin-2 in neurodegeneration. ALS shares a disease spectrum with frontotemporal lobar degeneration (FTLD) and TDP- 43 pathology characterizes other neurodegenerative diseases. We will assess polyQ repeat lengths of ataxin-2 in other diseases to assess whether expansions occur in related disorders. Taken together, these findings will reveal key aspects of the TDP-43/ataxin-2 interaction central to ALS, the broader role of ataxin-2 in neurodegenerative disease, and the foundation for novel therapeutic insights. PUBLIC HEALTH RELEVANCE: ALS and other neurodegenerative diseases are devastating for individuals and families involved;there are no cures and few treatments. Our studies have revealed a new gene, ataxin-2, whose mutation is a risk factor for ALS. The studies proposed will reveal insight into the role of ataxin-2 in this and other neurodegenerative diseases, providing mechanistic detail and the foundation for novel therapeutic approaches.

DESCRIPTION (provided by applicant): The neurodegenerative disease amyotrophic lateral sclerosis (ALS) and acquired immunodeficiency syndrome (AIDS) are diseases with quite different etiologies and symptoms yet they share an important connection to the human RNA debranching enzyme Dbr1. The main cellular role of Dbr1 is to help recycle intron RNAs following pre-mRNA splicing by cleaving the 2'-5' bond at intron lariat RNA branch points. Interestingly, loss of Dbr1 activity inhibits replication of Human Immunodeficiency Virus (HIV), the retroviral agent of AIDS. Surprisingly, it was recently discovered that accumulation of intron RNA lariats resulting from loss of Dbr1 activity also alleviates toxicity caused by cytoplasmic aggregation of the ALS associated RNA binding protein TDP-43. A consequence of these findings is that Dbr1 inhibitors have potential as anti-ALS and anti-HIV/AIDS drugs. The long-term goal of our research is to innovate novel Dbr1-based therapeutic strategies for treating ALS and HIV infection. The objective of this application is to develop a high-throughput phenotypic screen for Dbr1 inhibitors. Our central hypothesis is that a phenotypic screen using the budding yeast Saccharomyces cerevisiae can identify human Dbr1 inhibitors. Taking advantage of a novel yeast lariat intron reporter strain, research will focus on two specific aims. Aim 1 is to identify yeast Dbr1 inhibitors. For this aim a pilot high-throughput phenotypic screen of small molecule libraries will be performed, assaying for the intron RNA lariat signal in our yeast reporter strain as a proxy for Dbr1 activity. Hit compounds will be validated with independent assays for intron RNA lariat accumulation and Dbr1 enzyme activity. Select hit compounds will be further assessed for their effects on yeast models for ALS and HIV. Aim2 will be to identify human Dbr1 inhibitors by testing yeast Dbr1 inhibitors on human cells and the human Dbr1 enzyme. Follow up studies will examine the effects of human Dbr1 inhibitors on TDP-43 cytotoxicity and HIV-1 replication in human cells. The significance of the work is it will create the means to identify lead compounds that have potential as completely novel treatments for ALS and HIV/AIDS.

? DESCRIPTION (provided by applicant): The recent discovery of a mutation in the C9orf72 gene as the most common genetic cause of ALS and FTD (c9FTD/ALS) has opened up many new and exciting areas of investigation in the quest to understand ALS and FTD disease mechanisms and to develop effective disease-modifying strategies. The C9orf72 gene contains a polymorphic hexanucleotide repeat, GGGGCC, located in an intron. The repeat tract length in unaffected individuals, although variable, is typically between five and ten repeats and almost always fewer than 23 repeats. In c9FTD/ALS cases, the hexanucleotide repeat tract is expanded to hundreds or even thousands of repeats. Given the major contribution of this mutation to neurodegenerative disease, there is intense interest in defining the mechanism(s) by which GGGGCC repeat expansions in the C9orf72 gene cause ALS and FTD. An exciting new hypothesis has emerged to explain how the GGGGCC repeat expansions in C9orf72 could cause disease: Repeat-Associated Non-ATG (RAN) translation, which generates polymers of dipeptides derived from the sense and antisense C9orf72 RNA. These dipeptide repeat proteins are aggregation-prone and accumulate in the brain of affected C9orf72 mutation carriers. We have used a yeast model to explore the mechanisms by which C9orf72-derived dipeptide proteins cause cellular toxicity. In Preliminary Studies, we have performed two unbiased genome-wide screens and discovered potent modifiers of toxicity for one out of the five possible dipeptide products, proline-arginine (PR). Among the strongest modifiers are several karyopherin proteins, which mediate nuclear import of proteins, including FUS/TLS. This Co-PI research proposal employs complementary types of research that will allow for intellectual synergism between the Gitler laboratory at Stanford and the Petrucelli laboratory at the Mayo Clinic. We will use a combination of yeast genetics and cell biological experiments and validation in mammalian cells, mice, primary neurons, and human patient samples with the goal to test novel hypotheses about the mechanism by which C9orf72 dipeptide proteins cause neurodegeneration. In Aim 1, we will perform genetic screens in yeast to identify modifiers of C9orf72 dipeptide repeat protein toxicity. In Aim 2, we will validate findings from yeast in primar neurons and in human neurons generated by direct re-programming of patient cells. In Aim 3, we will perform mechanistic experiments to test the novel hypothesis that C9orf72-derived dipeptide repeat proteins interfere with karyopherin-mediated nuclear impairments and that this underlies the pathogenesis of c9FTD/ALS. Taken together, these findings will reveal key aspects of C9orf72 dipeptide repeat proteotoxicity central to ALS and FTD, and lay the foundation for novel therapeutic insights.

This is a proposal to combine our three NINDS funded R01 projects into one larger R35 award, enabling us the time and flexibility to explore important biological questions relevant to human neurodegenerative diseases. Over the past eight years, my laboratory has used a combination of yeast and human genetics to define novel mechanisms of ALS, FTD, and Parkinson's disease. These experiments have led to the discovery of ataxin 2 intermediate-length polyglutamine expansions as a major genetic risk factor for ALS, the discovery of RNA lariat debranching enzyme as a powerful therapeutic target for TDP-43 proteinopathies, a new cellular pathway to explain how C9orf72 mutations could cause neurodegeneration and unexpected connections between seemingly distinct Parkinson's disease genes. In preliminary studies, we have found that genetic reduction of ataxin 2 in mouse profoundly extends survival of TDP-43 transgenic mice (>80% increase in lifespan). We propose studies to explore how ataxin 2 protects against TDP-43 proteinopathy, test ataxin 2 in other mouse models (e.g., C9orf72 and FUS/TLS), and to pursue antisense oligonucleotides targeting ataxin 2 in human cell models. While our previous work has stemmed from yeast models and genetic modifier screens, we now propose an ambitious advance ? performing genomewide modifier screens in human cells using CRISPR/Cas9 gene activation and inactivation libraries. We have already performed three pilot screens with C9orf72 models and have identified several potent modifiers, which we will validate in primary neurons and mouse models. I plan to take my lab into this new direction by performing CRISPR screens in human cells with TDP-43, FUS, alpha-synuclein, ataxin 2, and further C9orf72 models (e.g., RNA vs. DPRs). We are also interested in the process of RAN (repeat-associated non-ATG) translation, which has emerged as a powerful facet of several nucleotide-repeat diseases (including c9ALS/FTD). We propose experiments to discover the molecular mechanisms of RAN translation in order to design specific inhibitors and we have already identified at least two genes that seem to be required for RAN translation. Together, we present an ambitious research program aimed at defining novel mechanisms of human neurodegenerative diseases and then intensely working to translate those mechanisms to novel therapies to help treat these devastating conditions.

PROJECT SUMMARY Amyotrophic lateral sclerosis (ALS) and Frontotemporal dementia (FTD) are devastating neurological diseases with no effective therapies. Mutation in the C9orf72 gene is the most common genetic cause of ALS and FTD (c9FTD/ALS). The C9orf72 gene contains a polymorphic hexanucleotide repeat, GGGGCC, located in an intron, which in unaffected individuals is typically between 5 and 10 repeats but expands to hundreds or thousands in ALS. The repeat-containing mRNAs have splicing defects and are translated cytoplasmically through a mechanism called repeat associated non-AUG (RAN) translation to make toxic dipeptide repeat proteins. The mechanism of RAN translation, and how RNA structure and exogenous factors dictate non-canonical initiation and elongation through the repeats remains unresolved. Our preliminary data show that (1) RAN translation occurs through a cap-dependent scanning mechanism, (2) RAN translation occurs in distinct reading frames, leading to different dipeptide repeats, and (3), there are specific genetic modulators of RAN translation. Most importantly, through an unbiased genetic screen we discovered deletion of ribosomal protein RPS25, a known modulator of non-canonical translation initiation, specifically inhibits RAN translation in yeast and human cells, and is neuroprotective in animal models expressing C9orf72 repeat expansions. Here we build on these data, combining genetic approaches and expertise in ALS (Gitler) with structural and biophysical approaches to translation (Puglisi) to delineate the mechanism of RAN translation in 3 specific aims. In Aim 1, we will use biochemical and single- molecule methods to determine the mechanistic pathways of RAN translation initiation and elongation, and delineate the role of repeat expansion mRNA structure, likely formed by G- quadruplexes, in start site selection during initiation, and subsequent reading frame selection and maintenance during elongation. We will apply cryoEM and other structural methods to describe stable intermediates identified in this aim. In Aim 2, we will use these methods to understand the specific role of RPS25 and other factors identified by genetic screens for inhibitors or enhancers of RAN translation. We will focus on RPS25, using bulk and single- molecule methods, coupled with structural/biochemical analyses on RPS25 knockout ribosomes. In Aim 3, we will use genetic screens to identify further modifiers of RAN translation, and characterize further those identified by our past screens, validating them across yeast, human and animal models. Our results should illuminate how RAN translation leads to neurotoxic protein production, providing potential therapeutic targets for FTD/ALS.

An exciting new area in neurodegenerative disease research is the emerging phenomenon of prion-like spreading of neurodegenerative disease proteins. Prions are well established as the protein-based infectious agent underlying the spongioform encephalopathies. In these rare, albeit devastating, diseases the prion protein converts from the normal soluble form to the aggregated self-templating infectious form. This process initiates an inexorable spread of pathology and contingent neurodegeneration throughout the brain. But could this disease mechanism extend to the more common neurodegenerative diseases like Parkinson?s disease (PD) and Alzheimer?s disease and related dementia? If so, it represents a game changer in terms of understanding disease mechanisms and opens many new avenues for therapeutic development. However, any therapeutic or mechanistic investigation into prion-like spreading will require the development of powerful new imaging approaches to track the path of prion-like spread and understand how these protein aggregates alter brain function as they spread. We will need to understand why they take some routes but not others and how this impacts brain function. To address this challenge, we have formed an interdisciplinary team, consisting of an engineer and a geneticist. First, we will use state of the art brain clearing technology to obtain high-resolution images of prion-like protein propagation of the Parkinson?s disease protein ?-synuclein. We will monitor these aggregates as they spread from one neuron to the next, tracking their paths. These high-resolution brain wide 3D maps of alpha-synuclein spreading will empower us to identify gene expression patterns associated with spreading paths and to nominate genes for functional studies. Then, we will utilize advanced high-resolution optogenetic functional magnetic resonance imaging (ofMRI) to reveal the longitudinal effects of prion-like spreading on brain network activity and likewise the impact of neural activity on prion-like spreading. Our experiments will provide fundamental mechanistic insight into prion-like spread of neurodegenerative disease. The tools we apply and the lessons we learn will likely be broadly applicable to neurodegenerative diseases including Alzheimer?s disease and related dementias.

Project Summary The overall goal and singular focus of our proposed Center Without Walls is to unravel the mechanisms of FTLD-TDP. We have formed a diverse interdisciplinary team to tackle this challenge. Our team brings together experts in genetics, genomics, neuroscience, neurology, and pathology. We have FTLD experts as well as outsiders who bring new perspectives and key resources and approaches to the field. Our team has also recently made an unexpected discovery of a new splicing target of TDP-43, which provides a direct and surprising connection to FTD human genetics and will be a launching pad for defining the mechanisms of FTLD-TDP. We posit that mis-splicing events caused by TDP-43 dysfunction may well be the earliest events in the process. Our vision is to create a Center dedicated to providing unprecedented access to TDP- 43 function, even before it is depleted from the nucleus. Rather than have human genetics as an afterthought or addendum, we endeavor to have the genetics deeply integrated in our program from Day 1. Our Center will make all of the data and code we generate freely available via a web portal that contains high resolution images of human brains across different subtypes of FTLD- TDP showing, at cellular resolution, TDP-43 localization along with a panel of cryptic splicing readouts as sensitive beacons of TDP-43 activity in different brain regions. This will empower the broad FTLD research community to generate (and test) new hypotheses about disease mechanisms and to have at their disposal sensitive biomarkers. Our Center will launch multimodal efforts to 1) comprehensively discover the TDP-43 splicing targets relevant to human FTLD-TDP; 2) define the mechanisms by which TDP-43-dependent cryptic exon splicing events contribute to neurodegeneration, using model systems and human tissues; 3) harness these novel cryptic exons to generate highly sensitive and specific biomarkers for the FTD field; 4) innovate genomics analysis methods to integrate human genetics data and RNA sequencing data and make these resources available to the community to discover how genetic risk factors for FTD contribute to cryptic exon splicing and vice versa. We strongly suspect that we will discover the cryptic exon splicing code that serves as the Achilles? heel to drive neurodegeneration in FTLD-TDP.

Project Summary The overall goal and singular focus of our proposed Center Without Walls is to unravel the mechanisms of FTLD-TDP. We have formed a diverse interdisciplinary team to tackle this challenge. Our team brings together experts in genetics, genomics, neuroscience, neurology, and pathology. We have FTLD experts as well as outsiders who bring new perspectives and key resources and approaches to the field. Our team has also recently made an unexpected discovery of a new splicing target of TDP-43, which provides a direct and surprising connection to FTD human genetics and will be a launching pad for defining the mechanisms of FTLD-TDP. We posit that mis-splicing events caused by TDP-43 dysfunction may well be the earliest events in the process. Our vision is to create a Center dedicated to providing unprecedented access to TDP- 43 function, even before it is depleted from the nucleus. Rather than have human genetics as an afterthought or addendum, we endeavor to have the genetics deeply integrated in our program from Day 1. Our Center will make all of the data and code we generate freely available via a web portal that contains high resolution images of human brains across different subtypes of FTLD- TDP showing, at cellular resolution, TDP-43 localization along with a panel of cryptic splicing readouts as sensitive beacons of TDP-43 activity in different brain regions. This will empower the broad FTLD research community to generate (and test) new hypotheses about disease mechanisms and to have at their disposal sensitive biomarkers. Our Center will launch multimodal efforts to 1) comprehensively discover the TDP-43 splicing targets relevant to human FTLD-TDP; 2) define the mechanisms by which TDP-43-dependent cryptic exon splicing events contribute to neurodegeneration, using model systems and human tissues; 3) harness these novel cryptic exons to generate highly sensitive and specific biomarkers for the FTD field; 4) innovate genomics analysis methods to integrate human genetics data and RNA sequencing data and make these resources available to the community to discover how genetic risk factors for FTD contribute to cryptic exon splicing and vice versa. We strongly suspect that we will discover the cryptic exon splicing code that serves as the Achilles? heel to drive neurodegeneration in FTLD-TDP.