Stefan M. Pulst - US grants

The overall goal of the UCLA UDALL center is to elucidate mechanisms of early dysfunction in cellular and animal models of Parkinson disease (PD) that can eventually be translated into novel neuroprotective treatments for PD in humans. Basic and clinical research evidence is accumulating that synaptic and cellular dysfunctions not only precede dopaminergic cell loss but also affect non-dopaminergic neurons. The identification of genetic mutations responsible for familial forms of PD offers the potential to glean new insight into the mechanisms underlying the more common sporadic forms of the disease. Mutations in the parkin gene cause an early-onset autosomal recessive form of PD, but are also found in patients with late- onset forms of PD indistinguishable from idiopathic PD. This project builds upon our discovery of four novel parkin-binding proteins, synaptotagmin (syt)1 and syt11, a synapsin-like-protein (SLP), and ataxin-2, a polyQ domain protein. SLP, syt11, and ataxin-2 are found in Lewy bodies of PD patients. The interaction of parkin with ataxin-2 is of particular importance in light of recent findings that polyQ expansion in the SCA2 (ataxin-2) gene are found in patients with late-onset tremor-predominant PD without ataxia or slow saccadic eye movements. We will test the following hypotheses: 1) Parkin ubiquitinates and facilitates degradation of these parkin interactors. 2) Over-expression of these parkin interactors causes neurotransmitter homeostatic dysfunction that progresses to cell death in vitro. 3) Dysfunction and death are prevented by co-expression of parkin, but not by co-expression of parkin mutants. 4) These effects are not restricted to primary dopaminergic neurons, but are also seen in hippocampal glutamatergic neurons. Three specific aims are proposed to test these hypotheses: 1) We will characterize the interaction of parkin with these novel proteins and determine whether parkin accelerates degradation of the proteins in cell-lines. We will also investigate whether mutant parkins lose the ability to bind these proteins or to ubiquitinate them. 2) In cultured cell-lines, we will determine whether exogenous expression of parkin interactors results in alterations in dopamine release and metabolism and cell death and whether co-expression of parkin can prevent these changes. 3) We will examine the mechanism by which these parkin interactors may regulate synaptic transmission and cell survival in primary dopaminergic and glutamatergic neurons utilizing measures of synaptic vesicle recycling. This project is closely integrated with Projects 2 and 3, in which parkin-mediated synaptic dysregulation will be studied in vivo, in brain slices and in primary cultured neurons. Our experiments will be particularly informative for the translation of our basic research findings to the diagnosis and treatment of PD (Project 5), hopefully preventing significant neuronal loss.

DESCRIPTION (provided by applicant): Neurodegenerative diseases represent an ever-increasing societal and economic burden with WHO estimates indicating that they will replace cancer as the second leading cause of death by 2040. Degenerative ataxias are a common form of neurodegeneration affecting cerebellar Purkinje cells (PCs) and other neurons in the CNS. Spinocerebellar ataxia type 2 (SCA2) is an autosomal dominant form of ataxia caused by expansion of a coding CAG repeat and belongs to the group of polyglutamine (polyQ) diseases. Mutations in the SCA2 (ATXN2) gene can also cause or contribute to the development of Parkinson disease and ALS, two common forms of neurodegeneration. Neither symptomatic nor neuroprotective agents have been identified for the treatment of human ataxias. In the previous funding period we developed a multidisciplinary approach to characterize several SCA2 mouse models combining morphologic, biochemical, physiologic, and behavioral techniques. We used the cerebellar slice preparation to show that a decrease in PC firing frequency closely mirrored the decline in motor function. Importantly, significant transcriptome and neurophysiological changes occurred before any dendritic or cell loss was apparent in the cerebellum. We discovered that mutant ATXN2 induced abnormal calcium release from the ER via enhanced interaction with the inositol-triphosphate receptor (ITPR1). Based on these findings, two specific aims are proposed. In Aim 1, we will test the hypothesis that the mGluR1-ITPR1 axis is hyperactive in cerebella from SCA2 mice expressing polyQ expanded ATXN2. We will use the cerebellar slice preparation to evaluate mGluR1 signaling and the relationship between PC firing and intracellular calcium. We will also test the mGluR1-ITPR1 axis in vivo by genetic interaction with mGluR1 haploinsufficient mouse lines. In aim 2, we will use transcriptome profiling at presymptomatic time points to identify novel genes involved in SCA2 pathogenesis. To increase sensitivity and the ability to identify changes in less abundantly PC-expressed genes we have established laser-capture microdissection (LCM) of cerebellar regions. Transcriptomes will be analyzed by WGCNA, GO and KEGG pathway analysis with a particular emphasis on mGluR1-ERK downstream targets. Changes in key genes will be verified by qPCR and ICC/ western blot analysis. We hypothesize that some expression changes will be homeostatic, but others will contribute to pathogenesis. We will differentiate between these alternatives by normalizing expression of the respective gene using AAV- transduction in vivo. Five genes will be chosen based on presence of early changes and presence of changes in multiple SCA2 mouse models. Mice will be evaluated by behavioral testing as well as analysis in the cerebellar slice. The proposed experiments address two significant questions relating to SCAs: the importance of intracellular calcium levels on PC function and survival and the identification of novel path- ways that are shared across multiple SCA2 rodent models. Answers to these questions will help in the identification of new avenues towards treatments of SCA2 and other degenerative ataxias.

DESCRIPTION (provided by applicant): Neurodegenerative diseases represent an ever-increasing societal and economic burden with WHO estimates indicating that they will replace cancer as the 2nd leading cause of death by 2040. In neurodegenerative disease research, a wealth of pathways has been uncovered, but their direct and primary relevance to the respective human disease have been difficult to prove and pathways have remained difficult to target. For polyglutamine diseases, there is clear evidence that expression levels of the respective genes affect the phenotype and shutting-off expression in mouse models reverses already established motor phenotypes. We have chosen an autosomal dominant polyglutamine (polyQ) disease to demonstrate the proof-of-principle that small molecules can be used to downregulate expression of a disease gene at the transcriptional or mRNA stability level. Spinocerebellar ataxia type 2 (SCA2) is a multisystem neurodegenerative disease caused by a dominantly-acting mutation leading to expansion of a polyQ domain in the ataxin-2 protein. SCA2 patients develop progressive ataxia and later lose function in other neuronal systems. Prominent parkinsonian signs develop in some. Similar to many neurodegenerative disorders, no symptomatic or disease-modifying treatments are known. The primary objective of the proposed research is to identify compounds inhibiting ATXN2 expression and to test them for efficacy in SCA2 mouse models. To this end, we have developed a cell-based assay paired with an in vivo mouse model using identical luciferase expression constructs. This will allow us to progress compounds rapidly from a high-throughput screen conducted at NIH Chemical Genomics Center (NCGC) to efficacy and toxicity testing in mice. Four specific aims are proposed: 1) HTS with 300,000 compounds at the NCGC, 2) In vitro testing of compounds in SCA2 patient lymphoblasts, 3) In vivo testing of compounds in ATXN2-luciferase transgenic mice, and 4) testing the ability for compounds to ameliorate an ATXN2 phenotype in a humanized SCA2 mouse BAC model. Our approach takes advantage of an ATXN2-luciferase transgene with luciferase gene flanked by the upstream and downstream portions of the ATXN2 gene. Mouse models are in place to test rapidly the efficacy of compounds in vivo including passage of the blood brain barrier, and further progression of compounds to test in mouse models with morphological, biochemical, and motor phenotypes replicating human SCA2. The proposed work will break new ground for treatment of neurodegenerative diseases by demonstrating feasibility of targeting dominant-acting mutated genes with compounds. We aim to identify small molecules not only targeting gene expression levels, but also mRNA stability via the 3'-UTR. Our focus is on small molecules and extensive and rapid post HTS-testing in a number of rodent models as well as human cell lines. PUBLIC HEALTH RELEVANCE: Neurodegenerative diseases are not only a major worldwide health problem, but owing to the large number affected and long course of illness, a significant economic threat. We are using SCA2, a debilitating and terminal disorder, as a model to examine novel approaches to identify disease-modifying compounds. Our approach of tightly coupling in vitro and in vivo screens can used to target other neurodegenerative diseases caused by dominant-acting gene mutations.

DESCRIPTION (provided by applicant): Neurodegenerative diseases represent an ever-increasing societal and economic burden with WHO estimates indicating that they will replace cancer as the 2nd leading cause of death by 2040. In neurodegenerative disease research, a wealth of pathways has been uncovered, but their direct and primary relevance to the respective human disease has been difficult to prove and targeting of pathways has remained difficult. The proposed work will identify a treatment for spinocerebellar ataxia type 2 (SCA2), a hereditary neurodegenerative disease affecting cerebellar Purkinje neurons (PNs) and other neurons in the cerebellum, brainstem and cerebrum. The cause of SCA2 is a gain-of-function CAG expansion in the ATXN2 gene resulting in an expanded polyglutamine (polyQ) in ataxin-2. Our objective is identification of antisense oligonucleotides (ASOs) that lower ATXN2 expression. Our rationale is based on observations in model organisms and humans indicating that higher dosages of the mutant allele/protein worsen disease severity and that down-regulation of mutant polyQ protein expression in rodents reverses clinical manifestations even after mice have become symptomatic. Additionally, complete knock-out of ATXN2 in mice does not cause neurodegeneration or premature death. Merit for this study is supported by positive results in clinical trials for multiple ASOs, and ongoing clinical trials to test ASOs for the treatment of amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA). The feasibility is based on established resources including lead ASOs identified in vitro and a well-characterized human-BAC transgenic mouse model with late onset progressive motor phenotypes. Three specific aims are proposed: 1) Identification of the 4 most effective ASOs for lowering ATXN2 expression, from 15 lead ASOs that we have already identified in a preliminary ASO screen, 2) testing the ASO leads for amelioration of a biochemical endophenotype that includes indication of maintained glial health, and 3) testing the two most effective ASOs for ameliorating a motor phenotype in an SCA2 mouse BAC model. The proposed work will break new ground for treatment of neurodegenerative diseases by demonstrating feasibility of targeting dominant-acting mutated polyQ genes with antisense oligonucleotides.

DESCRIPTION (provided by applicant): Degenerative ataxias are a group of neurological disorder associated with dysfunction of cerebellum and its connection. The clinical manifestations include progressive incoordination of movements and gait leading to complete disability and eventually to death. In humans, the prevalence of hereditary ataxias range from 6 to 20 cases for every 100,000 which is comparable to the prevalence of ALS or multiple sclerosis in the US. Rodent models of human ataxias have been limited to mice. The Shaker rat is a naturally occurring X- linked model for Purkinje cell degeneration in the Wistar Furth (WF) background. In contrast to most rodent models of human ataxias in which neuronal loss is not pronounced, the shaker rat progresses from a normal number of Purkinje cells at birth to almost complete loss at 1 year. Three specific aims are proposed: We will fine-map the shaker locus using F2's from an intercross of WF shaker rates with wildtype Brown Norway rats. A panel of 44 genetic markers that distinguish WF and BN alleles, informative in this cross, has been established. A second aim will identify the shaker mutation by RNA sequencing of shaker and wildtype RNAs isolated from pre-symptomatic and symptomatic cerebella. The third aim will employ whole genome sequencing for the case that the shaker mutation does not cause reduced abundance of the shaker transcript or is intronic. The overall goal of this proposal is the identification of the first gene in the rat leading to cerebellar PC degeneration and the establishment of the rat as a model system in which to test novel treatment strategies.

DESCRIPTION (provided by applicant): The Muscular dystrophies and Spinal Muscular Atrophy are neuromuscular disorders that account for an increasing burden of medical disability and healthcare costs. All of these disorders have some evidence to suggest that early detection and aggressive preventative care management may improve the morbidity and mortality. To that end, each disorder has an existing or nearly complete standard of care guideline. The implementation of such care requires early detection of affected individuals or those at-risk. In many disorders, such as Duchenne Muscular Dystrophy or Myotonic Dystrophy, there is a documented delay in diagnosis that impairs qualified individuals from delivering such care. This is underscored by promising new treatments being developed for Duchenne Muscular Dystrophy, Myotonic Dystrophy, or Spinal Muscular Atrophy. It is very likely that treatment effectiveness may hinge on early delivery. This application proposes to develop a surveillance network in the states of Utah and Nevada to detect all cases of muscular dystrophy and spinal muscular atrophy. These states include a diverse population of around 6 million persons. The surveillance program proposes uses an innovative tool, natural language processing, to more efficiently and reliably detect such cases from the states' electronic medical records. Secondly, this surveillance program utilizes a unique resource, The Utah Population Database, to better understand the between family variation and to confirm those cases identified from electronic health records. Finally, this proposal seeks to identify care disparities in underserved communities, particularly through guideline adherence, and address these disparities. Once complete, this proposal will achieve a better understanding of the prevalence, morbidity, and mortality in those individuals with muscular dystrophy or spinal muscular atrophy. This information is critical for future disease- modifying therapeutic trials, and for the detection and care of those individuals who may not currently have access to the standard of care.

! Degenerative cerebellar ataxias affect as many as 1 in 5,000 people worldwide, commonly leading to severe incoordination of gait, tremor, and falls. Current treatment options are not widely effective, and most patients are unable to experience substantial symptom relief. Additionally, the underlying electrophysiological changes that drive symptoms are not fully understood. We aim to develop a novel therapeutic strategy in the context of a genetic rat model of degenerative cerebellar ataxia and use it as a research tool to clarify the network changes that directly encode motor symptoms. Although etiologies vary, ranging from dozens of hereditary forms to sporadic ataxias, most, if not all, feature widespread, progressive Purkinje cell loss, resulting in modified inputs relevant to motor control in the dentate nucleus. As the dorsal dentate nucleus represents the major motor cerebellar output, we propose to develop a deep brain stimulation-like therapeutic strategy aimed at re-modulating dentate nucleus activity to reduce ataxic symptoms. We will carry out a pre- clinical test of deep cerebellar stimulation using the Wistar Furth shaker rat, which features progressive Purkinje cell loss, cerebellar tremor, profound incoordination of gait, and frequent falls. We have developed novel methodologies and software to automate the direct quantification of tremor, incoordination of gait, and fall rates in rats, and we have used them to rigorously quantify the progression of motor symptoms in our model. We have additionally collected preliminary data to strongly support the efficacy of deep cerebellar stimulation in reducing each of tremor, incoordination, and frequency of falls. Further, the ability to quickly and effectively modulate motor symptom severity will be a highly useful tool for electrophysiological study. Thus, we will carry out two specific aims. First, we will optimize deep cerebellar stimulation of the dorsal dentate nucleus to treat tremor, incoordination, and falls in the Wistar Furth shaker rat model of degenerative cerebellar ataxia. Second, we will quantify the network properties that directly underlie motor symptoms in the shaker rat. Given that the loss of Purkinje cells disinhibits the dentate nucleus, one might expect their loss to result in increased motor drive, given the excitatory nature of the dentatothalmocortical pathway. However, involuntary motor symptoms are not typically observed in ataxias, indicating that the study of patterning within dentate signaling is warranted. Indeed, we will expand on recent work that showed that irregularity in cerebellar signaling is linked, and that deep cerebellar oscillations at frequencies specific to tremors cohere strongly with motor acceleration during tremor. Using deep cerebellar stimulation as a research tool, we will test whether irregularity and increased oscillatory power not only covary with motor symptoms, but directly encode them, using electrical stimulation to encode electrophysiological signals in wild type rats, testing the hypothesis that modifying patterning with high specificity can directly lead to ataxic symptoms.

Neurodegenerative diseases represent an ever-increasing societal and economic burden with WHO estimates indicating that they will replace cancer as the 2nd leading cause of death by 2040. In neurodegenerative disease research, a wealth of pathways has been uncovered, but their direct and primary relevance to the respective human disease has been difficult to prove and targeting of pathways has remained difficult. The proposed work will identify a treatment for spinocerebellar ataxia type 2 (SCA2), a hereditary neurodegenerative disease affecting cerebellar Purkinje cells (PCs) and other neurons in the cerebellum, the subcortical grey matter and spinal cord. The cause of SCA2 is a gain-of-function CAG expansion in the ATXN2 gene resulting in an expanded polyglutamine (polyQ) domain in ataxin-2. Our objective is identification of highly-potent antisense oligonucleotides (ASOs) that lower ATXN2 expression. Our rationale is based on observations in model organisms and humans indicating that higher dosages of the mutant allele/protein worsen disease severity and that down-regulation of mutant polyQ protein expression in rodents reverses clinical manifestations even after mice have become symptomatic. Additionally, complete knock-out of ATXN2 in mice does not cause neurodegeneration or premature death. Merit for this study is supported by positive results in ongoing clinical trials to test ASOs for treating amyotrophic lateral sclerosis (ALS) and myotonic dystrophy type 1 (DM1), as well as the success of SPINRAZA? (Nusinersen) for spinal muscular atrophy (SMA), the first ever FDA approved ASO drug for a neurodegenerative disorder. The feasibility of this study is based on our positive proof-of-concept data demonstrating that our lead ATXN2 ASO delays progressive rodent SCA2 motor, molecular, and neurophysiological phenotypes after symptom onset. Five specific aims are proposed: 1) an intensive in vitro screen of ASOs targeting throughout the ATXN2 pre-mRNA including cultured SCA2 patient cells, 2) in vivo screens to identify leads lowering cerebellar ATXN2 in mice, 3) safety toxicity testing in rodents and other species, 4) testing the ASO leads for delaying established SCA2 mouse motor, molecular, and neurophysiological phenotypes, and 5) GMP manufacturing of the single most potent ASO candidate and pre-investigative new drug (IND) meetings with members of the FDA. The proposed work will break new ground for treatment of neurodegenerative diseases by demonstrating feasibility of targeting dominant-acting mutated polyQ genes with antisense oligonucleotides.

Project Summary/Abstract Neurodegenerative diseases represent an ever-increasing societal and economic burden with WHO estimates indicating that they will replace cancer as the 2nd leading cause of death by 2040. In neurodegenerative disease research, a wealth of pathways has been uncovered, but their direct and primary relevance to the respective human disease has been difficult to prove and targeting of pathways has remained difficult. The proposed work will characterize ATXN2 and pathways regulated by ATXN2 as therapeutic targets for amyotrophic lateral sclerosis (ALS), a disease burdening 1 out of 25,000 individuals. The ATXN2 gene is mutated in spinocerebellar ataxia type 2 (SCA2), a hereditary neurodegenerative disease affecting cerebellar Purkinje cells (PCs) and other neurons in the cerebellum and brainstem. Studies by us and others have demonstratedthat long normal CAG-repeat expansions in ATXN2 significantly increase the risk of ALS. More recently, survival (or lifespan) of some ALS mouse models and cultured ALS neuronal models were shown to be modified in association with ATXN2 mutation or overall level of expression. ATXN2 functions in mRNA metabolism based on its interactions with multiple RNA binding proteins (RBPs) including A2BP1/RBFOX1, DDX6, PABP1, and the ALS proteins TDP-43, FUS. Our most recent data demonstrate that mutant ATXN2 interacts with the stress granule (SG) RNA interacting protein Staufen1, and that this interaction has specific downstream effects on SGs. Since ALS motor neurons are also characterized by RNA granules, ATXN2 functions regulating staufen positive RNA granules in motor neurons may be relevant to ALS. The objective of this grant is to investigate ATXN2 as a therapeutic target for ALS using human pluripotent stem-cell-derived motor neurons from ALS and SCA2 patients. Three specific aims are proposed: 1) We will perform gene editing a panel of pluripotent stem cell lines (?CRISPR editing?) creating multiple pairs of cells that are genetically identical (?isogenic?) except for the edited change at of the ATXN2 gene. 2) We will then extensively characterize the phenotype of these cells vs ALS cells when differentiated to motor neurons neurons. 3) We will determine how lowering the expression of ATXN2 using our existing ATXN2 antisense oligonucleotide DNA drug alters the phenotypes of patient motor neurons generated in aim 1, including the dynamics of RNA granules in the presence of ATXN2 mutation. The proposed work will clarify the role for SGs in neurodegeneration and will aid in the identification of new avenues toward treatments of ALS and other degenerative ataxias including SCA2.