Leonard Petrucelli, PhD - US grants

DESCRIPTION: (Applicant's abstract) Alzheimer's disease (AD) is a progressive neurodegenerative disease, affecting memory and cognitive functions. The cholinergic neurons that project into the cerebral cortex and hippocampus are lost in AD. Since acetylcholine transmission is involved with learning and memory, restoration of a cholinergic influence might have therapeutic benefit. Unfortunately, clinical trials using medications to enhance cholinergic function have reported little to no improvement in memory. Failure is likely due to the eventual loss of these neurons. Since survival of cholinergic neurons requires nerve growth factor (NGF) and NGF receptors, NGF-mediated neurotrophic effects may be reduced in AD. TrkA and p75 are receptors on cholinergic neurons that bind to NGF. Activation of TrkA has been linked with cell survival, while activation of p75 may be linked to apoptosis. Abeta accumulates in regions containing cholinergic cell bodies. It is neurotoxic and may interfere with such NGF mediated responses as the regulation of TrkA levels. Increase in p75/TrkA ratio could affect the downstream signaling mechanisms and promote cell death. Alternatively Abeta may interact directly with p75 and activate death signaling mechanisms. I propose to study whether the alterations in the p75/TrkA ratio are involved with he mechanisms that lead to the loss of cholinergic neurons in AD.

DESCRIPTION (Provided by Applicant): Parkinson's disease (PD) is a major public health problem in North America. Although PD has been regarded as having only a minor heritable component, recent discoveries have highlighted the contribution of genetics to this disease, with the description of dominant and recessive inherited forms of PD in several families. A wide variety of mutations in the parkin gene are associated with autosomal recessive forms of PD, especially "juvenile" onset forms where symptoms start before the fourth decade of life. Inheritance of certain parkin mutations result in greatly reduced parkin protein expression. The function of the parkin protein is unknown but recent results strongly suggest that it is a ubiquitin-protein ligase, or at least a component of a multi subunit ligase. Ubiquitination is a major way in which cells dispose of misfolded or damaged proteins, as well as a way in which the cell regulates levels of specific proteins. A point mutation in a second gene that may affect ubiquitination, UCH-L1, has been reported to be associated with autosomal dominant familial PD. The major pathological hallmark of PD is loss of dopamine containing cells in the substantia nigra (SN). How loss of parkin function causes damage to dopaminergic cells is not yet known, but a prediction of the hypothesis that parkin is an ubiquitin-protein ligase is that parkin is involved in the removal of misfolded and/or potentially toxic proteins from cells. We hypothesize that lack of parkin expression in dopaminergic cells of the SN causes an accumulation of an unidentified protein or set of proteins (protein x) which is/are toxic. The effects of UCH-L1 mutations may be similar, in that these would be predicted to also affect ubiquitination and hence increase the concentration of toxic protein(s) within the cell. The studies in this proposal aim to test this hypothesis in cellular systems which have been carefully chosen to be relevant to dopaminergic cells. Specifically we will perform functional studies of the potential role of parkin and UCH-L1 protein in ubiquitination. We will attempt to understand how mutations in parkin result in altered function of this protein and how it is regulated within the cell. We will also look at the effects of loss of parkin function on cell death and on ubiquitination reactions within the cell. We will also attempt to address the idea that mutations in parkin and UCH-L1 may interact in the same pathway, and examine the balance between use of ubiquitin by reactions catalyzed by parkin, and the production of free ubiquitin via UCH-L1.

DESCRIPTION (provided by applicant): An autosomal recessive form of juvenile Parkinsonism (ARJP) is the major cause of early-onset Parkinson's disease (PD) which results from mutations of the PARK2 (parkin) gene. Parkin is an E3 ligase that is responsible for the addition of poly-ubiquitin chains on specific substrates, which is recognized by the proteasome for degradation. Most disease-causing mutations of parkin are thought to be loss of function mutations (1), leading to the failure of parkin substrates to be ubiquitinated and degraded by the proteasome (2). Presumably, one or several of these proteins subsequently accumulate, ultimately causing neuronal degeneration in ARJP. To date, nine putative substrates degraded by parkin have been reported, two of which, p38 and Pael-R, interact with CHIP (carboxyl terminus of Hsc70-interacting protein). CHIP, like parkin, is an ubiquitin ligase, which acts in concert with parkin to modulate binding and activity of these substrates despite having separate targets (3,4). Recent work from our laboratory has shown that the levels of parkin increase in a gene dose-dependent manner in CHIP knockout mice. Moreover, the levels the parkin substrate, p38, are also elevated in these mice. Several investigators have generated parkin knockout mice in attempt to mimic ARJP in a mouse model. Unexpectedly, these mice lack frank nigral neuronal loss perhaps due to a compensatory mechanism involving CHIP. Based on the above results, we hypothesize that parkin and CHIP share common substrates such as p38, which are pathologically relevant to the disease and contribute to selective neuronal loss. We postulate that CHIP is a key factor in the regulation of parkin cell biology such that depletion of CHIP expression will result in loss of the dopaminergic neurons in the parkin knockout mice. CHIP may exert a central and unique role in PD in fine-tuning the response to proteotoxic stress at multiple levels by compensating for and regulating parkin substrates. The unifying hypothesis in this proposal is that parkin and CHIP are functionally and mechanistically linked by common effects associated with the ubiquitin proteasome system. It is our focus to resolve the mechanisms by which CHIP regulates parkin cell biology in vivo, and how this may be applicable to the clinical/neuropathological spectrum of presentation in ARJP.

[unreadable] DESCRIPTION (provided by applicant): Frontotemporal dementia (FTD) is a heterogeneous condition characterized by early behavioral change, cognitive decline and atrophy of the frontal and temporal lobes. The microscopic pathology varies markedly in different forms of the disease with some cases having tau-positive neuronal inclusions. However, the majority of FTD cases (approximately 60%) lack tau-positive lesions displaying mainly a microvacuolization of the superficial neuropil in the cortex. A proportion of these cases (10-15%) however do have ubiquitin-positive inclusions in motor neurons and show evidence of motor neuron degeneration (MND) leading to their designation as FTD-MND cases. Genetic linkage studies in FTD families have revealed three loci on chromosomes 17, 3 and 9. Over 30 mutations in the tau gene account for the majority of autosomal-dominant chromosome 17-linked cases (FTDP-17). FTDP-17 patients with identified tau mutations develop tau neurofibrillary pathology and many families also develop tau inclusions in glial cells. It is becoming increasingly clear that a proportion of chromosome 17q21-linked families lack any apparent mutations in the tau gene and, moreover, do possess the neurofibrillary pathology seen in families with defined tau mutations. Importantly, these chromosome 17q21-linked families exhibit ubiquitin positive neuronal inclusions, and a intranuclear ubiquitin positive inclusions are also seen in certain pedigrees. It is possible that the genetic cause of these families results from an unidentified mutation in the tau gene e.g. deep within an intron or from gross alterations of the tau locus, such as duplication. Alternatively, this disease could be caused by an alternative gene in this region. The overall aim of this proposal is to identify gene mutations associated with tau-negative FTD with neuronal and intranuclear ubiqutin positive inclusion linked to chr17q21 and to study the genotype/phenotype relationship and pathogenic mechanism of mutations in this gene. The identification of this gene will be a crucial step towards understanding the etiology of FTD as well as determining how this disease relates to MND. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Hereditary mutations within the MAPT (microtubule-associated protein tau) gene result in the pathologic and intra-neuronal aggregation of the tau protein that leads to frontotemporal dementia (FTD). The development of therapeutics for misfolded and abnormally processed (i.e. hyper-phosphorylated) tau species is necessary for sufferers of these autosomal tauopathic disorders and other neurological disorders which present with abnormal tau accumulation such as Alzheimer's disease (AD) and Parkinson's disease (PD). Molecular chaperones that are induced following a heat shock stimulus direct either the refolding of misfolded proteins and/or their targeting to the proteasome for degradation. Heat shock protein 90 (HSP90) inhibitors such as geldanamycin (GA) and its derivatives (17-allylamino-17-demethoxygeldanamycin; 17-AAG) can pharmacologically modulate chaperone levels and have recently been implicated for the treatment of cancer; sponsor NCI, Phase II) and mouse models of neurodegenerative disorders. In fact, Waza and colleagues recently showed that systemic administration of 17-AAG can markedly ameliorate motor impairments and life span in the spinal and bulbar muscular atrophy transgenic mouse model without detectable toxicity, by reducing amounts of monomeric and aggregated mutant androgen receptor. This study illustrates that Hsp90 inhibitors can be administrated chronically (20 weeks) and is well-tolerated. After screening a small panel of novel HSP90 inhibitors in a novel In-Cell Western assay that allows for direct intracellular quantitation of native and aberrant tau protein species, we identified a potent, blood brain barrier permeable HSP90 inhibitor (EC102) that significantly reduces abnormal tau species in vitro. In addition, we found that affected regions from human AD brain tissue have a significantly lower nanomolar binding affinity of Hsp90 for this novel inhibitor similar to tumor cells, while micromolar affinity was demonstrated in unaffected regions, comparable to normal cells. This suggests for the first time that in neurons, which progressively accumulate abnormal proteins in neurodegenerative disorders, and in this case AD, Hsp90 becomes engaged in active chaperoning and stabilization of these proteins and the Hsp90 adopts a novel high-affinity state. Thus these studies provide compelling evidence for the use of Hsp90 inhibitors to enhance Hsp90-mediated phospho-tau degradation in AD. We therefore propose that treatment with this compound may delay the progression of pathology and restore memory loss in mice that express a mutant form of human tau (P301L) driven by the tetracycline operator (rTg4510). [unreadable] [unreadable] [unreadable]

PROJECT/SUMMARY ABSTRACT We recently found that null mutations in the gene encoding the secreted growth factor progranulin (PGRN) are a frequent cause of frontotemporal dementia, particularly in patients affected with the pathological subtype referred to as frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U) 9-11. Subsequently, the major protein that makes up the inclusions found in FTLD-U (and in amyotrophic lateral sclerosis, ALS) was identified as TAR DNA-binding protein-43 (TDP-43), an obscure nuclear protein known to be involved in exon splicing 12. Subsequent to this finding, mutations in the gene encoding TDP-43 (TARDBP) were identified as a direct cause of neurodegeneration in sporadic and familial patients with ALS. We have shown that decreasing PGRN expression leads to pathological processing of TDP-43 by caspases in cell culture models 13. Moreover, recent preliminary data revealed that deletion of progranulin in C. elegans leads to increased cleavage of endogenous TDP-43 strongly supporting our in vitro findings. Without PGRN or mutations in TARDBP, TDP-43 gets cleaved, which leads to translocation from the nucleus to the cytosol, a pathologic phenotype which resembles what happens to TDP-43 in patients with FTLD-U or ALS. Thus, loss of TDP-43 function due to inappropriate cleavage, translocation, or inclusion formation could play an important role in neurodegeneration. We also hypothesize that PGRN mutations other than null mutations, such as missense mutations, or causal TARDBP mutations can sufficiently abolish PGRN and TDP-43 functions to cause neurodegeneration. The overall goals of our proposal are 1) to provide additional mechanistic insight into the PGRN/TDP-43 axis in neurodegenerative diseases;2) to determine whether the shorter TDP-43 fragments are more fibrillogenic, are neurotoxic and can sequester nuclear TDP-43, a property that would explain the formation of inclusions, redistribution and neurodegeneration seen in FTLD-U and ALS;and 3) to explore neurodegenerative disease mechanism associated with TARDBP mutations. Our hypothesis is that loss of functional PGRN leads to loss of functional TDP-43, which leads to cell death.

DESCRIPTION (provided by applicant): Frontotemporal lobar degeneration (FTLD) is a group of dementing illnesses with devastating consequences that preferentially affect individuals in late middle age. Neocortical involvement in the frontal and temporal lobes leads to major alterations in behavior and affects cognition, including language function. Unfortunately, there are currently no substantial therapies for the treatment of these diseases. The recent identification that mutations in tau can cause FTLD and that it is the hyperphosphorylation of the mutant tau that leads to aggregation, has established an entirely novel drug target for treatment of FTLD and similar disorders such as progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD), It has long been known that small molecule inhibitors of Hsp90 can lead to the enhanced degradation of mutated and misfolded proteins. In fact, natural product and synthetic Hsp90 inhibitors are in various stages of clinical development as anti- cancer agents. One such drug, SNX-5422, is a potent inhibitor of Hsp90 and is particularly attractive in that it can achieve, following oral dosing, substantial and sustained plasma levels. It appears to be well tolerated to date and is currently being evaluated in multiple phase I trials. More recently, it has been discovered that Hsp90 inhibitors can cause the preferential degradation of abnormally phosphorylated tau both in vitro and in vivo. Unfortunately, SNX-5422 does not appear to cross the blood brain barrier at appreciable levels making this and closely related compounds not likely to be useful for treating CNS disorders such as FTLD. Clearly, novel analogues are necessary for the treatment of CNS disorders. Fundamentally, therefore, we propose in this application to exploit the known chemical space with respect to inhibitors of Hsp90 to identify compounds that maintain inhibition while simultaneously having physicochemical properties compatible with crossing the blood brain barrier (BBB). The initial positive results provide a proof of concept and suggest that a dedicated and funded drug discovery effort offers the real possibility of identifying Hsp90 inhibitors suitable for clinical development targeting FTLD. The ultimate goal of this overall proposal is to provide enough data to determine a go / no go decision for the filing of an Investigational New Drug application (IND) with the FDA. Public Health Relevance: We propose to develop and characterize small compounds called Hsp90 inhibitors as potential therapeutics for the treatment of tauopathies including frontotemporal degeneration. If these studies are successful, we can move into Clinical Phase I trials. Disclaimer: Please note that the following critiques were prepared by the reviewers prior to the Study Section meeting and are provided in an essentially unedited form. While there is opportunity for the reviewers to update or revise their written evaluation, based upon the group's discussion, there is no guarantee that individual critiques have been updated subsequent to the discussion at the meeting. Therefore, the critiques may not fully reflect the final opinions of the individual reviewers at the close of group discussion or the final majority opinion of the group. Thus the Resume and Summary of Discussion is the final word on what the reviewers actually considered critical at the meeting.

DESCRIPTION (provided by applicant): We found that null mutations in the gene encoding the secreted growth factor progranulin (PGRN) are a frequent cause of frontotemporal dementia, particularly in patients affected with the pathological subtype referred to as frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U). Subsequently, the major protein that makes up the inclusions found in FTLD-U (and in amyotrophic lateral sclerosis, ALS) was identified as TAR DNA-binding protein-43 (TDP-43), an obscure nuclear protein known to be involved in exon splicing. Subsequent to this finding, mutations in the gene encoding TDP-43 (TARDBP) were identified as a direct cause of neurodegeneration in sporadic and familial patients with ALS. We have shown that decreasing PGRN expression activates cell death pathways leading to pathological processing of TDP-43 by caspases in cell culture models. Recent preliminary data revealed that deletion of progranulin in mice and in knockdown experiments in cells lead to enhanced sensitivity to ER stressors and increased levels of the transcription factor CHOP (C/EBP homologous protein), a protein that is induced by ER stress and promotes apoptosis. Without PGRN or in the presence of mutations in TARDBP, TDP-43 gets cleaved, which leads to translocation from the nucleus to the cytosol, a pathologic phenotype which resembles what happens to TDP-43 in patients with FTLD-U or ALS. Thus, loss of TDP-43 function due to inappropriate cleavage, translocation, or inclusion formation could play an important role in neurodegeneration. We also hypothesize that PGRN mutations other than null mutations, such as missense mutations, or causal TARDBP mutations can sufficiently abolish PGRN and TDP-43 functions to cause neurodegeneration. The overall goals of our proposal are 1) to provide additional mechanistic insight into the signaling pathways associated with PGRN and neuronal survival 2) to determine whether the shorter TDP-43 fragments are more fibrillogenic and are neurotoxic, a property that would explain the formation of inclusions, redistribution and neurodegeneration seen in FTLD-U and ALS; and 3) to explore neurodegenerative disease mechanism associated with TARDBP mutations. Our hypothesis is that loss of functional PGRN leads to loss of functional TDP-43, which leads to cell death.

DESCRIPTION (provided by applicant): TDP-43 is the principal component of neuronal inclusions in frontotemporal lobar degeneration with TDP-43-positive inclusions (FTLD-TDP) and amyotrophic lateral sclerosis (ALS). Since the major actions of TDP-43 derive from its RNA binding activity, determining if TDP-43 RNA targets are altered in disease will provide insight on the mechanisms of TDP-43 toxicity. RNA-immunoprecipitation studies in human HEK293T cells identified sortilin (SORT1), a neuronal progranulin (PGRN) receptor, as a TDP-43 target. Mutations in GRN are a major cause of FTLD-TDP. That TDP-43 regulates SORT1 RNA now provides a putative link between TDP-43 dysfunction and altered PGRN signaling in sporadic FTLD-TDP and ALS. Preliminary data shows that Tdp-43 regulates Sort1 mRNA splicing in murine primary neurons and mouse brain. Tdp-43 knockdown leads to the retention of 99 base pairs within intron 17 of Sort1, referred to as exon 17b. Exon 17b is highly conserved between mouse and human, so it is very probable that human SORT1 is regulated by TDP-43. Indeed, an alternatively spliced SORT1 variant lacking exon 18 (SORT1 Ex18) was identified as a TDP-43-bound transcript in human neuroblastoma cells. It encodes a SORT1 (SORT1 Ex18) isoform with a truncated C-terminus. Given that the C-terminus is required for PGRN endocytosis, expression of SORT1 Ex18 is likely to have detrimental consequences on PGRN signaling. Likewise, Sort1+Ex17b expression may alter Pgrn function. We have shown that neurite outgrowth and branching are decreased in Pgrn-/- cortical neurons, a phenotype rescued by recombinant human PGRN. Overall, we presume there exists a bidirectional relationship between TDP-43 and SORT1/PGRN, such that abnormalities in TDP-43 promote SORT1/PGRN dysregulation and, conversely, SORT1/PGRN dysregulation causes TDP-43 pathology, thus perpetuating a neurotoxic cycle. The latter is supported by the finding that TDP-43 aggregation is attenuated in cultured cells when SORT1 is overexpressed, suggestion that loss of SORT1 function may indeed contribute to TDP-43 pathology. Our objective is thus to investigate this relationship in more detail. To this end, we will use a combination of in vitro and in vivo models to investigate: 1) the functional mechanisms underlying TDP-43-mediated SORT1 regulation; 2) whether SORT1 RNA expression and/or splicing, as well as SORT1 protein expression, are altered in FTLD-TDP; 3) whether various SORT1 isoforms differentially influence PGRN signaling; and 4) the influence of SORT1 isoforms on TDP-43 aggregation and toxicity.

DESCRIPTION (provided by applicant): Frontotemporal lobar degeneration with TDP-43-positive inclusions (FTLD-TDP) and amyotrophic lateral sclerosis (ALS) are devastating neurodegenerative diseases with significant clinical and neuropathological overlap. A recent groundbreaking study now highlights a genetic link - hexanucleotide (GGGGCC) repeat expansion in C9ORF72 - as the most common cause of ALS and FTLD-TDP. This discovery raises an important question: how do the expanded repeats in a non-coding region of C9ORF72 contribute to disease pathogenesis? We hypothesize that RNA-mediated toxicity is a likely mechanism given that transcripts containing the expanded GGGGCC repeat accumulate as nuclear RNA foci in the frontal cortex and spinal cord of C9ORF72 mutation carriers. RNA foci formation, and the subsequent sequestration and altered activity of RNA-binding proteins by the foci, are emerging as a common pathogenic mechanism in many neurodegenerative diseases caused by non-coding repeat expansions. To determine whether RNA foci formation is a primary neurotoxic mechanism in c9FTD/ALS, novel in vitro and in vivo models of C9ORF72 hexanucleotide repeat expansions are required. To this end, we created two constitutive expression constructs to drive expression of RNA containing 18 GGGGCC repeats (18R) or 51 GGGGCC repeats (51R), the latter to mimic hexanucleotide repeat expansion. Notably, expression of the 51R transcript, but not the 18R transcript, leads to the formation of distinct, intranuclear RNA foci in HeLa and COS1 cell lines reminiscent of the foci observed in C9FTD/ALS cases. We also found foci formation is associated with enhanced cytotoxicity, as well as the sequestration of the RNA-binding proteins SAM68 and hnRNP-K. These findings not only illustrate that we can model the RNA foci formation observed in C9FTD/ALS, but also show that we have generated a valuable tool to study the mechanisms by which they exert toxicity. On the heels of these exciting findings, the goals of this project are: 1) to generate and characterize transgenic mice overexpressing non-pathogenic (non-foci-forming) and pathogenic (foci-forming) C9ORF72 repeat expansions; 2) to evaluate whether RNA foci formation results in behavioral deficits and neurodegeneration in our foci-forming (GGGGCC)51 mouse model; 3) to identify RNA-binding proteins sequestered by the foci; 4) and to evaluate whether RNA targets of the sequestered proteins are altered in our novel cell and animal models, as well as in brain tissue and fibroblasts of C9FTD/ALS cases. Overall, we believe the proposed studies examining RNA foci formation consequences will lead to a better understanding of C9FTD/ALS-related mechanisms and help to uncover promising therapeutic targets.

PROJECT SUMMARY/ABSTRACT Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are neurodegenerative diseases with clinical, pathological and genetic overlap. The expansion of a hexanucleotide repeat in a non-coding region of C9ORF72 is the major genetic cause of FTD and ALS. Elucidating how these expanded repeats cause c9FTD/ALS has since become an important goal of the field. Our recent study identifying a new pathological hallmark of c9FTD/ALS - the accumulation of neuronal inclusions composed of peptides produced by repeat associated non-ATG (RAN) translation of sense and antisense transcripts of the expanded repeat - implicates RAN translation as a mechanism of disease. This unconventional mode of translation was first described for expanded CAG?CTG repeats and accounts for the accumulation of polyA or polyQ proteins in spinocerebellar ataxia type 8 and myotonic dystrophy type 1, respectively. Based on our findings that RAN translation of expanded repeats similarly occurs in c9FTD/ALS, we believe that peptides thusly produced play a role in disease pathogenesis. This project aims to investigate this pressing question, as well as several others. Specifically, we will evaluate whether: 1) the various peptides produced via RAN translation show a differential pattern of expression in c9FTD/ALS; 2) there is differential toxicity among RAN translated peptides or among soluble and insoluble species; 3) toxicity is enhanced by loss of C9ORF72 function; and 4) expression of RAN translated peptides influences disease phenotype or depend upon repeat length. To investigate these questions we will utilize novel antibodies, develop new cell culture and animal models, and examine fibroblasts and tissues from c9FTD/ALS patients. In addition to being subject to RAN translation, accumulating sense (GGGGCC)exp and antisense (CCCCGG)exp RNA transcripts may result in the sequestration of RNA-binding proteins in c9FTD/ALS. Nuclear RNA foci of expanded RNA transcripts accumulate in frontal cortex, spinal cord and cerebellum of C9ORF72 repeat expansion carriers. The sequestration and inactivation of RNA- binding proteins by RNA foci is observed in several repeat expansion diseases and is thought to account for some features of disease. The identification of RNA-binding proteins sequestered by transcripts of expanded repeats in C9ORF72, and determining whether the function of these proteins are altered in c9FTD/ALS, will increase our understanding of this potential mechanism of disease. Overall, the goals of Project 2 are to test the hypothesis that transcripts of expanded repeats contribute to c9FTD/ALS pathogenesis via two mechanisms: by RAN translation and by the mis-regulation of RNA-binding proteins.

DESCRIPTION (provided by applicant): In this P01 resubmission proposal entitled Pathobiology of Neurodegeneration in C9ORF72 Repeat Expansion, we seek to evaluate the mechanisms of C9ORF72 expanded repeats, the most common cause of amyotrophic lateral sclerosis (ALS) and front temporal dementia (FTD), to improve the diagnosis of and prognosis for patients suffering from c9FTD/ALS. We have assembled a world-class team combining expertise in neurology, genetics, neuropathology, and cell biology that has worked closely together and has all resources in place. Our significant progress to elucidate how expanded repeat RNA transcripts and epigenetic changes may respectively drive toxicity and haploinsufficiency in c9FTD/ALS has led to the discovery that repeat expansion size does affect disease severity, and the identification of a potential biomarker detectable in blood of c9FTD/ALS patients. We now present evidence that aberrant methylation of histone 3 at lysine 9 is detectable in brain tissue, fibroblasts and blood of C9ORF72 mutation carriers. We also identified TMEM106B as the first genetic modifier of disease phenotype in C9FTD/ALS. In drawing upon the strengths of the Mayo Clinic Neurology Department, we have begun longitudinal studies of 44 C9ALS pedigrees to determine whether expansion size, tri-methylation of histone lysine residues, mRNA expression levels and TMEM106B genotypes, correlate with phenotypic variability in c9FTD/ALS. In addition, we have since produced and characterized antibodies critical for detecting each of the five repeat-associated non-ATG (RAN) translation peptides [poly(GP), poly(GA), poly(GR), poly(PA) and poly(PR)] in cell and animal models as well as human tissue. We also provide evidence that ubiquilin-2, tau and p62/sequestosome are present in neuronal inclusions in various brain regions and spinal cord, indicating that these proteins, in addition to TDP-43, may play a role in pathogenesis of front temporal lobar degeneration with TDP-43 pathology (FTLD-TDP). We provide preliminary data that TDP-43-negative/C9RANT-positive neuronal inclusions can also be detected with antibodies to dimethylarginine, suggesting a new disease mechanism involving non-histone protein methylation. Simply put, our multi-disciplinary studies will improve understanding of C9ORF72-related neurodegeneration, identify potential biomarkers and therapeutic targets, and develop a compelling brain, biofluid and biopsy resource to aid future drug discovery.

PROJECT SUMMARY / ABSTRACT Hyperphosphorylated tau is the primary component of neurofibrillary tangles (NFTs), a pathological hallmark of several neurodegenerative diseases, including Alzheimer's disease (AD), frontotemporal dementia with parkinsonism associated with chromosome 17 (FTDP-17), progressive supranuclear palsy (PSP), and corticobasal degeneration (CBD). Since accumulation of hyperphosphorylated tau (p-tau) correlates with the onset of cognitive symptoms, we aim to evaluate and validate inhibition of cytosolic histone deacetylase 6 (HDAC6), which modulates the accumulation of hyperphosphorylated tau, as a lead strategy for the treatment of Alzheimer's disease (AD) and related neurodegenerative tauopathies. We seek to target HDAC6, through the use of a novel blood-brain-barrier-permeable inhibitor, as a means to delay emergence of tau pathology. We have already identified HDAC6 as a highly drug-able target and present new evidence that inhibiting HDAC6 leads to hyperacetylation and enhanced degradation of tau. We propose that acetylation of tau within KXGS motif regions (detectable with our novel site-specific antibody ac-KIGS), which are critical for tau to bind microtubules, counter balances phosphorylation on this same motif (recognized by 12E8). Given that tau species phosphorylated on KXGS motifs accumulate in neurofibrillary tangles, fail to bind and stabilize microtubules, and are not recognized by cellular degradation machinery, we hypothesize that loss of HDAC6 activity will increase the ratio of acetylation to phosphorylation on KXGS motifs to favor tau clearance, thereby preventing neuropathology and disease progression. There are no obvious, known counter indications regarding HDAC6 inhibition: HDAC6 knockout mice exhibit no overt deleterious phenotypes; we have not observed obvious toxicity with our lead HDAC6 inhibitors in cells or in mice; and selective inhibition of HDAC6 versus other HDAC isoforms has been shown to preserve normal gene expression in cells, thereby minimizing toxicity to patients. As such, we envision the use of HDAC6 modulation, in concert with the advent of techniques designed to detect and image amyloid beta peptide (Ab) pathology years to decades before the onset of cognitive symptoms, as a means to interrupt the development and progression of p-tau accumulation initially triggered by Ab deposition. In so doing, we aim to provide a greater understanding of the value of HDAC6 inhibition in slowing the onset or progression of tauopathies and, upon completion, will deliver a very compelling data package to warrant further clinical development of this novel therapeutic strategy for AD and other tauopathies.

PROJECT SUMMARY/ABSTRACT The overarching theme of this new program project proposal entitled Pathobiology of Neurodegeneration in C9ORF72 Repeat Expansion is to evaluate the mechanisms of C9ORF72 expanded repeats, the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), to improve the diagnosis of and prognosis for patients suffering from c9FTD/ALS. Core A will facilitate these goals by providing scientific and fiscal oversight. Core A will be responsible for managing fiscal responsibilities for the program project, ensuring ethical and responsible conduct of research, providing scientific direction and accountability, and reporting progress to the NIH and to the public. In order to assure the success of this research proposal, Core A will also leverage Mayo Clinic resources to foster a multidisciplinary program that advances research, generates valuable biological resources, and promotes education. Among institutional resources that Core A will avail itself on behalf of the P01 are administrative assistance with grants administration and all aspects of human subject research; access to biostatistics and bioinformatics; access to the latest in technology for biobanking (e.g., Nexus Universal BioStore); and institutional commitment to regenerative medicine (Center for Regenerative Medicine) for transformation of skin biopsies to fibroblast cell lines to be used by Projects 2 and 3. To accomplish these goals, the Core A will pursue the following specific aims. Specific Aim 1: Provide administrative structure and fiscal oversight for the program project grant. Specific Aim 2: Organize regular Executive Committee meetings composed of PLs of projects and cores, as well as other key personnel to assist in scientific administration and to facilitate integration and progress on research. Specific Aim 3: Establish an External Advisory Committee, conduct annual meetings, and report progress to the NIH. Specific Aim 4: Assume responsibility for quality control of program project activities and ensure the safety of human subjects and confidentiality of their data. Specific Aim 5: Ensure that research conforms to the standards of the ethical conduct of research and is compliant with HIPAA guidelines. Specific Aim 6: Promote compliance with the NIH Public Access publication policy to make the results of research funded by this proposal widely accessible to the general public. Specific Aim 7: Promote education of ALS and related disorders by leveraging Mayo Clinic resources for the invited speaker seminar series, scientific symposia, contribution to Mayo Clinic related social media, and encouragement of P01 scientists and clinicians to participate in patient and caregiver support groups.

DESCRIPTION (provided by applicant): We seek to define the molecular mechanisms by which histone deacetylase 6 (HDAC6) and stress granules (SGs) interactively drive the pathophysiology of tauopathies, such as Frontotemporal Dementia. In so doing, we will test the hypothesis that loss of HDAC6 activity augments site-specific tau acetylation, preventing the pathological recruitment of tau to stress granules, and slowing disease progression. We recently reported that HDAC6-mediated deacetylation of tau enhances accumulation and aggregation, and we have now identified the acetylation sites on tau that are regulated by HDAC6. We also recently reported that tau pathology develops in concert with SG pathology in human and mouse models. Understanding the role of HDAC6 in regulating tau acquires added significance because HDAC6 regulates SGs, which are protein/RNA complexes that form in response to translational inhibition. Thus, HDAC6 functions as a nexus, linking acetylation of tau, SG formation and disease progression. The work in this proposal will elucidate the mechanisms by which HDAC6 regulates tau recruitment to SGs, with a particular focus on tau acetylation, and also assess whether decreasing HDAC6 expression with antisense oligonucleotides (ASOs) is sufficient to alleviate both tau and SG pathology. We have already identified ASOs targeting HDAC6 that effectively decrease expression in mouse primary neurons, and through an active collaboration with Isis Pharmaceuticals, are working to identify ASOs that will progress to in vivo testing. Given HDAC6 inhibition or knockout produces no obvious toxicity in cells or in mice, HDAC6 as a therapeutic target possesses strong translational appeal. Our proposal is structured to provide a greater understanding of the intricate and dynamic relationship between HDAC6 and tau, as well as elucidate the role of HDAC6 and tau acetylation in SG dynamics. We will generate acetyl tau antibodies on HDAC6-responsive sites to investigate how acetylation on these key sites affects tau biology and SG dynamics in our cell culture and rTg4510 mouse models as well as postmortem brain tissue from tauopathy cases (Aim 1). We will also determine whether modulation of HDAC6, through the use of ASOs, diminishes tauopathy and preserves cognition in vivo (Aim 2). Using mouse primary neurons, fibroblast-derived neurons from human tauopathy patients, which we show exhibit robust SG formation in response to stress, and rTg4510 mice treated with HDAC6 ASOs, we will further determine the roles of HDAC6, tau and acetylation in regulating stress granule dynamics (Aim 3). Completion of these studies in tandem will fundamentally expand our knowledge of the pathophysiology of tauopathies, and provide key insights into the pre-clinical efficacy of HDAC6 ASOs as a novel therapeutic strategy.

PROJECT SUMMARY/ABSTRACT In order to uncover novel genetic variants that influence tau toxicity, and also recapitulate the genetic diversity characteristic of the human population, Project 2 aims to take advantage of the observation that severity of tauopathy in transgenic mouse models is significantly impacted by altering genetic background. While most transgenic lines have been created on a standard genetic background, a single inbred strain does not incorporate one of the major sources of phenotypic variation in human populations: genetic diversity. The lack of genetic diversity also limits the translational utility of standard mouse models because it grossly underestimates the variation of responses that will be seen in the human population. Therefore a genetically- diverse mouse model of tauopathy will be created in the current study, using AAV to drive mutant tau expression in a panel of Collaborative Cross (CC) recombinant inbred mice, as well as Diversity Outbred (DO) mice produced from random repeated outcrossing of CC strains. Collectively, the CC and DO mouse populations offer high mapping resolution and broad allelic diversity, carrying 45 million SNPs and structural variants and thus providing a unique opportunity to discover new genes that regulate tau toxicity in vivo. Although there are currently no genetically diverse models of tauopathy that are representative of the diversity present within the human population, the development of such a model would enable researchers to test the efficacy and toxicity of new therapeutic strategies, improving translational relevance by incorporating the genetic variation missing from traditional models. Here, based on the compelling evidence presented in Project 3 demonstrating that antibody-induced elevation of plasma tau fluctuates with disease progression, we will evaluate the extent to which genetic background and disease severity influence plasma tau levels in our AAV-injected, CC and DO mice. These findings will not only provide key insight into the potential significance of changes in plasma tau concentrations to progression of tauopathy, but also offer an indication of the magnitude of variability that could be anticipated in the human population. Collectively, the studies proposed will not only increase our understanding of underlying pathogenic mechanisms and pathways, but also drive the identification of new therapeutic targets for the treatment of tauopathy.

PROJECT SUMMARY/ABSTRACT The overarching theme of this new Center without Walls (CWOW) proposal entitled ?Identifying genes and pathways that impact tau toxicity in FTD? is to discover novel genetic modifiers of tauopathy to provide unique insight into disease mechanisms, as well as to develop and validate an innovative approach to measure tau burden in patients using plasma samples. Core A will facilitate these goals by providing scientific and fiscal oversight and promoting the Center's scientific governance strategy to ensure the synergy and integration of each core and project. Core A will be responsible for managing fiscal responsibilities for the Center, ensuring ethical and responsible conduct of research, providing scientific direction and accountability, and reporting progress to the NIH and to the public. In order to ensure the success of this research proposal, Core A will also leverage Mayo Clinic resources to foster a multidisciplinary and cross-institutional program that advances research, generates valuable biological resources, and promotes education and resource sharing with the field. Core A will avail itself of Mayo Clinic institutional resources on behalf of the Center for administrative assistance with grants administration and aspects of human subject research, as well as coordinate and leverage the institutional resources at the other sites, including the various relationships and memberships the project and core leaders have with many external organizations and consortia. To accomplish these goals, Core A will pursue the following specific aims. Specific Aim 1: Provide administrative structure and fiscal oversight for the CWOW. Specific Aim 2: Assume responsibility for the quality control of the Center's activities by ensuring responsible conduct of research. Specific Aim 3: Promote the scientific direction and integration of the Center components by organizing regular Executive Steering Committee meetings and overseeing external collaborations that support the Center cores and projects. Specific Aim 4: Establish an External Advisory Committee and report progress to the NIH. Specific Aim 5: Establish and maintain the Center website. Specific Aim 6: Promote education on FTLD and related tauopathies, and encourage CWOW investigators to participate in patient and caregiver support groups.

PROJECT SUMMARY/ABSTRACT In this highly-integrated and multi-disciplinary Center without Walls entitled ?Identifying genes and pathways that modulate tau toxicity in FTD?, we seek to discover novel genetic modifiers of tauopathy to provide unique insight into disease mechanisms, as well as support the development and validation of an innovative approach to measure tau burden in patients using plasma samples. Taking advantage of the genome sequencing data available from PSP patients, Project 1 will identify genetic variants that influence disease risk, and in collaboration with the Human Biology Validation Core determine how key variants are associated with tau burden. As a complementary but alternative approach to identify genetic modifiers, Project 2 will utilize the Collaborative Cross and Diversity Outbred mouse strains developed at Jax Labs to uncover genetic variants that determine sensitivity or resistance to AAV-induced tauopathy. Project 3 will investigate the hypothesis that assessment of plasma tau levels following the administration of anti-tau antibodies provides a sensitive and specific biomarker for tau aggregation in the brain, employing samples from both murine models of tauopathy as well as patients enrolled in ARTFL and LEFFTDS. Project 4 will construct a disease signature for FTD-tau and mine public databases and systems analyses to predict pharmacologic and/or genetic interventions to reverse the signature, as well as elucidate how genetic modifiers identified in Projects 1 and 2 impact tau metabolism and secretion. The four research projects will be supported by the Administrative Core (Core A), the Viral Production and Cloning Core (Core B), the Human Biology Validation Core (Core C), and the Data Coordination Core (Core D). We envision that at the conclusion of the funding period, we will have: (i) identified key genes that determine either sensitivity or resistance to tau-mediated neurodegeneration; (ii) developed a sensitive and specific blood-based biomarker for tau deposition in the brain; (iii) discovered novel therapeutic targets predicted to reverse the transcriptional signature that defines tau mutation carriers; and (iv) determined how tau mutations and genetic modifiers of tauopathy impact tau metabolism, protein homeostasis and cell viability. Therefore, our Center is uniquely poised to enable the identification of patient populations at risk, while simultaneously enhancing diagnostic capabilities and expanding therapeutic possibilities.

Frontotemporal lobar degeneration (FTLD), a common cause of early-onset dementia, encompasses a group of disorders with significant genetic, clinical and neuropathological heterogeneity. Understanding the diverse underlying mechanisms of FTLD pathogenesis is a fundamental area of interest of my research program. To accelerate scientific discovery, we have adopted a comprehensive approach that investigates multiple FTLD mechanisms driven by key molecular players (e.g., TDP-43, progranulin, and tau). We also place great emphasis on translational research geared towards identifying much needed biomarkers and therapies, an area of particular importance given that there exists no treatment for FTLD. Our endeavors to uncover the pathomechanisms associated with TDP-43, tau and FTLD-causing mutations have yielded seminal findings published in high-impact journals. For instance, in the five years since the discovery of the G4C2 repeat expansions in C9ORF72 as the most common known cause of FTLD, my group identified a new neuropathological hallmark specific to this mutation, namely the accumulation of proteins of repeating dipeptides synthesized from the expansion; made significant strides in elucidating mechanisms of disease relating to these so-called c9RAN proteins; identified promising therapeutic strategies and potential biomarkers for C9ORF72 repeat expansion carriers; and developed the first mouse model to recapitulate both neuropathological and clinical features of patients. Our productivity is influenced by the excellent research environment fostered at Mayo Clinic, which brings together highly interactive and devoted neurobiologists, geneticists, neuropathologists and physician scientists, as well as the numerous collaborations we have forged with world-renowned experts in the field. Herein, we propose to extend our discoveries by addressing impactful questions, some of which may be high risk, but all with clear potential to be transformative to the field. Of importance, the nature of the R35 mechanism will allow us the flexibility to explore intriguing new directions that emerge from our findings and those of others, ensuring that our studies remain timely and relevant. Overall, with the goal of advancing our understanding and developing therapies for FTLD, we propose mechanistic and translational investigations relating to three main areas: 1) C9ORF72-linked FTLD-TDP; 2) GRN-linked FTLD-TDP; and 3) FTLD-Tau. We will explore various therapeutic approaches, seek to identify disease-relevant biomarkers in cerebrospinal fluid and blood, and develop and improve FTLD animal models.

The development of an atomic model of tau filaments in Alzheimer?s disease (AD) has opened several new opportunities, including the ability to identify high-affinity ligands or antibodies to detect AD tau filaments (i.e., PET ligands, small molecule compounds, immunotherapy, biomarkers) and providing a clear directive about essential features that need to be recapitulated from a modeling perspective. Given that tauopathies are complex, pathogenic entities with significant heterogeneity in clinical presentation, disease course, and underlying pathology, it is essential that the tools under development are capable of detecting/recognizing the actual pathologies observed. As such, the overall goals of the current project are to characterize the heterogeneity of tau filaments across tauopathies, and to utilize structural features that define tau and TDP-43 filament morphology in sporadic and familial forms of disease to provide novel insight into pathogenesis and toxicity. In particular, we will assess and compare tau filament heterogeneity between AD and chronic traumatic encephalopathy (CTE) and develop novel resources to both detect and model tau in these distinct tauopathies. Similar methodology utilizing cryo-electron microscopy (cryo-EM) will also be employed to resolve filament structure in frontotemporal lobar degeneration (FTLD), which is the second most common cause of dementia after AD. As FTLD is usually characterized pathologically by the aberrant deposition of either tau (FTLD-tau) or TDP-43 (FTLD-TDP), we will develop atomic models of both tau and TDP-43 filaments in sporadic and familial forms of FTLD. Overlaying structural insights with our recent discovery of novel post-translational modifications (PTMs) of tau and TDP-43, we will assess the contribution of specific modifications to aggregation propensity, utilizing a combination of cryo-EM, mass spectrometry, molecular biology and biochemical approaches. These results will guide the continued development of resources to best detect and model particular aspects of pathology.

The intracellular aggregation of hyperphosphorylated tau in neurofibrillary tangles (NFTs) is a neuropathological hallmark of Alzheimer's Disease (AD). While tau pathology is correlated with both synaptic loss and neurodegeneration in AD, implicating a major role for tau accumulation in cognitive decline, the mechanisms of tau aggregation and cell toxicity remain unknown. We propose to provide novel insight into the extent to which abnormalities in nuclear pore complexes (NPCs) and the resulting nucleocytoplasmic transport (NCT) defects contribute to AD pathogenesis. NPCs are complex, molecular assemblies consisting of multiple copies of proteins called nucleoporins (Nups) that regulate the macromolecular trafficking of protein and RNA between the nucleus and cytoplasm. Previous reports have identified nuclear membrane irregularities that often associate with NFTs. However, the pathophysiology of tau and its relationship to nuclear function/integrity is not well understood. We have amassed a series of preliminary data demonstrating that disruptions of the NPC and functional nuclear transport are present in cells containing hyperphosphorylated tau in human AD brain, as well as in mouse and cellular models of tauopathy. Given the extent of NPC and NCT dysfunction in ALS and HD, our preliminary studies in AD/FTLD-tau suggest that impaired NPC function may represent a common mechanism of neurodegeneration. Specifically in AD, we find that a major nuclear pore component, Nup98, is mislocalized from the nuclear membrane and co-aggregates with tau in both human brain and transgenic tau mouse models. Tau and Nup98 directly interact and we have discovered that Nup98 can potently promote tau filament formation in vitro. Based on these key findings, we will test the overall hypothesis that tau-Nup98 interactions underlie a pathologically important disruption of nuclear pore function and represent a key mediator of neurotoxicity in AD and related tauopathies. As the ultimate goal of our project is to develop a disease- modifying therapeutic strategy, we will assess whether either clearing tau/Nup98 inclusions or rescuing the NCT defects are necessary and/or sufficient to ameliorate tau toxicity. Our studies specifically will: 1) Investigate alterations in nuclear pore complexes in human AD/FTD and model systems. These studies will establish NPC defects as a novel, pathological feature of tauopathy; 2) Determine the functional alterations in nucleocytoplasmic transport resulting from nuclear pore abnormalities in AD/FTD. These studies will determine if aberrant functional NCT is characteristic of cells containing tau lesions.; and finally, 3) Examine whether disaggregases rescue NPC/NCT pathway defects in vivo. These studies will help to test the hypothesis that NPC defects can contribute to tau toxicity

Hexanucleotide (GGGGCC) repeat expansions in C9orf72 are the leading genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). In this proposal, we seek to identify new therapeutic targets by using a genetically diverse mouse model of C9-FTD/ALS. In this study design, we will be using adeno-associated virus (AAV) to drive mutant C9-repeat expression (AAV-149R) in a panel of Collaborative Cross (CC) recombinant inbred mice, as well as Diversity Outbred (DO) mice produced from random repeated outcrossing of CC strains. Collectively, the CC and DO mouse populations offer high mapping resolution and broad allelic diversity, carrying 45 million SNPs and structural variants. This provides a unique opportunity to discover new genes that regulate C9-repeat toxicity in vivo. These modifiers will be compared to a parallel study of CC/DO mice that will be injected with AAV-TauP301L to determine modifiers of tau toxicity. In Aim 1 we will use CC mice to identify strains that are sensitive or resistant to C9 or tau toxicity while in Aim 2 we will use the DO mice to finely map these potential modifiers. In Aim 3 we will perform single- cell RNAseq studies from AAV-149R and AAV-TauP301L mice at stages of pre-symptomatic, as well as moderate and severe disease to determine the evolution of the brain response to these pathologies at the individual cell level. Also in Aim 3, we will perform single-nucleus RNAseq on postmortem human tissue from healthy controls, c9-ALS, c9-FTD, and FTLD-TauP301L from frontal cortex. Top hits from these datasets will be used to prioritize candidate genes/loci from Aims 1 and 2, and followed up with validation studies on postmortem tissue at the histological and biochemical level. At the conclusion of this comprehensive study, we hope to have identified numerous modifiers of both diseases that can be followed up for more in depth functional studies by our laboratories or the broader scientific community. It is likely that some of these genetic modifiers could also impact other neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, etc. and could be followed up by targeted testing in those disease models.

PROJECT SUMMARY/ABSTRACT: PROJECT 3 A major hurdle to identifying disease mechanisms in amyotrophic lateral sclerosis (ALS) is the heterogeneous nature of the disease. People with ALS exhibit a wide variety of clinical presentations, rates of progression and underlying genetic risk factors. ALS is more likely a syndrome rather than a single disease that leads to a recognizable clinical phenotype that we call ALS. Our approach to unraveling the pathophysiology of ALS is to focus on a specific genetically-defined population where the disease is defined by a common underlying mechanism. Here, people carrying the C9orf72 G4C2 hexanucleotide repeat expansion mutation (c9ALS) are the genetically defined population. As opposed to sporadic ALS (sALS), where an array of genetic, environmental, and unknown factors drive disease, a common disease-causing pathophysiology underlies c9ALS. The proposition is that investigating this relatively homogenous ALS genetic subgroup will provide a window of opportunity for understanding disease mechanisms and developing treatments. The hope is that discoveries in this smaller group of patients will be applicable to the larger ALS population. In this project, we will use next generation mass spectroscopy and systems biology analysis to discover biomarkers of disease pathways and progression. In Specific Aim 1, we will interrogate human motor cortex from c9ALS and sALS patients to generate proteomic networks and modules that define c9ALS. In Specific Aim 2, we will partner with the investigators of Project 1 to define proteomic networks in a C9orf72 G4C2 mouse model; this will allow us to analyze proteomic changes in brain at several time points during disease progression. In addition, proteomic analysis of brains from mice treated with an antisense oligonucleotide (ASO) targeting G4C2 transcripts will allow for the identification of protein signatures associated with a disease-modifying drug. In Specific Aim 3, proteomic signatures common to both human and mouse disease will be targeted in CSF samples from c9ALS patients and asymptomatic C9orf72 expansion carriers, both in cross sectional and longitudinal analyses. The ultimate goal of this project is the discovery of novel biomarkers of disease pathways and progression that will be important for ongoing research into disease pathogenesis and also for future clinical trials.

PROJECT SUMMARY/ABSTRACT There is a critical need to identify the molecular signatures that can define cell states and predict disease progression in C9orf72-associated amyotrophic lateral sclerosis and frontotemporal dementia (c9ALS/FTD) at a systems level. This P01 application proposes to investigate c9ALS/FTD pathomechanisms that can inform new therapeutic targets and identify biomarkers for diagnosing and prognosticating disease. Functional genomics approaches such as those taken in Projects 1-3 will generate hundreds of thousands of data points requiring sophisticated analytical methods for their biological interpretation. The adoption of network-based approaches has been a natural step as most biological systems can be accurately modeled and represented using these methods. The Bioinformatics and Biostatistics Core (Core D) will therefore support investigators in developing strategies for multi-omics data analysis and data integration of transcriptomic and proteomic data generated in Projects 1-3, and in applying additional state of the art data analytics to individual projects. Core D will also continue to assist Projects 1 and 3 by performing power analysis to address sample sizes as described in the proposed projects. In addition, Core D will promote uniform standards for data reporting through a common data dictionary and a web server to enable data exchange among the investigators. Specifically, Core D will work with investigators across Projects 1-3 and Cores B and C to develop a standardized set of descriptors and parameters appropriate for data harmonization and standard formats for raw and processed data. The Specific Aims are the following: Specific Aim 1. Provide a framework for standardized data-sharing and analyses of transcriptomics and proteomics studies. To provide infrastructure and harmonization among all projects, Core D will: 1) design and maintain a web-portal on a secure web server; 2) work with investigators to develop a standardized set of descriptors and parameters appropriate for data harmonization; and 3) perform power analysis to justify sample size and assist with statistical modeling of clinical information with confounding variables. Specific Aim 2. Construct multi-scale gene/protein networks and integrate with single-cell transcriptomics to identify core networks altered in c9ALS/FTD. To support bioinformatic analyses for all Projects, Core D will carry-out data integration. Specific Aim 3. Prioritize networks and bioinformatically validate the transcriptomic and proteomic discoveries by integrating with external published and unpublished datasets. To prioritize key drivers of c9ALS and bioinformatically validate the findings, Core D will: 1) develop and implement bioinformatic approaches to validate major findings of the Projects with multiple datasets encompassing human neurodegenerative disease cases and corresponding mouse models; and 2) define core regulators of c9ALS by integrating multi-dimensional data generated in Projects 1-3.

PROJECT SUMMARY/ABSTRACT: CORE A The overarching theme of this program project renewal entitled ?Pathobiology of Neurodegeneration in C9ORF72 Repeat Expansion? is to determine the mechanisms by which C9orf72 repeat expansions cause amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), and identify novel therapeutic targets and biomarkers in order to improve the diagnosis of and prognosis for patients suffering from c9ALS/FTD. Core A will facilitate these goals by providing scientific and fiscal oversight. Core A will be responsible for managing fiscal responsibilities for the program project, ensuring ethical and responsible conduct of research, providing scientific direction and accountability, and reporting progress to the NIH and to the public. To assure the success of this research proposal, Core A will also foster a multidisciplinary program that advances research, generates valuable biological resources, and promotes education. To accomplish its goals, Core A will pursue the following specific aims. Specific Aim 1: Provide administrative structure and fiscal oversight for the program project grant. Specific Aim 2: Organize regular meetings of the Executive Committee to assist in the scientific administration and to facilitate integration and continuing progress on research aims. Specific Aim 3: Establish an External Advisory Committee, conduct annual meetings, and report progress to the NINDS. Specific Aim 4: Ensure that the clinical research conforms to the standards of the ethical conduct of research and the safety of human subjects. Specific Aim 5: Report progress to NIH and ensure compliance with the NIH Public Access policy. Specific Aim 6: Promote education on ALS and related disorders, and encourage P01 scientists and clinicians to participate in patient and caregiver support groups.