PROJECT SUMMARY/ABSTRACT Amyotrophic lateral sclerosis (ALS) is a fatal disease characterized by death of motor neurons. A key pathologic feature is the cytoplasmic mislocalization of a nuclear transcription and splice regulator, Tar-DNA binding protein of 43kDa (TDP-43). TDP-43 is aggregated in cytoplasmic stress granules (SGs) along with nuclear import/export factors, and its toxicity is thought to be due to both cytoplasmic gain- and nuclear loss-of-function mechanisms. Relocating it to the nucleus has the potential to address both forms of toxicity. Inhibiting formation of SGs is one promising strategy, and downregulating the SG-associated protein Ataxin-2 (Atxn2) using antisense oligonucleotides (ASOs) prolongs strength and survival in a mouse model of ALS. However, ASOs require frequent CNS readministration, and a preferable approach would be to achieve knockdown after one treatment. A second approach is enhancing nuclear import, a strategy with success in dipeptide repeat (DPR) toxicity models of ALS in vitro. Extending this strategy to non-DPR forms of ALS has the potential to make a broad impact on the disease. In addition, potential synergy between the two approaches has great therapeutic potential. If successful, these strategies could be used to treat the vast majority of ALS. In preliminary work, RNAi delivered using a novel viral vector achieves robust knockdown of Atxn2 in the key areas of the nervous system affected in ALS. Aim 1 of this proposal is to determine if sustained Atxn2 knockdown in these regions reverses TDP-43 mislocalization and improves neuron survival in two distinct mouse models of ALS. In other preliminary work, cell lines overexpressing a nuclear import factor show reductions in TDP-43. Aim 2 is to test if augmenting nuclear import corrects TDP-43 localization and improves cell survival under conditions of stress. My central hypothesis is that targeting both cytoplasmic aggregation and nuclear loss of TDP-43 using viral-mediated approaches will result in sustained neuroprotection. This work fits squarely in NINDS’ mission to further our knowledge about the brain and nervous system and to use this knowledge to reduce the burden of disease, specifically targeting one of neurology’s most devastating afflictions. Dr. Amado is a passionate, highly-trained clinician-scientist uniquely poised to make a fundamental impact on ALS. Her mentor Dr. Beverly Davidson, a renowned neurodegenerative disease expert continually pushing the boundaries of vector-based therapeutics, and her advisory committee of deeply experienced and dedicated neurologists and neuroscientists, will provide the guidance and mentorship to ensure her success, backed by enthusiastic institutional support. The University of Pennsylvania, with its innumerable resources and facilities, is an outstanding place to launch a neuroscience career. Dr. Amado will use this 5-year mentored opportunity to build on her gene therapy training and merge it with her clinical expertise to become an independent, R01-funded physician-scientist developing novel therapies for patients with ALS.

PROJECT NARRATIVE Amyotrophic lateral sclerosis is a progressive neurodegenerative disease whose pathologic hallmark is cytoplasmic mislocalization of the nuclear protein TDP-43 in motor neurons, where it is bound up in cytoplasmic stress granules. Both cytoplasmic toxicity and loss of nuclear function of TDP-43 contribute to neuronal death. The goal of this proposal is to determine the effects of reversing TDP-43 mislocalization, by sustainably knocking down Ataxin 2, a key stress granule component, and by enhancing nuclear import, both alone and in combination with Ataxin 2 knockdown.