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Grant

Spatiotemporal Analysis of TDP-43 Toxicity and Endolysosomal Turnover Mechanisms

Sponsored by National Institute of Neurological Disorders and Stroke

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$393K Funding
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Abstract

Amyotrophic Lateral Sclerosis (ALS) is a devastating disease in which progressive degeneration of motorneurons leads to paralysis usually resulting in death 2-5 years after diagnosis. No therapies exist thatsignificantly increase quality of life or life expectancy. Mutations in >30 genes are linked to ALS onset albeit>90% of all ALS cases are sporadic. However a unifying cellular hallmark in >95% of all ALS cases is thecytoplasmic mis-localization accumulation and aggregation of the nuclear RNA binding protein TDP-43 (TARDNA/RNA binding protein 43) in motor neurons and support cells. Similar TDP-43 pathology is observed in otherneurodegenerative diseases including in forebrain neurons of ~50% of Frontotemporal dementia patients (FTD).TDP-43 pathology confers a toxicity to neurons but the nature of this toxicity remains fiercely debated. Loss ofnuclear function (LOF) and gain of cytoplasmic function (GOF) mechanisms have been proposed thoughseparating such mechanisms and identifying the earliest impacts of TDP-43 pathology has remained elusive.Regardless a therapeutic strategy that has shown promise in some ALS models is promoting the degradationof cytoplasmic TDP-43. Recently cytoplasmic TDP-43 was shown to be degraded via a novel endolysosomalpathway which when induced suppresses TDP-43 toxicity. However understanding of this degradationpathway remains limited. Key gaps in understanding include determining in an ALS-relevant neuronal modelthe earliest and most disease-relevant impacts of TDP-43 pathology and defining how endolysosomal TDP-43degradation occurs. The aims of this grant are: 1.) Establish a novel endogenous TDP-43 reporter system inneurons that allows precise control of TDP-43 abundance and cellular localization via small molecule andoptogenetic means. Using this system the impacts of altered TDP-43 levels and localization on TDP-43 itselfALS phenotypes and gene expression focusing on RNA abundance and translation will be examined. 2.) Testan endolysosomal degradation model involving TDP-43 ubiquitination and endosomal membrane invaginationin neurons using an optical pulse labelling approach. TDP-43 degradation mechanisms will also be defined inpatient-derived ALS models using similar means. Finally a novel high throughput yeast dot-blot assay will beused to identify genetic and chemical regulators of TDP-43 and Fused in Sarcoma (FUS) abundance which isalso implicated in ALS and FTD pathology. This grant is innovative in that a novel approach to exertspatiotemporal control of TDP-43 expression which promises separation of TDP43 LOF and GOF toxicityeffects and a means to identify regulators of TDP-43 and FUS abundance via dot blot are proposed. Finallymechanistically defining endolysosomal-based means of cytoplasmic TDP-43 degradation promises new basicinsight into proteostasis for TDP-43 and other substrates. In summary TDP-43 and FUS are logical entry pointsfor the study of ALS and FTD. This grant will enhance disease understanding and may lead to identification ofnew therapeutic targets with broad patient applicability.

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