INVESTIGATING THE ROLE OF RSP5/NEDD4 IN REGULATING TDP-43 PROTEOSTASIS AND PROTEINOPATHY

Abstract

Amyotrophic lateral sclerosis (ALS) is a devastating disease characterized by progressive degeneration of upper and lower motor neurons. An established molecular hallmark uniting >97% of all ALS cases is the cytoplasmic mislocalization and aggregation of the nuclear RNA-binding protein TDP-43. Upregulating clearance of cytoplasmic TDP-43 reduces toxicity in various ALS models, though how TDP-43 degradation is regulated remains controversial. To address this, we conducted an unbiased genome-wide screen in yeast, using high-throughput dot blots to identify genes that affect TDP-43 levels. Our screen identified factors involved in the ESCRT complex and K63-linked ubiquitination (particularly co-factors of the E3-Ubiquitin ligase Rsp5 (yeast)/NEDD4 (human) as primary drivers of TDP-43 clearance. Our subsequent work highlights the role of an Rsp5/NEDD4 in modulating TDP-43 toxicity and turnover. In both yeast and human (HEK293A) models, we establish that TDP-43 co-localizes and physically interacts with Rsp5/NEDD4 and that increasing Rsp5/NEDD4 expression attenuates TDP-43 cytotoxicity. Mechanistically, we find that OE Rsp5/NEDD4 accelerates TDP-43 turnover and enhances its solubility making it more susceptible to endolysosomal degradation. Our preliminary experiments affirm the role of endolysosomal flux in mediating TDP-43 clearance and suggest that Rsp5/NEDD4 is critically implicated in this pathway. These findings offer new perspectives on proteostasis and could have implications for the development of novel therapeutics for ALS and related TDP-43 proteinopathies.