Defining Endogenous Clearance Mechanisms of Full-Length TDP-43 and Its Disease-Prone Isoforms

Abstract

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by motor neuron atrophy, resulting in paralysis and respiratory failure. A pathological hallmark in >95% of ALS patients is the cytoplasmic mislocalization and aggregation of the RNA-binding protein TDP-43. Pathological N- or C-terminal truncated TDP-43 isoforms are associated with ALS, and reducing their levels, along with full-length cytoplasmic TDP-43, improves viability in various ALS cell models. However, the mechanisms by which cells normally degrade full-length and truncated TDP-43 isoforms remain incompletely understood. Here, I demonstrate that in addition to previously identified degradation pathways, the endolysosomal pathway plays a significant role in TDP-43 clearance. In yeast, I identified adaptors of the E3 Ub ligase Rsp5 that aid in TDP-43 degradation. In HEK293 cells, I confirmed that NEDD4 (Rsp5 homolog) ubiquitinates TDP-43 with K63-linked ubiquitin chains, consistent with endolysosomal degradation. To further investigate endogenous full-length TDP-43 and isoform-specific clearance mechanisms, I have constructed various full-length TDP-43 and isoform-expressing yeast strains under β-estradiol inducible promoters for use in genome-wide dot blot screens. These screens will identify which genes or drugs have the most significant effect on full-length TDP-43/isoform levels within the cell, which can then be further explored in human cells. This approach may allow for the identification of isoform-specific degradation pathways that do not target full-length TDP-43, uncovering new therapeutic strategies for ALS that mitigate toxicity without compromising the essential functions of full-length TDP-43.