Rsp5/NEDD4 and ESCRT regulate TDP-43 toxicity and turnover via an endolysosomal clearance mechanism.

The Journal of cell biology 2026

A pathological hallmark in >97% of amyotrophic lateral sclerosis (ALS) cases is the cytoplasmic mislocalization and aggregation of TDP-43, a nuclear RNA-binding protein, in motor neurons. Driving clearance of cytoplasmic TDP-43 reduces toxicity in ALS models, though how TDP-43 clearance is regulated remains controversial. We conducted an unbiased yeast screen using high-throughput dot blotting to identify genes that affect TDP-43 levels. We identified ESCRT complex genes, which induce membrane invagination (particularly at multivesicular bodies; MVBs) and genes linked to K63 ubiquitination (particularly cofactors of the E3 ubiquitin ligase Rsp5; NEDD4 in humans), as drivers of TDP-43 endolysosomal clearance. TDP-43 colocalized and bound Rsp5/NEDD4 and ESCRT proteins, and perturbations to either increased TDP-43 aggregation, stability, and toxicity. NEDD4 also ubiquitinates TDP-43. Lastly, TDP-43 accumulation induces giant MVB-like vesicles, within which TDP-43 accumulates in a NEDD4-dependent manner. Our studies shed light on endolysosomal-mediated cytoplasmic protein clearance, a poorly understood proteostasis mechanism, which may help identify novel ALS therapeutic strategies.

14 Figures Extracted

Figure 1.

A dot-blot genetic screen in yeast identifies genes regulating TDP-43 steady-state levels. (A) Schematic representation of the dot-blot genetic scree...

Figure 2.

TDP-43 clearance and aggregation depend on ESCRT function. (A) Indicated strains were transformed with TDP-43-mRuby2 plasmid (pRB194) and grown to mi...

Figure 3.

TDP-43 toxicity, aggregation, and toxicity depend on the E3 ubiquitin ligase Rsp5. (A) Representative serial dilution growth assay of WT and rsp5-3 ...

Figure S1.

Knockout of the Rsp5 adaptor Rim8 increases TDP-43-YFP levels. Indicated strains were transformed with TDP-43-YFP plasmid (pRB109) and grown to mid-l...

Figure 4.

VPS4A E228Q increases TDP-43 stability and promotes cytoplasmic mislocalization. (A) HEK293A WT cells were transfected with EGFP-VPS4A E228Q ...

Figure 5.

NEDD4 regulates TDP-43 turnover, toxicity, and ubiquitination. (A) HEK-TDP-43-GFP cells were transfected with siRNAs targeting NEDD4 or noncoding con...

Figure S2.

HEK293A cells were transfected with HA-ubiquitin (pRB506) or both HA-Ub and NEDD4-mCherry (pRB334) and grown for 2 days before TDP-43 was immunoprecip...

Figure S3.

Endogenous TDP-43 and VPS4A interact. TDP-43 was immunoprecipitated in HEK293A cells in the presence or absence of CHQ treatment and/or NEDD4 knockdo...

Figure 6.

NEDD4 knockdown stabilizes TDP-43 in iNeurons. (A) Schematic of optical pulse labeling strategy for measuring TDP-43-Dendra2 half-life. (B) NEDD4 k...

Figure 7.

Mild TDP-43 overexpression causes accumulation of giant Rab7-positive vesicles. (A) HEK293A WT, HEK-TDP-43-GFP, and HEK-TDP-35-GFP cells were grown i...

Figure S4.

Giant Rab7 vesicular structures exhibit CD63 colocalization and CHQ-sensitive intraluminal CD63 bodies, suggesting an endolysosomal origin. (A) HEK29...

Figure 8.

Giant Rab7-positive vesicles are proteolytically active compartments. (A) HEK-TDP-43-GFP cells were grown in media containing solvent control (DMSO) ...

Figure S5.

Giant Rab7-positive vesicles are proteolytically active compartments. (A) HEK-TDP-35-GFP cells were grown in media containing solvent control (DMSO; ...

Figure 9.

Potential model of Rsp5/NEDD4 and ESCRT-dependent clearance of cytoplasmic TDP-43. We speculate that TDP-43 is subject to K63 polyubiquitination by R...