Proteasome inhibition by VR23 enhances autophagic clearance of FUS^P525L-mediated persistent stress granule in SH-SY5Y cells

Autophagy is a conserved catabolic pathway that preserves cellular homeostasis through lysosomal degradation. Beyond its general role in proteostasis, selective autophagy mediates the clearance of selective cellular targets such as persistent stress granules (SGs), in a process termed granulophagy. SGs are dynamic cytoplasmic assemblies that normally disassemble after stress relief; however, their aberrant persistence has arisen as a pathological feature of neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). However, the molecular regulation of granulophagy remains incompletely understood. Here, we established a tandem fluorescent SG reporter system with mCherry-pHluorin-FUS^P525L, enabling live-cell visualization of granulophagic flux. Using this system, we screened a chemical library and identified VR23, a proteasome inhibitor, as a potent inducer of granulophagy. VR23 promoted SG clearance through autophagic mechanisms, as evidenced by enhanced LC3 colocalization, lysosome-dependent degradation, and Bafilomycin A1-sensitive flux. Notably, disruption of SG assembly via G3BP1 inhibition abolished VR23-induced clearance, confirming its SG selectivity. These findings suggest a link between proteasome inhibition and granulophagy, highlighting VR23 as a valuable tool compound to dissect the mechanisms of SG turnover, and provide a platform for discovering modulators of pathological SG clearance in protein aggregation.