Proteasome and autophagy inhibition reshapes extracellular vesicle cargo and spreads proteotoxicity

Protein quality control (PQC) preserves proteostasis through the coordinated action of the chaperone proteins, the ubiquitin–proteasome system (UPS) and the autophagy–lysosome pathway (ALP). Disruption of these pathways occurs in TDP-43–associated neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Extracellular vesicles (EVs) can mediate the export of misfolded proteins, but how PQC dysfunction reshapes EV cargo and contributes to disease propagation remains unclear. Large (20K) and small (100K) EVs released by motor neuron–like NSC-34 cells were isolated under basal conditions or following pharmacological inhibition of UPS or ALP. EVs were characterized by using nanoparticle tracking analysis, electron and super-resolution microscopy, biochemical assays, and biophysical spectroscopies. EV-associated miRNAs were profiled by small RNA sequencing, and the functional effects of EVs were assessed in recipient cells. PQC impairment markedly reshaped EV cargo. Both UPS and ALP inhibition enhanced the extracellular release of misfolded TDP-43 C-terminal fragments, SQSTM1/p62, and ubiquitinated proteins. Interestingly, ALP inhibition also increased the EV-associated release of insoluble phosphorylated TDP-43 (Ser409/410) together with MAP1LC3. Moreover, UPS inhibition favored the secretion of compact, large β -sheet–rich aggregates, whereas ALP inhibition promoted the release of smaller aggregated species with increased surface hydrophobicity, consistent with intermediate aggregates. PQC dysfunction also induced EV-associated miRNA remodeling, particularly in 100K EVs, enriching pathways linked to ER stress and prion-related processes. Functionally, EVs generated under condition of PQC impairment reduced cell viability in recipient cells and triggered proteotoxic responses linked to ER stress and prion-related pathways, while also activating Notch1–c-Myc signaling and cell death-associated transcriptional programs. Our findings identify EV secretion as a protective regulated response to proteostasis imbalance and support the concept that PQC dysfunction reprograms EV cargo, with the undesired consequence of propagating non–cell-autonomous proteotoxicity in TDP-43 proteinopathies.