Differential roles of proteasome and autophagy in α-synuclein and E46K oligomer clearance: insight into the modulatory effects of the dopamine metabolite DOPAC.

The build-up of misfolded α-synuclein (Syn) proteins plays a key role in diseases such as Parkinson's disease. Here, we compared the cytotoxicity and intracellular processing of wild-type and E46K mutant Syn aggregates in SH-SY5Y neuroblastoma cells and investigated the modulatory effects of the dopamine metabolite, 3,4-dihydroxyphenylacetic acid (DOPAC). E46K aggregates displayed markedly higher toxicity than wild-type counterparts, promoting mitochondrial dysfunction and elevated reactive oxygen species (ROS) production in a time-dependent manner. This effect is consistent with the mutation's higher affinity for cellular membranes, which fosters early and sustained aggregate-membrane interactions. Strikingly, co-incubation with DOPAC during aggregation significantly reduced both toxicity and oxidative stress in wild-type and E46K aggregates. DOPAC shifted Syn into less fibrillogenic conformations, favouring smaller oligomers that were less membrane-active and more effectively processed by cellular clearance systems. Mechanistic studies revealed that E46K/DOPAC aggregates were preferentially degraded via the ubiquitin-proteasome system (UPS), as proteasome inhibition with MG132 enhanced toxicity and intracellular accumulation. In contrast, autophagy inhibition by chloroquine paradoxically reduced toxicity, indicating redirection toward UPS-mediated degradation. Analysis of lysosomal markers showed that DOPAC-containing aggregates colocalized with LAMP1 but not LAMP2A, suggesting processing through macroautophagy rather than chaperone-mediated autophagy. Furthermore, p62 accumulation, indicative of impaired autophagic flux, was evident with untreated aggregates but absent when DOPAC was present. Overall, our results demonstrate that DOPAC reshapes the biophysical and toxicological properties of Syn aggregates, especially E46K species, by promoting less harmful oligomers and enhancing proteostatic clearance. These findings highlight DOPAC as a promising modulator of Syn aggregation and pathology.