To evaluate the role of vacuolar protein sorting 35 (VPS35) in α-synuclein (αS) degradation pathways using a novel retromer stabilizer, guanidine hydrazone (2a).  

The D620N VPS35 mutation is associated with increased αS aggregates and neuronal death in familial Parkinson’s disease (PD). Reducing αS levels in neurons is a potential neuroprotective strategy in PD. Recent studies suggest that VPS35 regulates αS degradation by impacting the endosomal-lysosomal system (ELS), macroautophagy (MA), chaperone-mediated autophagy (CMA), and the ubiquitin-proteasome system (UPS).

We overexpressed mutant A53T αS by lentiviral infection or transient transfection in SH-SY5Y cells, treated with 2a (or saline control) at 20 uM and analyzed αS levels, VPS35, and αS degradation pathway markers by immunoblot. We then looked at the effect of 2a on αS levels after selective blockage of αS degradation pathways. MA was inhibited for 1 day with bafilomycin A1 (BfA1) at 50 nM and UPS was blocked for 8 hours with MG-132 at 10 uM. 

2a increased VPS35 protein levels (~75%) and retromer function, based on increased VPS35 cargoes VMAT2 (~50%) and Transferrin Receptor (~75%). This correlated with a ~ 50% reduction of phospho-ser129 αS with transient transfection and total αS with lentiviral infection, in the soluble fraction. We found increases in ELS markers (LAMP1, Rab5), CMA markers (LAMP2a, Hsc70), UPS markers (PSMB5), and macroautophagy (increased LC3b, decreased p62), highlighting the 2a effect on boosting these pathways. Treatment with 2a was able to induce a ~75% reduction in phospho-ser129αSyn despite a block of MA with BfA1 or a block of UPS with MG-132. These results could suggest that other degradation pathways predominate in mediating the 2a-induced αS clearance, but it is also possible that compensatory upregulation occurs in the non-blocked pathways.

These findings highlight the role of VPS35 in the regulation of αS degradation pathways.