To test the impact of a novel retromer stabilizer, guanidine hydrazone (2a), on levels of vacuolar protein sorting 35 (VPS35) and on α-synuclein (αS) clearance pathways in human neuroblastoma SH-SY5Y cells. 

Reducing αS levels in dopaminergic neurons may have neuroprotective potential in Parkinson’s disease (PD). Mutations in the VPS35 gene are a rare cause of familial PD. VPS35 is a key component of the retromer, a heteropentameric complex involved in protein trafficking. VPS35 regulates the trafficking of proteins involved in three αS degradation pathways: macroautophagy (MA), chaperone-mediated autophagy (CMA), and the endosomal-lysosomal system (ELS).

We overexpressed exogenous mutant A53T αS by transient transfection in SH-SY5Y cells, co-treated this group with 2a (or vehicle) at 20 uM for 2, 3, 4 or 5 days, and analyzed levels of αS, VPS35, and αS degradation pathway markers by western blot and ELISA. We then looked at the effect of selective MA blockage for 1-day with bafilomycin A1 (BfA1) at 50 nM on the impact of 2a on αS levels.

2a increased VPS35 protein levels (~75%) and retromer function, with increased levels of the VPS35 cargo Sortilin. This correlated with a reduction of phospho-Ser129 αS (~ 80%), an important pathological form of αS, in the cytosolic and soluble fractions and oligomeric αS in the soluble fraction (~ 50%). Furthermore, we found a 2a-induced increase in CMA and MA markers, highlighting its effect on boosting the main αS clearance pathways. After blocking MA, there was a ~60% reduction of phospho-Ser129 αS in the A53T αS-transfected group treated with 2a. Thus, 2a can decrease aS levels even when MA is blocked, potentially by compensatory upregulation of CMA and ELS.

These findings link VPS35 to the regulation of multiple aS degradation pathways, and highlight 2a as a promising candidate for neuroprotection in PD.