The SUMO Conjugase Ubc9 Protects Dopaminergic Cells from Cytotoxicity and Enhances the Stability of α-Synuclein in Parkinson's Disease Models.

ABSTRACT

Small ubiquitin-like modifier (SUMO) is a widespread regulatory mechanism of post-translational modification (PTM) that induces rapid and reversible changes in protein function and stability. Using SUMO conjugase Ubc9-overexpressing or knock-down cells in Parkinson's disease (PD) models, we demonstrate that SUMOylation protects dopaminergic cells against MPP+ or preformed fibrils (PFFs) of α-synuclein (α-syn)-induced toxicities in cell viability and cytotoxicity assays. In the mechanism of protection, Ubc9 overexpression significantly suppressed the MPP+ or PFF-induced reactive oxygen species (ROS) generation, while Ubc9-RNAi enhanced the toxicity-induced ROS production. Further, PFF-mediated protein aggregation was exacerbated by Ubc9-RNAi in thioflavin T staining, compared with NC1 controls. In cycloheximide (Chx)-based protein stability assays, higher protein level of α-syn was identified in Ubc9-enhanced green fluorescent protein (EGFP) than in EGFP cells. Since there was no difference in endogenous mRNA levels of α-syn between Ubc9 and EGFP cells in quantitative real-time PCR (qRT-PCR), we assessed the mechanisms of SUMO-mediated delayed α-syn degradation via MG132, proteasomal inhibitor, and PMA, lysosomal degradation inducer. Ubc9-mediated SUMOylated α-syn avoided PMA-induced lysosomal degradation because of its high solubility. Our results suggest that Ubc9 enhances the levels of SUMO1 and ubiquitin on α-syn and interrupts SUMO1 removal from α-syn. In immunohistochemistry, dopaminergic axon tips in the striatum and cell bodies in the substantia nigra from Ubc9-overexpressing transgenic mice were protected from MPTP toxicities compared with wild-type (WT) siblings. Our results support that SUMOylation can be a regulatory target to protect dopaminergic neurons from oxidative stress and protein aggregation, with the implication that high levels of SUMOylation in dopaminergic neurons can prevent the pathologic progression of PD.