USP14 regulates pS129 α-synuclein levels and oxidative stress in human SH-SY5Y dopaminergic cells

Ubiquitin specific protease-14 (USP14) is critical for controlling protein homeostasis disturbed in human disorders like Parkinson′s disease (PD). Here we investigated the role of USP14 in regulating proteasome and autophagy pathways, and their influence on α-synuclein (α-syn) degradation. Data showed that α-syn and phosphorylated serine129 α-syn (pS129 α-syn) were elevated in USP14 gene deleted SH-SY5Y dopaminergic cells with concomitant reduction in proteasome activity. Inhibition of proteasomes using MG132 particularly elevated pS129 α-syn in these cells, but the levels were not influenced by inhibiting autophagy using chloroquine. In contrast, autophagy and the CLEAR (Coordinated Lysosomal Expression and Regulation) pathways were elevated in USP14 lacking cells with an upregulation of the transcription factor TFEB. USP14-ablated cells also exhibited increases in reactive oxidative species (ROS) and elongation of mitochondria. The addition of N-Acetylcysteine amide (NACA) to counteract oxidative stress, reduced pS129 α-syn and α-syn levels in USP14 deficient cells. Phospho-proteomic analyses revealed that USP14 is phosphorylated at S143 affecting its function and structure as shown by molecular modeling, and protein interaction studies. Re-expression of wild-type and the phospho-mimetic S143D-USP14 mutant decreased ROS, pS129 α-syn, and α-syn in USP14 lacking cells. These results demonstrate that pS129 α-syn levels are sensitive to oxidative stress in SH-SY5Y dopaminergic cells. USP14 by stimulating the proteasome activity and reducing oxidative stress is a promising factor for targeting α-syn and its pathogenic variants in PD.