Silencing of UCP5 enhances MPP+-induced neurotoxicity in SH-SY5Y cells

Abstract

OBJECTIVE: To investigate the role of UCP5 in MPP+-induced neurotoxicity by down-regulating UCP5 in SH-SY5Y cells. BACKGROUND: Uncoupling proteins (UCPs) are proteins that uncouple oxidative phosphorylation from respiration by controlling the leakage of protons across the inner mitochondrion. Oxidative stress resulting from the generation of reactive oxygen species (ROS) is associated with neuronal cell death in Parkinson’s disease (PD). UCP5 is mainly expressed in the brain. We explore the role of UCP5 in the brain, especially its regulation in response to the production of ROS under oxidative stress. METHODS: Endogenous UCP5 was knocked down by the transfection of siRNA against UCP5 in SH-SY5Y cells in normal and MPP+-induced toxicity conditions. Cell viability markers like LDH activity, ATP level, and mitochondrial membrane potential by flow cytometric detection of JC-1 were measured to investigate the role of UCP5 under oxidative stress conditions. Western blot analysis of other neuronal UCPs was also performed to test whether this UCP5 knockdown also affected other UCPs. RESULTS: A depletion of UCP5 protein by 56% (P_0.01) in SH-SY5Y cells was observed after siRNA transfection. The down-regulation of UCP5 alone increased LDH release by 37% (P_0.05) but did not change ATP level. MPP+ exposure reduced the ATP level by 32% (P<0.01). The combination of MPP+ and siRNA treatment further increased the LDH release by 24% (P<0.05) when compared to the MPP+ treatment group only. Down-regulation of UCP5 also partially restored the depolarization of mitochondrial membrane potential after MPP+ treatment by 64% (P<0.05 vs. MPP+ only). There was no significant change in the regulation of UCP2 observed. CONCLUSIONS: Down-regulation of UCP5 increased neuronal cell death and partially restored the depolarized membrane potential, but did not alter ATP levels or UCP2 expression.