Human uncoupling protein-4 protects neuronal cell death from MPP+ induced toxicity by regulating mitochondrial membrane potential, reducing ROS and maintaining ATP levels

Abstract

OBJECTIVE: To investigate the effects of UCP4 overexpression on mitochondrial functions and oxidative stress in SH-SY5Ycells. Background: Mitochondrial dysfunction, ATP deficiency, and oxidative stress are associated with neuronal cell death in Parkinson’s disease (PD). UCP4, a novel member of uncoupling proteins, is exclusively expressed in the brain, but its function is unclear. We hypothesize that UCP4 is involved to regulate neuronal energy homeostasis by modulating mitochondrial membrane potential and ATP production. METHODS: A full-length human UCP4 was stable expressed in SHSY5Y cells. Subcellular localization of UCP4 was examined by immunocytochemistry and Western analysis after cytosolic and mitochondrial fractionation. Stable lines and vector were incubated for 24hr with either MPP+ (0.5mM, 1mM). Cytotoxicity was measured by 3H thymidine incorporation assay. ATP levels were determined by luciferase-luciferin bioassay. MMP and oxidative stress were measured by FACS after staining of JC-1 and DHE, respectively. RESULTS: Recombinant UCP4 is exclusively expressed in the mitochondria in SH-SY5Y cells. No difference in cell viability was observed among the vector or UCP4-expressing constructs. After exposure of 0.5mM and 1mM MPP+ for 24 hours, UCP4 expressing cells showed 15% higher of survival rate compare with the vector control. Vector control was incubated MPP+ for 24hr induced significant oxidative stress, whereas UCP4 overexpressing cells did not. The relative mitochondrial membrane potential of UCP4 overexpressing group with 1.0 mM MPP+ was significantly restored towards levels seen in vector control with MPP+ group by 195% . In addition, the intracellular ATP level in UCP4 expressing cells after treated with MPP+ is significantly higher than that in the vector control treated with MPP+ in 38%. CONCLUSIONS: We report that UCP4 can protect cell death from MPP+ induced toxicity by restoring the depolarization of MMP, reducing production of ROS, and maintaining intracellular ATP levels. These findings might contribute to the understanding of physiological functions of neuronal UCP4 and provide insights on therapeutic possibilities against neurodegenerative diseases involving mitochondrial dysfunction.