Knock-down of uncoupling protein-5 (UCP5) in neuronal cells reduces mitochondrial membrane depolarization and increases oxidative stress induced by MPP+.

Abstract

OBJECTIVE: We explored UCP5 and its link to oxidative stress, mitochondrial membrane potential (MMP), ATP levels and cell viability under normal conditions and in MPP+-induced toxicity. BACKGROUND: Oxidative stress associated with mitochondrial dysfunction are found in Parkinson’s disease. Uncoupling proteins (UCPs) uncouple oxidative phosphorylation from ATP synthesis by dissipating MMP across mitochondrial inner membrane. Five isoforms (UCP1-5) have been identified. The role of neuronal specific UCP5 in mitochondrial dysfunction is unknown. METHODS: SH-SY5Y neuronal cells were transfected with UCP5 RNAi before MPP+ exposure 24 hrs later. At 48 hrs, cell viability, oxidative stress and ATP levels, and MMP were determined under normal conditions and in MPP+-induced toxicity. Cell viability and ATP level were measured using MTT assay and luciferin-luciferase bioluminescene assay, respectively. MMP and oxidative stress were determined by JC-1 and dihydroethidium (DHE) staining in flow cytometry, respectively. Western and real-time RT-PCR analyses were used to determine UCP expression. RESULTS: UCP5 protein expression knockdown by 56% (p_0.01) was associated with decreased (21%; p<0.01) cell viability compared with controls transfected with scrambled siRNA. UCP2 and UCP4 mRNA levels did not change after UCP5 knockdown. UCP5 knockdown before MPP+ exposure further decreased cell viability (14%) but did not modify ATP levels. MPP+ increased oxidative stress by three-fold and mitochondrial membrane depolarization by 15-fold. Oxidative stress levels did not alter after UCP5 knockdown under normal conditions but was elevated (40%; p<0.01) in MPP+-induced toxicity compared to controls without knockdown. Similarly, MMP did not change under normal conditions after UCP5 knockdown but it was reduced in MPP+-induced depolarization (57%; p<0.01). CONCLUSION: UCP5 knockdown did not affect basal levels of ATP, oxidative free radicals or mitochondrial membrane potential. Under MPP+-induced oxidative stress, UCP5 knockdown reduced the depolarizing effects of MPP+ and further increased oxidative stress, indicating that UCP5 might be neuroprotective in reducing MPP+-induced cytotoxicity. The lack of change in UCP2 and UCP4 expression indicated that there were no compensatory effects from these isoforms after UCP knockdown.