Localization and distribution of uncoupling protein 5 (UCP5) in rat brain

Abstract

OBJECTIVE: To investigate the expression pattern of UCP5 in rat brain and to overexpress UCP5 in MCF-7 cells. BACKGROUND: Evidences have shown that mitochondrial dysfunction and oxidative stress are linked to the pathogenesis of the Parkinson’s disease. Recent findings also suggested that neuronal uncoupling activity in mitochondria can help to prevent cell death by reducing ROS production. UCP5 is highly expressed in brain but its function is still unclear, although it is believed UCP5 plays a role in regulating the membrane potential in the mitochondria. In this study we performed an immunohistological experiment on the localization of UCP5 in rat brain slides and overexpressed UCP5 in MCF-7 cells so as to investigate the function of UCP5 in mitochondria. METHODS: Mitochondrial and cytosolic fractions of SHSY5Y cells were isolated and the expressions of UCP2 and UCP5 were determined by Western Blot; UCP5 mRNA detection was performed on a human multi-tissue northern blot membrane (MTN) with radio-labeled UCP5 probes; For cellular localization of UCP5, SHSY5Y cells were fixed and incubated with anti-UCP5 antibody and counterstained with fluorescent MitoTracker or DAPI; For immunolocalization of UCP5 in paraffin-fixed rat brain slides, they were first incubated with anti-UCP5 antibody and then with horse-radish peroxidase conjugated anti-rabbit IgG. The slides were visualized by DAB staining; For overexpression of UCP5, three FLAG-tagged human UCP5 inserts were made (full length UCP5, 5’UTR-UCP5 and 5’UTR-MTS-UCP5) and cloned into the vector pcDNA3.1(+) and transfect into the MCF-7 cells. Western blot analysis was performed to confirm the overexpression of FLAG-tagged UCP5 after 48 hours of transfection. RESULTS: UCP5 was mainly localized in the inner mitochondrial membrane and its mRNA transcripts were mostly detected in cortex, hippocampus and putamen. Recombinant UCP5 proteins were found to be overexpressed in MCF-7 cells after 48 hours of transfection. CONCLUSIONS: The expression of UCP5 in the brain regions suggests it has a protective role of decreasing the production of ROS in neuronal cells and the overexpression of UCP5 in MCF-7 cells can be one of the tools to elucidate the function of UCP5 in mitochondria.