Role of PARP-1 in mitochondroal DNA damage induced by rotenone in PC12 cells

Abstract

Objective To explore the effect of poly (ADP-ribose) polymerase-1 (PARP-1) on mitochondrial DNA (mtDNA) damage induced by rotenone in rat adrenal medulla pheochromocytoma PC12 cells. Methods PC12 cells were treated with various dose of rotenone. The mtDNA common deletion (CDs) was determined by TaqMan assay, and mtDNA copy number was detected by quantitative polymerase chain reaction (qPCR). Western blotting was performed to detect the protein levels of PARP-1 in whole cell and mitochondria. After the rotenone exposed PC12 cells were treated with PJ34 (PARP-1 selective inhibitor), cell viability was detected by CCK-8 assay, mitochondrial membrane potential was examined by TMRM staining, intracellular reactive oxygen species (ROS) level was tested by 2, 7-dichlorofluorescin diacetate (DCFH-DA) probe. Results TaqMan and qPCR assays showed that rotenone treatment induced increased mtDNA deletion and decreased mtDNA copy number in PC12 cells when compared to control cells (P < 0.05). Meanwhile, PARP-1 was activated by rotenone treatment in the whole cells and mitochondria, and Western boltting showed that the protein level of PARP-1 was upregulated in a dose-dependent manner. Furthermore, compared with the rotenone group, PJ34 pretreatment enhanced the cell viability significantly in PC12 cells (P < 0.05), abolished mtDNA CDs and increased mtDNA copy number (P < 0.05). Additionally, PJ34 pretreatment caused the improvement of mitochondrial membrane potential and reduced ROS production when compared with the rotenone cells(P < 0.05). Conclusion Rotenone induces mitochondrial DNA damage, which further activates the expression of PARP-1 protein, especially mitochondria in PC12 cells. And PJ34, as the PARP-1 selective inhibitor has a protective effect on rotenone-induced mitochondrial DNA damage in PC12 cells.

Keywords

• rotenone
• parkinson's disease
• poly (adp-ribose) polymerase-1
• pj34
• mtdna