43.: Mitochondrial dysfunction in PINK1 mutant human olfactory neurospheres is rescued by overexpression of PGC-1a

Abstract

This study aimed to (1) characterise the mitochondrial phenotype in PINK1 mutant human olfactory neurospheres (HON), and (2) rescue mitochondrial dysfunction in PINK1 mutant HON with overexpression of PGC-1a. We generated HON cultures from a patient harbouring the homozygous c1309T>G, W437G PINK1 mutation, and an age and sex-matched control. Real time polymerase chain reaction was performed for relative expression of PGC-1a, Tfam and SOD1, and Western blot was performed for PGC-1a and b-actin. Assessment of mitochondrial mass, mitochondrial membrane potential and reactive oxygen species were performed. Adenosine triphosphate (ATP) synthesis rates were detected luminometrically, using a previously published protocol. For transfection of lentivirus, cells were plated at 600,000/plate for 24 hours, then transfected with adenovirus (60 million/plate) expressing PGC-1a and incubated for 5 days. Gene expression of PGC-1a in PINK1 HON was significantly lower than control HON. Western blot for PGC-1a protein revealed decreased protein levels in PINK1 compared with control. Mitochondrial mass, mitochondrial membrane potential and ATP production was significantly reduced in PINK1 HON compared with control HON, whereas ROS production was significantly increased. Lentiviral transfection increased PGC-1a gene expression in both PINK1 and control HON. PGC-1a overexpression resulted in increased mitochondrial mass and mitochondrial membrane potential in PINK1 HON. There was also a trend towards decreased ROS production and increased ATP production. This study provides evidence of mitochondrial dysfunction in PINK1 HON, associated with abnormalities in the gene expression of PCG-1a, the master regulator of mitochondrial biogenesis. Overexpression of PGC-1a lead to statistically significant improvements in mitochondrial mass and mitochondrial membrane potential, with a trend towards improvement in production in ATP and reactive oxygen species. Further studies are required to define the mechanisms underlying the abnormalities in mitochondrial function in PINK1 HON, and characterise the role of PGC-1a in PINK1 cells.