Role Of PINK1 In The Protection Against α-Synuclein-induced Mitochondrial Dysfunction

Parkinson’s disease(PD) is a neurodegenerative disorder characterized by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta. Mutations in α-Synuclein(αS) are linked to dominant familial PD, while mutations in PTEN-induced kinase 1(PINK1) that functions in mitochondrial quality control cause recessive familial Parkinsonism. Mitochondrial dysfunction is a common pathogenic mechanism in PD, and it has been suggested that αS contributes to mitochondrial impairments in both sporadic and familial PD. The overall aim of my thesis project was to investigate the influence of αS overexpression on mitochondrial function and the importance of PINK1 in the protection against αS-induced mitochondrial deficits.In the first part, a lentiviral vector plasmid encoding A53T-αS(a mutant of αS linked to familial PD) was constructed and co-transfected with plasmids coding for vesicular stomatitis virus G-protein(VSV-G), HIV-gag/pol and Rev into 293 T cells to produce recombinant retrovirus. Subsequently, the A53T-αS retrovirus was used to infect wild-type(WT) and PINK1-deficient(PINK1-KO) mouse embryonic fibroblasts(MEFs) that were previously immortalized by SV40 large T, and MEF cell lines stably over-expressing A53T-αS were obtained as confirmed by flow cytometry and immunochemistry.In the next project phase, I compared mitochondrial function between WTA53T-αS and PINK1-KO-A53T-αS to study whether PINK1 influences or protects against A53T-αS-induced mitochondrial deficits. First, I measured mitochondrial membrane potential(Δψm). Surprisingly, A53T-αS over-expression significantly increased Δψm in both genotypes of MEFs. However, loss of PINK1 reduced membrane potential in normal MEFs(not overexpressing A53T-αS), such that PINK1-KO-A53T-αS MEFs had overall lower Δψm compared to their WT counterparts. Second, A53T-αS over-expression increased the levels of reactive oxygen species(ROS) independent of genotype in both the cytoplasm and in the mitochondrial matrix. Lack of PINK1 alone also increased ROS levels in mitochondria but not in the cytoplasm. Third, I analyzed mitochondrial respiration(oxygen consumption) by extracellular flux analysis. A53T-αS increased basal respiration in PINK-KO MEFs but not in WT MEFs. In addition, loss of PINK1 reduced basal OCR in normal MEFs. We also measured the spare respiratory capacity(SRC) that is required for cells to increase mitochondrial energy production in response to extracellular signals and/or stress. We found that A53T-αS reduced SRC in both WT and PINK1-KO MEFs. However, because PINK1 deficiency alone also reduced SRC, PINK1-KO MEFs overexpressing A53T-αS lost their SRC entirely(no SRC left), while WT-A53T-αS MEFs maintained a significant amount of SRC. In summary, A53T-αS affects mitochondrial function in complex ways that are influenced by the loss of PINK1. Although not all A53T-αS-induced mitochondrial deficits were exacerbated by PINK1 deficiency, A53T-αS-dependent elevations of cytosolic ROS and the decline in SRC were both enhanced by the lack of PINK1. Taken together, these results suggest that PINK1 deficiency may increase neuronal loss in PD by worsening(increasing) specific A53T-αS-induced mitochondrial deficits and cytoplasmic ROS.