Molecular mechanism underlying mutant PINK1-induced degeneration of substantia nigra dopaminergic neurons

• Main Authors: Ai Shun Wu, 吳艾瑄
• Other Authors: H. L. Wang
• Format: Others
• Published: 2009
• Online Access: http://ndltd.ncl.edu.tw/handle/10982021337048845171

碩士 === 長庚大學 === 生物醫學研究所 === 97 === Parkinson’s disease (PD) is the most common neurodegenerative motor disorder. Missense or truncating mutations of phosphatase and tesin homologue (PTEN)-induced kinase 1 (PINK1) gene are implicated in the pathogenesis of familial type 6 of Parkinson’s disease (PARK6) and that PINK1 is the second most frequent causative gene in early-onset Parkinson’s disease. PINK1 protein is mainly expressed in the mitochondria and has been proposed to function as a mitochondrial Ser/Thr protein kinase and exert neuroprotective effects against various cellular stresses. Better understanding the molecular basis of mutant PINK1-induced degeneration of substantia nigra (SN) dopaminergic neurons could lead to the development of effective PARK6 therapy. The autosomal recessive inheritance mode indicates a loss-of-function caused by PINK1 mutations is involved in the pathogenesis of PARK6. Thus, PINK1-deficient mice should be a valuable animal model to investigate the molecular pathogenesis of PARK6 and physiological functions of mitochondrial PINK1. In the present study, we have generated homozygous PINK1-/- mice using gene targeting strategy. Subsequently, primary SN dopaminergic neuronal culture of PINK1-null mice was prepared to study molecular mechanisms by which PINK1 exerts its neuroprotective effect within mitochondria and mutant PINK1 induces neurotoxicity of SN dopaminergic cells. PINK1 is believed to play an important role in regulating mitochondrial functions. Therefore, absence of PINK1 expression is likely to cause mitochondrial dysfunction of SN dopaminergic neurons. To test this hypothesis, confocal microcopy imaging was performed to visualize mitochondrial membrane potential, mitochondrial morphology and ROS (reactive oxygen species) formation of cultured SN dopaminergic neurons prepared from wild-type or PINK1-/- mice. Confocal imaging of potential sensitive dye tetramethylrhodamine methyl (TMRM) indicated that compared to cultured wild-type SN dopaminergic cells, a significant reduction in TMRM fluorescent signal and depolarized mitochondrial membrane potential was observed from SN dopaminergic neurons of PINK1-/- mice. Confocal MitoTracker Green staining was performed to visualize mitochondrial morphology of wild-type or PINK1-/- SN dopaminergic neurons. In contrast to filamentous and long thread-like mitochondria of wild-type SN dopaminergic neurons, fragmented mitochondria were observed from PINK1-deficient SN dopaminergic neurons. Compared to wild-type SN dopaminergic neurons, basal level of mitochondrial ROS formation, visualized by confocal MitoSOX staining, was significantly increased in PINK1-deficient SN dopaminergic neurons. Besides, the oxidative stressor H2O2-induced ROS production was greatly augmented in PINK1-null dopaminergic neurons than wild-type dopaminergic neurons. Our results indicate that PINK1 is required for maintaining mitochondrial membrane potential of SN dopaminergic neurons, which is the driving force behind oxidative phosphorylation and ion transportation, and exerts neuroprotective effects by inhibiting ROS formation. PINK1 is also involved in maintaining the structural integrity and modulating fission-fusion of mitochondria. Overexpression of wild-type PINK1 inhibited ROS formation, restored mitochondrial membrane potential and thread-like morphology of mitochondria in PINK1-deficient SN dopaminergic neurons. In contrast to wild-type PINK1, overexpression of PARK6 mutant (G309D), (E417G) or C-terminal truncated (C delta 145) PINK1 failed to rescue mitochondrial dysfunction and inhibit oxidative stress in PINK1-null SN dopaminergic cells. Our results provide the evidence that loss-of-function PARK6 mutation of PINK1 causes mitochondrial dysfunction, elevated oxidative stress and resulting neurotoxicity of SN dopaminergic cells.