Abstract
SummaryMutations in PINK1 cause autosomal recessive Parkinson's disease. PINK1 is a mitochondrial kinase of unknown function. We investigated calcium homeostasis and mitochondrial function in PINK1-deficient mammalian neurons. We demonstrate physiologically that PINK1 regulates calcium efflux from the mitochondria via the mitochondrial Na+/Ca2+ exchanger. PINK1 deficiency causes mitochondrial accumulation of calcium, resulting in mitochondrial calcium overload. We show that calcium overload stimulates reactive oxygen species (ROS) production via NADPH oxidase. ROS production inhibits the glucose transporter, reducing substrate delivery and causing impaired respiration. We demonstrate that impaired respiration may be restored by provision of mitochondrial complex I and II substrates. Taken together, reduced mitochondrial calcium capacity and increased ROS lower the threshold of opening of the mitochondrial permeability transition pore (mPTP) such that physiological calcium stimuli become sufficient to induce mPTP opening in PINK1-deficient cells. Our findings propose a mechanism by which PINK1 dysfunction renders neurons vulnerable to cell death.
Highlights
Mitochondrial dysfunction has been implicated in a range of neurodegenerative diseases, in particular, Parkinson’s disease (PD)
We report that loss of PINK1 function causes dysregulation of mitochondrial calcium handling, resulting in mitochondrial calcium overload which sensitizes the mitochondria to opening of the permeability transition pore (PTP)
Using stable expression of shRNA constructs, we were able to reduce PINK1 gene expression by >90% in (1) a human dopaminergic neuroblastoma cell line (SH-SY5Y), and (2) human neurons derived from fetal mesencephalic stem cells that represent the closest in vitro model of primary human neurons
Summary
Mitochondrial dysfunction has been implicated in a range of neurodegenerative diseases, in particular, Parkinson’s disease (PD). Several genes have been identified in which mutations cause forms of familial PD that are clinically and pathologically indistinguishable from sporadic PD. A number of these genes, namely PINK1, DJ-I, and Omi/HtrA2, encode mitochondrially located proteins, further implicating mitochondrial dysfunction as the primary event sufficient to cause PD. PINK1 is a 581 amino acid protein consisting of a mitochondrial targeting motif and a serine/threonine kinase domain homologous to the Ca2+/calmodulin family. Studies both in vitro (Silvestri et al, 2005; Muqit et al, 2006) and in vivo (Gandhi et al, 2006) show PINK1 localized to mitochondria. It is well established that PINK1 protects neurons from oxidative stress, and plays a role in mitochondrial morphology in mammalian cells and Drosophila models (Haque et al, 2008; Exner et al, 2007; Yang et al, 2006)
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