SUMMARY Cells keep their energy balance and avoid oxidative stress by regulating mitochondrial movement, distribution, and clearance. We report here that two Parkinson’s disease proteins, the Ser/Thr-kinase PINK1 and ubiquitin-ligase Parkin, participate in this regulation by arresting mitochondrial movement. PINK1 phosphorylates Miro, a component of the primary motor/adaptor complex that anchors kinesin to the mitochondrial surface. The phosphorylation of Miro activates proteasomal degradation of Miro in a Parkin-dependent manner. Removal of Miro from the mitochondrion also detaches kinesin from its surface. By preventing mitochondrial movement, the PINK1/Parkin pathway may quarantine damaged mitochondria prior to their clearance. PINK1 has been shown to act upstream of Parkin but the mechanism corresponding to this relationship has not been known. We propose that PINK1 phosphorylation of substrates triggers the subsequent action of Parkin and the proteasome.

The Parkinson’s disease–associated proteins PINK1 and Parkin mediate local mitophagy of dysfunctional mitochondria in distal neuronal axons, abrogating the need for retrograde organelle transport and ensuring rapid neuroprotection.

Loss-of-function mutations in the parkin gene (PARK2) and PINK1 gene (PARK6) are associated with autosomal recessive parkinsonism. PINK1 deficiency was recently linked to mitochondrial pathology in human cells and Drosophila melanogaster, which can be rescued by parkin, suggesting that both genes play a role in maintaining mitochondrial integrity. Here we demonstrate that an acute down-regulation of parkin in human SH-SY5Y cells severely affects mitochondrial morphology and function, a phenotype comparable with that induced by PINK1 deficiency. Alterations in both mitochondrial morphology and ATP production caused by either parkin or PINK1 loss of function could be rescued by the mitochondrial fusion proteins Mfn2 and OPA1 or by a dominant negative mutant of the fission protein Drp1. Both parkin and PINK1 were able to suppress mitochondrial fragmentation induced by Drp1. Moreover, in Drp1-deficient cells the parkin/PINK1 knockdown phenotype did not occur, indicating that mitochondrial alterations observed in parkin-or PINK1-deficient cells are associated with an increase in mitochondrial fission. Notably, mitochondrial fragmentation is an early phenomenon upon PINK1/parkin silencing that also occurs in primary mouse neurons and Drosophila S2 cells. We propose that the discrepant findings in adult flies can be explained by the time of phenotype analysis and suggest that in mammals different strategies may have evolved to cope with dysfunctional mitochondria.Many lines of evidence suggest that mitochondrial dysfunction plays a central role in the pathogenesis of Parkinson disease, starting from the early observation that the complex I inhibitor 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine induced acute and irreversible parkinsonism in young drug addicts (for review, see Refs. 1-3). In support of a crucial role of mitochondria in Parkinson disease, several Parkinson diseaseassociated gene products directly or indirectly impinge on mitochondrial integrity (for review, see . A clear link between Parkinson disease genes and mitochondria has recently emerged from studies on PINK1 (PTEN-induced putative kinase 1), a mitochondrial serine/threonine kinase, and parkin, a cytosolic E3 ubiquitin ligase. Drosophila parkin null mutants displayed reduced life span, male sterility, and locomotor defects due to apoptotic flight muscle degeneration (7). The earliest manifestation of muscle degeneration and defective spermatogenesis was mitochondrial pathology, exemplified by swollen mitochondria and disintegrated cristae. Remarkably, Drosophila PINK1 null mutants shared marked phenotypic similarities with parkin mutants, and parkin could compensate for the PINK1 loss-of-function phenotype but not vice versa, leading to the conclusion that PINK1 and parkin function in a common genetic pathway with parkin acting downstream of PINK1 (8 -10). We recently demonstrated that PINK1 deficiency in cultured human cells causes alterations in mitochondrial morphology, which can be rescued by wild type parkin but not by pathogenic parkin mutant...

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Parkinson disease (PD)2 is the second most common neurodegenerative disease after Alzheimer disease. Although most PD cases occur sporadically, familial variants share important features with sporadic PD, most notably the demise of dopaminergic neurons in the substantia nigra pars compacta. Consequently, insight into the function of PD-associated genes might promote our understanding of pathogenic mechanisms not only in familial, but also in sporadic PD. Five genes have unambiguously been linked to PD over the past decade, the genes encoding ␣-synuclein and LRRK2 for autosomal dominant PD, and those encoding Parkin, PINK1, and DJ-1 for autosomal recessive PD (reviewed in Refs. 1-3). So far, over a hundred different pathogenic mutations in the parkin gene have been identified, which account for the majority of autosomal recessive PD cases. Parkin is a member of the RBR (ring between ring fingers) protein family, characterized by the presence of two RING domains (really interesting new gene), which flank a cysteine-rich in-between RINGs (IBR) domain. Similarly to other RBR proteins, Parkin has an E3 ubiquitin ligase activity, mediating the attachment of ubiquitin to substrate proteins (4 -6). Parkin can obviously mediate different modes of ubiquitylation, including monoubiquitylation, multiple monoubiquitylation, and polyubiquitylation both via lysine 48 and lysine 63, depending on the experimental conditions and the putative Parkin substrate analyzed (reviewed in Refs. 7 and 8). Importantly, the neuroprotective activity of Parkin seems to be associated with its ability to promote degradation-independent ubiquitylation (9, 10).Different lines of evidence indicate that pathogenic parkin mutations result in a loss of Parkin function. Our initial studies revealed that misfolding and aggregation is characteristic for C-terminal deletion mutants of Parkin based on the following biochemical features specific for mutant Parkin: 1) insolubility in non-ionic and ionic detergents; 2) sedimentation in a sucrose step gradient; 3) resistance to a limited proteolytic digestion; 4) loss of membrane association; and 5) formation of scattered aggregates in cells determined by immunocytochemistry (11,12). Alterations in the detergent solubility of Parkin and formation of Parkin aggregates/inclusion bodies have also been reported for various Parkin missense mutants (13-18). We also observed that even wild-type Parkin is prone to misfolding under severe oxidative stress (12). Remarkably, insoluble, catechol-modified Parkin could be detected in the substantia nigra of patients suffering from sporadic PD, suggesting a more general role of Parkin in the pathogenesis of PD (19). In support of this concept, the E3 ligase activity of Parkin has been shown to be impaired by nitrosative stress, and there is indeed evidence for the presence of S-nitrosylated Parkin in the brains of PD patients (20,21).Based on our finding that the deletion of C-terminal amino acids results in misfolding and aggregation of Parkin, we performed a comparative an...