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...

BackgroundMutations in the gene encoding the E3 ubiquitin ligase parkin (PARK2) are responsible for the majority of autosomal recessive parkinsonism. Similarly to other knockout mouse models of PD-associated genes, parkin knockout mice do not show a substantial neuropathological or behavioral phenotype, while loss of parkin in Drosophila melanogaster leads to a severe phenotype, including reduced lifespan, apoptotic flight muscle degeneration and male sterility. In order to study the function of parkin in more detail and to address possible differences in its role in different species, we chose Danio rerio as a different vertebrate model system.Methodology/Principal FindingsWe first cloned zebrafish parkin to compare its biochemical and functional aspects with that of human parkin. By using an antisense knockdown strategy we generated a zebrafish model of parkin deficiency (knockdown efficiency between 50% and 60%) and found that the transient knockdown of parkin does not cause morphological or behavioral alterations. Specifically, we did not observe a loss of dopaminergic neurons in parkin-deficient zebrafish. In addition, we established transgenic zebrafish lines stably expressing parkin by using a Gal4/UAS-based bidirectional expression system. While parkin-deficient zebrafish are more vulnerable to proteotoxicity, increased parkin expression protected transgenic zebrafish from cell death induced by proteotoxic stress.Conclusions/SignificanceSimilarly to human parkin, zebrafish parkin is a stress-responsive protein which protects cells from stress-induced cell death. Our transgenic zebrafish model is a novel tool to characterize the protective capacity of parkin in vivo.