Bioenergetic consequences of PINK1 mutations in Parkinson disease

Background

Mutations of the gene for PTEN-induced kinase 1 (PINK1) are a cause of familial Parkinson''s disease (PD). PINK1 protein has been localised to mitochondria and PINK1 gene knockout models exhibit abnormal mitochondrial function. The purpose of this study was to determine whether cells derived from PD patients with a range of PINK1 mutations demonstrate similar defects of mitochondrial function, whether the nature and severity of the abnormalities vary between mutations and correlate with clinical features.

Methodology

We investigated mitochondrial bioenergetics in live fibroblasts from PINK1 mutation patients using single cell techniques. We found that fibroblasts from PINK1 mutation patients had significant defects of bioenergetics including reduced mitochondrial membrane potential, altered redox state, a respiratory deficiency that was determined by substrate availability, and enhanced sensitivity to calcium stimulation and associated mitochondrial permeability pore opening. There was an increase in the basal rate of free radical production in the mutant cells. The pattern and severity of abnormality varied between different mutations, and the less severe defects in these cells were associated with later age of onset of PD.

Conclusions

The results provide insight into the molecular pathology of PINK1 mutations in PD and also confirm the critical role of substrate availability in determining the biochemical phenotype – thereby offering the potential for novel therapeutic strategies to circumvent these abnormalities.     

The PINK1-Parkin pathway is involved in the regulation of mitochondrial remodeling process

The two Parkinson’s disease (PD) genes, PTEN-induced kinase 1 (PINK1) and parkin, are linked in a common pathway which affects mitochondrial integrity and function. However, it is still not known what this pathway does in the mitochondria. Therefore, we investigated its physiological function in Drosophila. Because Drosophila PINK1 and parkin mutants show changes in mitochondrial morphology in both indirect flight muscles and dopaminergic neurons, we here investigated whether the PINK1-Parkin pathway genetically interacts with the regulators of mitochondrial fusion and fission such as Drp1, which promotes mitochondrial fission, and Opa1 or Marf, which induces mitochondrial fusion. Surprisingly, DrosophilaPINK1 and parkin mutant phenotypes were markedly suppressed by overexpression of Drp1 or downregulation of Opa1 or Marf, indicating that the PINK1-Parkin pathway regulates mitochondrial remodeling process in the direction of promoting mitochondrial fission. Therefore, we strongly suggest that mitochondrial fusion and fission process could be a prominent therapeutic target for the treatment of PD.     

Mitochondrial defect and PGC-1α dysfunction in parkin-associated familial Parkinson's disease

Mutations in the parkin gene are expected to play an essential role in autosomal recessive Parkinson's disease. Recent studies have established an impact of parkin mutations on mitochondrial function and autophagy. In primary skin fibroblasts from two patients affected by an early onset Parkinson's disease, we identified a hitherto unreported compound heterozygous mutation del exon2-3/del exon3 in the parkin gene, leading to the complete loss of the full-length protein. In both patients, but not in their heterozygous parental control, we observed severe ultrastructural abnormalities, mainly in mitochondria. This was associated with impaired energy metabolism, deregulated reactive oxygen species (ROS) production, resulting in lipid oxidation, and peroxisomal alteration. In view of the involvement of parkin in the mitochondrial quality control system, we have investigated upstream events in the organelles' biogenesis. The expression of the peroxisome proliferator-activated receptor gamma-coactivator 1-alpha (PGC-1α), a strong stimulator of mitochondrial biogenesis, was remarkably upregulated in both patients. However, the function of PGC-1α was blocked, as revealed by the lack of its downstream target gene induction. In conclusion, our data confirm the role of parkin in mitochondrial homeostasis and suggest a potential involvement of the PGC-1α pathway in the pathogenesis of Parkinson's disease. This article is part of a Special Issue entitled: Translating nuclear receptors from health to disease.     

Drosophila Trap1 protects against mitochondrial dysfunction in a PINK1/parkin model of Parkinson's disease

Mitochondrial dysfunction caused by protein aggregation has been shown to have an important role in neurological diseases, such as Parkinson''s disease (PD). Mitochondria have evolved at least two levels of defence mechanisms that ensure their integrity and the viability of their host cell. First, molecular quality control, through the upregulation of mitochondrial chaperones and proteases, guarantees the clearance of damaged proteins. Second, organellar quality control ensures the clearance of defective mitochondria through their selective autophagy. Studies in Drosophila have highlighted mitochondrial dysfunction linked with the loss of the PTEN-induced putative kinase 1 (PINK1) as a mechanism of PD pathogenesis. The mitochondrial chaperone TNF receptor-associated protein 1 (TRAP1) was recently reported to be a cellular substrate for the PINK1 kinase. Here, we characterise Drosophila Trap1 null mutants and describe the genetic analysis of Trap1 function with Pink1 and parkin. We show that loss of Trap1 results in a decrease in mitochondrial function and increased sensitivity to stress, and that its upregulation in neurons of Pink1 mutant rescues mitochondrial impairment. Additionally, the expression of Trap1 was able to partially rescue mitochondrial impairment in parkin mutant flies; and conversely, expression of parkin rescued mitochondrial impairment in Trap1 mutants. We conclude that Trap1 works downstream of Pink1 and in parallel with parkin in Drosophila, and that enhancing its function may ameliorate mitochondrial dysfunction and rescue neurodegeneration in PD.     

The Use of Primary Human Fibroblasts for Monitoring Mitochondrial Phenotypes in the Field of Parkinson's Disease

Parkinson''s disease (PD) is the second most common movement disorder and affects 1% of people over the age of 60 ¹. Because ageing is the most important risk factor, cases of PD will increase during the next decades ². Next to pathological protein folding and impaired protein degradation pathways, alterations of mitochondrial function and morphology were pointed out as further hallmark of neurodegeneration in PD ^3-11.After years of research in murine and human cancer cells as in vitro models to dissect molecular pathways of Parkinsonism, the use of human fibroblasts from patients and appropriate controls as ex vivo models has become a valuable research tool, if potential caveats are considered. Other than immortalized, rather artificial cell models, primary fibroblasts from patients carrying disease-associated mutations apparently reflect important pathological features of the human disease.Here we delineate the procedure of taking skin biopsies, culturing human fibroblasts and using detailed protocols for essential microscopic techniques to define mitochondrial phenotypes. These were used to investigate different features associated with PD that are relevant to mitochondrial function and dynamics. Ex vivo, mitochondria can be analyzed in terms of their function, morphology, colocalization with lysosomes (the organelles degrading dysfunctional mitochondria) and degradation via the lysosomal pathway. These phenotypes are highly relevant for the identification of early signs of PD and may precede clinical motor symptoms in human disease-gene carriers. Hence, the assays presented here can be utilized as valuable tools to identify pathological features of neurodegeneration and help to define new therapeutic strategies in PD.     

Mitochondrial dysfunction induced by knockdown of mortalin is rescued by Parkin

Mutations in the parkin gene are the most common cause of autosomal recessive Parkinson’s disease (PD). As an E3-ubiquitin ligase, Parkin is associated with mitochondrial dynamics and mitophagy. Mortalin, a molecular chaperone, is located primarily in mitochondria, where it functions to maintain mitochondrial homeostasis and antagonize oxidative stress injury. A reduced expression level of mortalin has been observed in the affected brain regions of PD patients. Mortalin also interacts with a variety of PD-related proteins and plays an indispensible role in helping native protein refolding and importing proteins into the mitochondrial matrix. Thus, the main aims of the present study were to investigate mitochondrial dysfunction induced by knockdown of mortalin and to test whether Parkin overexpression could rescue this effect. We found that lentivirus-mediated knockdown of mortalin in HeLa cells resulted in a collapse of mitochondrial membrane potential, an abnormal accumulation of reactive oxygen species and apparent alterations in mitochondrial morphology under H₂O₂-induced stress conditions. Remarkably, Parkin overexpression rescued these mitochondrial abnormalities. In HeLa cells expressing Parkin, co-immunoprecipitation of endogenous mortalin and wild-type Parkin was detected when they were treated with carbonyl cyanide 3-chlorophenylhydrazone (CCCP). In conclusion, we indicate that the relatively decreased mortalin expression level and its impaired interaction with Parkin could affect its roles in mitochondrial function.     

Glucocerebrosidase inhibition causes mitochondrial dysfunction and free radical damage

Mutations of the gene for glucocerebrosidase 1 (GBA) cause Gaucher disease (GD), an autosomal recessive lysosomal storage disorder. Individuals with homozygous or heterozygous (carrier) mutations of GBA have a significantly increased risk for the development of Parkinson’s disease (PD), with clinical and pathological features that mirror the sporadic disease. The mechanisms whereby GBA mutations induce dopaminergic cell death and Lewy body formation are unknown. There is evidence of mitochondrial dysfunction and oxidative stress in PD and so we have investigated the impact of glucocerebrosidase (GCase) inhibition on these parameters to determine if there may be a relationship of GBA loss-of-function mutations to the known pathogenetic pathways in PD. We have used exposure to a specific inhibitor (conduritol-β-epoxide, CβE) of GCase activity in a human dopaminergic cell line to identify the biochemical abnormalities that follow GCase inhibition. We show that GCase inhibition leads to decreased ADP phosphorylation, reduced mitochondrial membrane potential and increased free radical formation and damage, together with accumulation of alpha-synuclein. Taken together, inhibition of GCase by CβE induces abnormalities in mitochondrial function and oxidative stress in our cell culture model. We suggest that GBA mutations and reduced GCase activity may increase the risk for PD by inducing these same abnormalities in PD brain.     

Assessing the prevalence of PINK1 genetic variants in South African patients diagnosed with early- and late-onset Parkinson’s disease

Mutations in the PINK1 gene are the second most common cause after parkin of autosomal recessive early-onset Parkinson’s disease (PD). PINK1 is a protein kinase that is localized to the mitochondrion and is ubiquitously expressed in the human brain. Recent studies aimed at elucidating the function of PINK1, have found that it has neuroprotective properties against mitochondrial dysfunction and proteasomally-induced apoptosis. In the present study, we aimed to investigate the prevalence of PINK1 genetic variants in 154 South African PD patients from all ethnic groups. Mutation screening was performed using the High-Resolution Melt technique and direct sequencing. A total of 16 sequence variants were identified: one known homozygous mutation (Y258X), two heterozygous missense variants (P305A and E476K), and 13 polymorphisms of which five were novel. No homozygous exonic deletions were detected. The novel P305A variant was found in a female patient of Black Xhosa ethnicity who has a positive family history of the disease and an age at onset of 30 years. This variant has the potential to modulate enzymatic activity due to its location in the kinase domain. This is the first report on mutation screening of PINK1 in the South African population. Results from the present study showed that point mutations and homozygous exonic deletions in PINK1 are not a common cause of PD in the South African population.     

How could Parkin-mediated ubiquitination of mitofusin promote mitophagy?

Much evidence links mitochondrial dysfunction to the death of neurons in Parkinson disease (PD), and is particularly emphasized by our growing understanding of the function of genes linked to recessively inherited PD such as PINK1, parkin and DJ-1. Recent work has revealed an exciting link between the PINK1-Parkin pathway and the autophagic turnover of dysfunctional mitochondrial (mitophagy). We have recently shown that mitofusin is ubiquitinated by Parkin when it is recruited to dysfunctional mitochondria. Recent work also shows that regulated fission and fusion events help segregate dysfunctional mitochondria prior to mitophagy. Here we hypothesize how Parkin-mediated ubiquitination of Mfn may play a role in this mechanism.     

Knockdown of Hsc70-5/mortalin Induces Loss of Synaptic Mitochondria in a Drosophila Parkinson’s Disease Model

Mortalin is an essential component of the molecular machinery that imports nuclear-encoded proteins into mitochondria, assists in their folding, and protects against damage upon accumulation of dysfunctional, unfolded proteins in aging mitochondria. Mortalin dysfunction associated with Parkinson’s disease (PD) increases the vulnerability of cultured cells to proteolytic stress and leads to changes in mitochondrial function and morphology. To date, Drosophila melanogaster has been successfully used to investigate pathogenesis following the loss of several other PD-associated genes. We generated the first loss-of-Hsc70-5/mortalin-function Drosophila model. The reduction of Mortalin expression recapitulates some of the defects observed in the existing Drosophila PD-models, which include reduced ATP levels, abnormal wing posture, shortened life span, and reduced spontaneous locomotor and climbing ability. Dopaminergic neurons seem to be more sensitive to the loss of mortalin than other neuronal sub-types and non-neuronal tissues. The loss of synaptic mitochondria is an early pathological change that might cause later degenerative events. It precedes both behavioral abnormalities and structural changes at the neuromuscular junction (NMJ) of mortalin-knockdown larvae that exhibit increased mitochondrial fragmentation. Autophagy is concomitantly up-regulated, suggesting that mitochondria are degraded via mitophagy. Ex vivo data from human fibroblasts identifies increased mitophagy as an early pathological change that precedes apoptosis. Given the specificity of the observed defects, we are confident that the loss-of-mortalin model presented in this study will be useful for further dissection of the complex network of pathways that underlie the development of mitochondrial parkinsonism.     

Mitochondrial remodeling in human skin fibroblasts from sporadic male Parkinson's disease patients uncovers metabolic and mitochondrial bioenergetic defects

Parkinson's Disease (PD) is characterized by dopaminergic neurodegeneration in the substantia nigra. The exact mechanism by which dopaminergic neurodegeneration occurs is still unknown; however, mitochondrial dysfunction has long been implicated in PD pathogenesis. To investigate the sub-cellular events that lead to disease progression and to develop personalized interventions, non-neuronal cells which are collected in a minimally invasive manner can be key to test interventions aimed at improving mitochondrial function. We used human skin fibroblasts from sporadic PD (sPD) patients as a cell proxy to detect metabolic and mitochondrial alterations which would also exist in a non-neuronal cell type. In this model, we used a glucose-free/galactose- glutamine- and pyruvate-containing cell culture medium, which forces cells to be more dependent on oxidative phosphorylation (OXPHOS) for energy production, in order to reveal hidden metabolic and mitochondrial alterations present in fibroblasts from sPD patients.We demonstrated that fibroblasts from sPD patients show hyperpolarized and elongated mitochondrial networks and higher mitochondrial ROS concentration, as well as decreased ATP levels and glycolysis-related ECAR. Our results also showed that abnormalities of fibroblasts from sPD patients became more evident when stimulating OXPHOS. Under these culture conditions, fibroblasts from sPD cells presented decreased basal respiration, ATP-linked OCR and maximal respiration, and increased mitochondria-targeting phosphorylation of DRP1 when compared to control cells.Our work validates the relevance of using fibroblasts from sPD patients to study cellular and molecular changes that are characteristic of dopaminergic neurodegeneration of PD, and shows that forcing mitochondrial OXPHOS uncovers metabolic defects that were otherwise hidden.     

Mitochondrial Targeting of Cytochrome P450 (CYP) 1B1 and Its Role in Polycyclic Aromatic Hydrocarbon-induced Mitochondrial Dysfunction

We report that polycyclic aromatic hydrocarbon (PAH)-inducible CYP1B1 is targeted to mitochondria by sequence-specific cleavage at the N terminus by a cytosolic Ser protease (polyserase 1) to activate the cryptic internal signal. Site-directed mutagenesis, COS-7 cell transfection, and in vitro import studies in isolated mitochondria showed that a positively charged domain at residues 41–48 of human CYP1B1 is part of the mitochondrial (mt) import signal. Ala scanning mutations showed that the Ser protease cleavage site resides between residues 37 and 41 of human CYP1B1. Benzo[a]pyrene (BaP) treatment induced oxidative stress, mitochondrial respiratory defects, and mtDNA damage that was attenuated by a CYP1B1-specific inhibitor, 2,3,4,5-tetramethoxystilbene. In support, the mitochondrial CYP1B1 supported by mitochondrial ferredoxin (adrenodoxin) and ferredoxin reductase showed high aryl hydrocarbon hydroxylase activity. Administration of benzo[a]pyrene or 2,3,7,8-tetrachlorodibenzodioxin induced similar mitochondrial functional abnormalities and oxidative stress in the lungs of wild-type mice and Cyp1a1/1a2-null mice, but the effects were markedly blunted in Cyp1b1-null mice. These results confirm a role for CYP1B1 in inducing PAH-mediated mitochondrial dysfunction. The role of mitochondrial CYP1B1 was assessed using A549 lung epithelial cells stably expressing shRNA against NADPH-cytochrome P450 oxidoreductase or mitochondrial adrenodoxin. Our results not only show conservation of the endoprotease cleavage mechanism for mitochondrial import of family 1 CYPs but also reveal a direct role for mitochondrial CYP1B1 in PAH-mediated oxidative and chemical damage to mitochondria.     

<Emphasis Type="Italic">parkin</Emphasis>mutation dosage and the phenomenon of anticipation: a molecular genetic study of familial parkinsonism

Background  

parkin mutations are a common cause of parkinsonism. Possessing two parkin mutations leads to early-onset parkinsonism, while having one mutation may predispose to late-onset disease. This dosage pattern suggests that some parkin families should exhibit intergenerational variation in age at onset resembling anticipation. A subset of familial PD exhibits anticipation, the cause of which is unknown. The aim of this study was to determine if anticipation was due to parkin mutation dosage.     

Characterization of PINK1 (PTEN-induced Putative Kinase 1) Mutations Associated with Parkinson Disease in Mammalian Cells and Drosophila

Mutations in PINK1 (PTEN-induced putative kinase 1) are tightly linked to autosomal recessive Parkinson disease (PD). Although more than 50 mutations in PINK1 have been discovered, the role of these mutations in PD pathogenesis remains poorly understood. Here, we characterized 17 representative PINK1 pathogenic mutations in both mammalian cells and Drosophila. These mutations did not affect the typical cleavage patterns and subcellular localization of PINK1 under both normal and damaged mitochondria conditions in mammalian cells. However, PINK1 mutations in the kinase domain failed to translocate Parkin to mitochondria and to induce mitochondrial aggregation. Consistent with the mammalian data, Drosophila PINK1 mutants with mutations in the kinase domain (G426D and L464P) did not genetically interact with Parkin. Furthermore, PINK1-null flies expressing the transgenic G426D mutant displayed defective phenotypes with increasing age, whereas L464P mutant-expressing flies exhibited the phenotypes at an earlier age. Collectively, these results strongly support the hypothesis that the kinase activity of PINK1 is essential for its function and for regulating downstream Parkin functions in mitochondria. We believe that this study provides the basis for understanding the molecular and physiological functions of various PINK1 mutations and provides insights into the pathogenic mechanisms of PINK1-linked PD.     

MPV17 Loss Causes Deoxynucleotide Insufficiency and Slow DNA Replication in Mitochondria

MPV17 is a mitochondrial inner membrane protein whose dysfunction causes mitochondrial DNA abnormalities and disease by an unknown mechanism. Perturbations of deoxynucleoside triphosphate (dNTP) pools are a recognized cause of mitochondrial genomic instability; therefore, we determined DNA copy number and dNTP levels in mitochondria of two models of MPV17 deficiency. In Mpv17 ablated mice, liver mitochondria showed substantial decreases in the levels of dGTP and dTTP and severe mitochondrial DNA depletion, whereas the dNTP pool was not significantly altered in kidney and brain mitochondria that had near normal levels of DNA. The shortage of mitochondrial dNTPs in Mpv17^-/- liver slows the DNA replication in the organelle, as evidenced by the elevated level of replication intermediates. Quiescent fibroblasts of MPV17-mutant patients recapitulate key features of the primary affected tissue of the Mpv17^-/- mice, displaying virtual absence of the protein, decreased dNTP levels and mitochondrial DNA depletion. Notably, the mitochondrial DNA loss in the patients’ quiescent fibroblasts was prevented and rescued by deoxynucleoside supplementation. Thus, our study establishes dNTP insufficiency in the mitochondria as the cause of mitochondrial DNA depletion in MPV17 deficiency, and identifies deoxynucleoside supplementation as a potential therapeutic strategy for MPV17-related disease. Moreover, changes in the expression of factors involved in mitochondrial deoxynucleotide homeostasis indicate a remodeling of nucleotide metabolism in MPV17 disease models, which suggests mitochondria lacking functional MPV17 have a restricted purine mitochondrial salvage pathway.     

Mitochondrial abnormalities in dermatomyositis: characteristic pattern of neuropathology

The objective of the work described in this paper was to evaluate mitochondrial abnormalities in perifascicular atrophic fibers in muscle biopsies from patients with dermatomyositis (DM). We localized cytochrome c oxidase (COX) and succinate dehydrogenase (SDH) histochemically in muscle biopsies of 12 patients with DM, and 12 control patients with neurogenic atrophy. These two histochemical techniques were also combined on single tissue sections in order to accentuate any COX-negative fibers. Eleven out of 12 patients (91.6%) with DM showed histochemical evidence of mitochondrial dysfunction in perifascicular distribution. Similar abnormalities in histochemical staining were not seen in comparably sized myofibers that were atrophic due to denervation. It is concluded that abnormal SDH and COX histochemical activities in atrophic perifascicular fibers are characteristic of dermatomyositis. These abnormal staining characteristics could not be accounted for solely by myofiber atrophy, or by generalized abnormalities in histochemical staining.     

Parkinson Phenotype in Aged PINK1-Deficient Mice Is Accompanied by Progressive Mitochondrial Dysfunction in Absence of Neurodegeneration

Background

Parkinson''s disease (PD) is an adult-onset movement disorder of largely unknown etiology. We have previously shown that loss-of-function mutations of the mitochondrial protein kinase PINK1 (PTEN induced putative kinase 1) cause the recessive PARK6 variant of PD.

Methodology/Principal Findings

Now we generated a PINK1 deficient mouse and observed several novel phenotypes: A progressive reduction of weight and of locomotor activity selectively for spontaneous movements occurred at old age. As in PD, abnormal dopamine levels in the aged nigrostriatal projection accompanied the reduced movements. Possibly in line with the PARK6 syndrome but in contrast to sporadic PD, a reduced lifespan, dysfunction of brainstem and sympathetic nerves, visible aggregates of α-synuclein within Lewy bodies or nigrostriatal neurodegeneration were not present in aged PINK1-deficient mice. However, we demonstrate PINK1 mutant mice to exhibit a progressive reduction in mitochondrial preprotein import correlating with defects of core mitochondrial functions like ATP-generation and respiration. In contrast to the strong effect of PINK1 on mitochondrial dynamics in Drosophila melanogaster and in spite of reduced expression of fission factor Mtp18, we show reduced fission and increased aggregation of mitochondria only under stress in PINK1-deficient mouse neurons.

Conclusion

Thus, aging Pink1^−/− mice show increasing mitochondrial dysfunction resulting in impaired neural activity similar to PD, in absence of overt neuronal death.     

Decreased amount of vimentin N-terminal truncated proteolytic products in parkin-mutant skin fibroblasts

Vimentin, a member of cytoskeleton intermediate filaments proteins, plays a critical role in cell structure and dynamics.The present proteomic study reveals reduced amount of six different lengths, N-terminal truncated proteolytic products of vimentin, in the primary skin fibroblasts from two unrelated PD patients, as compared to control fibroblasts. The decreased amount of N-terminal truncated forms of vimentin in parkin-mutant fibroblasts, could contribute to impairment of cellular function, potentially contributing to the pathogenesis of Parkinson disease.     

The PINK1/Parkin pathway: a mitochondrial quality control system?

Significant insight into the mechanisms that contribute to dopaminergic neurodegeneration in Parkinson disease has been gained from the analysis of genes linked to rare heritable forms of parkinsonism such as PINK1 and parkin, loss-of-function mutations of which cause autosomal recessive parkinsonism. PINK1 encodes a mitochondrially targeted Ser/Thr kinase and parkin encodes a ubiquitin-protein ligase. Functional studies of PINK1 and Parkin in animal and cellular model systems have shown that both proteins play important roles in maintaining mitochondrial integrity. Genetic studies of PINK1 and Parkin orthologs in flies have shown that PINK1 acts upstream from Parkin in a common pathway that appears to regulate mitochondrial morphology. Mitochondrial morphology is regulated by mitochondrial fission and fusion-promoting proteins, and is important in a variety of contexts, including mitochondrial trafficking and mitochondrial quality control. In particular, mitochondrial fission appears to promote the segregation of terminally dysfunctional mitochondria for degradation in the lysosome through a process termed mitophagy. Recent work has shown that Parkin promotes the degradation of dysfunctional mitochondria in vertebrate cell culture. Here we postulate a model whereby the PINK1/Parkin pathway regulates mitochondrial dynamics in an effort to promote the turnover of damaged mitochondria.     

PARK6 PINK1 mutants are defective in maintaining mitochondrial membrane potential and inhibiting ROS formation of substantia nigra dopaminergic neurons

Mutations in PTEN-induced kinase 1 (PINK1) gene cause recessive familial type 6 of Parkinson's disease (PARK6). PINK1 is believed to exert neuroprotective effect on SN dopaminergic cells by acting as a mitochondrial Ser/Thr protein kinase. Autosomal recessive inheritance indicates the involvement of loss of PINK1 function in PARK6 pathogenesis. In the present study, confocal imaging of cultured SN dopaminergic neurons prepared from PINK1 knockout mice was performed to investigate physiological importance of PINK1 in maintaining mitochondrial membrane potential (ΔΨ_m) and mitochondrial morphology and test the hypothesis that PARK6 mutations cause the loss of PINK1 function. PINK1-deficient SN dopaminergic neurons exhibited a depolarized ΔΨ_m. In contrast to long thread-like mitochondria of wild-type neurons, fragmented mitochondria were observed from PINK1-null SN dopaminergic cells. Basal level of mitochondrial superoxide and oxidative stressor H₂O₂-induced ROS generation were significantly increased in PINK1-deficient dopaminergic neurons. Overexpression of wild-type PINK1 restored hyperpolarized ΔΨ_m and thread-like mitochondrial morphology and inhibited ROS formation in PINK1-null dopaminergic cells. PARK6 mutant (G309D), (E417G) or (CΔ145) PINK1 failed to rescue mitochondrial dysfunction and inhibit oxidative stress in PINK1-deficient dopaminergic neurons. Mitochondrial toxin rotenone-induced cell death of dopaminergic neurons was augmented in PINK1-null SN neuronal culture. These results indicate that PINK1 is required for maintaining normal ΔΨ_m and mitochondrial morphology of cultured SN dopaminergic neurons and exerts its neuroprotective effect by inhibiting ROS formation. Our study also provides the evidence that PARK6 mutant (G309D), (E417G) or (CΔ145) PINK1 is defective in regulating mitochondrial functions and attenuating ROS production of SN dopaminergic cells.