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.     

Mitochondrial dynamics and mitophagy in Parkinson's disease: disordered cellular power plant becomes a big deal in a major movement disorder

Parkinson's disease (PD), the most common movement disorder, is characterized by age-dependent degeneration of dopaminergic neurons in the substantia nigra of the mid-brain. Non-motor symptoms of PD, however, precede the motor features caused by dysfunction of the dopaminergic system, suggesting that PD is a systemic disorder. Mitochondrial dysfunction has long been observed in PD patients and animal models, but the mechanistic link between mitochondrial dysfunction and PD pathogenesis is not well understood. Recent studies have revealed that genes associated with autosomal recessive forms of PD such as PINK1 and Parkin are directly involved in regulating mitochondrial morphology and maintenance, abnormality of which is also observed in the more common, sporadic forms of PD, although the autosomal recessive PDs lack Lewy-body pathology that is characteristic of sporadic PD. These latest findings suggest that at least some forms of PD can be characterized as a mitochondrial disorder. Whether mitochondrial dysfunction represents a unifying pathogenic mechanism of all PD cases remains a major unresolved question.     

Short Mitochondrial ARF Triggers Parkin/PINK1-dependent Mitophagy

Parkinson disease (PD) is a complex neurodegenerative disease characterized by the loss of dopaminergic neurons in the substantia nigra. Multiple genes have been associated with PD, including Parkin and PINK1. Recent studies have established that the Parkin and PINK1 proteins function in a common mitochondrial quality control pathway, whereby disruption of the mitochondrial membrane potential leads to PINK1 stabilization at the mitochondrial outer surface. PINK1 accumulation leads to Parkin recruitment from the cytosol, which in turn promotes the degradation of the damaged mitochondria by autophagy (mitophagy). Most studies characterizing PINK1/Parkin mitophagy have relied on high concentrations of chemical uncouplers to trigger mitochondrial depolarization, a stimulus that has been difficult to adapt to neuronal systems and one unlikely to faithfully model the mitochondrial damage that occurs in PD. Here, we report that the short mitochondrial isoform of ARF (smARF), previously identified as an alternate translation product of the tumor suppressor p19ARF, depolarizes mitochondria and promotes mitophagy in a Parkin/PINK1-dependent manner, both in cell lines and in neurons. The work positions smARF upstream of PINK1 and Parkin and demonstrates that mitophagy can be triggered by intrinsic signaling cascades.     

Drp1的调控对PINK1突变转基因果蝇的保护作用

帕金森病（PD）是以黑质致密部多巴胺神经元选择性减少和胞浆内路易小体的形成为特征的神经退行性疾病。研究发现,PTEN诱导激酶1(PINK1)基因突变导致家族性早发型帕金森病的发生。在转基因果蝇中,PINK1功能丢失导致间接飞行肌缺陷,线粒体结构、功能障碍,多巴胺神经元丢失。本研究在PINK1突变PD转基因果蝇中,进行发动蛋白相关蛋白1（Drp1）过表达和敲低,探索Drp1对PD转基因果蝇的保护作用及其可能机制。本研究选用MHC-Gal4/UAS系统的PD转基因果蝇模型,特异性启动PINK1B9基因于果蝇肌肉组织中表达;运用Drp1基因过表达和RNA干扰干预PINK1B9转基因果蝇,研究其对PD转基因果蝇的作用。结果显示,不论过表达Drp1还是Drp1敲低均可挽救PINK1突变转基因果蝇,降低翅膀异常率,改善飞行能力,恢复间接飞行肌排列,调节线粒体形态,提高ATP生成量,上调NDUFS3蛋白表达水平。本文结果提示,Drp1的调控挽救PINK1突变转基因果蝇与线粒体呼吸链有关。     

Mitochondrial DNA heteroplasmy rises in substantial nigra of aged PINK1 KO mice

Mutations in PINK1 and Parkin result in early-onset autosomal recessive Parkinson’s disease (PD). PINK1/Parkin pathway maintain mitochondrial function by mediating the clearance of damaged mitochondria. However, the role of PINK1/Parkin in maintaining the balance of mtDNA heteroplasmy is still unknown. Here, we isolated mitochondrial DNA (mtDNA) from cortex, striatum and substantia nigra of wildtype (WT), PINK1 knockout (PINK1 KO) and Parkin knockout (Parkin KO) mice to analyze mtDNA heteroplasmy induced by PINK1/Parkin deficiency or aging. Our results showed that the Single Nucleotide Variants (SNVs) of late-onset somatic variants mainly increased with aging. Conversely, the early-onset somatic variants exhibited significant increase in the cortex and substantia nigra of PINK1 KO mice than WT mice of the same age. Increased average variant allele frequency was observed in aged PINK1 KO mice and in substantial nigra of aged Parkin KO mice than in WT mice. Cumulative variant allele frequency in the substantia nigra of PINK1 KO mice was significantly higher than that in WT mice, further supporting the pivotal role of PINK1 in mtDNA maintenance.This study presented a new evidence for PINK1 and Parkin in participating in mitochondrial quality control and provided clues for further revealing the role of PINK1 and Parkin in the pathogenesis of PD.     

BAG5 Protects against Mitochondrial Oxidative Damage through Regulating PINK1 Degradation

Mutations in PTEN-induced kinase 1 (PINK1) gene cause PARK6 familial Parkinsonism, and loss of the stability of PINK1 may also contribute to sporadic Parkinson''s disease (PD). Degradation of PINK1 occurs predominantly through the ubiquitin proteasome system (UPS), however, to date, few of the proteins have been found to regulate the degradation of PINK1. Using the yeast two-hybrid system and pull-down methods, we identified bcl-2-associated athanogene 5 (BAG5), a BAG family member, directly interacted with PINK1. We showed that BAG5 stabilized PINK1 by decreasing the ubiquitination of PINK1. Interestingly, BAG5 rescued MPP⁺- and rotenone-induced mitochondria dysfunction by up-regulating PINK1 in vitro. In PINK1-null mice and MPTP-treated mice, BAG5 significantly increased in the substantia nigra pars compacta (SNpc) although PINK1 was decreased. Our findings indicated that BAG5, as a key protein to stabilize PINK1, is a promising therapeutic tool for preventing mitochondrial dysfunction following oxidative stress.     

Quantitative expression proteomics and phosphoproteomics profile of brain from PINK1 knockout mice: insights into mechanisms of familial Parkinson's disease

Parkinson's disease (PD) is an age‐related, neurodegenerative motor disorder characterized by progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta and presence of α‐synuclein‐containing protein aggregates. Mutations in the mitochondrial Ser/Thr kinase PTEN‐induced kinase 1 (PINK1) are associated with an autosomal recessive familial form of early‐onset PD. Recent studies have suggested that PINK1 plays important neuroprotective roles against mitochondrial dysfunction by phosphorylating and recruiting Parkin, a cytosolic E3 ubiquitin ligase, to facilitate elimination of damaged mitochondria via autophagy‐lysosomal pathways. Loss of PINK1 in cells and animals leads to various mitochondrial impairments and oxidative stress, culminating in dopaminergic neuronal death in humans. Using a 2‐D polyacrylamide gel electrophoresis proteomics approach, the differences in expressed brain proteome and phosphoproteome between 6‐month‐old PINK1‐deficient mice and wild‐type mice were identified. The observed changes in the brain proteome and phosphoproteome of mice lacking PINK1 suggest that defects in signaling networks, energy metabolism, cellular proteostasis, and neuronal structure and plasticity are involved in the pathogenesis of familial PD.

     

Mitochondrial dynamics,cell death and the pathogenesis of Parkinson’s disease

The structure and function of the mitochondrial network is regulated by mitochondrial biogenesis, fission, fusion, transport and degradation. A well-maintained balance of these processes (mitochondrial dynamics) is essential for neuronal signaling, plasticity and transmitter release. Core proteins of the mitochondrial dynamics machinery play important roles in the regulation of apoptosis, and mutations or abnormal expression of these factors are associated with inherited and age-dependent neurodegenerative disorders. In Parkinson’s disease (PD), oxidative stress and mitochondrial dysfunction underlie the development of neuropathology. The recessive Parkinsonism-linked genes PTEN-induced kinase 1 (PINK1) and Parkin maintain mitochondrial integrity by regulating diverse aspects of mitochondrial function, including membrane potential, calcium homeostasis, cristae structure, respiratory activity, and mtDNA integrity. In addition, Parkin is crucial for autophagy-dependent clearance of dysfunctional mitochondria. In the absence of PINK1 or Parkin, cells often develop fragmented mitochondria. Whereas excessive fission may cause apoptosis, coordinated induction of fission and autophagy is believed to facilitate the removal of damaged mitochondria through mitophagy, and has been observed in some types of cells. Compensatory mechanisms may also occur in mice lacking PINK1 that, in contrast to cells and Drosophila, have only mild mitochondrial dysfunction and lack dopaminergic neuron loss. A better understanding of the relationship between the specific changes in mitochondrial dynamics/turnover and cell death will be instrumental to identify potentially neuroprotective pathways steering PINK1-deficient cells towards survival. Such pathways may be manipulated in the future by specific drugs to treat PD and perhaps other neurodegenerative disorders characterized by abnormal mitochondrial function and dynamics.     

Parkin blushed by PINK1

Mutations in the PTEN-induced putative kinase 1 (PINK1) are a common cause of autosomal recessive Parkinson's disease. In a recent issue of Nature, two independent reports by and show that loss of Drosophila PINK1 leads to defects in mitochondrial function resulting in male sterility, apoptotic muscle degeneration, and minor loss of dopamine neurons that is rescued by overexpression of the ubiquitin E3 ligase, parkin. Thus, PINK1 and parkin appear to function in a common pathway suggesting a convergence of the two genes most commonly associated with autosomal recessive PD.     

The TOMM machinery is a molecular switch in PINK1 and PARK2/PARKIN-dependent mitochondrial clearance

Loss-of-function mutations in PARK2/PARKIN and PINK1 cause early-onset autosomal recessive Parkinson disease (PD). The cytosolic E3 ubiquitin-protein ligase PARK2 cooperates with the mitochondrial kinase PINK1 to maintain mitochondrial quality. A loss of mitochondrial transmembrane potential (ΔΨ) leads to the PINK1-dependent recruitment of PARK2 to the outer mitochondrial membrane (OMM), followed by the ubiquitination and proteasome-dependent degradation of OMM proteins, and by the autophagy-dependent clearance of mitochondrial remnants. We showed here that blockade of mitochondrial protein import triggers the recruitment of PARK2, by PINK1, to the TOMM machinery. PD-causing PARK2 mutations weakened or disrupted the molecular interaction between PARK2 and specific TOMM subunits: the surface receptor, TOMM70A, and the channel protein, TOMM40. The downregulation of TOMM40 or its associated core subunit, TOMM22, was sufficient to trigger OMM protein clearance in the absence of PINK1 or PARK2. However, PARK2 was required to promote the degradation of whole organelles by autophagy. Furthermore, the overproduction of TOMM22 or TOMM40 reversed mitochondrial clearance promoted by PINK1 and PARK2 after ΔΨ loss. These results indicated that the TOMM machinery is a key molecular switch in the mitochondrial clearance program controlled by the PINK1-PARK2 pathway. Loss of functional coupling between mitochondrial protein import and the neuroprotective degradation of dysfunctional mitochondria may therefore be a primary pathogenic mechanism in autosomal recessive PD.     

PINK1 Defect Causes Mitochondrial Dysfunction,Proteasomal Deficit and α-Synuclein Aggregation in Cell Culture Models of Parkinson's Disease

Mutations in PTEN induced kinase 1 (PINK1), a mitochondrial Ser/Thr kinase, cause an autosomal recessive form of Parkinson''s disease (PD), PARK6. Here, we report that PINK1 exists as a dimer in mitochondrial protein complexes that co-migrate with respiratory chain complexes in sucrose gradients. PARK6 related mutations do not affect this dimerization and its associated complexes. Using in vitro cell culture systems, we found that mutant PINK1 or PINK1 knock-down caused deficits in mitochondrial respiration and ATP synthesis. Furthermore, proteasome function is impaired with a loss of PINK1. Importantly, these deficits are accompanied by increased α-synclein aggregation. Our results indicate that it will be important to delineate the relationship between mitochondrial functional deficits, proteasome dysfunction and α-synclein aggregation.     

PINK1 as a molecular checkpoint in the maintenance of mitochondrial function and integrity

Parkinson's disease (PD), the most prevalent neurodegenerative movement disorder, is characterized by an age-dependent selective loss of dopaminergic (DA) neurons. Although most PD cases are sporadic, more than 20 responsible genes in familial cases were identified recently. Genetic studies using Drosophila models demonstrate that PINK1, a mitochondrial kinase encoded by a PD-linked gene PINK1, is critical for maintaining mitochondrial function and integrity. This suggests that mitochondrial dysfunction is the main cause of PD pathogenesis. Further genetic and cell biological studies revealed that PINK1 recruits Parkin, an E3 ubiquitin ligase encoded by another PD-linked gene parkin, to mitochondria and regulates the mitochondrial remodeling process via the Parkin-mediated ubiquitination of various mitochondrial proteins. PINK1 also directly phosphorylates the mitochondrial proteins Miro and TRAP1, subsequently inhibiting mitochondrial transport and mitochondrial oxidative damage, respectively. Moreover, recent Drosophila genetic analyses demonstrate that the neuroprotective molecules Sir2 and FOXO specifically complement mitochondrial dysfunction and DA neuron loss in PINK1 null mutants, suggesting that Sir2 and FOXO protect mitochondria and DA neurons downstream of PINK1. Collectively, these recent results suggest that PINK1 plays multiple roles in mitochondrial quality control by regulating its mitochondrial, cytosolic, and nuclear targets.     

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.     

The mitochondrial kinase PINK1, stress response and Parkinson’s disease

Mitochondrial dysfunction is well documented in presymptomatic brain tissue with Parkinson’s disease (PD). Identification of the autosomal recessive variant PARK6 caused by loss-of-function mutations in the mitochondrial kinase PINK1 provides an opportunity to dissect pathogenesis. Although PARK6 shows clinical differences to PD, the induction of alpha-synuclein “Lewy” pathology by PINK1-deficiency proves that mitochondrial pathomechanisms are relevant for old-age PD. Mitochondrial dysfunction is induced by PINK1 deficiency even in peripheral tissues unaffected by disease, consistent with the ubiquitous expression of PINK1. It remains unclear whether this dysfunction is due to PINK1-mediated phosphorylation of proteins inside or outside mitochondria. Although PINK1 deficiency affects the mitochondrial fission/fusion balance, cell stress is required in mammals to alter mitochondrial dynamics and provoke apoptosis. Clearance of damaged mitochondria depends on pathways including PINK1 and Parkin and is critical for postmitotic neurons with high energy demand and cumulative stress, providing a mechanistic concept for the tissue specificity of disease.     

Absence of nigral degeneration in aged parkin/DJ-1/PINK1 triple knockout mice

Recessively inherited loss-of-function mutations in the parkin , DJ-1 , or PINK1 gene are linked to familial cases of early-onset Parkinson's diseases (PD), and heterozygous mutations are associated with increased incidence of late-onset PD. We previously reported that single knockout mice lacking Parkin, DJ-1, or PINK1 exhibited no nigral degeneration, even though evoked dopamine release from nigrostriatal terminals was reduced and striatal synaptic plasticity was impaired. In this study, we tested whether inactivation of all three recessive PD genes, each of which was required for nigral neuron survival in the aging human brain, resulted in nigral degeneration during the lifespan of mice. Surprisingly, we found that triple knockout mice lacking Parkin, DJ-1, and PINK1 have normal morphology and numbers of dopaminergic and noradrenergic neurons in the substantia nigra and locus coeruleus, respectively, at the ages of 3, 16, and 24 months. Interestingly, levels of striatal dopamine in triple knockout mice were normal at 16 months of age but increased at 24 months. These results demonstrate that inactivation of all three recessive PD genes is insufficient to cause significant nigral degeneration within the lifespan of mice, suggesting that these genes may be protective rather than essential for the survival of dopaminergic neurons during the aging process. These findings also support the notion that mammalian Parkin and PINK1 may function in the same genetic pathway as in Drosophila .     

Imaging mitophagy in the fruit fly

Loss-of-function mutations in the genes encoding PRKN/parkin and PINK1 cause autosomal recessive Parkinson disease (PD). Seminal work in Drosophila revealed that loss of park/parkin and Pink1 causes prominent mitochondrial pathology in flight muscle and, to a lesser extent, in dopaminergic neurons. Subsequent studies in cultured mammalian cells discovered a crucial role for PRKN/PARK2 and PINK1 in selective macroautophagic removal of mitochondria (mitophagy). However, direct evidence for the existence of a PINK1-PRKN/PARK2-mediated mitophagy pathway in vivo is still scarce. Recently, we engineered Drosophila that express the mitophagy reporter mt-Keima. We demonstrated that mitophagy occurs in flight muscle cells and dopaminergic neurons in vivo and increases with aging. Moreover, this age-dependent rise depends on park and Pink1. Our data also suggested that some aspects of the mitochondrial phenotype of park- and Pink1-deficient flies are independent of the mitophagy defect, and that park and Pink1 may have multiple functions in the regulation of the integrity of these organelles. Here, we discuss implications of these findings as well as possible future applications of the mt-Keima fly model.     

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.     

Alpha-Synuclein and Mitochondrial Dysfunction in Parkinson’s Disease

Parkinson’s disease (PD) is one of the most common neurodegenerative diseases. The development of pathology is associated with the loss of dopaminergic neurons, mainly in substantia nigra pars compacta. Dopamine deficiency causes a whole range of severe motor symptoms, including bradykinesia, postural instability, muscle rigidity, and tremor. Studies have shown the primary role of the alpha-synuclein protein in this neurodegenerative disease. A large amount of data indicates different mechanisms of the toxic effect of alpha-synuclein. The process of neurodegeneration in PD is the result of significant disturbances in mitochondrial functions and/or genetic mutations. The number of mutated genes in hereditary and sporadic forms of Parkinson’s disease includes genes encoding PINK1 and Parkin, which are the main participants in the mitochondrial “quality control” system. The earliest biochemical hallmarks of the disease are disturbances of the mitochondrial interaction with endoplasmic reticulum, mitochondrial dynamics, Ca²⁺ homeostasis, and an increase in the level of mitochondrial reactive oxygen species. All these factors exert damaging effects on dopaminergic neurons.     

Genome-wide RNAi screen identifies ATPase inhibitory factor 1 (ATPIF1) as essential for PARK2 recruitment and mitophagy

Mitochondrial dysfunction is a hallmark of aging and numerous human diseases, including Parkinson disease (PD). Multiple homeostatic mechanisms exist to ensure mitochondrial integrity, including the selective autophagic program mitophagy, that is activated during starvation or in response to mitochondrial dysfunction. Following prolonged loss of potential across the inner mitochondrial membrane (ΔΨ), PTEN-induced putative kinase 1 (PINK1) and the E3-ubiquitin ligase PARK2 work in the same pathway to trigger mitophagy of dysfunctional mitochondria. Mutations in PINK1 and PARK2, as well as PARK7/DJ-1, underlie autosomal recessive Parkinsonism and impair mitochondrial function and morphology. In a genome-wide RNAi screen searching for genes that are required for PARK2 translocation to the mitochondria, we identified ATPase inhibitory factor 1 (ATPIF1/IF1) as essential for PARK2 recruitment and mitophagy in cultured cells. During uncoupling, ATPIF1 promotes collapse of ΔΨ and activation of the PINK-PARK2 mitophagy pathway by blocking the ATPase activity of the F₁-F_o ATP synthase. Restoration of ATPIF1 in Rho0 cells, which lack mtDNA and a functional electron transport chain, lowers ΔΨ and triggers PARK2 recruitment. Our findings identified ATPIF1 and the ATP synthase as novel components of the PINK1-PARK2 mitophagy pathway and provide genetic evidence that loss of ΔΨ is an essential trigger for mitophagy.     

Mitochondrial Alterations in PINK1 Deficient Cells Are Influenced by Calcineurin-Dependent Dephosphorylation of Dynamin-Related Protein 1

PTEN-induced novel kinase 1 (PINK1) mutations are associated with autosomal recessive parkinsonism. Previous studies have shown that PINK1 influences both mitochondrial function and morphology although it is not clearly established which of these are primary events and which are secondary. Here, we describe a novel mechanism linking mitochondrial dysfunction and alterations in mitochondrial morphology related to PINK1. Cell lines were generated by stably transducing human dopaminergic M17 cells with lentiviral constructs that increased or knocked down PINK1. As in previous studies, PINK1 deficient cells have lower mitochondrial membrane potential and are more sensitive to the toxic effects of mitochondrial complex I inhibitors. We also show that wild-type PINK1, but not recessive mutant or kinase dead versions, protects against rotenone-induced mitochondrial fragmentation whereas PINK1 deficient cells show lower mitochondrial connectivity. Expression of dynamin-related protein 1 (Drp1) exaggerates PINK1 deficiency phenotypes and Drp1 RNAi rescues them. We also show that Drp1 is dephosphorylated in PINK1 deficient cells due to activation of the calcium-dependent phosphatase calcineurin. Accordingly, the calcineurin inhibitor FK506 blocks both Drp1 dephosphorylation and loss of mitochondrial integrity in PINK1 deficient cells but does not fully rescue mitochondrial membrane potential. We propose that alterations in mitochondrial connectivity in this system are secondary to functional effects on mitochondrial membrane potential.