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Mitochondrial Protein Kinase A (PKA) and PTEN-induced kinase 1 (PINK1), which is linked to Parkinson's disease, are two neuroprotective serine/threonine kinases that regulate dendrite remodeling, and mitochondrial function. We have previously shown that PINK1 regulates dendrite morphology by enhancing PKA activity. Here, we show the molecular mechanisms by which PINK1 and PKA in the mitochondrion interact to regulate dendrite remodeling, mitochondrial morphology, content, and trafficking in dendrites. PINK1-deficient cortical neurons exhibit impaired mitochondrial trafficking, reduced mitochondrial content, fragmented mitochondria, and a reduction in dendrite outgrowth compared to wild-type neurons. Transient expression of wild-type, but not a PKA-binding deficient mutant of the PKA-mitochondrial scaffold dual-specificity A Kinase Anchoring Protein 1 (D-AKAP1), restores mitochondrial trafficking, morphology, and content in dendrites of PINK1-deficient cortical neurons suggesting that recruiting PKA to the mitochondrion reverses mitochondrial pathology in dendrites induced by loss of PINK1. Mechanistically, full-length and cleaved forms of PINK1 increase the binding of the regulatory subunit β of PKA (PKA/RIIβ) to D-AKAP1 to enhance the autocatalytic-mediated phosphorylation of PKA/RIIβ and PKA activity. D-AKAP1/PKA governs mitochondrial trafficking in dendrites via the Miro-2/TRAK2 complex and by increasing the phosphorylation of Miro-2. Our study identifies a new role of D-AKAP1 in regulating mitochondrial trafficking through Miro-2, and supports a model in which PINK1 and mitochondrial PKA participate in a similar neuroprotective signaling pathway to maintain dendrite connectivity. This article is protected by copyright. All rights reserved.

Parkinson's disease (PD) is one of the most common neurodegenerative disorders, affecting 1-1.5% of the total population. While progress has been made in understanding the neurodegenerative mechanisms that lead to cell death in late stages of PD, mechanisms for early, causal pathogenic events are still elusive. Recent developments in PD genetics increasingly point at endolysosomal (E-L) system dysfunction as the early pathomechanism and key pathway affected in PD. Clathrin-mediated synaptic endocytosis, an integral part of the neuronal E-L system, is probably the main early target as evident in auxilin, RME-8, and synaptojanin-1 mutations that cause PD. Autophagy, another important pathway in the E-L system, is crucial in maintaining proteostasis and a healthy mitochondrial pool, especially in neurons considering their inability to divide and requirement to function an entire life-time. PINK1 and Parkin mutations severely perturb autophagy of dysfunctional mitochondria (mitophagy), both in the cell body and synaptic terminals of dopaminergic neurons, leading to PD. Endolysosomal sorting and trafficking is also crucial, which is complex in multi-compartmentalized neurons. VPS35 and VPS13C mutations noted in PD target these mechanisms. Mutations in GBA comprise the most common risk factor for PD and initiate pathology by compromising lysosomal function. This is also the case for ATP13A2 mutations. Interestingly, α-synuclein and LRRK2, key proteins involved in PD, function in different steps of the E-L pathway and target their components to induce disease pathogenesis. In this review, we discuss these E-L system genes that are linked to PD and how their dysfunction results in PD pathogenesis. This article is part of the Special Issue "Synuclein".

Parkinson's disease (PD) is one of the most prevalent neurodegenerative disorders, characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta. PD mostly occurs sporadically and its cause remains unknown, nevertheless the discovery of familiar forms of PD, characterized by mutations of genes encoding proteins associated with mitochondria homeostasis, suggests a strong implication of the mitochondrial quality control system in PD. We investigated the effect of dopamine cytosolic accumulation in undifferentiated SH-SY5Y cells, an in vitro model widely used to reproduce impairment of dopamine homeostasis, an early step in PD pathogenesis. A strong depolarization of the mitochondrial membrane was observed after dopamine exposure. Nevertheless, mitochondrial network resulted to assume a peculiar morphology with a distinct pattern of OPA1 and MFN1, key regulators of mitochondrial dynamics. Moreover, selective elimination of dysfunctional mitochondria did not take place, suggesting an impairment of the mitophagic machinery induced by dopamine. Indeed, PINK1 did not accumulate on the outer mitochondrial membrane, nor was parkin recruited to depolarized mitochondria. Altogether, our results indicate that an improper handling of dysfunctional mitochondria may be a leading event in PD pathogenesis. Impaired dopamine (DA) homeostasis and oxidative stress play a key role in the pathogenesis of Parkinson's disease. Free cytosolic dopamine undergoes spontaneous oxidation and generates semiquinonic and quinonic species (DAQ) with the concurrent production of reactive oxygen species (ROS). Dopamine dissipates mitochondrial potential (Δψm) with a peculiar alteration of the mitochondrial network. However, PINK1-dependent mitophagy is not activated by dopamine toxicity and dysfunctional mitochondria accumulate inside the cell.

Impairment of the autophagy–lysosome pathway is implicated with the changes in α‐synuclein and mitochondrial dysfunction observed in Parkinson's disease (PD). Damaged mitochondria accumulate PINK1, which then recruits parkin, resulting in ubiquitination of mitochondrial proteins. These can then be bound by the autophagic proteins p62/SQSTM1 and LC3, resulting in degradation of mitochondria by mitophagy. Mutations in PINK1 and parkin genes are a cause of familial PD. We found a significant increase in the expression of p62/SQSTM1 mRNA and protein following mitophagy induction in human neuroblastoma SH‐SY5Y cells. p62 protein not only accumulated on mitochondria, but was also greatly increased in the cytosol. Increased p62/SQSMT1 expression was prevented in PINK1 knock‐down cells, suggesting increased p62 expression was a consequence of mitophagy induction. The transcription factors Nrf2 and TFEB, which play roles in mitochondrial and lysosomal biogenesis, respectively, can regulate p62/SQSMT1. We report that both Nrf2 and TFEB translocate to the nucleus following mitophagy induction and that the increase in p62 mRNA levels was significantly impaired in cells with Nrf2 or TFEB knockdown. TFEB translocation also increased expression of itself and lysosomal proteins such as glucocerebrosidase and cathepsin D following mitophagy induction. We also report that cells with increased TFEB protein have significantly higher PGC‐1α mRNA levels, a regulator of mitochondrial biogenesis, resulting in increased mitochondrial content. Our data suggests that TFEB is activated following mitophagy to maintain autophagy–lysosome pathway and mitochondrial biogenesis. Therefore, strategies to increase TFEB may improve both the clearance of α‐synuclein and mitochondrial dysfunction in PD. Damaged mitochondria are degraded by the autophagy–lysosome pathway and is termed mitophagy. Following mitophagy induction, the transcription factors Nrf2 and TFEB translocate to the nucleus, inducing the transcription of genes encoding for autophagic proteins such as p62, as well as lysosomal and mitochondrial proteins. We propose that these events maintain autophagic flux, replenish lysosomes and replace mitochondria.

Contributions of damaged mitochondria to neuropathologies have stimulated interest in mitophagy. We investigated triggers of neuronal mitophagy by disruption of mitochondrial energy metabolism in primary neurons. Mitophagy was examined in cultured murine cerebellar granule cells after inhibition of mitochondrial respiratory chain by drugs rotenone, 3-nitropropionic acid, antimycin A, and potassium cyanide, targeting complexes I, II, III, and IV, respectively. Inhibitor concentrations producing slow cellular demise were determined from analyses of cellular viability, morphology of neuritic damage, plasma membrane permeability, and oxidative phosphorylation. Live cell imaging of dissipation of mitochondrial membrane potential (ΔΨm ) by drugs targeting mitochondrial complexes was referenced to complete depolarization by carbonyl cyanide m-chlorophenyl hydrazone. While inhibition of complexes I, III and IV effected rapid dissipation of ΔΨm , inhibition of complex II using 3-nitropropionic acid led to minimal depolarization of mitochondria. Nonetheless, all respiratory chain inhibitors triggered mitophagy as indicated by increased aggregation of mitochondrially localized PINK1. Mitophagy was further analyzed using a dual fluorescent protein biosensor reporting mitochondrial relocation to acidic lysosomal environment. Significant acidification of mitochondria was observed in neurons treated with rotenone or 3-nitropropionic acid, revealing mitophagy at distal processes. Neurons treated with antimycin A or cyanide failed to show mitochondrial acidification. Minor dissipation of ΔΨm by 3-nitropropionic acid coupled with vigorous triggering of mitophagy suggested depolarization of mitochondria is not a necessary condition to trigger mitophagy. Moreover, weak elicitation of mitophagy by antimycin A, subsequent to loss of ΔΨm , suggested that mitochondrial depolarization is not a sufficient condition for triggering robust neuronal mitophagy. Our findings provide new insight into complexities of mitophagic clearance of neuronal mitochondria.

Iron dyshomeostasis and mitochondrial impairments are both vitally important for the progression of many neurodegenerative diseases, including Parkinson's disease and Alzheimer's disease. Nevertheless, how these two pathological phenomena are linked with one another remains unclear, especially in neurons. To address the question, a model of iron overload was established with exposure of rat primary cortical neurons to excessive iron. We first verified that iron overload resulted in a decrease in adenosine triphosphate (ATP) production in neurons. Meanwhile, the release of mitochondrial cytochrome c was significantly increased after iron overload and consequently triggered an apoptosis signal, as revealed by Caspase 3 cleavage. To explore the potential underlying molecular mechanisms, an unlabeled quantitative proteomics approach was applied to primary neurons. Gene Ontology enrichment analysis revealed that 58 mitochondria-associated proteins were significantly altered, including three subunits of mitochondrial complex I and optic atrophy 1(OPA1). Increased NADH-ubiquinone oxidoreductase 75 kDa subunit and decreased NADH-ubiquinone oxidoreductase subunit A10 levels were further validated by a western blot, and more importantly, complex I activity markedly declined. Iron-induced down-regulation on the OPA1 level was also validated by a western blot, which was not reversed by the anti-oxidant but was reversed by the iron chelator. Moreover, an OPA1-associated key downstream effect, mitochondrial fragmentation, was found to be aggravated in neurons exposed to excessive iron, which is consistent with the down-regulation of OPA1. Furthermore, the protein level of PTEN-induced putative kinase 1, an important protein closely related to complex I activity and mitochondrial fragmentation, also significantly declined in neurons by iron overload. Thus, our findings may shed new light on the linkage between iron toxicity and mitochondrial impairments, such as energy supply deficiency and mitochondrial fragmentation, and further expand the toxic repertoire of iron in the central nerve system. Cover Image for this issue: doi: 10.1111/jnc.14205.

Loss of function mutations in the PTEN-induced putative kinase 1 (Pink1) gene have been linked with an autosomal recessive familial form of early onset Parkinson's disease (PD). However, the underlying mechanism(s) responsible for degeneration remains elusive. Presently, using co-immunoprecipitation in HEK (Human embryonic kidney) 293 cells, we show that Pink1 endogenously interacts with FK506-binding protein 51 (FKBP51 or FKBP5), FKBP5 and directly phosphorylates FKBP5 at Serine in an in vitro kinase assay. Both FKBP5 and Pink1 have been previously associated with protein kinase B (AKT) regulation. We provide evidence using primary cortical cultured neurons from Pink1-deficient mice that Pink1 increases AKT phosphorylation at Serine 473 (Ser473) challenged by 1-methyl-4-phenylpyridinium (MPP+ ) and that over-expression of FKBP5 using an adeno-associated virus delivery system negatively regulates AKT phosphorylation at Ser473 in murine-cultured cortical neurons. Interestingly, FKBP5 over-expression promotes death in response to MPP+ in the absence of Pink1. Conversely, shRNA-mediated knockdown of FKBP5 in cultured cortical neurons is protective and this effect is reversed with inhibition of AKT signaling. In addition, shRNA down-regulation of PH domain leucine-rich repeat protein phosphatase (PHLPP) in Pink1 WT neurons increases neuronal survival, while down-regulation of PHLPP in Pink1 KO rescues neuronal death in response to MPP+ . Finally, using co-immunoprecipitation, we show that FKBP5 interacts with the kinase AKT and phosphatase PHLPP. This interaction is increased in the absence of Pink1, both in Mouse Embryonic Fibroblasts (MEF) and in mouse brain tissue. Expression of kinase dead Pink1 (K219M) enhances FKBP5 interaction with both AKT and PHLPP. Overall, our results suggest a testable model by which Pink1 could regulate AKT through phosphorylation of FKBP5 and interaction of AKT with PHLPP. Our results suggest a potential mechanism by which PINK1-FKBP5 pathway contributes to neuronal death in PD. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

Drosophila melanogaster has contributed significantly to the understanding of disease mechanisms in Parkinson's disease (PD) as it is one of the very few PD model organisms that allow the study of age-dependent behavioral defects, physiology and histology, and genetic interactions among different PD-related genes. However, there have been contradictory results from a number of recent reports regarding the loss of dopaminergic neurons in different PD fly models. In an attempt to re-evaluate and clarify this issue, we have examined three different genetic (α-synuclein, Pink1, parkin) and two toxin-based (rotenone and paraquat) models of the disease for neuronal cell loss. Our results showed no dopaminergic neuronal loss in all models tested. Despite this surprising result, we found additional phenotypes showing the dysfunctional status of the dopaminergic neurons in most of the models analyzed. A common feature found in most models is a quantifiable decrease in the fluorescence of a green-fluorescent protein reporter gene in dopaminergic neurons that correlates well with other phenotypes found for these models and can be reliably used as a hallmark of the neurodegenerative process when modeling diseases affecting the dopaminergic system in Drosophila. Analyzing three genetic and two toxin-based Drosophila models of Parkinson's disease (PD) through green fluorescent protein reporter and α-tyrosine hydroxylase staining, we have found the number of dopaminergic neurons to remain unchanged. Despite the lack of neuronal loss, we have detected a remarkable decrease in a reporter green-fluorescent protein (GFP) signal in dopaminergic neurons, suggesting an abnormal neuronal status that correlates with the phenotypes associated with those PD fly models.

The subcellular compartmentalization of kinase activity allows for regulation of distinct cellular processes involved in cell differentiation or survival. The PTEN-induced kinase 1 (PINK1), which is linked to Parkinson's disease, is a neuroprotective kinase localized to cytosolic and mitochondrial compartments. While mitochondrial targeting of PINK1 is important for its activities regulating mitochondrial homeostasis, the physiological role of the cytosolic pool of PINK1 remains unknown. Here, we demonstrate a novel role for cytosolic PINK1 in neuronal differentiation/neurite maintenance. Over-expression of wild-type PINK1, but not a catalytically inactive form of PINK1(K219M), promoted neurite outgrowth in SH-SY5Y cells and increased dendritic lengths in primary cortical and midbrain dopaminergic neurons. To identify the subcellular pools of PINK1 involved in promoting neurite outgrowth, we transiently transfected cells with PINK1 constructs designed to target PINK1 to the outer mitochondrial membrane (OMM-PINK1) or restrict PINK1 to the cytosol (ΔN111-PINK1). Both constructs blocked cell death associated with loss of endogenous PINK1. However, transient expression of ΔN111-PINK1, but not of OMM-PINK1 or ΔN111-PINK1(K219M), promoted dendrite outgrowth in primary neurons, and rescued the decreased dendritic arborization of PINK1-deficient neurons. Mechanistically, the cytosolic pool of PINK1 regulated neurite morphology through enhanced anterograde transport of dendritic mitochondria and amplification of protein kinase A-related signaling pathways. Our data support a novel role for PINK1 in regulating dendritic morphogenesis.

Parkinson's disease (PD) is the second most common neurodegenerative disease. About 2% of the population above the age of 60 is affected by the disease. The pathological hallmarks of the disease include the loss of dopaminergic neurons in the substantia nigra and the presence of Lewy bodies that are made of α-synuclein. Several theories have been suggested for the pathogenesis of PD, of which mitochondrial dysfunction plays a pivotal role in both sporadic and familial forms of the disease. Dysfunction of the mitochondria that is caused by bioenergetic defects, mutations in mitochondrial DNA, nuclear DNA gene mutations linked to mitochondria, and changes in dynamics of the mitochondria such fusion or fission, changes in size and morphology, alterations in trafficking or transport, altered movement of mitochondria, impairment of transcription, and the presence of mutated proteins associated with mitochondria are implicated in PD. In this review, we provide a detailed overview of the mechanisms that can cause mitochondrial dysfunction in PD. We bring to the forefront, new signaling pathways such as the retromer-trafficking pathway and its implication in the disease and also provide a brief overview of therapeutic strategies to improve mitochondrial defects in PD. Bioenergetic defects, mutations in mitochondrial DNA, nuclear DNA gene mutations, alterations in mitochondrial dynamics, alterations in trafficking/transport and mitochondrial movement, abnormal size and morphology, impairment of transcription and the presence of mutated proteins associated with mitochondria are implicated in PD. In this review, we focus on the mechanisms underlying mitochondrial dysfunction in PD and bring to the forefront new signaling pathways that may be involved in PD. We also provide an overview of therapeutic strategies to improve mitochondrial defects in PD. This article is part of a special issue on Parkinson disease.

Mutations in the genes encoding the mitochondrial kinase PINK1 and the E3 ubiquitin ligase Parkin cause autosomal recessive Parkinson's disease (PD). Pioneering work in Drosophila melanogaster revealed that the loss of PINK1 or Parkin function causes similar phenotypes including dysfunctional mitochondria. Further research showed that PINK1 can act upstream of Parkin in a mitochondrial quality control pathway to induce removal of damaged mitochondria in a process called mitophagy. Albeit the PINK1/Parkin-induced mitophagy pathway is well established and has recently been elucidated in great detail, its pathophysiological relevance is being debated. Mounting evidence indicates that PINK1 has additional functions, for example, in regulating complex I activity and maintaining neuronal viability in response to stress. Here, we discuss mitophagy-dependent and -independent functions of PINK1 and their possible role in PD pathogenesis. Mutations in the PINK1 gene, encoding a mitochondrial kinase, are associated with autosomal recessive Parkinson's disease. In this review, we summarize and discuss the functional roles of PINK1 in maintaining mitochondrial integrity, eliminating damaged mitochondria, and promoting cell survival. This article is part of a special issue on Parkinson disease.

J. Neurochem. (2012) 122, 196–207. Abstract Many genes associated with familial Parkinson’s disease contribute to mitochondrial morphology and function. Some of these genes, for example, Pink1 and Parkin, are part of a common pathway. The presenilin-associated rhomboid-like (PARL) gene was recently linked to familial Parkinson’s disease. The PARL gene product is found in the inner mitochondrial membrane and cleaves the optic atrophy 1 protein, involved in mitochondrial morphology and apoptosis. In Drosophila, the PARL-related rhomboid-7 gene acts upstream of pink1 and parkin. However, such a genetic relationship is still unknown in vertebrates. Here, we show that the zebrafish genome comprises two parl paralogs: parla and parlb. Morpholino-mediated loss of parla and/or parlb function resulted in mild neurodegeneration, as evidenced by a lower density of dopaminergic neurons. Patterning of dopaminergic neurons was also perturbed in the ventral diencephalon. Morphants exhibited extensive cell death throughout the entire body as well as increased larval mortality. The morphant phenotype could be rescued by injection of human PARL mRNA, but not catalytically inactive PARL, suggesting functional conservation between the human and zebrafish proteins. More importantly, the zebrafish pink1 mRNA as well as the human PINK1 mRNA, but not kinase-dead nor Parkinson’s disease-linked mutant PINK1 mRNA, also rescued the morphant phenotype, providing evidence that Parl genes may function upstream of Pink1, as part of a conserved pathway in vertebrates.

Mutations in the parkin gene cause early-onset, autosomal recessive Parkinson's disease. Parkin functions as an E3 ubiquitin ligase to mediate the covalent attachment of ubiquitin monomers or linked chains to protein substrates. Substrate ubiquitination can target proteins for proteasomal degradation or can mediate a number of non-degradative functions. Parkin has been shown to preserve mitochondrial integrity in a number of experimental systems through the regulation of mitochondrial fission. Upon mitochondrial damage, parkin translocates to mitochondria to mediate their selective elimination by autophagic degradation. The mechanism underlying this process remains unclear. Here, we demonstrate that parkin interacts with and selectively mediates the atypical poly-ubiquitination of the mitochondrial fusion factor, mitofusin 1, leading to its enhanced turnover by proteasomal degradation. Our data supports a model whereby the translocation of parkin to damaged mitochondria induces the degradation of mitofusins leading to impaired mitochondrial fusion. This process may serve to selectively isolate damaged mitochondria for their removal by autophagy.

Intramembrane proteolysis is a conserved mechanism that regulates a variety of cellular processes ranging from transcription control to signaling. In mitochondria, the inner membrane rhomboid protease PARL has been implicated in the control of life span and apoptosis by a so far uncharacterized mechanism. Here, we show that PARL cleaves human Pink1, which is implicated in Parkinson's disease, within its conserved membrane anchor. Mature Pink1 is then free to be released into the cytosol or the mitochondrial intermembrane space. Upon depolarization of the mitochondrial membrane potential, the canonical import of Pink1 and PARL-catalyzed processing is blocked, leading to accumulation of the Pink1 precursor. As targeting of this precursor to the outer mitochondrial membrane has been shown to trigger mitophagy, we suggest that the PARL-catalyzed removal of the Pink1 signal sequence in the canonical import pathway acts as a cellular checkpoint for mitochondrial integrity. Furthermore, we show that two Parkinson's disease-causing mutations decrease the processing of Pink1 by PARL, with attendant implications for pathogenesis.

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.

Parkinson's disease (PD) is characterized by accumulation of alpha-synuclein (alpha-syn) and degeneration of neuronal populations in cortical and subcortical regions. Mitochondrial dysfunction has been considered a potential unifying factor in the pathogenesis of the disease. Mutations in genes linked to familial forms of PD, including SNCA encoding alpha-syn and Pten-induced putative kinase 1 (PINK1), have been shown to disrupt mitochondrial activity. We investigated the mechanisms through which mutant Pink1 might disrupt mitochondrial function in neuronal cells with alpha-syn accumulation. For this purpose, a neuronal cell model of PD was infected with virally-delivered Pink1, and was analyzed for cell survival, mitochondrial activity and calcium flux. Mitochondrial morphology was analyzed by confocal and electron microscopy. These studies showed that mutant (W437X) but not wildtype Pink1 exacerbated the alterations in mitochondrial function promoted by mutant (A53T) alpha-syn. This effect was associated with increased intracellular calcium levels. Co-expression of both mutant Pink1 and alpha-syn led to alterations in mitochondrial structure and neurite outgrowth that were partially ameliorated by treatment with cyclosporine A, and completely restored by treatment with the mitochondrial calcium influx blocker Ruthenium Red, but not with other cellular calcium flux blockers. Our data suggest a role for mitochondrial calcium influx in the mechanisms of mitochondrial and neuronal dysfunction in PD. Moreover, these studies support an important function for Pink1 in regulating mitochondrial activity under stress conditions.

Mutations found in PTEN-induced putative kinase 1 (PINK1), a putative mitochondrial serine/threonine kinase of unknown function, have been linked to autosomal recessive Parkinson’s disease. It is suggested that mutations can cause a loss of PINK1 kinase activity and eventually lead to mitochondrial dysfunction. In this report, we examined the subcellular localization of PINK1 and the dynamic kinetics of PINK1 processing and degradation. We also identified cytosolic chaperone heat-shock protein 90 (Hsp90) as an interacting protein of PINK1 by PINK1 co-immunoprecipitation. Immunofluorescence of PINK1 protein and mitochondrial isolation show that the precursor form of PINK1 translocates to the mitochondria and is processed into two cleaved forms of PINK1, which in turn localize more to the cytosolic than mitochondrial fraction. The cleavage does not occur and the uncleaved precursor stays associated with the mitochondria when the mitochondrial membrane potential is disrupted. Metabolic labeling analyses show that the PINK1 processing is rapid and the levels of cleaved forms are tightly regulated. Furthermore, cleaved forms of PINK1 are stabilized by Hsp90 interaction as the loss of Hsp90 activity decreases PINK1 level after mitochondrial processing. Lastly, we also find that cleaved forms of PINK1 are degraded by the proteasome, which is uncommon for mitochondrial proteins. Our findings support a dual subcellular localization, implying that PINK1 can reside in the mitochondria and the cytosol. This raises intriguing functional roles that bridge these two cellular compartments.

Following our identification of PTEN-induced putative kinase 1 (PINK1) gene mutations in PARK6-linked Parkinson's disease (PD), we have recently reported that PINK1 protein localizes to Lewy bodies (LBs) in PD brains. We have used a cellular model system of LBs, namely induction of aggresomes, to determine how a mitochondrial protein, such as PINK1, can localize to aggregates. Using specific polyclonal antibodies, we firstly demonstrated that human PINK1 was cleaved and localized to mitochondria. We demonstrated that, on proteasome inhibition with MG-132, PINK1 and other mitochondrial proteins localized to aggresomes. Ultrastructural studies revealed that the mechanism was linked to the recruitment of intact mitochondria to the aggresome. Fractionation studies of lysates showed that PINK1 cleavage was enhanced by proteasomal stress in vitro and correlated with increased expression of the processed PINK1 protein in PD brain. These observations provide valuable insights into the mechanisms of LB formation in PD that should lead to a better understanding of PD pathogenesis.

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. Mutations in PINK1 are associated with an early-onset form of Parkinson's disease (PD). This study examines changes in the proteome and phosphoproteome of the PINK1 knockout mouse brain. Alterations were noted in several key proteins associated with: increased oxidative stress, aberrant cellular signaling, altered neuronal structure, decreased synaptic plasticity, reduced neurotransmission, diminished proteostasis networks, and altered metabolism. 14-3-3e, 14-3-3 protein epsilon; 3-PGDH, phosphoglycerate dehydrogenase; ALDOA, aldolase A; APT1, acyl-protein thioesterase 1; CaM, calmodulin; CBR3, carbonyl reductase [NADPH] 3; ENO2, gamma-enolase; HPRT, hypoxanthine-guanine phosphoribosyltransferase; HSP70, heat-shock-related 70 kDa protein 2; IDHc, cytoplasmic isocitrate dehydrogenase [NADP+]; MAPK1, mitogen-activated protein kinase 1; MEK1, MAP kinase kinase 1; MDHc, cytoplasmic malate dehydrogenase; NFM, neurofilament medium polypeptide; NSF, N-ethylmaleimide-sensitive fusion protein; PHB, prohibitin; PINK1, PTEN-induced putative kinase 1; PPIaseA, peptidyl-prolyl cis-trans isomerase A; PSA2, proteasome subunit alpha type-2; TK, transketolase; VDAC-2, voltage-dependent anion-selective channel protein 2.

The etiology of Parkinson’s disease (PD) is multifactorial, and the pathogenic pathways involved are complex. Mitochondrial dysfunction has been linked to PD by the discovery of parkinsonism in patients who mistakenly took synthetic meperidine contaminated with the selective mitochondrial toxicant 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) (Langston et al., 1983). The subsequent discovery of PD-linked gene mutations further fueled the long-standing interest in the role of mitochondria in PD. Among these PD-linked genes, identification of mutations in the PTEN-induced putative kinase 1 (pink1) provided the first genetic evidence to support the involvement of mitochondrial dysfunction in PD. PINK1 has a mitochondrion-targeting signal at its N-terminus, and mutations in the pink1 gene have been found in autosomal recessive PD (Valente et al., 2004). Since these discoveries were made, the vital roles of PINK1 in mitochondrial dynamics, mitochondrial function, and mitophagy have been extensively studied. Whereas PINK1 is mainly localized to mitochondria, a fraction of the protein is found in the cytosol (Meissner et al., 2011). After PINK1 is imported to mitochondria, it is cleaved by the mitochondrial intra-membrane protease presenilin-associated rhomboid-like protein (PARL), to release most of the cleaved PINK1 into the cytosol (Meissner et al., 2011). However, under pathological conditions where mitochondrial membrane potential is disrupted, PINK1 remains uncleaved in the mitochondria (Lin and Kang, 2008). Despite the two pools of PINK1, the cytosolic form has been less examined than its mitochondrial counterpart. In this issue of Journal of Neurochemistry, Dagda and colleagues provide substantial evidence that cytosolic rather than mitochondrial PINK1 promotes dendritic outgrowth (Dagda et al., 2013). The authors first examined the effects of cleaved and uncleaved PINK1 on neurite outgrowth in both immortalized cells and primary neurons. In human SH-SY5Y cells and mouse cortical neurons, overexpression of full-length PINK1 caused morphological changes resembling dendritic outgrowth, and this morphological change required PINK1 kinase activity. Upon closer examination, these cells expressed higher levels of dendritic and postsynaptic markers. To tease out whether these changes were induced by mitochondrial or cytosolic PINK1, they generated PINK1 with an outer mitochondrial membrane-targeting sequence (OMM) in the N-terminus (OMM-PINK1) and another without OMM (ΔN111-PINK1) that remained in the cytosolic compartment. Full-length PINK1 exhibits both cytosolic and mitochondrial localization. They observed that full-length and cytosolic PINK1, but not mitochondrial PINK1, promoted dendritic outgrowth. This outgrowth effect was specific to dendrites, as axonal length did not change. It would be informative to determine if cytosolic PINK1 also affects axonal terminals, which were not quantified in this study. In contrast to the effects of PINK1 overexpression, PINK1 deficiency reduced the dendritic length of primary neurons isolated from PINK1-knockout mice. They next examined the effects of cytosolic PINK1 on mitochondrial mobility. Cytosolic but not mitochondrial PINK1 enhanced anterograde mitochondrion movement in dendrites, resulting in increased mitochondrial density in the dendrites. The authors also observed that cytosolic PINK1 prevented SH-SY5Y cells from PINK1 knockdown-induced apoptosis, a cell-protective effect previously reported in an MPTP mouse model (Haque et al., 2008). Like its mitochondrial partner, cytosolic PINK1 has neuroprotective effects. Based on their evidence, Dagda et al. proposed a mechanism by which cytosolic PINK1 promotes dendritic outgrowth. Because PINK1 knockdown selectively blocked neuronal differentiation in cAMP-treated SH-SY5Y cells but not in those treated with retinoic acid (a commonly used compound to differentiate this cell type), the authors reasoned that protein kinase A (PKA) is involved in PINK1’s dendritic outgrowth effect. Their hypothesis is verified by the observation that both PKA activity and nuclear phosphorylated cAMP response element-binding protein (CREB) levels were reduced in PINK1-knockdown cells. Furthermore, PINK1 lost its ability to promote dendritic outgrowth after PKA activity was blocked. Interestingly, Chang and Blackstone reported that activating cAMP-dependent PKA leads to the phosphorylation of dynamin-related protein 1 (Drp1), a key regulator of mitochondrial fission (Chang and Blackstone, 2007). Phosphorylated Drp1 loses its GTPase activity and thus its ability to promote mitochondrial fission. Inhibiting Drp1 activity has been shown to block cytochrome C release and apoptosis and promote the exchange of mitochondrial contents between functional and defective mitochondria, which attenuates their negative impact. PINK1 prevents Drp1 translocation from the cytosol to the mitochondria (Zhao et al., 2013), inhibiting Drp1-promoted mitochondrial fission. Taken together, the evidence indicates that cytosolic PINK1 promotes dendritic outgrowth and contributes to mitochondrial fusion in mammalian cells, likely through PKA. The study by Dagda and colleagues highlights a novel role of cytosolic PINK1 that has not yet received adequate attention as compared to mitochondrial PINK1. It also raises some important issues related to emerging concepts in PD research. One of these is synaptic dysfunction in PD. In recent years, synaptic dysfunction was found to be an early pathology prior to neurodegeneration in different neurological disorders, including PD (Picconi et al., 2012). Thus, restoring synaptic function at both pre- and post-synaptic sites has been suggested as an early therapeutic strategy for PD. In genetic animal studies, PINK1, parkin, DJ-1, α-synuclein, and LRRK2 have all been reported to play roles in striatal pre-synaptic dopamine (DA) release. Relevant to Dagda’s study, acute striatal slices prepared from PINK1-knockout mice exhibit impaired DA release (Kitada et al., 2007). Because striatal DA modulates corticostriatal glutamatergic input at striatal medium spiny neurons, a reduction in striatal DA levels results in reduced activation of post-synaptic D1 and D2 receptors, which in turn impairs long-term potentiation and long-term depression in these animals. Precisely how a loss of PINK1 function would lead to DA release deficits is not completely understood. Mitochondrial function is likely implicated in impaired DA release. Since the first ultrastructural analysis of synapses half a century ago, it is now clear that mitochondria are enriched in the synapses and play a critical role in pre-synaptic functions (Hollenbeck, 2005). For example, they regulate cytosolic Ca2+ levels during neurotransmission and supply energy to synaptic terminals to maintain synaptic membrane potential, ionic gradient restoration following synaptic transmission, and the reloading of synaptic vesicles with neurotransmitters. The correlation between PINK1, mitochondrial function, and synaptic function is another important research topic inspired by Dagda’s study. In summary, combined with evidence from other reports, the study by Dagda et al. has a significant implication regarding the effects of PINK1 on synaptic function. PINK1 is crucial to pre-synaptic DA release because it maintains mitochondrial function, morphology, and integrity. At postsynaptic sites, cytosolic PINK1 maintains dendritic mitochondrial density and promotes dendritic outgrowth. By improving synaptic function at both pre- and post-synaptic levels, PINK1 ensures proper synaptic transmission and signaling. However, additional studies are required to validate these novels roles of PINK1. For example, Dagda et al. performed their study in cultured cells. It is important to extend their findings to an in vivo model. Perhaps by using virus-mediated gene delivery, additional insights will be gained into how mitochondrial and cytosolic PINK1 modulate pre- and post-synaptic activities in the nigrostriatal pathway of an intact model system. In addition, Dagda et al. demonstrated that cytosolic PINK1 enhanced the anterograde transport of mitochondria in the dendrites of cultured mammalian cells (Dagda et al., 2013); however, an opposite effect of PINK1 on mitochondrial mobility was observed in Drosophila larval motor neurons (Liu et al., 2012). Overexpression of Drosophila PINK1 inhibits both the antero- and retrograde transport of mitochondria in motor neuron axons (Liu et al., 2012). Because the effect of PINK1 on axonal mitochondrion transport was not examined or compared with the observations in dendrites (Dagda et al., 2013), it is not known whether PINK1 (or cytosolic PINK1 alone) has differential effects on mitochondrial mobility in axons and dendrites in mammalian neurons under physiological conditions. These interesting questions need to be resolved with future studies.

Our previous study has shown that PTEN-induced novel kinase 1 (PINK1) knocking down significantly induced mitochondrial fragmentation. Although PINK1 is proved to be associated with autosomal recessive parkinsonism and its function in this chronic pathological process is widely studied, its role in acute energy crisis such as ischemic stroke is poorly known. In this study by employing an oxygen-glucose deprivation (OGD) neuronal model, we explored the function of PINK1 in cerebral ischemia. Human PINK1, two PINK1 mutants W437X and K219M, or Pink1 shRNA were transduced before OGD using lentiviral delivery. Our results showed that over-expression of wild-type PINK1 significantly ameliorated OGD induced cell death and energy disturbance including reduced ATP generation and collapse of mitochondrial membrane potential. PINK1 over-expression also reversed OGD increased mitochondrial fragmentation, and suppressed the translocation of the mitochondrial fission protein dynamin-related protein 1 (Drp1) from the cytosol to the mitochondria. Transduction of the mutant PINK1 failed to provide any protective effect, while knockdown of Pink1 significantly increased the severity of OGD-induced neuronal damage. Importantly, inhibition of Drp1 reversed the effects of knocking down Pink1 on neuronal death and ATP production in response to OGD. This study demonstrates that PINK1 prevents ischemic damage in neurons by attenuating mitochondrial translocation of Drp1, which maintains mitochondrial function and inhibits ischemia-induced mitochondrial fission. These novel findings implicate a pivotal role of PINK1 regulated mitochondrial dynamics in the pathology of ischemic stroke. In this study by employing an oxygen-glucose deprivation (OGD) neuronal model, we explored the function of PINK1 in cerebral ischemia. We indicated that PINK1 significantly ameliorated OGD induced cell death and energy disturbance including reduced ATP generation and collapse of mitochondrial membrane potential by attenuating mitochondrial translocation of Drp1, which maintains mitochondrial function and inhibits ischemia-induced mitochondrial fission.

Mutations in PTEN-induced kinase 1 (PINK1) gene cause PARK6 familial Parkinsonism. To decipher the role of PINK1 in pathogenesis of Parkinson's disease (PD), researchers need to identify protein substrates of PINK1 kinase activity that govern neuronal survival, and establish whether aberrant regulation and inactivation of PINK1 contribute to both familial Parkinsonism and idiopathic PD. These studies should take into account the several unique structural and functional features of PINK1. First PINK1 is a rare example of a protein kinase with a predicted mitochondrial-targeting sequence and a possible resident mitochondrial function. Second, bioinformatic analysis reveals unique insert regions within the kinase domain that are potentially involved in regulation of kinase activity, substrate selectivity and stability of PINK1. Third, the C-terminal region contains functional motifs governing kinase activity and substrate selectivity. Fourth, accumulating evidence suggests that PINK1 interacts with other signaling proteins implicated in PD pathogenesis and mitochondrial dysfunction. The most prominent examples are the E3 ubiquitin ligase Parkin, the mitochondrial protease high temperature requirement serine protease 2 and the mitochondrial chaperone tumor necrosis factor receptor-associated protein 1. How PINK1 may regulate these proteins to maintain neuronal survival is unclear. This review describes the unique structural features of PINK1 and their possible roles in governing mitochondrial import, processing, kinase activity, substrate selectivity and stability of PINK1. Based upon the findings of previous studies of PINK1 function in cell lines and animal models, we propose a model on the neuroprotective mechanism of PINK1. This model may serve as a conceptual framework for future investigation into the molecular basis of PD pathogenesis.

Parkinson's disease (PD) is characterized by a unique clinical constellation that includes: slowness, rigidity, gait difficulty, and tremor at rest. Pathological studies have linked this presentation to the loss of midbrain dopamine neurons (Gelb et al. 1999) although other neuronal populations are also targeted in PD. Epidemiological data implicate both genetic and environmental factors in the etiology of the disease. The identification of a series of genes that underlie relatively rare, familial forms of Parkinsonism (a clinical term that encompasses 'sporadic' PD, familial Parkinson's-like forms, as well as other related syndromes) has brought excitement to the field. Three of the mutated familial Parkinsonism (FP) genes: Parkin, DJ-1, and PINK1, typically present with apparent autosomal recessive inheritance and are implicated in mitochondria and oxidative stress-related survival pathways. Two other FP genes: alpha-Synuclein (alphaSyn) and LRRK2, present in an autosomal dominant pattern and are associated with prominent intracellular protein inclusions. A series of recent publications suggest novel pathways that may link the FP genes.