DJ-1 regulation of mitochondrial function and autophagy through oxidative stress

The dysregulation of mitochondrial function has been implicated in the pathogenesis of Parkinson disease. Mutations in the parkin, PINK1 and DJ-1 genes all result in recessive parkinsonism. Although the protein products of these genes have not been fully characterized, it has been established that all three contribute to the maintenance of mitochondrial function. PINK1 and parkin act in a common pathway to regulate the selective autophagic removal of depolarized mitochondria, but the relationship between DJ-1 and PINK1- and/or parkin-mediated effects on mitochondria and autophagy is less clear. We have shown that loss of DJ-1 leads to mitochondrial phenotypes including reduced membrane potential, increased fragmentation and accumulation of autophagic markers. Supplementing DJ-1-deficient cells with glutathione reverses both mitochondrial and autophagic changes suggesting that DJ-1 may act to maintain mitochondrial function during oxidative stress and thereby alter mitochondrial dynamics and autophagy indirectly.     

DJ-1 regulation of mitochondrial function and autophagy through oxidative stress

The dysregulation of mitochondrial function has been implicated in the pathogenesis of Parkinson disease.

Mutations in the parkin, PINK1 and DJ-1 genes all result in recessive parkinsonism. Although the protein products of these genes have not been fully characterized, it has been established that all three contribute to the maintenance of mitochondrial function. PINK1 and parkin act in a common pathway to regulate the selective autophagic removal of depolarized mitochondria, but the relationship between DJ-1 and PINK1- and/or parkin-mediated effects on mitochondria and autophagy is less clear. We have shown that loss of DJ-1 leads to mitochondrial phenotypes including reduced membrane potential, increased fragmentation and accumulation of autophagic markers. Supplementing DJ-1-deficient cells with glutathione reverses both mitochondrial and autophagic changes suggesting that DJ-1 may act to maintain mitochondrial function during oxidative stress and thereby alter mitochondrial dynamics and autophagy indirectly.     

Pink1, Parkin, DJ-1 and mitochondrial dysfunction in Parkinson's disease

Mutations in PARKIN, PTEN-induced kinase 1 (PINK1) and DJ-1 are found in autosomal recessive forms and some sporadic cases of Parkinson's disease. Recent work on these genes underscores the central importance of mitochondrial dysfunction and oxidative stress in Parkinson's disease. In particular, pink1 and parkin loss-of-function mutants in Drosophila show similar phenotypes, and pink1 acts upstream of parkin in a common genetic pathway to regulate mitochondrial function. DJ-1 has a role in oxidative stress protection, but a direct role of DJ-1 in mitochondrial function has not been fully established. Importantly, defects in mitochondrial function have also been identified in patients who carry both PINK1 and PARKIN mutations, and in those who have sporadic Parkinson's disease. Future studies of the biochemical interactions between Pink1 and Parkin, and identification of other components in this pathway, are likely to provide insight into Parkinson's disease pathogenesis, and might identify new therapeutic targets.     

Loss of Parkin or PINK1 Function Increases Drp1-dependent Mitochondrial Fragmentation

Loss-of-function mutations in the parkin gene (PARK2) and PINK1 gene (PARK6) are associated with autosomal recessive parkinsonism. PINK1 deficiency was recently linked to mitochondrial pathology in human cells and Drosophila melanogaster, which can be rescued by parkin, suggesting that both genes play a role in maintaining mitochondrial integrity. Here we demonstrate that an acute down-regulation of parkin in human SH-SY5Y cells severely affects mitochondrial morphology and function, a phenotype comparable with that induced by PINK1 deficiency. Alterations in both mitochondrial morphology and ATP production caused by either parkin or PINK1 loss of function could be rescued by the mitochondrial fusion proteins Mfn2 and OPA1 or by a dominant negative mutant of the fission protein Drp1. Both parkin and PINK1 were able to suppress mitochondrial fragmentation induced by Drp1. Moreover, in Drp1-deficient cells the parkin/PINK1 knockdown phenotype did not occur, indicating that mitochondrial alterations observed in parkin- or PINK1-deficient cells are associated with an increase in mitochondrial fission. Notably, mitochondrial fragmentation is an early phenomenon upon PINK1/parkin silencing that also occurs in primary mouse neurons and Drosophila S2 cells. We propose that the discrepant findings in adult flies can be explained by the time of phenotype analysis and suggest that in mammals different strategies may have evolved to cope with dysfunctional mitochondria.Many lines of evidence suggest that mitochondrial dysfunction plays a central role in the pathogenesis of Parkinson disease, starting from the early observation that the complex I inhibitor 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine induced acute and irreversible parkinsonism in young drug addicts (for review, see Refs. 1–3). In support of a crucial role of mitochondria in Parkinson disease, several Parkinson disease-associated gene products directly or indirectly impinge on mitochondrial integrity (for review, see Refs. 4–6). A clear link between Parkinson disease genes and mitochondria has recently emerged from studies on PINK1 (PTEN-induced putative kinase 1), a mitochondrial serine/threonine kinase, and parkin, a cytosolic E3 ubiquitin ligase. Drosophila parkin null mutants displayed reduced life span, male sterility, and locomotor defects due to apoptotic flight muscle degeneration (7). The earliest manifestation of muscle degeneration and defective spermatogenesis was mitochondrial pathology, exemplified by swollen mitochondria and disintegrated cristae. Remarkably, Drosophila PINK1 null mutants shared marked phenotypic similarities with parkin mutants, and parkin could compensate for the PINK1 loss-of-function phenotype but not vice versa, leading to the conclusion that PINK1 and parkin function in a common genetic pathway with parkin acting downstream of PINK1 (8–10). We recently demonstrated that PINK1 deficiency in cultured human cells causes alterations in mitochondrial morphology, which can be rescued by wild type parkin but not by pathogenic parkin mutants (11). We now present evidence that parkin plays an essential role in maintaining mitochondrial integrity. RNAi³-mediated knockdown of parkin increases mitochondrial fragmentation and decreases cellular ATP production. Notably, mitochondrial fragmentation induced by PINK1/parkin deficiency is observed not only in human neuroblastoma cells but also in primary mouse neurons and insect S2 cells. Alterations in mitochondrial morphology are early manifestations of parkin/PINK1 silencing that are not caused by an increase in apoptosis. The mitochondrial phenotype observed in parkin- or PINK1-deficient cells can morphologically and functionally be rescued by the increased expression of a dominant negative mutant of the fission-promoting protein Drp1. Moreover, manifestation of the PINK1/parkin knockdown phenotype is dependent on Drp1 expression, indicating that an acute loss of parkin or PINK1 function increases mitochondrial fission.     

Formation of parkin aggregates and enhanced PINK1 accumulation during the pathogenesis of Parkinson’s disease

Parkinson’s disease (PD) is a devastating neurodegenerative disease characterized by a distinct set of motor symptoms. Loss-of-function mutations in PTEN-induced kinase 1 (PINK1) or parkin have been linked to early-onset autosomal recessive forms of familial PD. We have recently shown that parkin (an E3 ubiquitin ligase) and PINK1 (a serine/threonine kinase) affect one other’s stability, solubility, and tendency to form cytoprotective aggresomes (Um et al., 2009, [16]). Here we validated the functional relevance of this mutual interaction under pathologic PD conditions, by investigating the changes of expression and solubility of these factors in response to PD-linked toxins. Consistent with our previous cell culture data, exposure of human dopaminergic neuroblastoma SH-SY5Y cells to PD-linked toxins (1-methyl-4-phenylpyridinium ion, 6-hydroxydopamine, or MG132) reduced Nonidet P-40-soluble parkin levels and induced PINK1 accumulation. Consistent with our previous findings from parkin knockout mice, rat models of PD (6-hydroxydopamine-, rotenone-, or MG132-induced PD) were also associated with an increase in soluble and insoluble PINK1 levels as well as enhanced formation of parkin aggregates. These findings suggest that both PINK1 and parkin play important roles in regulating the formation of Lewy bodies during the pathogenesis of sporadic and familial PD.     

Loss of parkin promotes lipid rafts-dependent endocytosis through accumulating caveolin-1: implications for Parkinson’s disease

Background

Parkinson’s disease (PD) is characterized by progressive loss of midbrain dopaminergic neurons, resulting in motor dysfunctions. While most PD is sporadic in nature, a significant subset can be linked to either autosomal dominant or recessive mutations. PARK2, encoding the E3 ubiquitin ligase, parkin, is the most frequently mutated gene in autosomal recessive early onset PD. It has recently been reported that PD-associated gene products such as PINK1, α-synuclein, LRRK2, and DJ-1, as well as parkin associate with lipid rafts, suggesting that the dysfunction of these proteins in lipid rafts may be a causal factor of PD. Therefore here, we examined the relationship between lipid rafts-related proteins and parkin.

Results

We identified caveolin-1 (cav-1), which is one of the major constituents of lipid rafts at the plasma membrane, as a substrate of parkin. Loss of parkin function was found to disrupt the ubiquitination and degradation of cav-1, resulting in elevated cav-1 protein level in cells. Moreover, the total cholesterol level and membrane fluidity was altered by parkin deficiency, causing dysregulation of lipid rafts-dependent endocytosis. Further, cell-to-cell transmission of α-synuclein was facilitated by parkin deficiency.

Conclusions

Our results demonstrate that alterations in lipid rafts by the loss of parkin via cav-1 may be a causal factor of PD, and cav-1 may be a novel therapeutic target for PD.

     

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.     

Enhanced parkin levels favor ER-mitochondria crosstalk and guarantee Ca2 + transfer to sustain cell bioenergetics

Loss-of-function mutations in PINK1 or parkin genes are associated with juvenile-onset autosomal recessive forms of Parkinson disease. Numerous studies have established that PINK1 and parkin participate in a common mitochondrial-quality control pathway, promoting the selective degradation of dysfunctional mitochondria by mitophagy. Upregulation of parkin mRNA and protein levels has been proposed as protective mechanism against mitochondrial and endoplasmic reticulum (ER) stress. To better understand how parkin could exert protective function we considered the possibility that it could modulate the ER–mitochondria inter-organelles cross talk. To verify this hypothesis we investigated the effects of parkin overexpression on ER–mitochondria crosstalk with respect to the regulation of two key cellular parameters: Ca^2 + homeostasis and ATP production. Our results indicate that parkin overexpression in model cells physically and functionally enhanced ER–mitochondria coupling, favored Ca^2 + transfer from the ER to the mitochondria following cells stimulation with an 1,4,5 inositol trisphosphate (InsP₃) generating agonist and increased the agonist-induced ATP production. The overexpression of a parkin mutant lacking the first 79 residues (ΔUbl) failed to enhance the mitochondrial Ca^2 + transients, thus highlighting the importance of the N-terminal ubiquitin like domain for the observed phenotype. siRNA-mediated parkin silencing caused mitochondrial fragmentation, impaired mitochondrial Ca^2 + handling and reduced the ER–mitochondria tethering. These data support a novel role for parkin in the regulation of mitochondrial homeostasis, Ca^2 + signaling and energy metabolism under physiological conditions.     

Silent information regulator 2 (Sir2) and Forkhead box O (FOXO) complement mitochondrial dysfunction and dopaminergic neuron loss in Drosophila PTEN-induced kinase 1 (PINK1) null mutant

PTEN-induced kinase 1 (PINK1), which is associated with early onset Parkinson disease, encodes a serine-threonine kinase that is critical for maintaining mitochondrial function. Moreover, another Parkinson disease-linked gene, parkin, functions downstream of PINK1 in protecting mitochondria and dopaminergic (DA) neuron. In our fly genetic screening, knockdown of Sir2 blocked PINK1 overexpression-induced phenotypes. Consistently, ectopic expression of Sir2 successfully rescued mitochondrial defects in PINK1 null mutants, but unexpectedly, failed in parkin mutants. In further genetic analyses, deletion of FOXO nullified the Sir2-induced mitochondrial restoration in PINK1 null mutants. Moreover, overexpression of FOXO or its downstream target gene such as SOD2 or Thor markedly ameliorated PINK1 loss-of-function defects, suggesting that FOXO mediates the mitochondrial protecting signal induced by Sir2. Consistent with its mitochondria-protecting role, Sir2 expression prevented the DA neuron loss of PINK1 null mutants in a FOXO-dependent manner. Loss of Sir2 or FOXO induced DA neuron degeneration, which is very similar to that of PINK1 null mutants. Furthermore, PINK1 deletion had no deleterious effect on the DA neuron loss in Sir2 or FOXO mutants, supporting the idea that Sir2, FOXO, and PINK1 protect DA neuron in a common pathway. Overall, these results strongly support the role of Sir2 and FOXO in preventing mitochondrial dysfunction and DA neuron loss, further suggesting that Sir2 and FOXO function downstream of PINK1 and independently of Parkin.     

Ubiquitination Increases Parkin Activity to Promote Autophagic α-Synuclein Clearance

Parkinson’s disease (PD) is a movement disorder associated with genetic and age related causes. Although autosomal recessive early onset PD linked to parkin mutations does not exhibit α-Synuclein accumulation, while autosomal dominant and sporadic PD manifest with α-Synuclein inclusions, loss of dopaminergic substantia nigra neurons is a common denominator in PD. Here we show that decreased parkin ubiquitination and loss of parkin stability impair interaction with Beclin-1 and alter α-Synuclein degradation, leading to death of dopaminergic neurons. Tyrosine kinase inhibition increases parkin ubiquitination and interaction with Beclin-1, promoting autophagic α-Synuclein clearance and nigral neuron survival. However, loss of parkin via deletion increases α-Synuclein in the blood compared to the brain, suggesting that functional parkin prevents α-Synuclein release into the blood. These studies demonstrate that parkin ubiquitination affects its protein stability and E3 ligase activity, possibly leading to α-Synuclein sequestration and subsequent clearance.     

Characterization of PINK1 processing, stability, and subcellular localization

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.     

The role of PTEN-induced kinase 1 in mitochondrial dysfunction and dynamics

Mutations in parkin, PTEN-induced kinase 1 (PINK1) and DJ-1 can all cause autosomal recessive forms of Parkinson's disease. Recent data suggest that these recessive parkinsonism-associated genes converge within a single pathogenic pathway whose dysfunction leads to the loss of substantia nigra pars compacta neurons. The major common functional effects of all three genes relate to mitochondrial and oxidative damage, with a possible additional involvement of the ubiquitin proteasome system. This review highlights the role of the mitochondrial kinase, PINK1, in protection against mitochondrial dysfunction and how this might relate to loss of substantia nigra neurons in recessive parkinsonism.     

Tickled PINK1: Mitochondrial homeostasis and autophagy in recessive Parkinsonism

Dysregulation of mitochondrial structure and function has emerged as a central factor in the pathogenesis of Parkinson's disease and related parkinsonian disorders (PD). Toxic and environmental injuries and risk factors perturb mitochondrial complex I function, and gene products linked to familial PD often affect mitochondrial biology. Autosomal recessive mutations in PTEN-induced kinase 1 (PINK1) cause an L-DOPA responsive parkinsonian syndrome, stimulating extensive interest in the normal neuroprotective and mitoprotective functions of PINK1. Recent data from mammalian and invertebrate model systems converge upon interactions between PINK1 and parkin, as well as DJ-1, α-synuclein and leucine rich repeat kinase 2 (LRRK2). While all studies to date support a neuroprotective role for wild type, but not mutant PINK1, there is less agreement on subcellular compartmentalization of PINK1 kinase function and whether PINK1 promotes mitochondrial fission or fusion. These controversies are reviewed in the context of the dynamic mitochondrial lifecycle, in which mitochondrial structure and function are continuously modulated not only by the fission–fusion machinery, but also by regulation of biogenesis, axonal/dendritic transport and autophagy. A working model is proposed, in which PINK1 loss-of-function results in mitochondrial reactive oxygen species (ROS), cristae/respiratory dysfunction and destabilization of calcium homeostasis, which trigger compensatory fission, autophagy and biosynthetic repair pathways that dramatically alter mitochondrial structure. Concurrent strategies to identify pathways that mediate normal PINK1 function and to identify factors that facilitate appropriate compensatory responses to its loss are both needed to halt the aging-related penetrance and incidence of familial and sporadic PD.     

PINK1/parkin通路调控线粒体自噬机制的研究进展

线粒体自噬指细胞通过自噬机制选择性除去损伤或多余的线粒体。真核生物通过线粒体自噬调控线粒体质量,维持供能细胞器的功能。大量研究表明,帕金森病相关基因PINK1和parkin可通过线粒体自噬参与并维持线粒体功能。PINK1与parkin能协同特异性识别损伤的线粒体,PINK1作为线粒体质量调控的探测器被活化,此过程中泛素化酶和去泛素化酶对维持parkin活性及线粒体自噬的效率有重要作用。本文主要总结PINK1/parkin通路在线粒体自噬中的功能与作用。     

Involvement and interplay of Parkin, PINK1, and DJ1 in neurodegenerative and neuroinflammatory disorders

The involvement of parkin, PINK1, and DJ1 in mitochondrial dysfunction, oxidative injury, and impaired functioning of the ubiquitin-proteasome system (UPS) has been intensively investigated in light of Parkinson's disease (PD) pathogenesis. However, these pathological mechanisms are not restricted to PD, but are common denominators of various neurodegenerative and neuroinflammatory disorders. It is therefore conceivable that parkin, PINK1, and DJ1 are also linked to the pathogenesis of other neurological diseases, including Alzheimer's disease (AD) and multiple sclerosis (MS). The importance of these proteins in mechanisms underlying neurodegeneration is reflected by the neuroprotective properties of parkin, DJ1, and PINK1 in counteracting oxidative stress and improvement of mitochondrial and UPS functioning. This review provides a concise overview on the cellular functions of the E3 ubiquitin ligase parkin, the mitochondrial kinase PINK1, and the cytoprotective protein DJ1 and their involvement and interplay in processes underlying neurodegeneration in common neurological disorders.     

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.     

Structural basis for feedforward control in the PINK1/Parkin pathway

PINK1 and parkin constitute a mitochondrial quality control system mutated in Parkinson’s disease. PINK1, a kinase, phosphorylates ubiquitin to recruit parkin, an E3 ubiquitin ligase, to mitochondria. PINK1 controls both parkin localization and activity through phosphorylation of both ubiquitin and the ubiquitin‐like (Ubl) domain of parkin. Here, we observed that phospho‐ubiquitin can bind to two distinct sites on parkin, a high‐affinity site on RING1 that controls parkin localization and a low‐affinity site on RING0 that releases parkin autoinhibition. Surprisingly, ubiquitin vinyl sulfone assays, ITC, and NMR titrations showed that the RING0 site has higher affinity for phospho‐ubiquitin than phosphorylated Ubl in trans. We observed parkin activation by micromolar concentrations of tetra‐phospho‐ubiquitin chains that mimic mitochondria bearing multiple phosphorylated ubiquitins. A chimeric form of parkin with the Ubl domain replaced by ubiquitin was readily activated by PINK1 phosphorylation. In all cases, mutation of the binding site on RING0 abolished parkin activation. The feedforward mechanism of parkin activation confers robustness and rapidity to the PINK1‐parkin pathway and likely represents an intermediate step in its evolutionary development.     

Parkin and PINK1 function in a vesicular trafficking pathway regulating mitochondrial quality control

Mitochondrial dysfunction has long been associated with Parkinson's disease (PD). Parkin and PINK1, two genes associated with familial PD, have been implicated in the degradation of depolarized mitochondria via autophagy (mitophagy). Here, we describe the involvement of parkin and PINK1 in a vesicular pathway regulating mitochondrial quality control. This pathway is distinct from canonical mitophagy and is triggered by the generation of oxidative stress from within mitochondria. Wild‐type but not PD‐linked mutant parkin supports the biogenesis of a population of mitochondria‐derived vesicles (MDVs), which bud off mitochondria and contain a specific repertoire of cargo proteins. These MDVs require PINK1 expression and ultimately target to lysosomes for degradation. We hypothesize that loss of this parkin‐ and PINK1‐dependent trafficking mechanism impairs the ability of mitochondria to selectively degrade oxidized and damaged proteins leading, over time, to the mitochondrial dysfunction noted in PD.