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-dependent mitophagy involves ubiquitination of mitofusins 1 and 2: Implications for Parkinson disease pathogenesis

Mitochondrial dysfunction has long been implicated in the pathogenesis of Parkinson disease (PD). Recent research has highlighted that two proteins encoded by genes linked to familial PD, PINK1 and parkin, play a role in the autophagic degradation of dysfunctional mitochondria (mitophagy). We have recently shown that mitochondrial dysfunction in PINK1-deficient human dopaminergic cells correlates with decreased autophagic flux and can be rescued by parkin expression. Further dissection of PINK1-parkin-dependent mitophagy indicates that the ubiquitination of mitofusins 1 and 2 is an early event. Here, we discuss how ubiquitination of the mitofusins might facilitate mitochondria degradation and the potential for activating mitophagy as a treatment for diseases affecting brain and muscle.     

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。     

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.     

Mitophagy and Parkinson's disease: The PINK1-parkin link

The study of rare, inherited mutations underlying familial forms of Parkinson's disease has provided insight into the molecular mechanisms of disease pathogenesis. Mutations in these genes have been functionally linked to several key molecular pathways implicated in other neurodegenerative disorders, including mitochondrial dysfunction, protein accumulation and the autophagic-lysosomal pathway. In particular, the mitochondrial kinase PINK1 and the cytosolic E3 ubiquitin ligase parkin act in a common pathway to regulate mitochondrial function. In this review we discuss the recent evidence suggesting that the PINK1/parkin pathway also plays a critical role in the autophagic removal of damaged mitochondria-mitophagy. This article is part of a Special Issue entitled Mitochondria: the deadly organelle.     

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.     

How could Parkin-mediated ubiquitination of mitofusin promote mitophagy?

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

Convergence of Parkin,PINK1, and α-Synuclein on Stress-induced Mitochondrial Morphological Remodeling

Mutations in PARKIN (PARK2), an ubiquitin ligase, cause early onset Parkinson disease. Parkin was shown to bind, ubiquitinate, and target depolarized mitochondria for destruction by autophagy. This process, mitophagy, is considered crucial for maintaining mitochondrial integrity and suppressing Parkinsonism. Here, we report that under moderate mitochondrial stress, parkin does not translocate to mitochondria to induce mitophagy; rather, it stimulates mitochondrial connectivity. Mitochondrial stress-induced fusion requires PINK1 (PARK6), mitofusins, and parkin ubiquitin ligase activity. Upon exposure to mitochondrial toxins, parkin binds α-synuclein (PARK1), and in conjunction with the ubiquitin-conjugating enzyme Ubc13, stimulates K63-linked ubiquitination. Importantly, α-synuclein inactivation phenocopies parkin overexpression and suppresses stress-induced mitochondria fission, whereas Ubc13 inactivation abrogates parkin-dependent mitochondrial fusion. The convergence of parkin, PINK1, and α-synuclein on mitochondrial dynamics uncovers a common function of these PARK genes in the mitochondrial stress response and provides a potential physiological basis for the prevalence of α-synuclein pathology in Parkinson disease.     

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.     

Mitochondrial autophagy as a compensatory response to PINK1 deficiency

Macroautophagy (hereafter, autophagy) plays a critical role in maintaining cellular homeostasis by degrading protein aggregates and dysfunctional/damaged organelles. We recently reported that silencing the recessive familial Parkinson disease gene encoding PTEN-induced kinase 1 (PINK1) leads to neuronal cell death accompanied by mitochondrial dysfunction and Drp1-dependent fragmentation. In this model, mitochondrial fission and Beclin 1-dependent autophagy play protective roles, cooperating to sequester and eliminate damaged mitochondria. We discuss the role of superoxide and other reactive oxygen species upstream of mitochondrial depolarization, fission, and autophagy in PINK1 knockdown lines. PINK1 deficiency appears to trigger several compensatory responses that together facilitate clearance of depolarized mitochondria, through a mechanism that is further enhanced by increased expression of parkin. These data offer additional insights that broaden the spectrum of potential interactions between PINK1 and parkin with respect to the regulation of mitochondrial homeostasis and mitophagy.     

The ubiquitin signal and autophagy: an orchestrated dance leading to mitochondrial degradation

The quality of mitochondria, essential organelles that produce ATP and regulate numerous metabolic pathways, must be strictly monitored to maintain cell homeostasis. The loss of mitochondrial quality control systems is acknowledged as a determinant for many types of neurodegenerative diseases including Parkinson's disease (PD). The two gene products mutated in the autosomal recessive forms of familial early‐onset PD, Parkin and PINK1, have been identified as essential proteins in the clearance of damaged mitochondria via an autophagic pathway termed mitophagy. Recently, significant progress has been made in understanding how the mitochondrial serine/threonine kinase PINK1 and the E3 ligase Parkin work together through a novel stepwise cascade to identify and eliminate damaged mitochondria, a process that relies on the orchestrated crosstalk between ubiquitin/phosphorylation signaling and autophagy. In this review, we highlight our current understanding of the detailed molecular mechanisms governing Parkin‐/PINK1‐mediated mitophagy and the evidences connecting Parkin/PINK1 function and mitochondrial clearance in neurons.     

PINK1/Parkin direct mitochondria to autophagy

Mutations in PTEN-induced putative kinase 1 (PINK1) and PARK2/Parkin cause autosomal recessive forms of Parkinson disease. In mammalian cells, cytosolic Parkin is selectively recruited to depolarized mitochondria, followed by a stimulation of mitochondrial autophagy. We show that Parkin translocation to mitochondria is mediated by PINK1, even in cells with normal mitochondrial membrane potential (ΔΨ_m). Once at the mitochondria, Parkin is in close proximity to PINK1, but Parkin does not catalyze PINK1 ubiquitination nor does PINK1 phosphorylate Parkin. However, co-overexpression of Parkin and PINK1 collapses the normal tubular mitochondrial network into large mitochondrial perinuclear clusters, many of which are surrounded by autophagic vacuoles. Our results suggest that Parkin and PINK1 modulate mitochondrial trafficking to the perinuclear region, a subcellular area associated with autophagy. Mutations in either Parkin or PINK1 impair this process and, consequently, mitochondrial turnover may be altered, inducing accumulation of defective mitochondria and, ultimately, causing neurodegeneration in Parkinson disease.     

Reduced Basal Autophagy and Impaired Mitochondrial Dynamics Due to Loss of Parkinson's Disease-Associated Protein DJ-1

Background

Mitochondrial dysfunction and degradation takes a central role in current paradigms of neurodegeneration in Parkinson''s disease (PD). Loss of DJ-1 function is a rare cause of familial PD. Although a critical role of DJ-1 in oxidative stress response and mitochondrial function has been recognized, the effects on mitochondrial dynamics and downstream consequences remain to be determined.

Methodology/Principal Findings

Using DJ-1 loss of function cellular models from knockout (KO) mice and human carriers of the E64D mutation in the DJ-1 gene we define a novel role of DJ-1 in the integrity of both cellular organelles, mitochondria and lysosomes. We show that loss of DJ-1 caused impaired mitochondrial respiration, increased intramitochondrial reactive oxygen species, reduced mitochondrial membrane potential and characteristic alterations of mitochondrial shape as shown by quantitative morphology. Importantly, ultrastructural imaging and subsequent detailed lysosomal activity analyses revealed reduced basal autophagic degradation and the accumulation of defective mitochondria in DJ-1 KO cells, that was linked with decreased levels of phospho-activated ERK2.

Conclusions/Significance

We show that loss of DJ-1 leads to impaired autophagy and accumulation of dysfunctional mitochondria that under physiological conditions would be compensated via lysosomal clearance. Our study provides evidence for a critical role of DJ-1 in mitochondrial homeostasis by connecting basal autophagy and mitochondrial integrity in Parkinson''s disease.     

Genetic analysis of mitochondrial protein misfolding in Drosophila melanogaster

Protein misfolding has a key role in several neurological disorders including Parkinson's disease. Although a clear mechanism for such proteinopathic diseases is well established when aggregated proteins accumulate in the cytosol, cell nucleus, endoplasmic reticulum and extracellular space, little is known about the role of protein aggregation in the mitochondria. Here we show that mutations in both human and fly PINK1 result in higher levels of misfolded components of respiratory complexes and increase in markers of the mitochondrial unfolded protein response. Through the development of a genetic model of mitochondrial protein misfolding employing Drosophila melanogaster, we show that the in vivo accumulation of an unfolded protein in mitochondria results in the activation of AMP-activated protein kinase-dependent autophagy and phenocopies of pink1 and parkin mutants. Parkin expression acts to clear mitochondria with enhanced levels of misfolded proteins by promoting their autophagic degradation in vivo, and refractory to Sigma P (ref(2)P), the Drosophila orthologue of mammalian p62, is a critical downstream effector of this quality control pathway. We show that in flies, a pathway involving pink1, parkin and ref(2)P has a role in the maintenance of a viable pool of cellular mitochondria by promoting organellar quality control.     

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.     

Mitochondrial quality control: insights on how Parkinson’s disease related genes PINK1, parkin,and Omi/HtrA2 interact to maintain mitochondrial homeostasis

Alterations in mitochondrial homeostasis have been implicated in the etiology of Parkinson disease (PD) as demonstrated by human tissue studies, cell culture and in vivo genetic and toxin models. Mutations in the genes encoding PTEN-induced kinase 1 (PINK1), Omi/HtrA2 and parkin contribute to rare forms of parkinsonian neurodegeneration. Recently, each of these proteins has been shown to play a normal role in regulating mitochondrial structure, function, fission-fusion dynamics, or turnover (autophagy and biogenesis), promoting neuronal survival. Here, we review the biochemical mechanisms of mitochondrial protection conferred by each of these PD associated gene products in neurons, neuronal cell lines and other cell types. Potential molecular interactions and mitoprotective signaling pathways involving these three PD associated gene products are discussed in the context of mitochondrial quality control, in response to increasing levels of mitochondrial damage. We propose that PINK1, Omi/HtrA2 and parkin participate at different levels in mitochondrial quality control, converging through some overlapping and some distinct steps to maintain a common phenotype of healthy mitochondrial networks.     

Mitophagy in cells with mtDNA mutations

Despite the emergence of autophagy as a key process for mitochondrial quality control, the existence and persistence of pathogenic mtDNA mutations in human disease suggests that the degradation of dysfunctional mitochondria does not occur widely in vivo. During macroautophagy, a double-membraned cup-shaped structure engulfs cytosolic content. This autophagic vesicle then fuses with lysosomes, allowing hydrolytic enzymes to degrade the contents. Mitochondrial autophagy, or mitophagy, is thought to degrade damaged or nonfunctioning mitochondria specifically. The Parkinson disease-related proteins PINK1 (a mitochondrially localized kinase) and PARK2 (PARKIN, a cytosolically-localized E3 ubiquitin ligase) are essential for targeting mitochondria for mitophagy. Upon chemical uncoupling of the mitochondrial transmembrane potential (Δψ_m), PINK1 located in the mitochondrial outer membrane recruits PARK2 from the cytosol to the mitochondria, followed by delivery of the organelle to the autophagic machinery for degradation.     

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.     

Mitophagy in cells with mtDNA mutations: being sick is not enough

Despite the emergence of autophagy as a key process for mitochondrial quality control, the existence and persistence of pathogenic mtDNA mutations in human disease suggests that the degradation of dysfunctional mitochondria does not occur widely in vivo. During macroautophagy, a double-membraned cup-shaped structure engulfs cytosolic content. This autophagic vesicle then fuses with lysosomes, allowing hydrolytic enzymes to degrade the contents. Mitochondrial autophagy, or mitophagy, is thought to degrade damaged or nonfunctioning mitochondria specifically. The Parkinson disease-related proteins PINK1 (a mitochondrially localized kinase) and PARK2 (PARKIN, a cytosolically-localized E3 ubiquitin ligase) are essential for targeting mitochondria for mitophagy. Upon chemical uncoupling of the mitochondrial transmembrane potential (Δψ(m)), PINK1 located in the mitochondrial outer membrane recruits PARK2 from the cytosol to the mitochondria, followed by delivery of the organelle to the autophagic machinery for degradation.     

The loss of PGAM5 suppresses the mitochondrial degeneration caused by inactivation of PINK1 in Drosophila

PTEN-induced kinase 1 (PINK1), which is required for mitochondrial homeostasis, is a gene product responsible for early-onset Parkinson's disease (PD). Another early onset PD gene product, Parkin, has been suggested to function downstream of the PINK1 signalling pathway based on genetic studies in Drosophila. PINK1 is a serine/threonine kinase with a predicted mitochondrial target sequence and a probable transmembrane domain at the N-terminus, while Parkin is a RING-finger protein with ubiquitin-ligase (E3) activity. However, how PINK1 and Parkin regulate mitochondrial activity is largely unknown. To explore the molecular mechanism underlying the interaction between PINK1 and Parkin, we biochemically purified PINK1-binding proteins from human cultured cells and screened the genes encoding these binding proteins using Drosophila PINK1 (dPINK1) models to isolate a molecule(s) involved in the PINK1 pathology. Here we report that a PINK1-binding mitochondrial protein, PGAM5, modulates the PINK1 pathway. Loss of Drosophila PGAM5 (dPGAM5) can suppress the muscle degeneration, motor defects, and shorter lifespan that result from dPINK1 inactivation and that can be attributed to mitochondrial degeneration. However, dPGAM5 inactivation fails to modulate the phenotypes of parkin mutant flies. Conversely, ectopic expression of dPGAM5 exacerbated the dPINK1 and Drosophila parkin (dParkin) phenotypes. These results suggest that PGAM5 negatively regulates the PINK1 pathway related to maintenance of the mitochondria and, furthermore, that PGAM5 acts between PINK1 and Parkin, or functions independently of Parkin downstream of PINK1.