Parkin uses the UPS to ship off dysfunctional mitochondria

Parkin is a ubiquitin E3 ligase that is implicated in familial Parkinson disease (PD). Previous studies have established its role in mitophagy, a pathway whereby dysfunctional mitochondria are targeted for autophagic degradation. We recently reported that a major function of Parkin in dysfunctional mitochondria is to activate the ubiquitin-proteasome system (UPS) for proteolysis of multiple outer membrane proteins, and that such activation of the UPS is a critical step in Parkin-mediated mitophagy. Here, we discuss the possible roles of the UPS in mitophagy and the pathogenesis of 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.     

Mutations in PINK1 and Parkin impair ubiquitination of Mitofusins in human fibroblasts

PINK1 and Parkin mutations cause recessive Parkinson's disease (PD). In Drosophila and SH-SY5Y cells, Parkin is recruited by PINK1 to damaged mitochondria, where it ubiquitinates Mitofusins and consequently promotes mitochondrial fission and mitophagy.Here, we investigated the impact of mutations in endogenous PINK1 and Parkin on the ubiquitination of mitochondrial fusion and fission factors and the mitochondrial network structure. Treating control fibroblasts with mitochondrial membrane potential (Δψ) inhibitors or H(2)O(2) resulted in ubiquitination of Mfn1/2 but not of OPA1 or Fis1. Ubiquitination of Mitofusins through the PINK1/Parkin pathway was observed within 1 h of treatment. Upon combined inhibition of Δψ and the ubiquitin proteasome system (UPS), no ubiquitination of Mitofusins was detected. Regarding morphological changes, we observed a trend towards increased mitochondrial branching in PD patient cells upon mitochondrial stress.For the first time in PD patient-derived cells, we demonstrate that mutations in PINK1 and Parkin impair ubiquitination of Mitofusins. In the presence of UPS inhibitors, ubiquitinated Mitofusin is deubiquitinated by the UPS but not degraded, suggesting that the UPS is involved in Mitofusin degradation.     

Melatonin activates Parkin translocation and rescues the impaired mitophagy activity of diabetic cardiomyopathy through Mst1 inhibition

Mitophagy eliminates dysfunctional mitochondria and thus plays a cardinal role in diabetic cardiomyopathy (DCM). We observed the favourable effects of melatonin on cardiomyocyte mitophagy in mice with DCM and elucidated their underlying mechanisms. Electron microscopy and flow cytometric analysis revealed that melatonin reduced the number of impaired mitochondria in the diabetic heart. Other than decreasing mitochondrial biogenesis, melatonin increased the clearance of dysfunctional mitochondria in mice with DCM. Melatonin increased LC3 II expression as well as the colocalization of mitochondria and lysosomes in HG‐treated cardiomyocytes and the number of typical autophagosomes engulfing mitochondria in the DCM heart. These results indicated that melatonin promoted mitophagy. When probing the mechanism, increased Parkin translocation to the mitochondria may be responsible for the up‐regulated mitophagy exerted by melatonin. Parkin knockout counteracted the beneficial effects of melatonin on the cardiac mitochondrial morphology and bioenergetic disorders, thus abolishing the substantial effects of melatonin on cardiac remodelling with DCM. Furthermore, melatonin inhibited Mammalian sterile 20‐like kinase 1 (Mst1) phosphorylation, thus enhancing Parkin‐mediated mitophagy, which contributed to mitochondrial quality control. In summary, this study confirms that melatonin rescues the impaired mitophagy activity of DCM. The underlying mechanism may be attributed to activation of Parkin translocation via inhibition of Mst1.     

Parkin,as a Regulator,Participates in Arsenic Trioxide-Triggered Mitophagy in HeLa Cells

Parkin is a well-established synergistic mediator of mitophagy in dysfunctional mitochondria. Mitochondria are the main target of arsenic trioxide (ATO) cytotoxicity, and the effect of mitophagy on ATO action remains unclear. In this study, we used stable Parkin-expressing (YFP-Parkin) and Parkin loss-of-function mutant (Parkin C431S) HeLa cell models to ascertain whether Parkin-mediated mitophagy participates in ATO-induced apoptosis/cell death. Our data showed that the overexpression of Parkin significantly sensitized HeLa cells to ATO-initiated proliferation inhibition and apoptosis; however, the mutation of Parkin C431S significantly weakened this Parkin-mediated responsiveness. Our further investigation found that ATO significantly downregulated two fusion proteins (Mfn1/2) and upregulated fission-related protein (Drp1). Autophagy was also activated as evidenced by the formation of autophagic vacuoles and mitophagosomes, increased expression of PINK1, and recruitment of Parkin to impaired mitochondria followed by their degradation, accompanied by the increased transformation of LC3-I to LC3-II, increased expression of Beclin1 and decreased expression of P62 in YFP-Parkin HeLa cells. Enhanced mitochondrial fragmentation and autophagy indicated that mitophagy was activated. Furthermore, during the process of mitophagy, the overproduction of ROS implied that ROS might represent a key factor that initiates mitophagy following Parkin recruitment to mitochondria. In conclusion, our findings indicate that Parkin is critically involved in ATO-triggered mitophagy and functions as a potential antiproliferative target in cancer cells.     

Parkin-induced mitophagy in the pathogenesis of Parkinson disease

Knockout of the ubiquitin ligase Parkin, the gene product of the Parkinson associated Park2, leads to loss of mitochondrial integrity and function in Drosophila melanogaster. Although Parkin is primarily cytosolic, we have found that Parkin is selectively recruited to dysfunctional mitochondria with low membrane potential and subsequently promotes their autophagy. Here we report that Parkin recruitment is voltage-dependent and independent of changes in ATP or pH. These findings suggest that Parkin promotes mitophagy of dysfunctional mitochondria following loss of mitochondrial membrane potential and implicate the targeted elimination of mitochondria in the pathogenesis of Parkinson disease.     

USP8 regulates mitophagy by removing K6‐linked ubiquitin conjugates from parkin

Mutations in the Park2 gene, encoding the E3 ubiquitin‐ligase parkin, are responsible for a familial form of Parkinson's disease (PD). Parkin‐mediated ubiquitination is critical for the efficient elimination of depolarized dysfunctional mitochondria by autophagy (mitophagy). As damaged mitochondria are a major source of toxic reactive oxygen species within the cell, this pathway is believed to be highly relevant to the pathogenesis of PD. Little is known about how parkin‐mediated ubiquitination is regulated during mitophagy or about the nature of the ubiquitin conjugates involved. We report here that USP8/UBPY, a deubiquitinating enzyme not previously implicated in mitochondrial quality control, is critical for parkin‐mediated mitophagy. USP8 preferentially removes non‐canonical K6‐linked ubiquitin chains from parkin, a process required for the efficient recruitment of parkin to depolarized mitochondria and for their subsequent elimination by mitophagy. This work uncovers a novel role for USP8‐mediated deubiquitination of K6‐linked ubiquitin conjugates from parkin in mitochondrial quality control.     

Spatial parkin translocation and degradation of damaged mitochondria via mitophagy in live cortical neurons

Mitochondria are essential for neuronal survival and function. Proper degradation of aged and damaged mitochondria through mitophagy is a key cellular pathway for mitochondrial quality control. Recent studies have indicated that PINK1/Parkin-mediated pathways ensure mitochondrial integrity and function. Translocation of Parkin to damaged mitochondria induces mitophagy in many nonneuronal cell types. However, evidence showing Parkin translocation in primary neurons is controversial, leaving unanswered questions as to how and where Parkin-mediated mitophagy occurs in neurons. Here, we report the unique process of dissipating mitochondrial Δψ(m)-induced and Parkin-mediated mitophagy in mature cortical neurons. Compared with nonneuronal cells, neuronal mitophagy is a much slower and compartmentally restricted process, coupled with reduced anterograde mitochondrial transport. Parkin-targeted mitochondria are accumulated in the somatodendritic regions where mature lysosomes are predominantly located. Time-lapse imaging shows dynamic formation and elimination of Parkin- and LC3-ring-like structures surrounding depolarized mitochondria through the autophagy-lysosomal pathway in the soma. Knocking down Parkin in neurons impairs the elimination of dysfunctional mitochondria. Thus, our study provides neuronal evidence for dynamic and spatial Parkin-mediated mitophagy, which will help us understand whether altered mitophagy contributes to pathogenesis of several major neurodegenerative diseases characterized by mitochondrial dysfunction and impaired transport.     

Short Mitochondrial ARF Triggers Parkin/PINK1-dependent Mitophagy

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

p62/SQSTM1 is required for Parkin-induced mitochondrial clustering but not mitophagy; VDAC1 is dispensable for both

Mitochondria sustain damage with aging, and the resulting mitochondrial dysfunction has been implicated in a number of diseases including Parkinson disease. We recently demonstrated that the E3 ubiquitin ligase Parkin, which is linked to recessive forms of parkinsonism, causes a dramatic increase in mitophagy and a change in mitochondrial distribution, following its translocation from the cytosol to mitochondria. Investigating how Parkin induces these changes may offer insight into the mechanisms that lead to the sequestration and elimination of damaged mitochondria. We report that following Parkin’s translocation from the cytosol to mitochondria, Parkin (but not a pathogenic mutant) promotes the K63-linked polyubiquitination of mitochondrial substrate(s) and recruits the ubiquitin- and LC3-binding protein, p62/SQSTM1, to mitochondria. After its recruitment, p62/SQSTM1 mediates the aggregation of dysfunctional mitochondria through polymerization via its PB1 domain, in a manner analogous to its aggregation of polyubiquitinated proteins. Surprisingly and in contrast to what has been recently reported for ubiquitin-induced pexophagy and xenophagy, p62 appears to be dispensable for mitophagy. Similarly, mitochondrial-anchored ubiquitin is sufficient to recruit p62 and promote mitochondrial clustering, but does not promote mitophagy. Although VDAC1 (but not VDAC2) is ubiquitinated following mitochondrial depolarization, we find VDAC1 cannot fully account for the mitochondrial K63-linked ubiquitin immunoreactivity observed following depolarization, as it is also observed in VDAC1/3-/- mouse embryonic fibroblasts. Additionally, we find VDAC1 and VDAC3 are dispensable for the recruitment of p62, mitochondrial clustering and mitophagy. These results demonstrate that mitochondria are aggregated by p62, following its recruitment by Parkin in a VDAC1-independent manner. They also suggest that proteins other than p62 are likely required for mitophagy downstream of Parkin substrates other than VDAC1.     

The PINK1/Parkin-mediated mitophagy is compromised by PD-associated mutations

Mitochondrial dysfunction is an early sign of many neurodegenerative diseases. Very recently, two Parkinson disease (PD) associated genes, PINK1 and Parkin, were shown to mediate the degradation of damaged mitochondria via selective autophagy (mitophagy). PINK1 kinase activity is needed for prompt and efficient Parkin recruitment to impaired mitochondria. PD-associated Parkin mutations interfere with the process of mitophagy at distinct steps. Here we show that whole mitochondria are turned over via macroautophagy. Moreover, disease-associated PINK1 mutations also compromise the selective degradation of depolarized mitochondria. This may be due to the decreased physical binding activity of PD-linked PINK1 mutations to Parkin. Thus, PINK1 mutations abrogate autophagy of impaired mitochondria upstream of Parkin. In addition to compromised PINK1 kinase activity, reduced binding of PINK1 to Parkin leads to failure in Parkin mitochondrial translocation, resulting in the accumulation of damaged mitochondria, which may contribute to disease pathogenesis.     

Ambra1

Mutations in the gene for the E3 ubiquitin ligase Parkin are the most prevalent cause of autosomal recessive Parkinson disease (PD), an incurable neurodegenerative disorder. Parkin surveys mitochondrial quality by translocating to depolarized mitochondria and inducing their selective macroautophagic removal (mitophagy). We recently reported that Parkin interacts with Ambra1 (activating molecule in Beclin 1-regulated autophagy), a protein that promotes autophagy in the vertebrate central nervous system. We discovered that prolonged mitochondrial depolarization strongly increases the interaction of Parkin with Ambra1. Ambra1 is recruited in a Parkin-dependent manner to perinuclear clusters of depolarized mitochondria, activates the class III phosphatidylinositol 3-kinase (PtdIns3K) complex around these mitochondria and contributes to their selective autophagic clearance. Here, we discuss these findings and suggest a model where translocated Parkin efficiently triggers mitophagy through combined recruitment of Ambra1 and ubiquitination of outer mitochondrial membrane proteins.     

Ambra1: a Parkin-binding protein involved in mitophagy

Mutations in the gene for the E3 ubiquitin ligase Parkin are the most prevalent cause of autosomal recessive Parkinson disease (PD), an incurable neurodegenerative disorder. Parkin surveys mitochondrial quality by translocating to depolarized mitochondria and inducing their selective macroautophagic removal (mitophagy). We recently reported that Parkin interacts with Ambra1 (activating molecule in Beclin 1-regulated autophagy), a protein that promotes autophagy in the vertebrate central nervous system. We discovered that prolonged mitochondrial depolarization strongly increases the interaction of Parkin with Ambra1. Ambra1 is recruited in a Parkin-dependent manner to perinuclear clusters of depolarized mitochondria, activates the class III phosphatidylinositol 3-kinase (PtdIns3K) complex around these mitochondria and contributes to their selective autophagic clearance. Here, we discuss these findings and suggest a model where translocated Parkin efficiently triggers mitophagy through combined recruitment of Ambra1 and ubiquitination of outer mitochondrial membrane proteins.     

p62/SQSTM1 is required for Parkin-induced mitochondrial clustering but not mitophagy; VDAC1 is dispensable for both

Mitochondria sustain damage with aging, and the resulting mitochondrial dysfunction has been implicated in a number of diseases including Parkinson disease. We recently demonstrated that the E3 ubiquitin ligase Parkin, which is linked to recessive forms of parkinsonism, causes a dramatic increase in mitophagy and a change in mitochondrial distribution, following its translocation from the cytosol to mitochondria. Investigating how Parkin induces these changes may offer insight into the mechanisms that lead to the sequestration and elimination of damaged mitochondria. We report that following Parkin’s translocation from the cytosol to mitochondria, Parkin (but not a pathogenic mutant) promotes the K63-linked polyubiquitination of mitochondrial substrate(s) and recruits the ubiquitin- and LC3-binding protein, p62/SQSTM1, to mitochondria. After its recruitment, p62/SQSTM1 mediates the aggregation of dysfunctional mitochondria through polymerization via its PB1 domain, in a manner analogous to its aggregation of polyubiquitinated proteins. Surprisingly and in contrast to what has been recently reported for ubiquitin-induced pexophagy and xenophagy, p62 appears to be dispensable for mitophagy. Similarly, mitochondrial-anchored ubiquitin is sufficient to recruit p62 and promote mitochondrial clustering, but does not promote mitophagy. Although VDAC1 (but not VDAC2) is ubiquitinated following mitochondrial depolarization, we find VDAC1 cannot fully account for the mitochondrial K63-linked ubiquitin immunoreactivity observed following depolarization, as it is also observed in VDAC1/3^-/- mouse embryonic fibroblasts. Additionally, we find VDAC1 and VDAC3 are dispensable for the recruitment of p62, mitochondrial clustering and mitophagy. These results demonstrate that mitochondria are aggregated by p62, following its recruitment by Parkin in a VDAC1-independent manner. They also suggest that proteins other than p62 are likely required for mitophagy downstream of Parkin substrates other than VDAC1.     

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.     

Mitophagy of damaged mitochondria occurs locally in distal neuronal axons and requires PINK1 and Parkin

To minimize oxidative damage to the cell, malfunctioning mitochondria need to be removed by mitophagy. In neuronal axons, mitochondrial damage may occur in distal regions, far from the soma where most lysosomal degradation is thought to occur. In this paper, we report that PINK1 and Parkin, two Parkinson’s disease–associated proteins, mediate local mitophagy of dysfunctional mitochondria in neuronal axons. To reduce cytotoxicity and mimic physiological levels of mitochondrial damage, we selectively damaged a subset of mitochondria in hippocampal axons. Parkin was rapidly recruited to damaged mitochondria in axons followed by formation of LC3-positive autophagosomes and LAMP1-positive lysosomes. In PINK1^−/− axons, damaged mitochondria did not accumulate Parkin and failed to be engulfed in autophagosomes. Similarly, initiation of mitophagy was blocked in Parkin^−/− axons. Our findings demonstrate that the PINK1–Parkin-mediated pathway is required for local mitophagy in distal axons in response to focal damage. Local mitophagy likely provides rapid neuroprotection against oxidative stress without a requirement for retrograde transport to the soma.     

MITOL promotes cell survival by degrading Parkin during mitophagy

Parkin promotes cell survival by removing damaged mitochondria via mitophagy. However, although some studies have suggested that Parkin induces cell death, the regulatory mechanism underlying the dual role of Parkin remains unknown. Herein, we report that mitochondrial ubiquitin ligase (MITOL/MARCH5) regulates Parkin‐mediated cell death through the FKBP38‐dependent dynamic translocation from the mitochondria to the ER during mitophagy. Mechanistically, MITOL mediates ubiquitination of Parkin at lysine 220 residue, which promotes its proteasomal degradation, and thereby fine‐tunes mitophagy by controlling the quantity of Parkin. Deletion of MITOL leads to accumulation of the phosphorylated active form of Parkin in the ER, resulting in FKBP38 degradation and enhanced cell death. Thus, we have shown that MITOL blocks Parkin‐induced cell death, at least partially, by protecting FKBP38 from Parkin. Our findings unveil the regulation of the dual function of Parkin and provide a novel perspective on the pathogenesis of PD.     

Cardiolipin remodeling by ALCAT1 links mitochondrial dysfunction to Parkinson’s diseases

Cardiolipin (CL) is a mitochondrial signature phospholipid that is required for membrane structure, respiration, dynamics, and mitophagy. Oxidative damage of CL by reactive oxygen species is implicated in the pathogenesis of Parkinson's disease (PD), but the underlying cause remains elusive. This work investigated the role of ALCAT1, an acyltransferase that catalyzes pathological remodeling of CL in various aging‐related diseases, in a mouse model of PD induced by 1‐methyl‐4‐phenyl‐1,2,4,6‐tetrahydropyridine (MPTP). We show that MPTP treatment caused oxidative stress, mtDNA mutations, and mitochondrial dysfunction in the midbrain. In contrast, ablation of the ALCAT1 gene or pharmacological inhibition of ALCAT1 prevented MPTP‐induced neurotoxicity, apoptosis, and motor deficits. ALCAT1 deficiency also mitigated mitochondrial dysfunction by modulating DRP1 translocation to the mitochondria. Moreover, pharmacological inhibition of ALCAT1 significantly improved mitophagy by promoting the recruitment of Parkin to dysfunctional mitochondria. Finally, ALCAT1 expression was upregulated by MPTP and by α‐synucleinopathy, a key hallmark of PD, whereas ALCAT1 deficiency prevented α‐synuclein oligomerization and S‐129 phosphorylation, implicating a key role of ALCAT1 in the etiology of mouse models of PD. Together, these findings identify ALCAT1 as a novel drug target for the treatment of PD.     

Long time-lapse imaging reveals unique features of PARK2/Parkin-mediated mitophagy in mature cortical neurons

Proper degradation of aged and damaged mitochondria through mitophagy is essential to ensure mitochondrial integrity and function. Translocation of PARK2/Parkin onto damaged mitochondria induces mitophagy in many non-neuronal cell types. However, direct evidence showing PARK2-mediated mitophagy in mature neurons is controversial, leaving unanswered questions as to how, where, and by what time course PARK2-mediated mitophagy occurs in neurons following mitochondrial depolarization. We applied long time-lapse imaging in live mature cortical neurons to monitor the slow but dynamic and spatial PARK2 translocation onto damaged mitochondria and subsequent degradation through the autophagy-lysosomal pathway. In comparison with non-neuronal cells, our study reveals unique features of PARK2-mediated mitophagy in mature neurons, which will advance our understanding of pathogenesis of several major neurodegenerative diseases characterized by damaged mitochondria or a dysfunctional autophagy-lysosomal system.     

Long time-lapse imaging reveals unique features of PARK2/Parkin-mediated mitophagy in mature cortical neurons

Proper degradation of aged and damaged mitochondria through mitophagy is essential to ensure mitochondrial integrity and function. Translocation of PARK2/Parkin onto damaged mitochondria induces mitophagy in many non-neuronal cell types. However, direct evidence showing PARK2-mediated mitophagy in mature neurons is controversial, leaving unanswered questions as to how, where, and by what time course PARK2-mediated mitophagy occurs in neurons following mitochondrial depolarization. We applied long time-lapse imaging in live mature cortical neurons to monitor the slow but dynamic and spatial PARK2 translocation onto damaged mitochondria and subsequent degradation through the autophagy-lysosomal pathway. In comparison with non-neuronal cells, our study reveals unique features of PARK2-mediated mitophagy in mature neurons, which will advance our understanding of pathogenesis of several major neurodegenerative diseases characterized by damaged mitochondria or a dysfunctional autophagy-lysosomal system.