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.     

Role of Glucose Metabolism and ATP in Maintaining PINK1 Levels during Parkin-mediated Mitochondrial Damage Responses

Mutations in several genes, including PINK1 and Parkin, are known to cause autosomal recessive cases of Parkinson disease in humans. These genes operate in the same pathway and play a crucial role in mitochondrial dynamics and maintenance. PINK1 is required to recruit Parkin to mitochondria and initiate mitophagy upon mitochondrial depolarization. In this study, we show that PINK1-dependent Parkin mitochondrial recruitment in response to global mitochondrial damage by carbonyl cyanide m-chlorophenylhydrazine (CCCP) requires active glucose metabolism. Parkin accumulation on mitochondria and subsequent Parkin-dependent mitophagy is abrogated in glucose-free medium or in the presence of 2-deoxy-d-glucose upon CCCP treatment. The defects in Parkin recruitment correlate with intracellular ATP levels and can be attributed to suppression of PINK1 up-regulation in response to mitochondria depolarization. Low levels of ATP appear to prevent PINK1 translation instead of affecting PINK1 mRNA expression or reducing its stability. Consistent with a requirement of ATP for elevated PINK1 levels and Parkin mitochondrial recruitment, local or individual mitochondrial damage via photoirradiation does not affect Parkin recruitment to damaged mitochondria as long as a pool of functional mitochondria is present in the photoirradiated cells even in glucose-free or 2-deoxy-d-glucose-treated conditions. Thus, our data identify ATP as a key regulator for Parkin mitochondrial translocation and sustaining elevated PINK1 levels during mitophagy. PINK1 functions as an AND gate and a metabolic sensor coupling biogenetics of cells and stress signals to mitochondria dynamics.     

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

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

PINK1 Is Dispensable for Mitochondrial Recruitment of Parkin and Activation of Mitophagy in Cardiac Myocytes

Myocyte function and survival relies on the maintenance of a healthy population of mitochondria. The PINK1/Parkin pathway plays an important role in clearing defective mitochondria via autophagy in cells. However, how the PINK1/Parkin pathway regulates mitochondrial quality control and whether it coordinates with other mitophagy pathways are still unclear. Therefore, the objective of this study was to investigate the effect of PINK1-deficiency on mitochondrial quality control in myocytes. Using PINK1-deficient (PINK1-/-) mice, we found that Parkin is recruited to damaged cardiac mitochondria in hearts after treatment with the mitochondrial uncoupler FCCP or after a myocardial infarction even in the absence of PINK1. Parkin recruitment to depolarized mitochondria correlates with increased ubiquitination of mitochondrial proteins and activation of mitophagy in PINK1-/- myocytes. In addition, induction of mitophagy by the atypical BH3-only protein BNIP3 is unaffected by lack of PINK1. Overall, these data suggest that Parkin recruitment to depolarized cardiac mitochondria and subsequent activation of mitophagy is independent of PINK1. Moreover, alternative mechanisms of Parkin activation and pathways of mitophagy remain functional in PINK1-/- myocytes and could compensate for the PINK1 deficiency.     

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.     

The many faces of mitophagy

Failure to maintain mitochondrial integrity is linked to age‐related conditions, such as neurodegeneration. Two genes linked to Parkinson's disease, PINK1 and Parkin, play a key role in targeting the degradation of dysfunctional mitochondria (mitophagy). However, the mechanisms regulating the PINK1/Parkin pathway and other processes that impinge on mitochondrial turnover are poorly understood. Two articles in EMBO reports, by the Przedborski and Ganley groups 1 2 , shed light on a new role for processed, cytoplasmic PINK1, and show that depletion of cellular iron levels stimulates PINK1/Parkin‐independent mitophagy.     

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.     

Phosphorylation of Mitochondrial Polyubiquitin by PINK1 Promotes Parkin Mitochondrial Tethering

The kinase PINK1 and the E3 ubiquitin (Ub) ligase Parkin participate in mitochondrial quality control. The phosphorylation of Ser65 in Parkin''s ubiquitin-like (UBl) domain by PINK1 stimulates Parkin activation and translocation to damaged mitochondria, which induces mitophagy generating polyUb chain. However, Parkin Ser65 phosphorylation is insufficient for Parkin mitochondrial translocation. Here we report that Ser65 in polyUb chain is also phosphorylated by PINK1, and that phosphorylated polyUb chain on mitochondria tethers Parkin at mitochondria. The expression of Tom70^MTS-4xUb SE, which mimics phospho-Ser65 polyUb chains on the mitochondria, activated Parkin E3 activity and its mitochondrial translocation. An E3-dead form of Parkin translocated to mitochondria with reduced membrane potential in the presence of Tom70^MTS-4xUb SE, whereas non-phospho-polyUb mutant Tom70^MTS-4xUb SA abrogated Parkin translocation. Parkin binds to the phospho-polyUb chain through its RING1-In-Between-RING (IBR) domains, but its RING0-linker is also required for mitochondrial translocation. Moreover, the expression of Tom70^MTS-4xUb SE improved mitochondrial degeneration in PINK1-deficient, but not Parkin-deficient, Drosophila. Our study suggests that the phosphorylation of mitochondrial polyUb by PINK1 is implicated in both Parkin activation and mitochondrial translocation, predicting a chain reaction mechanism of mitochondrial phospho-polyUb production by which rapid translocation of Parkin is achieved.     

Nrf2 activation induces mitophagy and reverses Parkin/Pink1 knock down-mediated neuronal and muscle degeneration phenotypes

The balanced functionality of cellular proteostatic modules is central to both proteome stability and mitochondrial physiology; thus, the age-related decline of proteostasis also triggers mitochondrial dysfunction, which marks multiple degenerative disorders. Non-functional mitochondria are removed by mitophagy, including Parkin/Pink1-mediated mitophagy. A common feature of neuronal or muscle degenerative diseases, is the accumulation of damaged mitochondria due to disrupted mitophagy rates. Here, we exploit Drosophila as a model organism to investigate the functional role of Parkin/Pink1 in regulating mitophagy and proteostatic responses, as well as in suppressing degenerative phenotypes at the whole organism level. We found that Parkin or Pink1 knock down in young flies modulated proteostatic components in a tissue-dependent manner, increased cell oxidative load, and suppressed mitophagy in neuronal and muscle tissues, causing mitochondrial aggregation and neuromuscular degeneration. Concomitant to Parkin or Pink1 knock down cncC/Nrf2 overexpression, induced the proteostasis network, suppressed oxidative stress, restored mitochondrial function, and elevated mitophagy rates in flies’ tissues; it also, largely rescued Parkin or Pink1 knock down-mediated neuromuscular degenerative phenotypes. Our in vivo findings highlight the critical role of the Parkin/Pink1 pathway in mitophagy, and support the therapeutic potency of Nrf2 (a druggable pathway) activation in age-related degenerative diseases.Subject terms: Mitophagy, Mechanisms of disease, Proteasome     

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.     

PINK1/Parkin介导的线粒体自噬

线粒体自噬（mitochondrial autophagy, or mitophagy）指的是细胞通过自吞噬作用,降解与清除受损线粒体或者多余线粒体,其对整个线粒体网络的功能完整性和细胞存活具有重要作用。线粒体自噬过程受多种途径调控,PINK1/Parkin通路是其中的一条,其异常与多种疾病的发生密切相关,如心血管疾病、肿瘤和帕金森病等。在去极化线粒体中,磷酸酶及张力蛋白同源物(PTEN)诱导的激酶1（PTEN-induced kinase 1,PINK1）作为受损线粒体的分子传感器,触发线粒体自噬的起始信号,并将Parkin募集至线粒体;Parkin作为线粒体自噬信号的“增强子”,通过对线粒体蛋白质进一步泛素化介导自噬信号的扩大;去泛素化酶和PTEN-long蛋白参与调控该过程,并对维持线粒体稳态具有重要作用。本文主要对PINK1与Parkin蛋白质的分子结构和其介导线粒体自噬发生的分子机制,以及参与调控该途径的关键蛋白质进行综述,为进一步研究以线粒体自噬缺陷为特征的疾病治疗提供理论基础。     

PINK1-Mediated Phosphorylation of Parkin Boosts Parkin Activity in Drosophila

Two genes linked to early onset Parkinson''s disease, PINK1 and Parkin, encode a protein kinase and a ubiquitin-ligase, respectively. Both enzymes have been suggested to support mitochondrial quality control. We have reported that Parkin is phosphorylated at Ser65 within the ubiquitin-like domain by PINK1 in mammalian cultured cells. However, it remains unclear whether Parkin phosphorylation is involved in mitochondrial maintenance and activity of dopaminergic neurons in vivo. Here, we examined the effects of Parkin phosphorylation in Drosophila, in which the phosphorylation residue is conserved at Ser94. Morphological changes of mitochondria caused by the ectopic expression of wild-type Parkin in muscle tissue and brain dopaminergic neurons disappeared in the absence of PINK1. In contrast, phosphomimetic Parkin accelerated mitochondrial fragmentation or aggregation and the degradation of mitochondrial proteins regardless of PINK1 activity, suggesting that the phosphorylation of Parkin boosts its ubiquitin-ligase activity. A non-phosphorylated form of Parkin fully rescued the muscular mitochondrial degeneration due to the loss of PINK1 activity, whereas the introduction of the non-phosphorylated Parkin mutant in Parkin-null flies led to the emergence of abnormally fused mitochondria in the muscle tissue. Manipulating the Parkin phosphorylation status affected spontaneous dopamine release in the nerve terminals of dopaminergic neurons, the survivability of dopaminergic neurons and flight activity. Our data reveal that Parkin phosphorylation regulates not only mitochondrial function but also the neuronal activity of dopaminergic neurons in vivo, suggesting that the appropriate regulation of Parkin phosphorylation is important for muscular and dopaminergic functions.     

PINK1 Is Selectively Stabilized on Impaired Mitochondria to Activate Parkin

Loss-of-function mutations in PINK1 and Parkin cause parkinsonism in humans and mitochondrial dysfunction in model organisms. Parkin is selectively recruited from the cytosol to damaged mitochondria to trigger their autophagy. How Parkin recognizes damaged mitochondria, however, is unknown. Here, we show that expression of PINK1 on individual mitochondria is regulated by voltage-dependent proteolysis to maintain low levels of PINK1 on healthy, polarized mitochondria, while facilitating the rapid accumulation of PINK1 on mitochondria that sustain damage. PINK1 accumulation on mitochondria is both necessary and sufficient for Parkin recruitment to mitochondria, and disease-causing mutations in PINK1 and Parkin disrupt Parkin recruitment and Parkin-induced mitophagy at distinct steps. These findings provide a biochemical explanation for the genetic epistasis between PINK1 and Parkin in Drosophila melanogaster. In addition, they support a novel model for the negative selection of damaged mitochondria, in which PINK1 signals mitochondrial dysfunction to Parkin, and Parkin promotes their elimination.     

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

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

The accumulation of misfolded proteins in the mitochondrial matrix is sensed by PINK1 to induce PARK2/Parkin-mediated mitophagy of polarized mitochondria

Defective mitochondria exert deleterious effects on host cells. To manage this risk, mitochondria display several lines of quality control mechanisms: mitochondria-specific chaperones and proteases protect against misfolded proteins at the molecular level, and fission/fusion and mitophagy segregate and eliminate damage at the organelle level. An increase in unfolded proteins in mitochondria activates a mitochondrial unfolded protein response (UPR^mt) to increase chaperone production, while the mitochondrial kinase PINK1 and the E3 ubiquitin ligase PARK2/Parkin, whose mutations cause familial Parkinson disease, remove depolarized mitochondria through mitophagy. It is unclear, however, if there is a connection between those different levels of quality control (QC). Here, we show that the expression of unfolded proteins in the matrix causes the accumulation of PINK1 on energetically healthy mitochondria, resulting in mitochondrial translocation of PARK2, mitophagy and subsequent reduction of unfolded protein load. Also, PINK1 accumulation is greatly enhanced by the knockdown of the LONP1 protease. We suggest that the accumulation of unfolded proteins in mitochondria is a physiological trigger of 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-mediated neuronal mitophagy deficiency in prion disease

A persistent accumulation of damaged mitochondria is part of prion disease pathogenesis. Normally, damaged mitochondria are cleared via a major pathway that involves the E3 ubiquitin ligase parkin and PTEN-induced kinase 1 (PINK1) that together initiate mitophagy, recognize and eliminate damaged mitochondria. However, the precise mechanisms underlying mitophagy in prion disease remain largely unknown. Using prion disease cell models, we observed PINK1-parkin-mediated mitophagy deficiency in which parkin depletion aggravated blocked mitochondrial colocalization with LC3-II-labeled autophagosomes, and significantly increased mitochondrial protein levels, which led to inhibited mitophagy. Parkin overexpression directly induced LC3-II colocalization with mitochondria and alleviated defective mitophagy. Moreover, parkin-mediated mitophagy was dependent on PINK1, since PINK1 depletion blocked mitochondrial Parkin recruitment and reduced optineurin and LC3-II proteins levels, thus inhibiting mitophagy. PINK1 overexpression induced parkin recruitment to the mitochondria, which then stimulated mitophagy. In addition, overexpressed parkin and PINK1 also protected neurons from apoptosis. Furthermore, we found that supplementation with two mitophagy-inducing agents, nicotinamide mononucleotide (NMN) and urolithin A (UA), significantly stimulated PINK1-parkin-mediated mitophagy. However, compared with NMN, UA could not alleviate prion-induced mitochondrial fragmentation and dysfunction, and neuronal apoptosis. These findings show that PINK1-parkin-mediated mitophagy defects lead to an accumulation of damaged mitochondria, thus suggesting that interventions that stimulate mitophagy may be potential therapeutic targets for prion diseases.Subject terms: Targeted gene repair, Target validation, Neurodegeneration, Neurodegeneration, Prion diseases     

Role of PINK1 binding to the TOM complex and alternate intracellular membranes in recruitment and activation of the E3 ligase Parkin

Mutations in the mitochondrial kinase PINK1 and the cytosolic E3 ligase Parkin can cause Parkinson's disease. Damaged mitochondria accumulate PINK1 on the outer membrane where, dependent on kinase activity, it recruits and activates Parkin to induce mitophagy, potentially maintaining organelle fidelity. How PINK1 recruits Parkin is unknown. We show that endogenous PINK1 forms a 700 kDa complex with the translocase of the outer membrane (TOM) selectively on depolarized mitochondria whereas PINK1 ectopically targeted to the outer membrane retains association with TOM on polarized mitochondria. Inducibly targeting PINK1 to peroxisomes or lysosomes, which lack a TOM complex, recruits Parkin and activates ubiquitin ligase activity on the respective organelles. Once there, Parkin induces organelle selective autophagy of peroxisomes but not lysosomes. We propose that the association of PINK1 with the TOM complex allows rapid reimport of PINK1 to rescue repolarized mitochondria from mitophagy, and discount mitochondrial-specific factors for Parkin translocation and activation.     

PINK1 protects against cell death induced by mitochondrial depolarization,by phosphorylating Bcl-xL and impairing its pro-apoptotic cleavage

Mutations in the PINK1 gene are a frequent cause of autosomal recessive Parkinson''s disease (PD). PINK1 encodes a mitochondrial kinase with neuroprotective activity, implicated in maintaining mitochondrial homeostasis and function. In concurrence with Parkin, PINK1 regulates mitochondrial trafficking and degradation of damaged mitochondria through mitophagy. Moreover, PINK1 can activate autophagy by interacting with the pro-autophagic protein Beclin-1. Here, we report that, upon mitochondrial depolarization, PINK1 interacts with and phosphorylates Bcl-xL, an anti-apoptotic protein also known to inhibit autophagy through its binding to Beclin-1. PINK1–Bcl-xL interaction does not interfere either with Beclin-1 release from Bcl-xL or the mitophagy pathway; rather it protects against cell death by hindering the pro-apoptotic cleavage of Bcl-xL. Our data provide a functional link between PINK1, Bcl-xL and apoptosis, suggesting a novel mechanism through which PINK1 regulates cell survival. This pathway could be relevant for the pathogenesis of PD as well as other diseases including cancer.