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.     

PINK1 controls mitochondrial localization of Parkin through direct phosphorylation

PTEN-induced putative kinase 1 (PINK1) and Parkin, encoded by their respective genes associated with Parkinson’s disease (PD), are linked in a common pathway involved in the protection of mitochondrial integrity and function. However, the mechanism of their interaction at the biochemical level has not been investigated yet. Using both mammalian and Drosophila systems, we here demonstrate that the PINK1 kinase activity is required for its function in mitochondria. PINK1 regulates the localization of Parkin to the mitochondria in its kinase activity-dependent manner. In detail, Parkin phosphorylation by PINK1 on its linker region promotes its mitochondrial translocation, and the RING1 domain of Parkin is critical for this occurrence. These results demonstrate the biochemical relationship between PINK1, Parkin, and the mitochondria and thereby suggest the possible mechanism of PINK-Parkin-associated PD pathogenesis.     

MicroRNA-137 Is a Novel Hypoxia-responsive MicroRNA That Inhibits Mitophagy via Regulation of Two Mitophagy Receptors FUNDC1 and NIX

Mitophagy receptors mediate the selective recognition and targeting of damaged mitochondria by autophagosomes. The mechanism for the regulation of these receptors remains unknown. Here, we demonstrated that a novel hypoxia-responsive microRNA, microRNA-137 (miR-137), markedly inhibits mitochondrial degradation by autophagy without affecting global autophagy. miR-137 targets the expression of two mitophagy receptors NIX and FUNDC1. Impaired mitophagy in response to hypoxia caused by miR-137 is reversed by re-expression of FUNDC1 and NIX expression vectors lacking the miR-137 recognition sites at their 3′ UTR. Conversely, miR-137 also suppresses the mitophagy induced by fundc1 (CDS+3′UTR) but not fundc1 (CDS) overexpression. Finally, we found that miR-137 inhibits mitophagy by reducing the expression of the mitophagy receptor thereby leads to inadequate interaction between mitophagy receptor and LC3. Our results demonstrated the regulatory role of miRNA to mitophagy receptors and revealed a novel link between miR-137 and mitophagy.     

Parkin stabilizes PINK1 through direct interaction

Parkinson disease (PD) is the most common movement disorder and is characterized by dopaminergic dysfunction. The majority of PD cases are sporadic; however, the discovery of genes linked to rare familial forms of the disease has provided crucial insight into the molecular mechanisms of disease pathogenesis. Multiple genes mediating familial forms of Parkinson’s disease (PD) have been identified, such as parkin (PARK2) and phosphatase and tensin homologue deleted on chromosome ten (PTEN)-induced putative kinase 1: PINK1 (PARK6). Here, we showed that Parkin directly interacts with PINK1, but did not bind to pathogenic PINK1 mutants. Parkin, but not its pathogenic mutants, stabilizes PINK1 by interfering with its degradation via the ubiquitin-mediated proteasomal pathway. In addition, the interaction between Parkin and PINK1 resulted in reciprocal reduction of their solubility. Our results indicate that Parkin regulates PINK1 stabilization via direct interaction with PINK1, and operates through a common pathway with PINK1 in the pathogenesis of early-onset 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。     

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.     

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 autophosphorylation is required for ubiquitin recognition

Mutations in PINK1 cause autosomal recessive Parkinson's disease (PD), a neurodegenerative movement disorder. PINK1 is a kinase that acts as a sensor of mitochondrial damage and initiates Parkin‐mediated clearance of the damaged organelle. PINK1 phosphorylates Ser65 in both ubiquitin and the ubiquitin‐like (Ubl) domain of Parkin, which stimulates its E3 ligase activity. Autophosphorylation of PINK1 is required for Parkin activation, but how this modulates the ubiquitin kinase activity is unclear. Here, we show that autophosphorylation of Tribolium castaneum PINK1 is required for substrate recognition. Using enzyme kinetics and NMR spectroscopy, we reveal that PINK1 binds the Parkin Ubl with a 10‐fold higher affinity than ubiquitin via a conserved interface that is also implicated in RING1 and SH3 binding. The interaction requires phosphorylation at Ser205, an invariant PINK1 residue (Ser228 in human). Using mass spectrometry, we demonstrate that PINK1 rapidly autophosphorylates in trans at Ser205. Small‐angle X‐ray scattering and hydrogen–deuterium exchange experiments provide insights into the structure of the PINK1 catalytic domain. Our findings suggest that multiple PINK1 molecules autophosphorylate first prior to binding and phosphorylating ubiquitin and Parkin.     

大鼠心肌缺血再灌注损伤模型线粒体自噬变化的研究

目的:观察大鼠心肌缺血再灌注损伤模型不同时间点线粒体及线粒体自噬的变化。方法:成年雄性SD大鼠40只,随机分为假手术对照组(sham组):开胸不进行冠状动脉左前降支(Left anterior descending coronary artery,LAD)血流阻断;缺血再灌注组2h组(I/R 2 h组)、24 h组(I/R 24 h组)及48 h组(I/R 48 h组),以上3组均阻断LAD 30 min,分别于再灌注后2 h、24 h、48 h观察心肌ATP含量,线粒体膜电位水平变化,透射电镜下观察线粒体及线粒体自噬超微结构变化,western blot法测定线粒体自噬蛋白PINK1、Parkin、p62、LC3B及线粒体膜蛋白Tom20表达水平。结果:与对照组相比,线粒体膜电位水平及心肌组织ATP含量于再灌注2 h开始下降,24 h下降最显著,48 h有所改善,线粒体超微结构损伤再灌注24 h最为明显,48 h有所改善。PINK1、Parkin、p62蛋白表达于损伤后2 h增强,于再灌注后24 h升高最显著,持续至48 h,LC3BⅡ表达于损伤后24 h增强,同样持续至48 h。透射电镜下可见线粒体自噬体于再灌注后24 h明显增多,并持续至48 h。结论:大鼠心肌缺血再灌注损伤后,线粒体功能与形态损伤以损伤后24 h最为显著,至损伤后48 h后好转;线粒体自噬水平升高以损伤后24 h最为显著,且维持至损伤后48 h,提示两者之间可能存在关联。     

Nitric Oxide Induction of Parkin Translocation in PTEN-induced Putative Kinase 1 (PINK1) Deficiency: FUNCTIONAL ROLE OF NEURONAL NITRIC OXIDE SYNTHASE DURING MITOPHAGY*

The failure to trigger mitophagy is implicated in the pathogenesis of familial Parkinson disease that is caused by PINK1 or Parkin mutations. According to the prevailing PINK1-Parkin signaling model, mitophagy is promoted by the mitochondrial translocation of Parkin, an essential PINK1-dependent step that occurs via a previously unknown mechanism. Here we determined that critical concentrations of NO was sufficient to induce the mitochondrial translocation of Parkin even in PINK1 deficiency, with apparent increased interaction of full-length PINK1 accumulated during mitophagy, with neuronal nitric oxide synthase (nNOS). Specifically, optimum levels of NO enabled PINK1-null dopaminergic neuronal cells to regain the mitochondrial translocation of Parkin, which appeared to be significantly suppressed by nNOS-null mutation. Moreover, nNOS-null mutation resulted in the same mitochondrial electron transport chain (ETC) enzyme deficits as PINK1-null mutation. The involvement of mitochondrial nNOS activation in mitophagy was further confirmed by the greatly increased interactions of full-length PINK1 with nNOS, accompanied by mitochondrial accumulation of phospho-nNOS (Ser¹⁴¹²) during mitophagy. Of great interest is that the L347P PINK1 mutant failed to bind to nNOS. The loss of nNOS phosphorylation and Parkin accumulation on PINK1-deficient mitochondria could be reversed in a PINK1-dependent manner. Finally, non-toxic levels of NO treatment aided in the recovery of PINK1-null dopaminergic neuronal cells from mitochondrial ETC enzyme deficits. In summary, we demonstrated the full-length PINK1-dependent recruitment of nNOS, its activation in the induction of Parkin translocation, and the feasibility of NO-based pharmacotherapy for defective mitophagy and ETC enzyme deficits in Parkinson disease.     

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.     

Loss of iron triggers PINK1/Parkin‐independent mitophagy

In this study, we develop a simple assay to identify mitophagy inducers on the basis of the use of fluorescently tagged mitochondria that undergo a colour change on lysosomal delivery. Using this assay, we identify iron chelators as a family of compounds that generate a strong mitophagy response. Iron chelation‐induced mitophagy requires that cells undergo glycolysis, but does not require PINK1 stabilization or Parkin activation, and occurs in primary human fibroblasts as well as those isolated from a Parkinson's patient with Parkin mutations. Thus, we have identified and characterized a mitophagy pathway, the induction of which could prove beneficial as a potential therapy for several neurodegenerative diseases in which mitochondrial clearance is advantageous.     

Cytosolic cleaved PINK1 represses Parkin translocation to mitochondria and mitophagy

PINK1 is a mitochondrial kinase proposed to have a role in the pathogenesis of Parkinson''s disease through the regulation of mitophagy. Here, we show that the PINK1 main cleavage product, PINK1_52, after being generated inside mitochondria, can exit these organelles and localize to the cytosol, where it is not only destined for degradation by the proteasome but binds to Parkin. The interaction of cytosolic PINK1 with Parkin represses Parkin translocation to the mitochondria and subsequent mitophagy. Our work therefore highlights the existence of two cellular pools of PINK1 that have different effects on Parkin translocation and mitophagy.     

PINK1 is activated by mitochondrial membrane potential depolarization and stimulates Parkin E3 ligase activity by phosphorylating Serine 65

Missense mutations in PTEN-induced kinase 1 (PINK1) cause autosomal-recessive inherited Parkinson's disease (PD). We have exploited our recent discovery that recombinant insect PINK1 is catalytically active to test whether PINK1 directly phosphorylates 15 proteins encoded by PD-associated genes as well as proteins reported to bind PINK1. We have discovered that insect PINK1 efficiently phosphorylates only one of these proteins, namely the E3 ligase Parkin. We have mapped the phosphorylation site to a highly conserved residue within the Ubl domain of Parkin at Ser(65). We show that human PINK1 is specifically activated by mitochondrial membrane potential (Δψm) depolarization, enabling it to phosphorylate Parkin at Ser(65). We further show that phosphorylation of Parkin at Ser(65) leads to marked activation of its E3 ligase activity that is prevented by mutation of Ser(65) or inactivation of PINK1. We provide evidence that once activated, PINK1 autophosphorylates at several residues, including Thr(257), which is accompanied by an electrophoretic mobility band-shift. These results provide the first evidence that PINK1 is activated following Δψm depolarization and suggest that PINK1 directly phosphorylates and activates Parkin. Our findings indicate that monitoring phosphorylation of Parkin at Ser(65) and/or PINK1 at Thr(257) represent the first biomarkers for examining activity of the PINK1-Parkin signalling pathway in vivo. Our findings also suggest that small molecule activators of Parkin that mimic the effect of PINK1 phosphorylation may confer therapeutic benefit for PD.     

PINK1- and PARK2-mediated local mitophagy in distal neuronal axons

Mutations in the PINK1 and PARK2/PARKIN genes are associated with hereditary early onset Parkinson disease (PD), and in cell lines the corresponding gene products play a critical role in mitophagic clearance of damaged mitochondria. In neurons, however, where the extraordinary cellular shapes pose particular challenges for maintaining healthy mitochondria, the pathways of mitophagy are less well understood. Both the location at which mitophagy occurs and the involvement of PINK1 and PARK2 have been controversial. Here we review our recent study where we found that selective damage to a subset of axonal mitochondria causes them to be engulfed within autophagosomes and cleared locally within the axon without the need for transport back to the soma. We also found this process to be completely dependent on neuronal PINK1 and PARK2.     

OGT Regulates Hematopoietic Stem Cell Maintenance via PINK1-Dependent Mitophagy

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An invisible ubiquitin conformation is required for efficient phosphorylation by PINK1

The Ser/Thr protein kinase PINK1 phosphorylates the well‐folded, globular protein ubiquitin (Ub) at a relatively protected site, Ser65. We previously showed that Ser65 phosphorylation results in a conformational change in which Ub adopts a dynamic equilibrium between the known, common Ub conformation and a distinct, second conformation wherein the last β‐strand is retracted to extend the Ser65 loop and shorten the C‐terminal tail. We show using chemical exchange saturation transfer (CEST) nuclear magnetic resonance experiments that a similar, C‐terminally retracted (Ub‐CR) conformation also exists at low population in wild‐type Ub. Point mutations in the moving β5 and neighbouring β‐strands shift the Ub/Ub‐CR equilibrium. This enabled functional studies of the two states, and we show that while the Ub‐CR conformation is defective for conjugation, it demonstrates improved binding to PINK1 through its extended Ser65 loop, and is a superior PINK1 substrate. Together our data suggest that PINK1 utilises a lowly populated yet more suitable Ub‐CR conformation of Ub for efficient phosphorylation. Our findings could be relevant for many kinases that phosphorylate residues in folded protein domains.     

PINK1 is recruited to mitochondria with parkin and associates with LC3 in mitophagy

Mutations in PTEN-induced putative kinase 1 (PINK1) cause recessive form of Parkinson’s disease (PD). PINK1 acts upstream of parkin, regulating mitochondrial integrity and functions. Here, we show that PINK1 in combination with parkin results in the perinuclear mitochondrial aggregation followed by their elimination. This elimination is reduced in cells expressing PINK1 mutants with wild-type parkin. Although wild-type PINK1 localizes in aggregated mitochondria, PINK1 mutants localization remains diffuse and mitochondrial elimination is not observed. This phenomenon is not observed in autophagy-deficient cells. These results suggest that mitophagy controlled by the PINK1/parkin pathway might be associated with PD pathogenesis.

Structured summary

MINT-7557195: PINK1 (uniprotkb:Q9BXM7) physically interacts (MI:0915) with LC3 (uniprotkb:Q9GZQ8) by anti tag coimmunoprecipitation (MI:0007)MINT-7557109: LC3 (uniprotkb:Q9GZQ8) and PINK1 (uniprotkb:Q9BXM7) colocalize (MI:0403) by fluorescence microscopy (MI:0416)MINT-7557121: tom20 (uniprotkb:Q15388) and PINK1 (uniprotkb:Q9BXM7) colocalize (MI:0403) by fluorescence microscopy (MI:0416)MINT-7557138: parkin (uniprotkb:O60260), PINK1 (uniprotkb:Q9BXM7) and tom20 (uniprotkb:Q15388) colocalize (MI:0403) by fluorescence microscopy (MI:0416)MINT-7557173: LC3 (uniprotkb:Q9GZQ8) physically interacts (MI:0915) with PINK1 (uniprotkb:Q9BXM7) by anti bait coimmunoprecipitation (MI:0006)