Parkin-mediated selective mitochondrial autophagy, mitophagy: Parkin purges damaged organelles from the vital mitochondrial network

Cellular homeostasis is linked tightly to mitochondrial functions. Some damage to mitochondrial proteins and nucleic acids can lead to the depolarization of the inner mitochondrial membrane, thereby sensitizing impaired mitochondria for selective elimination by autophagy. Mitochondrial dysfunction is one of the key aspects of the pathobiology of neurodegenerative disease. Parkin, an E3 ligase located in the cytosol and originally discovered as mutated in monogenic forms of Parkinson’s disease (PD), was found recently to translocate specifically to uncoupled mitochondria and to induce their autophagy.     

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。     

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.     

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.     

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.     

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。     

红景天苷通过PINK1-Parkin 通路在MPP+诱导的SH-SY5Y细胞中维持线粒体形态和功能

目的:研究红景天苷(Salidroside,Sal)对在MPP+诱导SH-SY5Y细胞线粒体形态和功能的影响及其机制。方法:采用3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐(3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide,MTT)检测细胞活性,Mito Tracker Red CMXRos进行线粒体染色,四甲基罗丹明乙酯(Tetramethylrhodamine ethyl ester,TMRE)检测线粒体膜电位,Western blot检测PINK1和Parkin蛋白表达水平。结果:单纯Sal处理24 h对细胞活性、线粒体形态和MMP无影响(P0.05)。MPP+(500μM)处理SH-SY5Y细胞24 h后,与正常组比较,细胞活性、MMP水平均降低,线粒体长度减短(P0.01),并发生碎片化。Sal(25μM)预处理24 h可以显著抑制MPP+诱导的细胞活性降低(P0.01),并维持线粒体长度和增加MMP水平(P0.01)。而且,Sal(25μM)预处理24 h可以显著恢复MPP+诱导的PINK1和Parkin蛋白表达水平下降(P0.01)。结论:体外实验证实Sal可以保护MPP+诱导的SH-SY5Y细胞活性降低、线粒体形态和功能异常,而PINK1-Parkin通路可能是其机制之一,为进一步临床开发Sal治疗PD的新药提供实验依据。     

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。     

Lysine 27 Ubiquitination of the Mitochondrial Transport Protein Miro Is Dependent on Serine 65 of the Parkin Ubiquitin Ligase

Mitochondrial transport plays an important role in matching mitochondrial distribution to localized energy production and calcium buffering requirements. Here, we demonstrate that Miro1, an outer mitochondrial membrane (OMM) protein crucial for the regulation of mitochondrial trafficking and distribution, is a substrate of the PINK1/Parkin mitochondrial quality control system in human dopaminergic neuroblastoma cells. Moreover, Miro1 turnover on damaged mitochondria is altered in Parkinson disease (PD) patient-derived fibroblasts containing a pathogenic mutation in the PARK2 gene (encoding Parkin). By analyzing the kinetics of Miro1 ubiquitination, we further demonstrate that mitochondrial damage triggers rapid (within minutes) and persistent Lys-27-type ubiquitination of Miro1 on the OMM, dependent on PINK1 and Parkin. Proteasomal degradation of Miro1 is then seen on a slower time scale, within 2–3 h of the onset of ubiquitination. We find Miro ubiquitination in dopaminergic neuroblastoma cells is independent of Miro1 phosphorylation at Ser-156 but is dependent on the recently identified Ser-65 residue within Parkin that is phosphorylated by PINK1. Interestingly, we find that Miro1 can stabilize phospho-mutant versions of Parkin on the OMM, suggesting that Miro is also part of a Parkin receptor complex. Moreover, we demonstrate that Ser-65 in Parkin is critical for regulating Miro levels upon mitochondrial damage in rodent cortical neurons. Our results provide new insights into the ubiquitination-dependent regulation of the Miro-mediated mitochondrial transport machinery by PINK1/Parkin and also suggest that disruption of this regulation may be implicated in Parkinson disease pathogenesis.     

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.     

Genetic Insights into Sporadic Parkinson's Disease Pathogenesis

Intensive research over the last 15 years has led to the identification of several autosomal recessive and dominantgenes that cause familial Parkinson’s disease (PD). Importantly, the functional characterization of these genes hasshed considerable insights into the molecular mechanisms underlying the etiology and pathogenesis of PD. Collectively;these studies implicate aberrant protein and mitochondrial homeostasis as key contributors to the development of PD, withoxidative stress likely acting as an important nexus between the two pathogenic events. Interestingly, recent genome-wideassociation studies (GWAS) have revealed variations in at least two of the identified familial PD genes (i.e. α-synucleinand LRRK2) as significant risk factors for the development of sporadic PD. At the same time, the studies also uncoveredvariability in novel alleles that is associated with increased risk for the disease. Additionally, in-silico meta-analyses ofGWAS data have allowed major steps into the investigation of the roles of gene-gene and gene-environment interactionsin sporadic PD. The emergent picture from the progress made thus far is that the etiology of sporadic PD is multi-factorialand presumably involves a complex interplay between a multitude of gene networks and the environment. Nonetheless,the biochemical pathways underlying familial and sporadic forms of PD are likely to be shared.     

Parkin mediates proteasome-dependent protein degradation and rupture of the outer mitochondrial membrane

Upon mitochondrial depolarization, Parkin, a Parkinson disease-related E3 ubiquitin ligase, translocates from the cytosol to mitochondria and promotes their degradation by mitophagy, a selective type of autophagy. Here, we report that in addition to mitophagy, Parkin mediates proteasome-dependent degradation of outer membrane proteins such as Tom20, Tom40, Tom70, and Omp25 of depolarized mitochondria. By contrast, degradation of the inner membrane and matrix proteins largely depends on mitophagy. Furthermore, Parkin induces rupture of the outer membrane of depolarized mitochondria, which also depends on proteasomal activity. Upon induction of mitochondrial depolarization, proteasomes are recruited to mitochondria in the perinuclear region. Neither proteasome-dependent degradation of outer membrane proteins nor outer membrane rupture is required for mitophagy. These results suggest that Parkin regulates degradation of outer and inner mitochondrial membrane proteins differently through proteasome- and mitophagy-dependent pathways.     

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.     

帕金森病相关蛋白Parkin 与PINK1的相互作用研究

帕金森病(Parkinson's disease,PD)是常见的神经系统变性疾病．分子遗传学研究发现,突变的Parkin蛋白及PINK1蛋白均参与了帕金森病的致病过程,但二者之间是否存在相互作用以及是否能够相互调节仍不十分清楚．为明确生理状态下Parkin蛋白与PINK1蛋白之间的相互作用,首先运用蛋白体外结合实验(GST pull-down)技术及免疫共沉淀技术证实了Parkin与PINK1在体外及体内均可相互结合．进一步构建PINK1的不同截短型,运用GST pull-down技术验证了PINK1与Parkin相互结合的区段为PINK1的蛋白激酶结构域．免疫细胞化学实验也证实Parkin与PINK1蛋白在细胞中存在共定位．进一步运用免疫共沉淀技术证实Parkin可减少PINK1通过泛素蛋白酶体系统(ubiquitin proteasome system,UPS)的降解,从而稳定PINK1．PINK1可增加Parkin通过UPS的降解,从而减少Parkin的水平,降低其稳定性．这些结果提示,帕金森病相关蛋白Parkin与PINK1能够直接结合,二者通过泛素蛋白酶体降解系统相互调节,可能协同作用参与了帕金森病的致病过程．