SREBF1 links lipogenesis to mitophagy and sporadic Parkinson disease

Mitochondrial quality control has an impact on many diseases, but intense research has focused on the action of 2 genes linked to heritable forms of Parkinson disease (PD), PINK1 and PARK2/parkin, which act in a common pathway to promote mitophagy. However, criticism has been raised that little evidence links this mechanism to sporadic PD. To gain a greater insight into the mechanisms of PINK1-PARK2 mediated mitophagy, we undertook a genome-wide RNAi screen in Drosophila and human cell models. Strikingly, we discovered several components of the lipogenesis pathway, including SREBF1, playing a conserved role in mitophagy. Our results suggest that lipids influence the stabilization of PINK1 during the initiation of mitophagy. Importantly, SREBF1 has previously been identified as a risk locus for sporadic PD, and thus implicates aberrant mitophagy as contributing to sporadic PD. Our findings suggest a role for lipid synthesis in PINK1-PARK2 mediated mitophagy, and propose a mechanistic link between familial and sporadic PD, supporting a common etiology.     

Characterization of PINK1 (PTEN-induced Putative Kinase 1) Mutations Associated with Parkinson Disease in Mammalian Cells and Drosophila

Mutations in PINK1 (PTEN-induced putative kinase 1) are tightly linked to autosomal recessive Parkinson disease (PD). Although more than 50 mutations in PINK1 have been discovered, the role of these mutations in PD pathogenesis remains poorly understood. Here, we characterized 17 representative PINK1 pathogenic mutations in both mammalian cells and Drosophila. These mutations did not affect the typical cleavage patterns and subcellular localization of PINK1 under both normal and damaged mitochondria conditions in mammalian cells. However, PINK1 mutations in the kinase domain failed to translocate Parkin to mitochondria and to induce mitochondrial aggregation. Consistent with the mammalian data, Drosophila PINK1 mutants with mutations in the kinase domain (G426D and L464P) did not genetically interact with Parkin. Furthermore, PINK1-null flies expressing the transgenic G426D mutant displayed defective phenotypes with increasing age, whereas L464P mutant-expressing flies exhibited the phenotypes at an earlier age. Collectively, these results strongly support the hypothesis that the kinase activity of PINK1 is essential for its function and for regulating downstream Parkin functions in mitochondria. We believe that this study provides the basis for understanding the molecular and physiological functions of various PINK1 mutations and provides insights into the pathogenic mechanisms of PINK1-linked 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.     

Parkin、PINK1、DJ-1和线粒体功能障碍与帕金森病

帕金森病(Parkinson’s disese,PD)是一种常见的神经退行性疾病,但到目前为止发病机制尚不明确,环境和遗传等因素与其发病有密切关系。研究表明,蛋白质异常积聚(泛素/蛋白酶体途径)和线粒体氧化损伤(线粒体途径),可能是导致PD患者发病的关键分子机制。Parkin、PINK1和DJ-1等基因突变与常染色体隐性的家族性PD有关,这些相关基因编码的蛋白对于维持线粒体形态和功能起着重要的作用。本文将主要从Parkin、PINK1、DJ-1和线粒体功能障碍与帕金森病的关系进行综述。     

Regulation of DNA repair by parkin

Mutation of parkin is one of the most prevalent causes of autosomal recessive Parkinson’s disease (PD). Parkin is an E3 ubiquitin ligase that acts on a variety of substrates, resulting in polyubiquitination and degradation by the proteasome or monoubiquitination and regulation of biological activity. However, the cellular functions of parkin that relate to its pathological involvement in PD are not well understood. Here we show that parkin is essential for optimal repair of DNA damage. Parkin-deficient cells exhibit reduced DNA excision repair that can be restored by transfection of wild-type parkin, but not by transfection of a pathological parkin mutant. Parkin also protects against DNA damage-induced cell death, an activity that is largely lost in the pathological mutant. Moreover, parkin interacts with the proliferating cell nuclear antigen (PCNA), a protein that coordinates DNA excision repair. These results suggest that parkin promotes DNA repair and protects against genotoxicity, and implicate DNA damage as a potential pathogenic mechanism in PD.     

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.     

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.     

A Dimeric PINK1-containing Complex on Depolarized Mitochondria Stimulates Parkin Recruitment

Parkinsonism typified by sporadic Parkinson disease is a prevalent neurodegenerative disease. Mutations in PINK1 (PTEN-induced putative kinase 1), a mitochondrial Ser/Thr protein kinase, or PARKIN, a ubiquitin-protein ligase, cause familial parkinsonism. The accumulation and autophosphorylation of PINK1 on damaged mitochondria results in the recruitment of Parkin, which ultimately triggers quarantine and/or degradation of the damaged mitochondria by the proteasome and autophagy. However, the molecular mechanism of PINK1 in dissipation of the mitochondrial membrane potential (ΔΨm) has not been fully elucidated. Here we show by fluorescence-based techniques that the PINK1 complex formed following a decrease in ΔΨm is composed of two PINK1 molecules and is correlated with intermolecular phosphorylation of PINK1. Disruption of complex formation by the PINK1 S402A mutation weakened Parkin recruitment onto depolarized mitochondria. The most disease-relevant mutations of PINK1 inhibit the complex formation. Taken together, these results suggest that formation of the complex containing dyadic PINK1 is an important step for Parkin recruitment onto damaged mitochondria.     

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.     

New insights into the structure of PINK1 and the mechanism of ubiquitin phosphorylation

Mutations in PINK1 cause early-onset recessive Parkinson’s disease. This gene encodes a protein kinase implicated in mitochondrial quality control via ubiquitin phosphorylation and activation of the E3 ubiquitin ligase Parkin. Here, we review and analyze functional features emerging from recent crystallographic, nuclear magnetic resonance (NMR) and mass spectrometry studies of PINK1. We compare the apo and ubiquitin-bound PINK1 structures and reveal an allosteric switch, regulated by autophosphorylation, which modulates substrate recognition. We critically assess the conformational changes taking place in ubiquitin and the Parkin ubiquitin-like domain in relation to its binding to PINK1. Finally, we discuss the implications of these biophysical findings in our understanding of the role of PINK1 in mitochondrial function, and analyze the potential for structure-based drug design.     

Parkin is an E3 ligase for the ubiquitin-like modifier FAT10, which inhibits Parkin activation and mitophagy

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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.     

miR-182靶向抑制FBXW7表达影响非小细胞肺癌细胞增殖研究

目的:研究microRNA-182(miR-182)在非小细胞肺癌(NSCLC)组织中的表达,并探讨其对NSCLC细胞增殖的影响及作用机制。方法:采用实时荧光定量PCR (qRT-PCR)检测miR-182在11例NSCLC及相应癌旁组织中的表达情况;Western blot检测FBXW7,c-Jun,c-Myc及cyclin D蛋白的表达;将miR-182模拟物,抑制物及相应空白对照瞬时转染H460细胞后,以细胞增殖与活性检测和克隆形成实验检测细胞系的增殖情况;流式细胞术检测细胞周期和凋亡变化;荧光素酶报告基因实验证实miR-182对FBXW7的靶向性作用。结果:NSCLC组织中miR-182的相对表达水平显著高于癌旁组织(P0.05)。转染组与对照组相比,H460细胞生长、克隆形成能力显著增强,细胞周期进程加快,细胞凋亡受到抑制(P0.05)。在NSCLC组织中,FBXW7蛋白的表达水平明显低于癌旁组织(P0.05)。miR-182 mimics显著降低野生型FBXW7质粒荧光素酶的活性,然而将结合位点突变后,miR-182 mimics则不再影响荧光素酶的活性。结论:miR-182在NSCLC组织中高表达,与FBXW7之间存在靶向关系,通过下调FBXW7蛋白表达促进NSCLC细胞的增殖,参与肿瘤的发生发展,预示其可能成为一种潜在的生物标志和治疗靶点。     

Parkin与α-酮戊二酸载体蛋白(OGCP)的相互作用研究

Parkin基因是帕金森病的致病基因之一,Parkin蛋白作为泛素-蛋白酶体通路(ubiquitin-proteasome pathway,UPP)的一种E3酶,介导了多种底物的泛素化过程,而后者与帕金森病的发病机制有着密切的联系．α-酮戊二酸载体蛋白(2-oxoglutarate carrier protein,OGCP) 是一种线粒体内膜蛋白．采用激光共聚焦技术和免疫共沉淀技术证实,在HEK293细胞中Parkin蛋白与OGCP共定位,且二者之间存在相互作用关系;通过体内、外泛素化实验发现Parkin蛋白能介导OGCP的泛素化．提示OGCP可能是Parkin蛋白的泛素化底物蛋白,且Parkin蛋白能促进OGCP的泛素化．     

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.