Functional interplay between Parkin and Drp1 in mitochondrial fission and clearance

Autosomal recessive early-onset Parkinson's disease is most often caused by mutations in the genes encoding the cytosolic E3 ubiquitin ligase Parkin and the mitochondrial serine/threonine kinase PINK1. Studies in Drosophila models and mammalian cells have demonstrated that these proteins regulate various aspects of mitochondrial physiology, including organelle transport, dynamics and turnover. How PINK1 and Parkin orchestrate these processes, and whether they always do so within a common pathway remain to be clarified.We have revisited the role of PINK1 and Parkin in mitochondrial dynamics, and explored its relation to the mitochondrial clearance program controlled by these proteins. We show that PINK1 and Parkin promote Drp1-dependent mitochondrial fission by mechanisms that are at least in part independent. Parkin-mediated mitochondrial fragmentation was abolished by treatments interfering with the calcium/calmodulin/calcineurin signaling pathway, suggesting that it requires dephosphorylation of serine 637 of Drp1. Parkinson's disease-causing mutations with differential impact on mitochondrial morphology and organelle degradation demonstrated that the pro-fission effect of Parkin is not required for efficient mitochondrial clearance. In contrast, the use of Förster energy transfer imaging microscopy revealed that Drp1 and Parkin are co-recruited to mitochondria in proximity of PINK1 following mitochondrial depolarization, indicating spatial coordination between these events in mitochondrial degradation. Our results also hint at a major role of the outer mitochondrial adaptor MiD51 in Drp1 recruitment and Parkin-dependent mitophagy. Altogether, our observations provide new insight into the mechanisms underlying the regulation of mitochondrial dynamics by Parkin and its relation to the mitochondrial clearance program mediated by the PINK1/Parkin pathway.     

The Mitochondrial Fusion-Promoting Factor Mitofusin Is a Substrate of the PINK1/Parkin Pathway

Loss-of-function mutations in the PINK1 or parkin genes result in recessive heritable forms of parkinsonism. Genetic studies of Drosophila orthologs of PINK1 and parkin indicate that PINK1, a mitochondrially targeted serine/threonine kinase, acts upstream of Parkin, a cytosolic ubiquitin-protein ligase, to promote mitochondrial fragmentation, although the molecular mechanisms by which the PINK1/Parkin pathway promotes mitochondrial fragmentation are unknown. We tested the hypothesis that PINK1 and Parkin promote mitochondrial fragmentation by targeting core components of the mitochondrial morphogenesis machinery for ubiquitination. We report that the steady-state abundance of the mitochondrial fusion-promoting factor Mitofusin (dMfn) is inversely correlated with the activity of PINK1 and Parkin in Drosophila. We further report that dMfn is ubiquitinated in a PINK1- and Parkin-dependent fashion and that dMfn co-immunoprecipitates with Parkin. By contrast, perturbations of PINK1 or Parkin did not influence the steady-state abundance of the mitochondrial fission-promoting factor Drp1 or the mitochondrial fusion-promoting factor Opa1, or the subcellular distribution of Drp1. Our findings suggest that dMfn is a direct substrate of the PINK1/Parkin pathway and that the mitochondrial morphological alterations and tissue degeneration phenotypes that derive from mutations in PINK1 and parkin result at least in part from reduced ubiquitin-mediated turnover of dMfn.     

Loss of Parkin or PINK1 Function Increases Drp1-dependent Mitochondrial Fragmentation

Loss-of-function mutations in the parkin gene (PARK2) and PINK1 gene (PARK6) are associated with autosomal recessive parkinsonism. PINK1 deficiency was recently linked to mitochondrial pathology in human cells and Drosophila melanogaster, which can be rescued by parkin, suggesting that both genes play a role in maintaining mitochondrial integrity. Here we demonstrate that an acute down-regulation of parkin in human SH-SY5Y cells severely affects mitochondrial morphology and function, a phenotype comparable with that induced by PINK1 deficiency. Alterations in both mitochondrial morphology and ATP production caused by either parkin or PINK1 loss of function could be rescued by the mitochondrial fusion proteins Mfn2 and OPA1 or by a dominant negative mutant of the fission protein Drp1. Both parkin and PINK1 were able to suppress mitochondrial fragmentation induced by Drp1. Moreover, in Drp1-deficient cells the parkin/PINK1 knockdown phenotype did not occur, indicating that mitochondrial alterations observed in parkin- or PINK1-deficient cells are associated with an increase in mitochondrial fission. Notably, mitochondrial fragmentation is an early phenomenon upon PINK1/parkin silencing that also occurs in primary mouse neurons and Drosophila S2 cells. We propose that the discrepant findings in adult flies can be explained by the time of phenotype analysis and suggest that in mammals different strategies may have evolved to cope with dysfunctional mitochondria.Many lines of evidence suggest that mitochondrial dysfunction plays a central role in the pathogenesis of Parkinson disease, starting from the early observation that the complex I inhibitor 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine induced acute and irreversible parkinsonism in young drug addicts (for review, see Refs. 1–3). In support of a crucial role of mitochondria in Parkinson disease, several Parkinson disease-associated gene products directly or indirectly impinge on mitochondrial integrity (for review, see Refs. 4–6). A clear link between Parkinson disease genes and mitochondria has recently emerged from studies on PINK1 (PTEN-induced putative kinase 1), a mitochondrial serine/threonine kinase, and parkin, a cytosolic E3 ubiquitin ligase. Drosophila parkin null mutants displayed reduced life span, male sterility, and locomotor defects due to apoptotic flight muscle degeneration (7). The earliest manifestation of muscle degeneration and defective spermatogenesis was mitochondrial pathology, exemplified by swollen mitochondria and disintegrated cristae. Remarkably, Drosophila PINK1 null mutants shared marked phenotypic similarities with parkin mutants, and parkin could compensate for the PINK1 loss-of-function phenotype but not vice versa, leading to the conclusion that PINK1 and parkin function in a common genetic pathway with parkin acting downstream of PINK1 (8–10). We recently demonstrated that PINK1 deficiency in cultured human cells causes alterations in mitochondrial morphology, which can be rescued by wild type parkin but not by pathogenic parkin mutants (11). We now present evidence that parkin plays an essential role in maintaining mitochondrial integrity. RNAi³-mediated knockdown of parkin increases mitochondrial fragmentation and decreases cellular ATP production. Notably, mitochondrial fragmentation induced by PINK1/parkin deficiency is observed not only in human neuroblastoma cells but also in primary mouse neurons and insect S2 cells. Alterations in mitochondrial morphology are early manifestations of parkin/PINK1 silencing that are not caused by an increase in apoptosis. The mitochondrial phenotype observed in parkin- or PINK1-deficient cells can morphologically and functionally be rescued by the increased expression of a dominant negative mutant of the fission-promoting protein Drp1. Moreover, manifestation of the PINK1/parkin knockdown phenotype is dependent on Drp1 expression, indicating that an acute loss of parkin or PINK1 function increases mitochondrial fission.     

PINK1/Parkin direct mitochondria to autophagy

Mutations in PTEN-induced putative kinase 1 (PINK1) and PARK2/Parkin cause autosomal recessive forms of Parkinson disease. In mammalian cells, cytosolic Parkin is selectively recruited to depolarized mitochondria, followed by a stimulation of mitochondrial autophagy. We show that Parkin translocation to mitochondria is mediated by PINK1, even in cells with normal mitochondrial membrane potential (ΔΨ_m). Once at the mitochondria, Parkin is in close proximity to PINK1, but Parkin does not catalyze PINK1 ubiquitination nor does PINK1 phosphorylate Parkin. However, co-overexpression of Parkin and PINK1 collapses the normal tubular mitochondrial network into large mitochondrial perinuclear clusters, many of which are surrounded by autophagic vacuoles. Our results suggest that Parkin and PINK1 modulate mitochondrial trafficking to the perinuclear region, a subcellular area associated with autophagy. Mutations in either Parkin or PINK1 impair this process and, consequently, mitochondrial turnover may be altered, inducing accumulation of defective mitochondria and, ultimately, causing neurodegeneration in Parkinson disease.     

Mitochondrial autophagy as a compensatory response to PINK1 deficiency

Macroautophagy (hereafter, autophagy) plays a critical role in maintaining cellular homeostasis by degrading protein aggregates and dysfunctional/damaged organelles. We recently reported that silencing the recessive familial Parkinson disease gene encoding PTEN-induced kinase 1 (PINK1) leads to neuronal cell death accompanied by mitochondrial dysfunction and Drp1-dependent fragmentation. In this model, mitochondrial fission and Beclin 1-dependent autophagy play protective roles, cooperating to sequester and eliminate damaged mitochondria. We discuss the role of superoxide and other reactive oxygen species upstream of mitochondrial depolarization, fission, and autophagy in PINK1 knockdown lines. PINK1 deficiency appears to trigger several compensatory responses that together facilitate clearance of depolarized mitochondria, through a mechanism that is further enhanced by increased expression of parkin. These data offer additional insights that broaden the spectrum of potential interactions between PINK1 and parkin with respect to the regulation of mitochondrial homeostasis and mitophagy.     

Characterization of PINK1 processing, stability, and subcellular localization

Mutations found in PTEN-induced putative kinase 1 (PINK1), a putative mitochondrial serine/threonine kinase of unknown function, have been linked to autosomal recessive Parkinson's disease. It is suggested that mutations can cause a loss of PINK1 kinase activity and eventually lead to mitochondrial dysfunction. In this report, we examined the subcellular localization of PINK1 and the dynamic kinetics of PINK1 processing and degradation. We also identified cytosolic chaperone heat-shock protein 90 (Hsp90) as an interacting protein of PINK1 by PINK1 co-immunoprecipitation. Immunofluorescence of PINK1 protein and mitochondrial isolation show that the precursor form of PINK1 translocates to the mitochondria and is processed into two cleaved forms of PINK1, which in turn localize more to the cytosolic than mitochondrial fraction. The cleavage does not occur and the uncleaved precursor stays associated with the mitochondria when the mitochondrial membrane potential is disrupted. Metabolic labeling analyses show that the PINK1 processing is rapid and the levels of cleaved forms are tightly regulated. Furthermore, cleaved forms of PINK1 are stabilized by Hsp90 interaction as the loss of Hsp90 activity decreases PINK1 level after mitochondrial processing. Lastly, we also find that cleaved forms of PINK1 are degraded by the proteasome, which is uncommon for mitochondrial proteins. Our findings support a dual subcellular localization, implying that PINK1 can reside in the mitochondria and the cytosol. This raises intriguing functional roles that bridge these two cellular compartments.     

6-Hydroxydopamine (6-OHDA) induces Drp1-dependent mitochondrial fragmentation in SH-SY5Y cells

Mitochondrial alterations have been associated with the cytotoxic effect of 6-hydroxydopamine (6-OHDA), a widely used neurotoxin to study Parkinson's disease. Herein we studied the potential effects of 6-OHDA on mitochondrial morphology in SH-SY5Y neuroblastoma cells. By immunofluorescence and time-lapse fluorescence microscopy we demonstrated that 6-OHDA induced profound mitochondrial fragmentation in SH-SY5Y cells, an event that was similar to mitochondrial fission induced by overexpression of Fis1p, a membrane adaptor for the dynamin-related protein 1 (DLP1/Drp1). 6-OHDA failed to induce any changes in peroxisome morphology. Biochemical experiments revealed that 6-OHDA-induced mitochondrial fragmentation is an early event preceding the collapse of the mitochondrial membrane potential and cytochrome c release in SH-SY5Y cells. Silencing of DLP1/Drp1, which is involved in mitochondrial and peroxisomal fission, prevented 6-OHDA-induced fragmentation of mitochondria. Furthermore, in cells silenced for Drp1, 6-OHDA-induced cell death was reduced, indicating that a block in mitochondrial fission protects SH-SY5Y cells against 6-OHDA toxicity. Experiments in mouse embryonic fibroblasts deficient in Bax or p53 revealed that both proteins are not essential for 6-OHDA-induced mitochondrial fragmentation. Our data demonstrate for the first time an involvement of mitochondrial fragmentation and Drp1 function in 6-OHDA-induced apoptosis.     

The Parkinson's disease‐associated gene PINK1 protects neurons from ischemic damage by decreasing mitochondrial translocation of the fission promoter Drp1

Our previous study has shown that PTEN‐induced novel kinase 1 (PINK1) knocking down significantly induced mitochondrial fragmentation. Although PINK1 is proved to be associated with autosomal recessive parkinsonism and its function in this chronic pathological process is widely studied, its role in acute energy crisis such as ischemic stroke is poorly known. In this study by employing an oxygen–glucose deprivation (OGD) neuronal model, we explored the function of PINK1 in cerebral ischemia. Human PINK1, two PINK1 mutants W437X and K219M, or Pink1 shRNA were transduced before OGD using lentiviral delivery. Our results showed that over‐expression of wild‐type PINK1 significantly ameliorated OGD induced cell death and energy disturbance including reduced ATP generation and collapse of mitochondrial membrane potential. PINK1 over‐expression also reversed OGD increased mitochondrial fragmentation, and suppressed the translocation of the mitochondrial fission protein dynamin‐related protein 1 (Drp1) from the cytosol to the mitochondria. Transduction of the mutant PINK1 failed to provide any protective effect, while knockdown of Pink1 significantly increased the severity of OGD‐induced neuronal damage. Importantly, inhibition of Drp1 reversed the effects of knocking down Pink1 on neuronal death and ATP production in response to OGD. This study demonstrates that PINK1 prevents ischemic damage in neurons by attenuating mitochondrial translocation of Drp1, which maintains mitochondrial function and inhibits ischemia‐induced mitochondrial fission. These novel findings implicate a pivotal role of PINK1 regulated mitochondrial dynamics in the pathology of ischemic stroke.

     

Endophilin B1 is required for the maintenance of mitochondrial morphology

We report that a fatty acyl transferase, endophilin B1, is required for maintenance of mitochondrial morphology. Down-regulation of this protein or overexpression of endophilin B1 lacking the NH(2)-terminal lipid-modifying domain causes striking alterations of the mitochondrial distribution and morphology. Dissociation of the outer mitochondrial membrane compartment from that of the matrix, and formation of vesicles and tubules of outer mitochondrial membrane, was also observed in both endophilin B1 knockdown cells and after overexpression of the truncated protein, indicating that endophilin B1 is required for the regulation of the outer mitochondrial membrane dynamics. We also show that endophilin B1 translocates to the mitochondria during the synchronous remodeling of the mitochondrial network that has been described to occur during apoptosis. Double knockdown of endophilin B1 and Drp1 leads to a mitochondrial phenotype identical to that of the Drp1 single knockdown, a result consistent with Drp1 acting upstream of endophilin B1 in the maintenance of morphological dynamics of mitochondria.     

PARK6 PINK1 mutants are defective in maintaining mitochondrial membrane potential and inhibiting ROS formation of substantia nigra dopaminergic neurons

Mutations in PTEN-induced kinase 1 (PINK1) gene cause recessive familial type 6 of Parkinson's disease (PARK6). PINK1 is believed to exert neuroprotective effect on SN dopaminergic cells by acting as a mitochondrial Ser/Thr protein kinase. Autosomal recessive inheritance indicates the involvement of loss of PINK1 function in PARK6 pathogenesis. In the present study, confocal imaging of cultured SN dopaminergic neurons prepared from PINK1 knockout mice was performed to investigate physiological importance of PINK1 in maintaining mitochondrial membrane potential (ΔΨ_m) and mitochondrial morphology and test the hypothesis that PARK6 mutations cause the loss of PINK1 function. PINK1-deficient SN dopaminergic neurons exhibited a depolarized ΔΨ_m. In contrast to long thread-like mitochondria of wild-type neurons, fragmented mitochondria were observed from PINK1-null SN dopaminergic cells. Basal level of mitochondrial superoxide and oxidative stressor H₂O₂-induced ROS generation were significantly increased in PINK1-deficient dopaminergic neurons. Overexpression of wild-type PINK1 restored hyperpolarized ΔΨ_m and thread-like mitochondrial morphology and inhibited ROS formation in PINK1-null dopaminergic cells. PARK6 mutant (G309D), (E417G) or (CΔ145) PINK1 failed to rescue mitochondrial dysfunction and inhibit oxidative stress in PINK1-deficient dopaminergic neurons. Mitochondrial toxin rotenone-induced cell death of dopaminergic neurons was augmented in PINK1-null SN neuronal culture. These results indicate that PINK1 is required for maintaining normal ΔΨ_m and mitochondrial morphology of cultured SN dopaminergic neurons and exerts its neuroprotective effect by inhibiting ROS formation. Our study also provides the evidence that PARK6 mutant (G309D), (E417G) or (CΔ145) PINK1 is defective in regulating mitochondrial functions and attenuating ROS production of SN dopaminergic cells.     

The PINK1-Parkin pathway is involved in the regulation of mitochondrial remodeling process

The two Parkinson’s disease (PD) genes, PTEN-induced kinase 1 (PINK1) and parkin, are linked in a common pathway which affects mitochondrial integrity and function. However, it is still not known what this pathway does in the mitochondria. Therefore, we investigated its physiological function in Drosophila. Because Drosophila PINK1 and parkin mutants show changes in mitochondrial morphology in both indirect flight muscles and dopaminergic neurons, we here investigated whether the PINK1-Parkin pathway genetically interacts with the regulators of mitochondrial fusion and fission such as Drp1, which promotes mitochondrial fission, and Opa1 or Marf, which induces mitochondrial fusion. Surprisingly, DrosophilaPINK1 and parkin mutant phenotypes were markedly suppressed by overexpression of Drp1 or downregulation of Opa1 or Marf, indicating that the PINK1-Parkin pathway regulates mitochondrial remodeling process in the direction of promoting mitochondrial fission. Therefore, we strongly suggest that mitochondrial fusion and fission process could be a prominent therapeutic target for the treatment of PD.     

Mitochondrial Safeguard: a stress response that offsets extreme fusion and protects respiratory function via flickering‐induced Oma1 activation

The connectivity of mitochondria is regulated by a balance between fusion and division. Many human diseases are associated with excessive mitochondrial connectivity due to impaired Drp1, a dynamin‐related GTPase that mediates division. Here, we report a mitochondrial stress response, named mitochondrial safeguard, that adjusts the balance of fusion and division in response to increased mitochondrial connectivity. In cells lacking Drp1, mitochondria undergo hyperfusion. However, hyperfusion does not completely connect mitochondria because Opa1 and mitofusin 1, two other dynamin‐related GTPases that mediate fusion, become proteolytically inactivated. Pharmacological and genetic experiments show that the activity of Oma1, a metalloprotease that cleaves Opa1, is regulated by short pulses of the membrane depolarization without affecting the overall membrane potential in Drp1‐knockout cells. Re‐activation of Opa1 and Mitofusin 1 in Drp1‐knockout cells further connects mitochondria beyond hyperfusion, termed extreme fusion, leading to bioenergetic deficits. These findings reveal an unforeseen safeguard mechanism that prevents extreme fusion of mitochondria, thereby maintaining mitochondrial function when the balance is shifted to excessive connectivity.     

A Human Dynamin-related Protein Controls the Distribution of Mitochondria

Mitochondria exist as a dynamic tubular network with projections that move, break, and reseal in response to local environmental changes. We present evidence that a human dynamin-related protein (Drp1) is specifically required to establish this morphology. Drp1 is a GTPase with a domain structure similar to that of other dynamin family members. To identify the function of Drp1, we transiently transfected cells with mutant Drp1. A mutation in the GTPase domain caused profound alterations in mitochondrial morphology. The tubular projections normally present in wild-type cells were retracted into large perinuclear aggregates in cells expressing mutant Drp1. The morphology of other organelles was unaffected by mutant Drp1. There was also no effect of mutant Drp1 on the transport functions of the secretory and endocytic pathways. By EM, the mitochondrial aggregates found in cells that were transfected with mutant Drp1 appear as clusters of tubules rather than a large mass of coalescing membrane. We propose that Drp1 is important for distributing mitochondrial tubules throughout the cell. The function of this new dynamin-related protein in organelle morphology represents a novel role for a member of the dynamin family of proteins.     

S6 kinase 1 plays a key role in mitochondrial morphology and cellular energy flow

Mitochondrial morphology, which is associated with changes in metabolism, cell cycle, cell development and cell death, is tightly regulated by the balance between fusion and fission. In this study, we found that S6 kinase 1 (S6K1) contributes to mitochondrial dynamics, homeostasis and function. Mouse embryo fibroblasts lacking S6K1 (S6K1-KO MEFs) exhibited more fragmented mitochondria and a higher level of Dynamin related protein 1 (Drp1) and active Drp1 (pS616) in both whole cell extracts and mitochondrial fraction. In addition, there was no evidence for autophagy and mitophagy induction in S6K1 depleted cells. Glycolysis and mitochondrial respiratory activity was higher in S6K1-KO MEFs, whereas OxPhos ATP production was not altered. However, inhibition of Drp1 by Mdivi1 (Drp1 inhibitor) resulted in higher OxPhos ATP production and lower mitochondrial membrane potential. Taken together the depletion of S6K1 increased Drp1-mediated fission, leading to the enhancement of glycolysis. The fission form of mitochondria resulted in lower yield for OxPhos ATP production as well as in higher mitochondrial membrane potential. Thus, these results have suggested a potential role of S6K1 in energy metabolism by modulating mitochondrial respiratory capacity and mitochondrial morphology.     

Genome-wide RNAi screen identifies ATPase inhibitory factor 1 (ATPIF1) as essential for PARK2 recruitment and mitophagy

Mitochondrial dysfunction is a hallmark of aging and numerous human diseases, including Parkinson disease (PD). Multiple homeostatic mechanisms exist to ensure mitochondrial integrity, including the selective autophagic program mitophagy, that is activated during starvation or in response to mitochondrial dysfunction. Following prolonged loss of potential across the inner mitochondrial membrane (ΔΨ), PTEN-induced putative kinase 1 (PINK1) and the E3-ubiquitin ligase PARK2 work in the same pathway to trigger mitophagy of dysfunctional mitochondria. Mutations in PINK1 and PARK2, as well as PARK7/DJ-1, underlie autosomal recessive Parkinsonism and impair mitochondrial function and morphology. In a genome-wide RNAi screen searching for genes that are required for PARK2 translocation to the mitochondria, we identified ATPase inhibitory factor 1 (ATPIF1/IF1) as essential for PARK2 recruitment and mitophagy in cultured cells. During uncoupling, ATPIF1 promotes collapse of ΔΨ and activation of the PINK-PARK2 mitophagy pathway by blocking the ATPase activity of the F₁-F_o ATP synthase. Restoration of ATPIF1 in Rho0 cells, which lack mtDNA and a functional electron transport chain, lowers ΔΨ and triggers PARK2 recruitment. Our findings identified ATPIF1 and the ATP synthase as novel components of the PINK1-PARK2 mitophagy pathway and provide genetic evidence that loss of ΔΨ is an essential trigger for mitophagy.     

Drp1的调控对PINK1突变转基因果蝇的保护作用

帕金森病（PD）是以黑质致密部多巴胺神经元选择性减少和胞浆内路易小体的形成为特征的神经退行性疾病。研究发现,PTEN诱导激酶1(PINK1)基因突变导致家族性早发型帕金森病的发生。在转基因果蝇中,PINK1功能丢失导致间接飞行肌缺陷,线粒体结构、功能障碍,多巴胺神经元丢失。本研究在PINK1突变PD转基因果蝇中,进行发动蛋白相关蛋白1（Drp1）过表达和敲低,探索Drp1对PD转基因果蝇的保护作用及其可能机制。本研究选用MHC-Gal4/UAS系统的PD转基因果蝇模型,特异性启动PINK1B9基因于果蝇肌肉组织中表达;运用Drp1基因过表达和RNA干扰干预PINK1B9转基因果蝇,研究其对PD转基因果蝇的作用。结果显示,不论过表达Drp1还是Drp1敲低均可挽救PINK1突变转基因果蝇,降低翅膀异常率,改善飞行能力,恢复间接飞行肌排列,调节线粒体形态,提高ATP生成量,上调NDUFS3蛋白表达水平。本文结果提示,Drp1的调控挽救PINK1突变转基因果蝇与线粒体呼吸链有关。     

膜蛋白ATAD3A在线粒体质量控制中的作用

线粒体质量控制对于线粒体网络的稳态和线粒体功能的正常发挥具有重要意义。三磷酸腺苷酶家族蛋白3A(ATAD3A)是同时参与调节线粒体结构功能、线粒体动力学和线粒体自噬等重要生物学过程的线粒体膜蛋白之一。近期研究表明,ATAD3A既可与Mic60/Mitofilin和线粒体转录因子A (TFAM)等因子相互作用以维持线粒体嵴的形态和氧化磷酸化功能,又能与发动蛋白相关蛋白1 (Drp1)结合而正性/负性调节线粒体分裂,还可作为线粒体外膜转位酶（TOM）复合物和线粒体内膜转位酶（TIM）复合物之间的桥接因子而介导PTEN诱导激酶（PINK1）输入线粒体进行加工,显示出促自噬或抗自噬活性。本文对ATAD3A在调控线粒体质量控制中的作用及其机制进行了综述。     

Role of the lipid transport protein StarD7 in mitochondrial dynamics

Mitochondria are dynamic organelles crucial for cell function and survival implicated in oxidative energy production whose central functions are tightly controlled by lipids. StarD7 is a lipid transport protein involved in the phosphatidylcholine (PC) delivery to mitochondria. Previous studies have shown that StarD7 knockdown induces alterations in mitochondria and endoplasmic reticulum (ER) with a reduction in PC content, however whether StarD7 modulates mitochondrial dynamics remains unexplored. Here, we generated HTR-8/SVneo stable cells expressing the precursor StarD7.I and the mature processed StarD7.II isoforms. We demonstrated that StarD7.I overexpression altered mitochondrial morphology increasing its fragmentation, whereas no changes were observed in StarD7.II-overexpressing cells compared to the control (Ct) stable cells. StarD7.I (D7.I) stable cells were able to transport higher fluorescent PC analog to mitochondria than Ct cells, yield mitochondrial fusions, maintained the membrane potential, and produced lower levels of reactive oxygen species (ROS). Additionally, the expression of Dynamin Related Protein 1 (Drp1) and Mitofusin (Mfn2) proteins were increased, whereas the amount of Mitofusin 1 (Mfn1) decreased. Moreover, transfections with plasmids encoding Drp1-K38A, Drp1-S637D or Drp1-S637A mutants indicated that mitochondrial fragmentation in D7.I cells occurs in a fission-dependent manner via Drp1. In contrast, StarD7 silencing decreased Mfn1 and Mfn2 fusion proteins without modification of Drp1 protein level. These cells increased ROS levels and presented donut-shape mitochondria, indicative of metabolic stress. Altogether our findings provide novel evidence indicating that alterations in StarD7.I expression produce significant changes in mitochondrial morphology and dynamics.     

PINK1 protects against oxidative stress by phosphorylating mitochondrial chaperone TRAP1

Mutations in the PTEN induced putative kinase 1 (PINK1) gene cause an autosomal recessive form of Parkinson disease (PD). So far, no substrates of PINK1 have been reported, and the mechanism by which PINK1 mutations lead to neurodegeneration is unknown. Here we report the identification of TNF receptor-associated protein 1 (TRAP1), a mitochondrial molecular chaperone also known as heat shock protein 75 (Hsp75), as a cellular substrate for PINK1 kinase. PINK1 binds and colocalizes with TRAP1 in the mitochondria and phosphorylates TRAP1 both in vitro and in vivo. We show that PINK1 protects against oxidative-stress-induced cell death by suppressing cytochrome c release from mitochondria, and this protective action of PINK1 depends on its kinase activity to phosphorylate TRAP1. Moreover, we find that the ability of PINK1 to promote TRAP1 phosphorylation and cell survival is impaired by PD-linked PINK1 G309D, L347P, and W437X mutations. Our findings suggest a novel pathway by which PINK1 phosphorylates downstream effector TRAP1 to prevent oxidative-stress-induced apoptosis and implicate the dysregulation of this mitochondrial pathway in PD pathogenesis.     

PINK1-Parkin Pathway Activity Is Regulated by Degradation of PINK1 in the Mitochondrial Matrix

Loss-of-function mutations in PINK1, which encodes a mitochondrially targeted serine/threonine kinase, result in an early-onset heritable form of Parkinson''s disease. Previous work has shown that PINK1 is constitutively degraded in healthy cells, but selectively accumulates on the surface of depolarized mitochondria, thereby initiating their autophagic degradation. Although PINK1 is known to be a cleavage target of several mitochondrial proteases, whether these proteases account for the constitutive degradation of PINK1 in healthy mitochondria remains unclear. To explore the mechanism by which PINK1 is degraded, we performed a screen for mitochondrial proteases that influence PINK1 abundance in the fruit fly Drosophila melanogaster. We found that genetic perturbations targeting the matrix-localized protease Lon caused dramatic accumulation of processed PINK1 species in several mitochondrial compartments, including the matrix. Knockdown of Lon did not decrease mitochondrial membrane potential or trigger activation of the mitochondrial unfolded protein stress response (UPR^mt), indicating that PINK1 accumulation in Lon-deficient animals is not a secondary consequence of mitochondrial depolarization or the UPR^mt. Moreover, the influence of Lon on PINK1 abundance was highly specific, as Lon inactivation had little or no effect on the abundance of other mitochondrial proteins. Further studies indicated that the processed forms of PINK1 that accumulate upon Lon inactivation are capable of activating the PINK1-Parkin pathway in vivo. Our findings thus suggest that Lon plays an essential role in regulating the PINK1-Parkin pathway by promoting the degradation of PINK1 in the matrix of healthy mitochondria.