A chemical genetic approach to probe the function of PINK1 in regulating mitochondrial dynamics.
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ABSTRACT:
SUBMITTER: Zhang C
PROVIDER: S-EPMC4349247 | biostudies-literature | 2015 Mar
REPOSITORIES: biostudies-literature
Publications
A chemical genetic approach to probe the function of PINK1 in regulating mitochondrial dynamics.
Zhang Conggang C Lee Schuyler S Peng Yinghua Y Bunker Eric E Shen Chong C Giaime Emilie E Shen Jie J Shen Jingshi J Zhou Zongyao Z Liu Xuedong X
Cell research 20141205 3
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Project description: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.
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Project description:Endotoxin-induced acute kidney injury (AKI) is commonly observed in clinical practice. Renal tubular epithelial cell (RTEC) pyroptosis is one of the main factors leading to the development of endotoxin-induced AKI. Mitochondrial dysfunction can lead to pyroptosis. However, the biological pathways involved in the potential lipopolysaccharide (LPS)-induced pyroptosis of RTECs, notably those associated with mitochondrial dysfunction, are poorly understood. Previous studies have demonstrated that heme oxygenase (HO)-1 confers cell protection via the induction of PTEN-induced putative kinase 1 (PINK1) expression through PTEN to regulate mitochondrial fusion/fission during endotoxin-induced AKI in vivo. Therefore, the present study investigated the role of HO-1/PINK1 in maintaining mitochondrial function and inhibiting the pyroptosis of RTECs exposed to LPS. Primary cultures of RTECs were obtained from wild-type (WT) and PINK1-knockout (PINK1KO) rats. An in vitro model of endotoxin-associated RTEC injury was established following treatment of the cells with LPS. The WT RTECs were divided into the control, LPS, Znpp + LPS and Hemin + LPS groups, and the PINK1KO RTECs were divided into the control, LPS and Hemin + LPS groups. RTECs were exposed to LPS for 6 h to assess cell viability, inflammation, pyroptosis and mitochondrial function. In the LPS-treated RTECs, the mRNA and protein expression levels of HO-1 and PINK1 were upregulated. Cell viability, adenosine triphosphate (ATP) levels and the mitochondrial oxygen consumption rate were decreased, whereas the inflammatory response, pyroptosis and mitochondrial reactive oxygen species (ROS) levels were increased. The cell inflammatory response and the induction of pyroptosis were inhibited, whereas the levels of mitochondrial ROS were decreased. In addition, the cell viability and ATP levels were increased in the WT RTECs following the upregulation of HO-1 expression. These effects were reversed by the downregulation of HO-1 expression. However, no statistically significant differences were noted between the LPS and the Hemin + LPS groups in the PINK1KO RTECs. Collectively, the findings of the present study indicate that HO-1 inhibits inflammation and regulates mitochondrial function by inhibiting the pyroptosis of LPS-exposed RTECs via PINK1.
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Project description:PTEN-induced putative kinase 1 (PINK1) and Parkin act in a common pathway to regulate mitochondrial dynamics, the involvement of which in the pathogenesis of Parkinson's disease (PD) is increasingly being appreciated. However, how the PINK1/Parkin pathway influences mitochondrial function is not well understood, and the exact role of this pathway in controlling mitochondrial dynamics remains controversial. Here we used mammalian primary neurons to examine the function of the PINK1/Parkin pathway in regulating mitochondrial dynamics and function. In rat hippocampal neurons, PINK1 or Parkin overexpression resulted in increased mitochondrial number, smaller mitochondrial size and reduced mitochondrial occupancy of neuronal processes, suggesting that the balance of mitochondrial fission/fusion dynamics is tipped toward more fission. Conversely, inactivation of PINK1 resulted in elongated mitochondria, indicating that the balance of mitochondrial fission/fusion dynamics is tipped toward more fusion. Furthermore, overexpression of the fission protein Drp1 (dynamin-related protein 1) or knocking down of the fusion protein OPA1 (optical atrophy 1) suppressed PINK1 RNAi-induced mitochondrial morphological defect, and overexpression of PINK1 or Parkin suppressed the elongated mitochondria phenotype caused by Drp1 RNAi. Functionally, PINK1 knockdown and overexpression had opposite effects on dendritic spine formation and neuronal vulnerability to excitotoxicity. Finally, we found that PINK1/Parkin similarly influenced mitochondrial dynamics in rat midbrain dopaminergic neurons. These results, together with previous findings in Drosophila dopaminergic neurons, indicate that the PINK1/Parkin pathway plays conserved roles in regulating neuronal mitochondrial dynamics and function.
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Project description:Recessive mutations in Pink1 lead to a selective degeneration of dopaminergic neurons in the substantia nigra that is characteristic of Parkinson disease. Pink1 is a kinase that is targeted in part to mitochondria, and loss of Pink1 function can alter mitochondrial morphology and dynamics, thus supporting a link between mitochondrial dysfunction and Parkinson disease etiology. Here, we report the unbiased identification and confirmation of a mitochondrial multiprotein complex that contains Pink1, the atypical GTPase Miro, and the adaptor protein Milton. Our screen also identified an interaction between Pink1 and Mitofilin. Based on previously established functions for Miro and Milton in the trafficking of mitochondria along microtubules, we postulate here a role for Pink1 in mitochondrial trafficking. Using subcellular fractionation, we show that the overexpression of Miro and Milton, both of which are known to reside at the outer mitochondrial membrane, increases the mitochondrial Pink1 pool, suggesting a function of Pink1 at the outer membrane. Further, we document that Pink1 expressed without a mitochondrial targeting sequence can still be targeted to a mitochondria-enriched subcellular fraction via Miro and Milton. The latter finding is important for the interpretation of a previously reported protective effect of Pink1 expressed without a mitochondrial targeting sequence. Finally, we find that Miro and Milton expression suppresses altered mitochondrial morphology induced by loss of Pink1 function in cell culture. Our findings suggest that Pink1 functions in the trafficking of mitochondria in cells.
| S-EPMC2693257 | biostudies-literature
Project description:Mitochondria form dynamic tubular networks that undergo frequent morphological changes through fission and fusion, the imbalance of which can affect cell survival in general and impact synaptic transmission and plasticity in neurons in particular. Some core components of the mitochondrial fission/fusion machinery, including the dynamin-like GTPases Drp1, Mitofusin, Opa1, and the Drp1-interacting protein Fis1, have been identified. How the fission and fusion processes are regulated under normal conditions and the extent to which defects in mitochondrial fission/fusion are involved in various disease conditions are poorly understood. Mitochondrial malfunction tends to cause diseases with brain and skeletal muscle manifestations and has been implicated in neurodegenerative diseases such as Parkinson's disease (PD). Whether abnormal mitochondrial fission or fusion plays a role in PD pathogenesis has not been shown. Here, we show that Pink1, a mitochondria-targeted Ser/Thr kinase linked to familial PD, genetically interacts with the mitochondrial fission/fusion machinery and modulates mitochondrial dynamics. Genetic manipulations that promote mitochondrial fission suppress Drosophila Pink1 mutant phenotypes in indirect flight muscle and dopamine neurons, whereas decreased fission has opposite effects. In Drosophila and mammalian cells, overexpression of Pink1 promotes mitochondrial fission, whereas inhibition of Pink1 leads to excessive fusion. Our genetic interaction results suggest that Fis1 may act in-between Pink1 and Drp1 in controlling mitochondrial fission. These results reveal a cell biological role for Pink1 and establish mitochondrial fission/fusion as a paradigm for PD research. Compounds that modulate mitochondrial fission/fusion could have therapeutic value in PD intervention.
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Project description:Mutations or deletions in PARKIN/PARK2, PINK1/PARK6, and DJ-1/PARK7 lead to autosomal recessive parkinsonism. In Drosophila, deletions in parkin and pink1 result in swollen and dysfunctional mitochondria in energy-demanding tissues. The relationship between DJ-1 and mitochondria, however, remains unclear. We now report that Drosophila and mouse mutants in DJ-1 show compromised mitochondrial function with age. Flies deleted for DJ-1 manifest similar defects as pink1 and parkin mutants: male sterility, shortened lifespan, and reduced climbing ability. We further found poorly coupled mitochondria in vitro and reduced ATP levels in fly and mouse DJ-1 mutants. Surprisingly, up-regulation of DJ-1 can ameliorate pink1, but not parkin, mutants in Drosophila; cysteine C104 (analogous to C106 in human) is critical for this rescue, implicating the oxidative functions of DJ-1 in this property. These results suggest that DJ-1 is important for proper mitochondrial function and acts downstream of, or in parallel to, pink1. These findings link DJ-1, pink1, and parkin to mitochondrial integrity and provide the foundation for therapeutics that link bioenergetics and parkinsonism.
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Project description:PTEN-induced putative kinase (PINK) 1 is regarded as a master regulator of cellular mitophagy such that loss of function mutations contribute to early onset Parkinson's disease, through aberrant mitochondrial control and function. Mitochondrial function is key to platelet procoagulant activity, controlling the haemostatic response to vessel injury, but can also predispose blood vessels to thrombotic complications. Here, we sought to determine the role of PINK1 in platelet mitochondrial health and function using PINK1 knockout (KO) mice. The data largely show an absence of such a role. Haematological analysis of blood counts from KO mice was comparable to wild type. Quantification of mitochondrial mass by citrate synthase activity assay or expression of mitochondrial markers were comparable, suggesting normal mitophagy in KO platelets. Analysis of mitochondrial permeability transition pore opening, changes in mitochondrial membrane potential and calcium signalling to platelet activation were unaffected by loss of PINK1, whereas subtle enhancements of activation-induced reactive oxygen species were detected. Platelet aggregation, integrin activation, α- and dense granule secretion and phosphatidylserine exposure were unaltered in KO platelets while mouse tail bleeding responses were similar to wild type. Together these results demonstrate that PINK1 does not regulate basal platelet mitophagy and is dispensable for platelet function.
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Project description: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.
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Project description:Transcriptional consequences of Pladienolide B, WX-02-23, and WX-02-43 treatment in 22Rv1 cells by RNA-sequencing
2023-04-19 | GSE185373 | GEO
Project description:Dynamic fission and fusion events regulate mitochondrial shape, distribution, and rejuvenation, and proper control of these processes is essential for neuronal homeostasis. Here, we report that Gas7, a known cytoskeleton regulator, controls mitochondrial dynamics within neurons of the central nervous system. In this study, we generated an improved Gas7-knockout mouse and evaluated its mitochondrial phenotype. We first identified Gas7 in mitochondrial fractions from wild-type brain tissue, and observed Gas7 colocalization with mitochondria in primary cortical neurons. In Gas7-deficient brain tissue and neuronal cultures mitochondria were elongated with perinuclear clustering. These morphological abnormalities were associated with increased levels mitochondrial fusion proteins and increased PKA-dependent phosphorylation of Drp-1 in brain tissues, suggesting an imbalance of mitochondrial fusion and fission. Moreover, expression of mitochondrial quality control kinase, PINK1, and PINK1-specific phosphorylation of Mfn-2 (S442), Parkin (S65), and ubiquitin (S65) were all reduced in the knockout cells. Ectopic expression of Gas7 restored mitochondrial morphology and distribution, as well as PINK1 expression in Gas7-null cortical neurons. Collectively, our results introduce a novel role of mouse Gas7 in determining the dynamics, morphology, and intracellular distribution of neuronal mitochondria, which are expected to be required for normal neuronal function.
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