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Find video protocols related to scientific articles indexed in Pubmed.
Lysine 63-Linked Polyubiquitination Is Dispensable for Parkin-Mediated Mitophagy.
J. Biol. Chem.
PUBLISHED: 10-23-2014
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PINK1/Parkin-mediated mitophagy is thought to ensure mitochondrial quality control in neurons as well as other cells. Upon the loss of mitochondrial membrane potential (??m)3, lysine 63 (K63)-linked polyubiquitin chains accumulate on the mitochondrial outer membrane in a Parkin-dependent manner. However, the physiological significance of K63-linked polyubiquitination during mitophagy is not fully understood. Here, we report that the suppression of K63-linked polyubiquitination through the removal of Ubc13 activity essentially affects neither PINK1 activation nor the degradation of depolarized mitochondria. Moreover, the inactivation of Ubc13 did not modulate the mitochondrial phenotypes of PINK1 knockdown Drosophila. Our data indicate that the formation of K63-linked polyubiquitin chains on depolarized mitochondria is not a key factor for the PINK1-Parkin pathway as was once thought.PINK1/Parkin-mediated mitophagy is thought to ensure mitochondrial quality control in neurons as well as other cells. Upon the loss of mitochondrial membrane potential (??m)3, lysine 63 (K63)-linked polyubiquitin chains accumulate on the mitochondrial outer membrane in a Parkin-dependent manner. However, the physiological significance of K63-linked polyubiquitination during mitophagy is not fully understood. Here, we report that the suppression of K63-linked polyubiquitination through the removal of Ubc13 activity essentially affects neither PINK1 activation nor the degradation of depolarized mitochondria. Moreover, the inactivation of Ubc13 did not modulate the mitochondrial phenotypes of PINK1 knockdown Drosophila. Our data indicate that the formation of K63-linked polyubiquitin chains on depolarized mitochondria is not a key factor for the PINK1-Parkin pathway as was once thought.
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PINK1-mediated phosphorylation of Parkin boosts Parkin activity in Drosophila.
PLoS Genet.
PUBLISHED: 06-01-2014
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Two genes linked to early onset Parkinson's disease, PINK1 and Parkin, encode a protein kinase and a ubiquitin-ligase, respectively. Both enzymes have been suggested to support mitochondrial quality control. We have reported that Parkin is phosphorylated at Ser65 within the ubiquitin-like domain by PINK1 in mammalian cultured cells. However, it remains unclear whether Parkin phosphorylation is involved in mitochondrial maintenance and activity of dopaminergic neurons in vivo. Here, we examined the effects of Parkin phosphorylation in Drosophila, in which the phosphorylation residue is conserved at Ser94. Morphological changes of mitochondria caused by the ectopic expression of wild-type Parkin in muscle tissue and brain dopaminergic neurons disappeared in the absence of PINK1. In contrast, phosphomimetic Parkin accelerated mitochondrial fragmentation or aggregation and the degradation of mitochondrial proteins regardless of PINK1 activity, suggesting that the phosphorylation of Parkin boosts its ubiquitin-ligase activity. A non-phosphorylated form of Parkin fully rescued the muscular mitochondrial degeneration due to the loss of PINK1 activity, whereas the introduction of the non-phosphorylated Parkin mutant in Parkin-null flies led to the emergence of abnormally fused mitochondria in the muscle tissue. Manipulating the Parkin phosphorylation status affected spontaneous dopamine release in the nerve terminals of dopaminergic neurons, the survivability of dopaminergic neurons and flight activity. Our data reveal that Parkin phosphorylation regulates not only mitochondrial function but also the neuronal activity of dopaminergic neurons in vivo, suggesting that the appropriate regulation of Parkin phosphorylation is important for muscular and dopaminergic functions.
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Regulation by mitophagy.
Int. J. Biochem. Cell Biol.
PUBLISHED: 04-27-2014
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Eukaryotes employ elaborate mitochondrial quality control to maintain the function of the power-generating organelle. Mitochondrial quality control is particularly important for the maintenance of neural and muscular tissues. Mitophagy is specialized version of the autophagy pathway. Mitophagy delivers damaged mitochondria to lysosomes for degradation. Recently, a series of elegant studies have demonstrated that two Parkinson's disease-associated genes PINK1 and parkin are involved in the maintenance of healthy mitochondria as mitophagy. Parkin in co-operation with PINK1 specifically recognizes damaged mitochondria with reduced mitochondrial membrane potential (??m), rapidly isolates them from the mitochondrial network and eliminates them through the ubiquitin-proteasome and autophagy pathways. Here we introduce and review recent studies that contribute to understanding the molecular mechanisms of mitophagy such as PINK1 and Parkin-mediated mitochondrial regulation. We also discuss how defects in the PINK1-Parkin pathway may cause neurodegeneration in Parkinson's disease.
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Tricornered/NDR kinase signaling mediates PINK1-directed mitochondrial quality control and tissue maintenance.
Genes Dev.
PUBLISHED: 01-26-2013
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Eukaryotes employ elaborate mitochondrial quality control (MQC) to maintain the function of the power-generating organelle. Parkinsons disease-associated PINK1 and Parkin actively participate in MQC. However, the signaling events involved are largely unknown. Here we show that mechanistic target of rapamycin 2 (mTORC2) and Tricornered (Trc) kinases act downstream from PINK1 to regulate MQC. Trc is phosphorylated in mTORC2-dependent and mTORC2-independent manners and is specifically localized to mitochondria in response to PINK1, which regulates mTORC2 through mitochondrial complex-I activity. Genetically, mTORC2 and Trc act upstream of Parkin. Thus, multiplex kinase signaling is acting between PINK1 and Parkin to regulate MQC, a process highly conserved in mammals.
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Mitochondrial dynamics and mitophagy in Parkinsons disease: disordered cellular power plant becomes a big deal in a major movement disorder.
Curr. Opin. Neurobiol.
PUBLISHED: 07-28-2011
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Parkinsons disease (PD), the most common movement disorder, is characterized by age-dependent degeneration of dopaminergic neurons in the substantia nigra of the mid-brain. Non-motor symptoms of PD, however, precede the motor features caused by dysfunction of the dopaminergic system, suggesting that PD is a systemic disorder. Mitochondrial dysfunction has long been observed in PD patients and animal models, but the mechanistic link between mitochondrial dysfunction and PD pathogenesis is not well understood. Recent studies have revealed that genes associated with autosomal recessive forms of PD such as PINK1 and Parkin are directly involved in regulating mitochondrial morphology and maintenance, abnormality of which is also observed in the more common, sporadic forms of PD, although the autosomal recessive PDs lack Lewy-body pathology that is characteristic of sporadic PD. These latest findings suggest that at least some forms of PD can be characterized as a mitochondrial disorder. Whether mitochondrial dysfunction represents a unifying pathogenic mechanism of all PD cases remains a major unresolved question.
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Activation of FoxO by LRRK2 induces expression of proapoptotic proteins and alters survival of postmitotic dopaminergic neuron in Drosophila.
Hum. Mol. Genet.
PUBLISHED: 07-12-2010
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Missense mutations in leucine-rich repeat kinase 2 (LRRK2)/Dardarin gene, the product of which encodes a kinase with multiple domains, are known to cause autosomal dominant late onset Parkinsons disease (PD). In the current study, we report that the gene product LRRK2 directly phosphorylates the forkhead box transcription factor FoxO1 and enhances its transcriptional activity. This pathway was found to be conserved in Drosophila, as the Drosophila LRRK2 homolog (dLRRK) enhanced the neuronal toxicity of FoxO. Importantly, FoxO mutants that were resistant to LRRK2/dLRRK-induced phosphorylation suppressed this neurotoxicity. Moreover, we have determined that FoxO targets hid and bim in Drosophila and human, respectively, are responsible for the LRRK2/dLRRK-mediated cell death. These data suggest that the cell death molecules regulated by FoxO are key factors during the neurodegeneration in LRRK2-linked PD.
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The loss of PGAM5 suppresses the mitochondrial degeneration caused by inactivation of PINK1 in Drosophila.
PLoS Genet.
PUBLISHED: 05-24-2010
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PTEN-induced kinase 1 (PINK1), which is required for mitochondrial homeostasis, is a gene product responsible for early-onset Parkinsons disease (PD). Another early onset PD gene product, Parkin, has been suggested to function downstream of the PINK1 signalling pathway based on genetic studies in Drosophila. PINK1 is a serine/threonine kinase with a predicted mitochondrial target sequence and a probable transmembrane domain at the N-terminus, while Parkin is a RING-finger protein with ubiquitin-ligase (E3) activity. However, how PINK1 and Parkin regulate mitochondrial activity is largely unknown. To explore the molecular mechanism underlying the interaction between PINK1 and Parkin, we biochemically purified PINK1-binding proteins from human cultured cells and screened the genes encoding these binding proteins using Drosophila PINK1 (dPINK1) models to isolate a molecule(s) involved in the PINK1 pathology. Here we report that a PINK1-binding mitochondrial protein, PGAM5, modulates the PINK1 pathway. Loss of Drosophila PGAM5 (dPGAM5) can suppress the muscle degeneration, motor defects, and shorter lifespan that result from dPINK1 inactivation and that can be attributed to mitochondrial degeneration. However, dPGAM5 inactivation fails to modulate the phenotypes of parkin mutant flies. Conversely, ectopic expression of dPGAM5 exacerbated the dPINK1 and Drosophila parkin (dParkin) phenotypes. These results suggest that PGAM5 negatively regulates the PINK1 pathway related to maintenance of the mitochondria and, furthermore, that PGAM5 acts between PINK1 and Parkin, or functions independently of Parkin downstream of PINK1.
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Pathogenic LRRK2 negatively regulates microRNA-mediated translational repression.
Nature
PUBLISHED: 05-20-2010
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Gain-of-function mutations in leucine-rich repeat kinase 2 (LRRK2) cause familial as well as sporadic Parkinsons disease characterized by age-dependent degeneration of dopaminergic neurons. The molecular mechanism of LRRK2 action is not known. Here we show that LRRK2 interacts with the microRNA (miRNA) pathway to regulate protein synthesis. Drosophila e2f1 and dp messenger RNAs are translationally repressed by let-7 and miR-184*, respectively. Pathogenic LRRK2 antagonizes these miRNAs, leading to the overproduction of E2F1/DP, previously implicated in cell cycle and survival control and shown here to be critical for LRRK2 pathogenesis. Genetic deletion of let-7, antagomir-mediated blockage of let-7 and miR-184* action, transgenic expression of dp target protector, or replacement of endogenous dp with a dp transgene non-responsive to let-7 each had toxic effects similar to those of pathogenic LRRK2. Conversely, increasing the level of let-7 or miR-184* attenuated pathogenic LRRK2 effects. LRRK2 associated with Drosophila Argonaute-1 (dAgo1) or human Argonaute-2 (hAgo2) of the RNA-induced silencing complex (RISC). In aged fly brain, dAgo1 protein level was negatively regulated by LRRK2. Further, pathogenic LRRK2 promoted the association of phospho-4E-BP1 with hAgo2. Our results implicate deregulated synthesis of E2F1/DP caused by the miRNA pathway impairment as a key event in LRRK2 pathogenesis and suggest novel miRNA-based therapeutic strategies.
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[Frontier researches for the development of molecular-targeted therapies for familial Parkinson disease].
Brain Nerve
PUBLISHED: 08-25-2009
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Parkinson disease (PD), is a movement disorder pathologically characterized by the progressive loss of dopaminergic neurons in the substantia nigra pars compacta. Although the inherited forms of PD account for only 5 to 10% of PD cases, the identification of gene mutations in the genes implicated in familial PD in the past 10 years, including the findings regarding the a-synuclein, Parkin, ubiquitin-C-terminal hydrolase-L1 (UCH-L1), PINK1, DJ-1 and the ATP13A2 genes, has advanced understanding of the molecular mechanisms in each case of genetic PD. Most familial forms of PD develop at an early onset. However, recent identification of the leucine-rich repeat kinase (LRRK) 2 gene for a late-onset PD, the clinicopathological feature of which closely resembles that of sporadic PD, is expected to enable the clarification of the underlying causes of general PD. Recent studies on the physiological and pathological functions of these identified gene products have revealed overlapping pathogenetic pathways. The common features of these aberrant pathways are impaired protein degradation/quality control, mitochondrial dysfunction, and altered vesicle transport. Several attempts have been made towards developing molecular-targeted therapies directed against mitochondria (e.g., antioxidants, permeability transition pore modulators, and mitochondrial biogenesis stimulators), protein quality control and vesicle transport (e.g., gene silencing, immunization of asynuclein, and protofibril-destabilizing reagents). To ensure the successful implementation of such strategies, it is important to understand the events occuring at an early stage of PD. Further, studies using mammalian PD models for pharmacological analysis combined with studies employing lower organisms for genetic analyses such as worm, fly, and yeast will be helpful to determine effective prevention and treatment strategies for PD, which will replace the conventional symptomatic treatments for PD.
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Leucine-Rich Repeat Kinase 2 interacts with Parkin, DJ-1 and PINK-1 in a Drosophila melanogaster model of Parkinsons disease.
Hum. Mol. Genet.
PUBLISHED: 08-19-2009
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Mutations in the LRRK2 gene are the most common genetic cause of familial Parkinsons disease (PD). However, its physiological and pathological functions are unknown. Therefore, we generated several independent Drosophila lines carrying WT or mutant human LRRK2 (mutations in kinase, COR or LRR domains, resp.). Ectopic expression of WT or mutant LRRK2 in dopaminergic neurons caused their significant loss accompanied by complex age-dependent changes in locomotor activity. Overall, the ubiquitous expression of LRRK2 increased lifespan and fertility of the flies. However, these flies were more sensitive to rotenone. LRRK2 expression in the eye exacerbated retinal degeneration. Importantly, in double transgenic flies, various indices of the eye and dopaminergic survival were modified in a complex fashion by a concomitant expression of PINK1, DJ-1 or Parkin. This evidence suggests a genetic interaction between these PD-relevant genes.
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PINK1-mediated phosphorylation of the Parkin ubiquitin-like domain primes mitochondrial translocation of Parkin and regulates mitophagy.
Sci Rep
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Parkinsons disease genes PINK1 and parkin encode kinase and ubiquitin ligase, respectively. The gene products PINK1 and Parkin are implicated in mitochondrial autophagy, or mitophagy. Upon the loss of mitochondrial membrane potential (??m), cytosolic Parkin is recruited to the mitochondria by PINK1 through an uncharacterised mechanism - an initial step triggering sequential events in mitophagy. This study reports that Ser65 in the ubiquitin-like domain (Ubl) of Parkin is phosphorylated in a PINK1-dependent manner upon depolarisation of ??m. The introduction of mutations at Ser65 suggests that phosphorylation of Ser65 is required not only for the efficient translocation of Parkin, but also for the degradation of mitochondrial proteins in mitophagy. Phosphorylation analysis of Parkin pathogenic mutants also suggests Ser65 phosphorylation is not sufficient for Parkin translocation. Our study partly uncovers the molecular mechanism underlying the PINK1-dependent mitochondrial translocation and activation of Parkin as an initial step of mitophagy.
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The synaptic function of LRRK2.
Biochem. Soc. Trans.
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Mutations in LRRK2 (leucine-rich repeat kinase 2) are the most frequent genetic lesions so far found in familial as well as sporadic forms of PD (Parkinsons disease), a neurodegenerative disease characterized by the dysfunction and degeneration of dopaminergic and other neuronal types. The molecular and cellular mechanisms underlying LRRK2 action remain poorly defined. Synaptic dysfunction has been increasingly recognized as an early event in the pathogenesis of major neurological disorders. Using Drosophila as a model system, we have shown that LRRK2 controls synaptic morphogenesis. Loss of dLRRK (Drosophila LRRK2) results in synaptic overgrowth at the Drosophila neuromuscular junction synapse, whereas overexpression of wild-type dLRRK, hLRRK2 (human LRRK2) or the pathogenic hLRRK2-G2019S mutant has the opposite effect. Alteration of LRRK2 activity also affects synaptic transmission in a complex manner. LRRK2 exerts its effects on synaptic morphology by interacting with distinct downstream effectors at the pre- and post-synaptic compartments. At the postsynapse, LRRK2 functionally interacts with 4E-BP (eukaryotic initiation factor 4E-binding protein) and the microRNA machinery, both of which negatively regulate protein synthesis. At the presynapse, LRRK2 phosphorylates and negatively regulates the microtubule-binding protein Futsch and functionally interacts with the mitochondrial transport machinery. These results implicate compartment-specific synaptic dysfunction caused by altered protein synthesis, cytoskeletal dynamics and mitochondrial transport in LRRK2 pathogenesis and offer a new paradigm for understanding and ultimately treating LRRK2-related PD.
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Parkinsons disease-associated kinase PINK1 regulates Miro protein level and axonal transport of mitochondria.
PLoS Genet.
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Mutations in Pten-induced kinase 1 (PINK1) are linked to early-onset familial Parkinsons disease (FPD). PINK1 has previously been implicated in mitochondrial fission/fusion dynamics, quality control, and electron transport chain function. However, it is not clear how these processes are interconnected and whether they are sufficient to explain all aspects of PINK1 pathogenesis. Here we show that PINK1 also controls mitochondrial motility. In Drosophila, downregulation of dMiro or other components of the mitochondrial transport machinery rescued dPINK1 mutant phenotypes in the muscle and dopaminergic (DA) neurons, whereas dMiro overexpression alone caused DA neuron loss. dMiro protein level was increased in dPINK1 mutant but decreased in dPINK1 or dParkin overexpression conditions. In Drosophila larval motor neurons, overexpression of dPINK1 inhibited axonal mitochondria transport in both anterograde and retrograde directions, whereas dPINK1 knockdown promoted anterograde transport. In HeLa cells, overexpressed hPINK1 worked together with hParkin, another FPD gene, to regulate the ubiquitination and degradation of hMiro1 and hMiro2, apparently in a Ser-156 phosphorylation-independent manner. Also in HeLa cells, loss of hMiro promoted the perinuclear clustering of mitochondria and facilitated autophagy of damaged mitochondria, effects previously associated with activation of the PINK1/Parkin pathway. These newly identified functions of PINK1/Parkin and Miro in mitochondrial transport and mitophagy contribute to our understanding of the complex interplays in mitochondrial quality control that are critically involved in PD pathogenesis, and they may explain the peripheral neuropathy symptoms seen in some PD patients carrying particular PINK1 or Parkin mutations. Moreover, the different effects of loss of PINK1 function on Miro protein level in Drosophila and mouse cells may offer one explanation of the distinct phenotypic manifestations of PINK1 mutants in these two species.
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The nitric oxide-cyclic GMP pathway regulates FoxO and alters dopaminergic neuron survival in Drosophila.
PLoS ONE
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Activation of the forkhead box transcription factor FoxO is suggested to be involved in dopaminergic (DA) neurodegeneration in a Drosophila model of Parkinsons disease (PD), in which a PD gene product LRRK2 activates FoxO through phosphorylation. In the current study that combines Drosophila genetics and biochemical analysis, we show that cyclic guanosine monophosphate (cGMP)-dependent kinase II (cGKII) also phosphorylates FoxO at the same residue as LRRK2, and Drosophila orthologues of cGKII and LRRK2, DG2/For and dLRRK, respectively, enhance the neurotoxic activity of FoxO in an additive manner. Biochemical assays using mammalian cGKII and FoxO1 reveal that cGKII enhances the transcriptional activity of FoxO1 through phosphorylation of the FoxO1 S319 site in the same manner as LRRK2. A Drosophila FoxO mutant resistant to phosphorylation by DG2 and dLRRK (dFoxO S259A corresponding to human FoxO1 S319A) suppressed the neurotoxicity and improved motor dysfunction caused by co-expression of FoxO and DG2. Nitric oxide synthase (NOS) and soluble guanylyl cyclase (sGC) also increased FoxOs activity, whereas the administration of a NOS inhibitor L-NAME suppressed the loss of DA neurons in aged flies co-expressing FoxO and DG2. These results strongly suggest that the NO-FoxO axis contributes to DA neurodegeneration in LRRK2-linked PD.
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