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1.  How could Parkin-mediated ubiquitination of mitofusin promote mitophagy? 
Autophagy  2010;6(5):660-662.
doi:10.4161/auto.6.5.12242
PMCID: PMC4196639  PMID: 20484985
Parkinson disease; neurodegeneration; Parkin; PINK1; Mfn; mitochondrial dynamics; ubiquitination; Drosophila
2.  The Complex I Subunit NDUFA10 Selectively Rescues Drosophila pink1 Mutants through a Mechanism Independent of Mitophagy 
PLoS Genetics  2014;10(11):e1004815.
Mutations in PINK1, a mitochondrially targeted serine/threonine kinase, cause autosomal recessive Parkinson's disease (PD). Substantial evidence indicates that PINK1 acts with another PD gene, parkin, to regulate mitochondrial morphology and mitophagy. However, loss of PINK1 also causes complex I (CI) deficiency, and has recently been suggested to regulate CI through phosphorylation of NDUFA10/ND42 subunit. To further explore the mechanisms by which PINK1 and Parkin influence mitochondrial integrity, we conducted a screen in Drosophila cells for genes that either phenocopy or suppress mitochondrial hyperfusion caused by pink1 RNAi. Among the genes recovered from this screen was ND42. In Drosophila pink1 mutants, transgenic overexpression of ND42 or its co-chaperone sicily was sufficient to restore CI activity and partially rescue several phenotypes including flight and climbing deficits and mitochondrial disruption in flight muscles. Here, the restoration of CI activity and partial rescue of locomotion does not appear to have a specific requirement for phosphorylation of ND42 at Ser-250. In contrast to pink1 mutants, overexpression of ND42 or sicily failed to rescue any Drosophila parkin mutant phenotypes. We also find that knockdown of the human homologue, NDUFA10, only minimally affecting CCCP-induced mitophagy, and overexpression of NDUFA10 fails to restore Parkin mitochondrial-translocation upon PINK1 loss. These results indicate that the in vivo rescue is due to restoring CI activity rather than promoting mitophagy. Our findings support the emerging view that PINK1 plays a role in regulating CI activity separate from its role with Parkin in mitophagy.
Author Summary
Two genes linked to heritable forms of the neurodegenerative movement disorder Parkinson's disease (PD), PINK1 and parkin, play important roles in mitochondrial homeostasis through mechanisms which include the degradation of dysfunctional mitochondria, termed mitophagy, and the maintenance of complex I (CI) activity. Here we report the findings of an RNAi based screen in Drosophila cells for genes that may regulate the PINK1-Parkin pathway which identified NDUFA10 (ND42 in Drosophila), a subunit of CI. Using a well-established cellular system and in vivo Drosophila genetics, we demonstrate that while NDUFA10/ND42 only plays a minimal role in mitophagy, restoration of CI activity through overexpression of either ND42 or its co-chaperone sicily is able to substantially rescue behavioral deficits in pink1 mutants but not parkin mutants. Moreover, while parkin overexpression is known to rescue pink1 mutants, it apparently achieves this without restoring CI activity. These results suggest that increasing CI activity or promoting mitophagy can be beneficial in pink1 mutants, and further highlights separable functions of PINK1 and Parkin.
doi:10.1371/journal.pgen.1004815
PMCID: PMC4238976  PMID: 25412178
3.  Increasing microtubule acetylation rescues axonal transport and locomotor deficits caused by LRRK2 Roc-COR domain mutations 
Nature Communications  2014;5:5245.
Leucine-rich repeat kinase 2 (LRRK2) mutations are the most common genetic cause of Parkinson’s disease. LRRK2 is a multifunctional protein affecting many cellular processes and has been described to bind microtubules. Defective microtubule-based axonal transport is hypothesized to contribute to Parkinson’s disease, but whether LRRK2 mutations affect this process to mediate pathogenesis is not known. Here we find that LRRK2 containing pathogenic Roc-COR domain mutations (R1441C, Y1699C) preferentially associates with deacetylated microtubules, and inhibits axonal transport in primary neurons and in Drosophila, causing locomotor deficits in vivo. In vitro, increasing microtubule acetylation using deacetylase inhibitors or the tubulin acetylase αTAT1 prevents association of mutant LRRK2 with microtubules, and the deacetylase inhibitor trichostatin A (TSA) restores axonal transport. In vivo knockdown of the deacetylases HDAC6 and Sirt2, or administration of TSA rescues both axonal transport and locomotor behavior. Thus, this study reveals a pathogenic mechanism and a potential intervention for Parkinson’s disease.
Mutations in the kinase LRRK2 are associated with Parkinson’s disease. Godena et al. find that disease-associated LRRK2 mutations promote its binding to deacetylated microtubules, and cause defects in axonal transport and Drosophila locomotor behaviour that can be reversed by enhancing tubulin acetylation.
doi:10.1038/ncomms6245
PMCID: PMC4208097  PMID: 25316291
4.  TRAP1 rescues PINK1 loss-of-function phenotypes 
Human Molecular Genetics  2013;22(14):2829-2841.
PTEN-induced kinase 1 (PINK1) is a serine/threonine kinase that is localized to mitochondria. It protects cells from oxidative stress by suppressing mitochondrial cytochrome c release, thereby preventing cell death. Mutations in Pink1 cause early-onset Parkinson's disease (PD). Consistently, mitochondrial function is impaired in Pink1-linked PD patients and model systems. Previously, in vitro analysis implied that the protective effects of PINK1 depend on phosphorylation of the downstream factor, TNF receptor-associated protein 1 (TRAP1). Furthermore, TRAP1 has been shown to mitigate α-Synuclein-induced toxicity, linking α-Synuclein directly to mitochondrial dysfunction. These data suggest that TRAP1 seems to mediate protective effects on mitochondrial function in pathways that are affected in PD. Here we investigated the potential of TRAP1 to rescue dysfunction induced by either PINK1 or Parkin deficiency in vivo and in vitro. We show that overexpression of human TRAP1 is able to mitigate Pink1 but not parkin loss-of-function phenotypes in Drosophila. In addition, detrimental effects observed after RNAi-mediated silencing of complex I subunits were rescued by TRAP1 in Drosophila. Moreover, TRAP1 was able to rescue mitochondrial fragmentation and dysfunction upon siRNA-induced silencing of Pink1 but not parkin in human neuronal SH-SY5Y cells. Thus, our data suggest a functional role of TRAP1 in maintaining mitochondrial integrity downstream of PINK1 and complex I deficits but parallel to or upstream of Parkin.
doi:10.1093/hmg/ddt132
PMCID: PMC3690968  PMID: 23525905
5.  The Parkinson’s disease genes Fbxo7 and Parkin interact to mediate mitophagy 
Nature neuroscience  2013;16(9):10.1038/nn.3489.
Compelling evidence indicates that two autosomal recessive Parkinson’s disease genes, PINK1 (PARK6) and Parkin (PARK2), co-operate to mediate the autophagic clearance of damaged mitochondria (mitophagy). Mutations in the F-box domain containing protein Fbxo7 (PARK15) also cause early onset autosomal recessive Parkinson’s disease by an unknown mechanism. Here we show that Fbxo7 participates in mitochondrial maintenance through direct interaction with PINK1 and Parkin and plays a role in Parkin-mediated mitophagy. Cells with reduced Fbxo7 expression show deficiencies in Parkin mitochondrial translocation, ubiquitination of mitofusin 1 and mitophagy. In Drosophila, ectopic overexpression of Fbxo7 rescued loss of Parkin supporting a functional relationship between the two proteins. Parkinson’s disease-causing mutations in Fbxo7 interfere with this process, emphasising the importance of mitochondrial dysfunction in Parkinson’s disease pathogenesis.
doi:10.1038/nn.3489
PMCID: PMC3827746  PMID: 23933751
Fbxo7; Parkin; PINK1; mitofusin 1; mitophagy; Drosophila; Parkinson’s disease
6.  Modeling Pathogenic Mutations of Human Twinkle in Drosophila Suggests an Apoptosis Role in Response to Mitochondrial Defects 
PLoS ONE  2012;7(8):e43954.
The human gene C10orf2 encodes the mitochondrial replicative DNA helicase Twinkle, mutations of which are responsible for a significant fraction of cases of autosomal dominant progressive external ophthalmoplegia (adPEO), a human mitochondrial disease caused by defects in intergenomic communication. We report the analysis of orthologous mutations in the Drosophila melanogaster mitochondrial DNA (mtDNA) helicase gene, d-mtDNA helicase. Increased expression of wild type d-mtDNA helicase using the UAS-GAL4 system leads to an increase in mtDNA copy number throughout adult life without any noteworthy phenotype, whereas overexpression of d-mtDNA helicase containing the K388A mutation in the helicase active site results in a severe depletion of mtDNA and a lethal phenotype. Overexpression of two d-mtDNA helicase variants equivalent to two human adPEO mutations shows differential effects. The A442P mutation exhibits a dominant negative effect similar to that of the active site mutant. In contrast, overexpression of d-mtDNA helicase containing the W441C mutation results in a slight decrease in mtDNA copy number during the third instar larval stage, and a moderate decrease in life span in the adult population. Overexpression of d-mtDNA helicase containing either the K388A or A442P mutations causes a mitochondrial oxidative phosphorylation (OXPHOS) defect that significantly reduces cell proliferation. The mitochondrial impairment caused by these mutations promotes apoptosis, arguing that mitochondria regulate programmed cell death in Drosophila. Our study of d-mtDNA helicase overexpression provides a tractable Drosophila model for understanding the cellular and molecular effects of human adPEO mutations.
doi:10.1371/journal.pone.0043954
PMCID: PMC3429445  PMID: 22952820
7.  Discovery of catalytically active orthologues of the Parkinson's disease kinase PINK1: analysis of substrate specificity and impact of mutations 
Open biology  2011;1(3):110012.
Missense mutations of the phosphatase and tensin homolog (PTEN)-induced kinase 1 (PINK1) gene cause autosomal-recessive Parkinson's disease. To date, little is known about the intrinsic catalytic properties of PINK1 since the human enzyme displays such low kinase activity in vitro. We have discovered that, in contrast to mammalian PINK1, insect orthologues of PINK1 we have investigated—namely Drosophila melanogaster (dPINK1), Tribolium castaneum (TcPINK1) and Pediculus humanus corporis (PhcPINK1)—are active as judged by their ability to phosphorylate the generic substrate myelin basic protein. We have exploited the most active orthologue, TcPINK1, to assess its substrate specificity and elaborated a peptide substrate (PINKtide, KKWIpYRRSPRRR) that can be employed to quantify PINK1 kinase activity. Analysis of PINKtide variants reveal that PINK1 phosphorylates serine or threonine, but not tyrosine, and we show that PINK1 exhibits a preference for a proline at the +1 position relative to the phosphorylation site. We have also, for the first time, been able to investigate the effect of Parkinson's disease-associated PINK1 missense mutations, and found that nearly all those located within the kinase domain, as well as the C-terminal non-catalytic region, markedly suppress kinase activity. This emphasizes the crucial importance of PINK1 kinase activity in preventing the development of Parkinson's disease. Our findings will aid future studies aimed at understanding how the activity of PINK1 is regulated and the identification of physiological substrates.
doi:10.1098/rsob.110012
PMCID: PMC3352081  PMID: 22645651
biochemistry; Parkinson's disease; kinase
8.  PINK1 cleavage at position A103 by the mitochondrial protease PARL 
Human Molecular Genetics  2010;20(5):867-879.
Mutations in PTEN-induced kinase 1 (PINK1) cause early onset autosomal recessive Parkinson's disease (PD). PINK1 is a 63 kDa protein kinase, which exerts a neuroprotective function and is known to localize to mitochondria. Upon entry into the organelle, PINK1 is cleaved to produce a ∼53 kDa protein (ΔN-PINK1). In this paper, we show that PINK1 is cleaved between amino acids Ala-103 and Phe-104 to generate ΔN-PINK1. We demonstrate that a reduced ability to cleave PINK1, and the consequent accumulation of full-length protein, results in mitochondrial abnormalities reminiscent of those observed in PINK1 knockout cells, including disruption of the mitochondrial network and a reduction in mitochondrial mass. Notably, we assessed three N-terminal PD-associated PINK1 mutations located close to the cleavage site and, while these do not prevent PINK1 cleavage, they alter the ratio of full-length to ΔN-PINK1 protein in cells, resulting in an altered mitochondrial phenotype. Finally, we show that PINK1 interacts with the mitochondrial protease presenilin-associated rhomboid-like protein (PARL) and that loss of PARL results in aberrant PINK1 cleavage in mammalian cells. These combined results suggest that PINK1 cleavage is important for basal mitochondrial health and that PARL cleaves PINK1 to produce the ΔN-PINK1 fragment.
doi:10.1093/hmg/ddq526
PMCID: PMC3033179  PMID: 21138942
9.  Modulation of mitochondrial function and morphology by interaction of Omi/HtrA2 with the mitochondrial fusion factor OPA1 
Experimental Cell Research  2010;316(7):1213-1224.
Loss of Omi/HtrA2 function leads to nerve cell loss in mouse models and has been linked to neurodegeneration in Parkinson's and Huntington's disease. Omi/HtrA2 is a serine protease released as a pro-apoptotic factor from the mitochondrial intermembrane space into the cytosol. Under physiological conditions, Omi/HtrA2 is thought to be involved in protection against cellular stress, but the cytological and molecular mechanisms are not clear. Omi/HtrA2 deficiency caused an accumulation of reactive oxygen species and reduced mitochondrial membrane potential. In Omi/HtrA2 knockout mouse embryonic fibroblasts, as well as in Omi/HtrA2 silenced human HeLa cells and Drosophila S2R+ cells, we found elongated mitochondria by live cell imaging. Electron microscopy confirmed the mitochondrial morphology alterations and showed abnormal cristae structure. Examining the levels of proteins involved in mitochondrial fusion, we found a selective up-regulation of more soluble OPA1 protein. Complementation of knockout cells with wild-type Omi/HtrA2 but not with the protease mutant [S306A]Omi/HtrA2 reversed the mitochondrial elongation phenotype and OPA1 alterations. Finally, co-immunoprecipitation showed direct interaction of Omi/HtrA2 with endogenous OPA1. Thus, we show for the first time a direct effect of loss of Omi/HtrA2 on mitochondrial morphology and demonstrate a novel role of this mitochondrial serine protease in the modulation of OPA1. Our results underscore a critical role of impaired mitochondrial dynamics in neurodegenerative disorders.
doi:10.1016/j.yexcr.2010.01.005
PMCID: PMC3063334  PMID: 20064504
ANT, adenine nucleotide translocator; Drp1, dynamin-related protein 1; Fis1, mitochondrial fission 1 protein; Hsp90, heat shock protein 90; HtrA2, high temperature requirement protein A2; KO, knockout; MEF, mouse embryonic fibroblast; Mfn2, mitofusin 2; MMP, mitochondrial membrane potential; PBS, phosphate-buffered saline; PD, Parkinson's disease; ROS, reactive oxygen species; SD, standard deviation; SEM, standard error of the mean; VDAC1, voltage dependent anion channel 1; WT, wild-type; Omi; HtrA2; Mitochondria; Fusion; OPA1; Parkinson's disease
10.  Rapamycin activation of 4E-BP prevents parkinsonian dopaminergic neuron loss 
Nature neuroscience  2009;12(9):1129-1135.
Mutations in PINK1 and parkin cause autosomal recessive parkinsonism, a neurodegenerative disorder characterized by the loss of dopaminergic neurons. To highlight potential therapeutic pathways we have identified factors that genetically interact with parkin/PINK1. Here we report that overexpression of the translation inhibitor 4E-BP can suppress all pathologic phenotypes including degeneration of dopaminergic neurons in Drosophila. 4E-BP is activated in vivo by the TOR inhibitor rapamycin, which we find can potently suppress pathology in PINK1/parkin mutants. Rapamycin also ameliorates mitochondrial defects in cells from parkin-mutant patients. Recently, 4E-BP was shown to be inhibited by the most common cause of parkinsonism, dominant mutations in LRRK2. Here we further show that loss of the Drosophila LRRK2 homolog activates 4E-BP and is also able to suppress PINK1/parkin pathology. Thus, in conjunction with recent findings our results suggest that pharmacologic stimulation of 4E-BP activity may represent a viable therapeutic approach for multiple forms of parkinsonism.
doi:10.1038/nn.2372
PMCID: PMC2745154  PMID: 19684592
parkinsonism; neurodegeneration; parkin; PINK1; 4E-BP; rapamycin; TOR; LRRK2
11.  Drosophila HtrA2 is dispensable for apoptosis but acts downstream of PINK1 independently from Parkin 
Cell death and differentiation  2009;16(8):1118-1125.
High Temperature Requirement A2 (HtrA2/Omi) is a mitochondrial protease that exhibits pro-apoptotic and cell protective properties and has been linked to Parkinson disease (PD). Impaired mitochondrial function is a common trait in PD patients, and is likely to play a significant role in pathogenesis of parkinsonism, but the molecular mechanisms remain poorly understood. Genetic studies in Drosophila have provided valuable insight into the function of other PD-linked genes, in particular PINK1 and parkin, and their role in maintaining mitochondrial integrity. Recently, HtrA2 was shown to be phosphorylated in a PINK1-dependent manner, suggesting it might act in the PINK1 pathway. Here, we describe the characterization of mutations in Drosophila HtrA2, and genetic analysis of its function with PINK1 and parkin. Interestingly, we find HtrA2 appears to be dispensable for developmental or stress-induced apoptosis. In addition, we found HtrA2 mutants share some phenotypic similarities with parkin and PINK1 mutants, suggesting that it may function in maintaining mitochondrial integrity. Our genetic interaction studies, including analysis of double-mutant combinations and epistasis experiments, suggest HtrA2 acts downstream of PINK1 but in a pathway parallel to Parkin.
doi:10.1038/cdd.2009.23
PMCID: PMC2711053  PMID: 19282869

Results 1-11 (11)