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Loss-Of-Function of Human PINK1 Results in Mitochondrial Pathology and Can Be Rescued by Parkin

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Loss-Of-Function of Human PINK1 Results in Mitochondrial Pathology and Can Be Rescued by Parkin The Journal of Neuroscience, November 7, 2007 • 27(45):12413–12418 • 12413 Neurobiology of Disease Loss-of-Function of Human PINK1 Results in Mitochondrial Pathology and Can Be Rescued by Parkin Nicole Exner,1 Bettina Treske,1 Dominik Paquet,1 Kira Holmstro¨m,2 Carola Schiesling,2 Suzana Gispert,3 Iria Carballo-Carbajal,2 Daniela Berg,2 Hans-Hermann Hoepken,3 Thomas Gasser,2 Rejko Kru¨ger,2 Konstanze F. Winklhofer,4 Frank Vogel,5 Andreas S. Reichert,6,7 Georg Auburger,3 Philipp J. Kahle,1,2 Bettina Schmid,1 and Christian Haass1 1Center for Integrated Protein Science Munich and Adolf-Butenandt-Institute, Department of Biochemistry, Laboratory for Alzheimer’s and Parkinson’s Disease Research, Ludwig-Maximilians-University, 80336 Munich, Germany, 2Department of Neurodegeneration, Hertie Institute for Clinical Brain Research, 72076 Tu¨bingen, Germany, 3Section of Molecular Neurogenetics, Department of Neurology, Johann Wolfgang Goethe University Medical School, 60590 Frankfurt, Germany, 4Adolf-Butenandt-Institute, Department of Biochemistry, Neurobiochemistry Group, Ludwig-Maximilians-University, 80336 Munich, Germany, 5Max-Delbru¨ck-Center for Molecular Medicine, 13092 Berlin, Germany, 6Adolf-Butenandt-Institute, Physiological Chemistry, Ludwig- Maximilians-University, 81377 Munich, Germany, and 7Cluster of Excellence Macromolecular Complexes, Mitochondrial Biology, Johann Wolfgang Goethe University, 60590 Frankfurt, Germany Degeneration of dopaminergic neurons in the substantia nigra is characteristic for Parkinson’s disease (PD), the second most common neurodegenerative disorder. Mitochondrial dysfunction is believed to contribute to the etiology of PD. Although most cases are sporadic, recentevidencepointstoanumberofgenesinvolvedinfamilialvariantsofPD.Amongthem,aloss-of-functionofphosphataseandtensin homolog-induced kinase 1 (PINK1; PARK6) is associated with rare cases of autosomal recessive parkinsonism. In HeLa cells, RNA interference-mediated downregulation of PINK1 results in abnormal mitochondrial morphology and altered membrane potential. Mor- phological changes of mitochondria can be rescued by expression of wild-type PINK1 but not by PD-associated PINK1 mutants. More- over, primary cells derived from patients with two different PINK1 mutants showed a similar defect in mitochondrial morphology. Human parkin but not PD-associated mutants could rescue mitochondrial pathology in human cells like wild-type PINK1. Our results may therefore suggest that PINK1 deficiency in humans results in mitochondrial abnormalities associated with cellular stress, a patho- logical phenotype, which can be ameliorated by enhanced expression of parkin. Key words: neurodegeneration; familial Parkinson’s disease; PINK1; loss-of-function; mitochondria; parkin Introduction Until recently, PD was believed to be exclusively a sporadic dis- Parkinson’s disease (PD) is the second most abundant neurode- ease associated with aging and environmental stress. However, generative disease. The disease is associated with the degenera- there is now very strong evidence for genetic inheritance in a tion of dopaminergic neurons in the substantia nigra pars com- small number of families (Gasser, 2005). Autosomal dominant pacta and other areas of the brain. Neuropathologically, PD is mutations were observed in the genes encoding ␣-synuclein characterized by accumulation of cytoplasmic deposits (Lewy (Polymeropoulos et al., 1997) and LRRK2/dardarin (Paisan-Ruiz bodies and Lewy neurites) (Shen and Cookson, 2004), with et al., 2004; Zimprich et al., 2004). Autosomal recessive mutants ␣-synuclein as the major fibrillar component (Spillantini et al., associated with juvenile parkinsonism were found in parkin 1997; Baba et al., 1998). It is currently unclear whether insoluble (Kitada et al., 1998), DJ-1 (Bonifati et al., 2003), and the phos- deposits or, rather, soluble neurotoxic oligomers contribute to phatase and tensin homolog-induced kinase 1 (PINK1) (Rogaeva disease manifestation and progression (Haass and Selkoe, 2007). et al., 2004; Valente et al., 2004). PINK1 is a putative serine/ threonine kinase (Valente et al., 2004), the in vivo substrate of which is not yet known. So far, homozygous missense mutations Received Feb. 16, 2007; revised Sept. 25, 2007; accepted Sept. 28, 2007. and nonsense mutations affecting the kinase domain were ob- This work was supported by the Gottfried Wilhelm Leibniz Award from the Deutsche Forschungsgemeinschaft served. The putative kinase domain is apparently active because (DFG) (C.H.), Collaborative Research Center Grant SFB596 (project A12), the Center for Integrated Protein Science Munich, DFG Grants AU96/10-1 and GI342/3-1 (G.A.), German Ministry for Education and Research Grant NGFN-2 at least in vitro autophosphorylation activity was observed (C.H. and P.J.K.), the Hertie Foundation (P.J.K. and T.G.), and a NEUROTRAIN Early Stage Research Training Fellow- (Beilina et al., 2005; Silvestri et al., 2005). However, it is currently ship funded through the European Union research program FP6 (K.H). C.H. was supported by a “Forschungsprofes- unclear whether the missense mutations affect the kinase activity sur” from the Ludwig-Maximilians-University. We thank Drs. H. Steiner and A. Yamamoto for critical discussion. or any other biologically important function of PINK1 in vivo. Correspondence should be addressed to Dr. Christian Haass, Department of Biochemistry, Adolf-Butenandt- Institute, Schillerstrasse 44, 80336 Munich, Germany. E-mail: [email protected]. PINK1 contains a putative N-terminal mitochondrial targeting DOI:10.1523/JNEUROSCI.0719-07.2007 sequence, and evidence exists that PINK1 is targeted to mito- Copyright © 2007 Society for Neuroscience 0270-6474/07/2712413-06$15.00/0 chondria (Valente et al., 2004; Beilina et al., 2005; Silvestri et al., 12414 • J. Neurosci., November 7, 2007 • 27(45):12413–12418 Exner et al. • Parkin Rescues PINK1 Deficiency in Human Cells 2005; Gandhi et al., 2006), although PINK1 was also observed in the cytoplasm, in Lewy bodies (Gandhi et al., 2006), and in aggresomes (Muqit et al., 2006). Thus, in vivo mitochondrial targeting of PINK1 and its final destination within mitochon- dria is not entirely settled. Missense mu- tants of PINK1 apparently do not affect mitochondrial targeting (Beilina et al., 2005). A large body of evidence suggests that mitochondrial abnormalities are di- rectly associated with PD pathology (Shen and Cookson, 2004). In Drosophila, but not in vertebrates, PINK1 deficiency was shown to affect mitochondrial function and morphology (Clark et al., 2006; Park et al., 2006; Yang et al., 2006). In addition, there is evidence from Drosophila that PINK1 is genetically linked to parkin (Clark et al., 2006; Park et al., 2006; Yang et al., 2006). Unfortunately, little is known about PINK1 and the consequences of its deficiency in vertebrates. We now show that impairment of PINK1 in human cells (including cells from patients with two dif- ferent PINK1 mutations) causes abnor- malities in mitochondrial morphology and demonstrate that this defect can be ameliorated by increased expression of Figure 1. RNA silencing of PINK1 causes alteration in mitochondrial morphology in human cells. A, siRNA-mediated down- wild-type but not mutant parkin. regulation of PINK1. Untransfected HeLa cells (Ϫ), or cells transfected with a control siRNA (control), or an siRNA against PINK1 were analyzed for endogenous PINK1 protein levels. Cells were transfected at two different cell densities with 5, 15, or 100 pmol Materials and Methods ofsiRNA(1ϫ,3ϫ,or20ϫ)(pPINK1,precursorform;mPINK1,matureform).Across-reactiveproteinismarkedbyanasterisk.B, cDNA constructs Representative examples of normal or altered (truncated or fragmented) mitochondrial morphologies including higher magnifi- Point mutations A377C and G926A were intro- cations are shown. duced by PCR based on the PINK1 wild-type construct using primers 5Ј-CGGCCTGTC- using HiPerFect (Qiagen). After 2 d, the cells were harvested using PBS Ј Ј Ј Ј CGGAGATCCAG-3 and 5 -CTGGATCTCCGGACAGGCCG-3 or 5 - with 2 mM EDTA and washed once with PBS, after which they were Ј Ј GAAGGCCTGGACCATGGCCG-3 and 5 -CGGCCATGGTCCAGG- stained with 200 nM tetramethyl rhodamine methyl ester (TMRM; In- CCTTC-3Ј for subcloning into pcDNA6A (Invitrogen, Carlsbad, CA). vitrogen) for 15 min at room temperature. The cells were then washed For small interfering RNA (siRNA)-resistant constructs, four silent mu- once with PBS and kept at 4°C for the measuring procedure. For each tations were introduced by PCR inside the siRNA target sequence. sample, ϳ50,000 cells were scored after excitation with a 488 nm argon laser and emission through the phycoerythrin filter (575 nm) of a CyAn RNA interference-mediated downregulation of PINK1 and flow cytometer (DakoCytomation, Hamburg, Germany). The percent- fluorescent staining of mitochondria age of cells above and below the threshold TMRM fluorescence signal was PINK1 gene silencing was established by transfection of H:Performance- determined with Summit version 4.2 software. validated siRNA (target region 928–978 of human PINK1) or chemically unmodified nontargeting control siRNA from Qiagen (Hilden, Ger- many) into HeLa cells. For analysis of mitochondrial morphology, 2 ϫ Electron microscopy 10 4 HeLa cells were seeded on coated coverslips and transfected with 5 or Cells were fixed with freshly prepared 4% formaldehyde and 0.5% glu- 15 pmol of siRNA using Lipofectamine 2000 (Invitrogen, Karlsruhe, taraldehyde in PBS adjusted
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