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• W2146081034 abstract "Article29 January 2014Open Access Source Data Ret rescues mitochondrial morphology and muscle degeneration of Drosophila Pink1 mutants Pontus Klein Pontus Klein Molecules – Signaling – Development, Max Planck Institute of Neurobiology, Martinsried, Germany Search for more papers by this author Anne Kathrin Müller-Rischart Anne Kathrin Müller-Rischart German Center for Neurodegenerative Diseases (DZNE), Munich, Germany Search for more papers by this author Elisa Motori Elisa Motori Neurobiochemistry, Adolf Butenandt Institute, Ludwig Maximilians University, Munich, Germany Department of Life Quality Studies - Alma Mater Studiorum, University of Bologna, Bologna, Italy Search for more papers by this author Cornelia Schönbauer Cornelia Schönbauer Max Planck Institute of Biochemistry, Martinsried, Germany Search for more papers by this author Frank Schnorrer Frank Schnorrer Max Planck Institute of Biochemistry, Martinsried, Germany Search for more papers by this author Konstanze F Winklhofer Konstanze F Winklhofer German Center for Neurodegenerative Diseases (DZNE), Munich, Germany Neurobiochemistry, Adolf Butenandt Institute, Ludwig Maximilians University, Munich, Germany Molecular Cell Biology, Institute of Physiological Chemistry, Ruhr University Bochum, Bochum, Germany Munich Cluster for Systems Neurology (Synergy), Munich, Germany Search for more papers by this author Rüdiger Klein Corresponding Author Rüdiger Klein Molecules – Signaling – Development, Max Planck Institute of Neurobiology, Martinsried, Germany Munich Cluster for Systems Neurology (Synergy), Munich, Germany Search for more papers by this author Pontus Klein Pontus Klein Molecules – Signaling – Development, Max Planck Institute of Neurobiology, Martinsried, Germany Search for more papers by this author Anne Kathrin Müller-Rischart Anne Kathrin Müller-Rischart German Center for Neurodegenerative Diseases (DZNE), Munich, Germany Search for more papers by this author Elisa Motori Elisa Motori Neurobiochemistry, Adolf Butenandt Institute, Ludwig Maximilians University, Munich, Germany Department of Life Quality Studies - Alma Mater Studiorum, University of Bologna, Bologna, Italy Search for more papers by this author Cornelia Schönbauer Cornelia Schönbauer Max Planck Institute of Biochemistry, Martinsried, Germany Search for more papers by this author Frank Schnorrer Frank Schnorrer Max Planck Institute of Biochemistry, Martinsried, Germany Search for more papers by this author Konstanze F Winklhofer Konstanze F Winklhofer German Center for Neurodegenerative Diseases (DZNE), Munich, Germany Neurobiochemistry, Adolf Butenandt Institute, Ludwig Maximilians University, Munich, Germany Molecular Cell Biology, Institute of Physiological Chemistry, Ruhr University Bochum, Bochum, Germany Munich Cluster for Systems Neurology (Synergy), Munich, Germany Search for more papers by this author Rüdiger Klein Corresponding Author Rüdiger Klein Molecules – Signaling – Development, Max Planck Institute of Neurobiology, Martinsried, Germany Munich Cluster for Systems Neurology (Synergy), Munich, Germany Search for more papers by this author Author Information Pontus Klein1, Anne Kathrin Müller-Rischart2, Elisa Motori3,4,8, Cornelia Schönbauer5, Frank Schnorrer5, Konstanze F Winklhofer2,3,6,7 and Rüdiger Klein 1,7 1Molecules – Signaling – Development, Max Planck Institute of Neurobiology, Martinsried, Germany 2German Center for Neurodegenerative Diseases (DZNE), Munich, Germany 3Neurobiochemistry, Adolf Butenandt Institute, Ludwig Maximilians University, Munich, Germany 4Department of Life Quality Studies - Alma Mater Studiorum, University of Bologna, Bologna, Italy 5Max Planck Institute of Biochemistry, Martinsried, Germany 6Molecular Cell Biology, Institute of Physiological Chemistry, Ruhr University Bochum, Bochum, Germany 7Munich Cluster for Systems Neurology (Synergy), Munich, Germany 8Present address: Department of Mitochondrial Biology, Max Planck Institute for Biology of Ageing, Cologne, Germany *Corresponding author. Tel: +49 89 85783150; Fax: +49 89 85783152; E-mail: [email protected] The EMBO Journal (2014)33:341-355https://doi.org/10.1002/embj.201284290 Correction(s) for this article Ret rescues mitochondrial morphology and muscle degeneration of Drosophila Pink1 mutants01 April 2014 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Parkinson's disease (PD)-associated Pink1 and Parkin proteins are believed to function in a common pathway controlling mitochondrial clearance and trafficking. Glial cell line-derived neurotrophic factor (GDNF) and its signaling receptor Ret are neuroprotective in toxin-based animal models of PD. However, the mechanism by which GDNF/Ret protects cells from degenerating remains unclear. We investigated whether the Drosophila homolog of Ret can rescue Pink1 and park mutant phenotypes. We report that a signaling active version of Ret (RetMEN2B) rescues muscle degeneration, disintegration of mitochondria and ATP content of Pink1 mutants. Interestingly, corresponding phenotypes of park mutants were not rescued, suggesting that the phenotypes of Pink1 and park mutants have partially different origins. In human neuroblastoma cells, GDNF treatment rescues morphological defects of PINK1 knockdown, without inducing mitophagy or Parkin recruitment. GDNF also rescues bioenergetic deficits of PINK knockdown cells. Furthermore, overexpression of RetMEN2B significantly improves electron transport chain complex I function in Pink1 mutant Drosophila. These results provide a novel mechanism underlying Ret-mediated cell protection in a situation relevant for human PD. Synopsis Glial cell line derived neurotrophic factor (GDNF) improves survival in toxin-models of Parkinson's disease and is currently undergoing clinical development, however the protective mechanism is elusive. This study provides evidence that the GDNF receptor Ret rescues defects of a genetic Parkinson model and proposes a new mechanism-of-action. Active Ret overexpression rescues muscle degeneration and mitochondrial morphology in muscles and dopamine neurons in Pink1 mutant Drosophila. In human neuroblastoma cells, GDNF treatment rescues mitochondrial fragmentation caused by Pink1 knockdown. Ret signaling improves mitochondrial respiration and activity of complex I, providing a potential novel mechanism for the protective effect of GDNF/Ret. Introduction The etiology of Parkinson's Disease (PD) is highly complex and largely unknown, involving both environmental and genetic risk factors. Mitochondrial dysfunction, oxidative stress and protein aggregation are believed to be central events in the pathological process, but their interconnection remains unclear (Schapira & Jenner, 2011; Exner et al, 2012; McCoy & Cookson, 2012). The first indications of a role for mitochondria came with the discovery that the toxin 1-methyl-4-phenyl-1,2,3,4-tetrahydropyridine (MPTP) causes Parkinsonism in humans and animal models (Burns et al, 1983; Langston et al, 1983). Its active metabolite, 1-methyl-4-phenylpyridinium ion (MPP+), is selectively imported into dopaminergic neurons via the dopamine transporter, and inhibits complex I of the electron transport chain (ETC). Several other mitochondrial toxins, including paraquat and rotenone, generating either mitochondrial reactive oxygen species (ROS) or specifically inhibiting complex I, have been linked to PD in epidemiological studies and animal models (de Lau & Breteler, 2006). Furthermore, patients with sporadic PD can have decreased activity of complex I in brain and other tissues (Schapira et al, 1989; Parker & Swerdlow, 1998), or less complex I proteins in the substantia nigra (Mizuno et al, 1989). Autosomal recessive PD-associated proteins Parkin, PINK1 and DJ-1 (OMIM #600116, 605909, 606324) have been shown to have functions related to mitochondrial integrity, (reviewed in Exner et al, 2012; Martin et al, 2011). In three seminal studies, Pink1 mutant Drosophila displayed mitochondrial abnormalities and muscle degeneration in a manner highly similar to park mutants, and Parkin overexpression largely rescued the phenotypes of Pink1 mutants, but not vice versa, suggesting that the two proteins act in a common linear pathway (Clark et al, 2006; Park et al, 2006; Yang et al, 2006). Manipulation of the mitochondrial remodeling machinery rescues some Pink1 and park mutant phenotypes in Drosophila and in mammalian cell lines. However, while increasing fission rescues the Drosophila phenotypes, shifting the fusion/fission balance in the opposite direction rescues mammalian cell lines, but the underlying mechanisms are not fully understood (Deng et al, 2008; Poole et al, 2008; Lutz et al, 2009). PINK1, a mitochondrial Ser/Thr kinase, and Parkin, an E3 Ubiquitin ligase, were found to regulate clearance of damaged mitochondria via mitophagy (Geisler et al, 2010; Narendra et al, 2010; Vives-Bauza et al, 2010), and microtubular transport (Weihofen et al, 2009; Wang et al, 2011). However, other studies have reported additional functions of Parkin in the regulation of stress response proteins and mitochondrial biogenesis (Bouman et al, 2011; Shin et al, 2011), in promoting NF-κB signaling (Henn et al, 2007; Muller-Rischart et al, 2013), and in controlling cytochrome-c release (Berger et al, 2009). PINK1 also has additional functions, unrelated to recruiting Parkin, such as regulating mitochondrial calcium buffering (Gandhi et al, 2009; Sandebring et al, 2009; Heeman et al, 2011). Furthermore, PINK1 mutant mitochondria have decreased activity of complex I of the ETC (Morais et al, 2009), and overexpression of a yeast substitute for complex I rescued many of the functional impairments of Pink1 mutant flies (Vilain et al, 2012). Additional studies are required to elucidate which of the functions reported for Parkin and PINK1 are critical for causing Parkinson pathology. The neurotrophic factor Glial cell line-derived neurotrophic factor (GDNF) promotes the survival of dopamine neurons (Lin et al, 1993) and protects nigral dopamine neurons from cell death in rodent and primate toxin-models of PD such as 6-hydroxydopamine (6-OHDA) and MPTP (Kearns & Gash, 1995; Sauer et al, 1995; Tomac et al, 1995; Gash et al, 1996). Several clinical trials have been performed with mixed outcomes, but ongoing research and development aims at improving delivery methods of GDNF (Deierborg et al, 2008). GDNF signals via the GPI-anchored co-receptor GFR-α1 and the receptor tyrosine kinase Ret (Airaksinen & Saarma, 2002). Endogenous Ret expression is required for long-term survival of a fraction of nigral dopamine neurons in aged mice (Kramer et al, 2007). Conversely, mice that express a constitutively active Ret receptor in dopamine neurons (RetMEN2B) show increased numbers of dopamine neurons (Mijatovic et al, 2007). The mechanism by which GDNF/Ret protects dopamine neurons from cell death is not fully elucidated. We hypothesized that Ret-activated signaling pathways converge with functions of proteins associated with familial PD. We recently reported that Ret and DJ-1 double loss-of-function in aged mice exacerbates the neuron loss observed in Ret single mutants (Aron et al, 2010). Here, we investigated whether Ret interacts genetically with park and Pink1 in Drosophila. We found that constitutively active RetMEN2B specifically rescues phenotypes of Pink1 mutants, including muscle degeneration, mitochondrial morphology and function, whereas park mutants remained unaffected. Moreover, Ret signaling rescued mitochondrial morphological and functional defects of PINK1-deficient human SH-SY5Y cells, without activating mitophagy. Mechanistically, Ret signaling restored the activity of complex I of the ETC, which is reduced in Pink1, but not park mutant flies. Thus our study indicates that Ret signaling can specifically ameliorate Pink1 loss-of-function deficiencies that are relevant to human Parkinson's disease. Results Active Ret rescues Pink1 but not park mutant muscle degeneration To study whether Ret can modify Pink1 and park phenotypes, we utilized the Drosophila indirect flight muscles (IFMs) as a model system. Here, Pink1 and park mutants undergo significant muscle degeneration, likely because of the high energy consumption of the IFMs, and display enlarged mitochondria with broken cristae. Late stage pupae display normal muscle morphology, but soon after eclosion, the muscle tissue degenerates (Greene et al, 2003; Clark et al, 2006; Park et al, 2006). In 3- to 5-day-old Pink1 and park mutant animals housed at 18°C, interrupted muscles were found, and one or several of the six muscles displayed degenerated, highly irregular myofibrils with abnormal sarcomere structure, hereafter referred to as “degenerated” (Fig 1I and K) in approximately 65% of the animals as compared to controls, which never displayed this phenotype (Fig 1A, B, E, F, L). To investigate whether Ret signaling could modify muscle degeneration, we utilized the constitutively active version, RetMEN2B, which has an activating point mutation in the kinase domain (M955T) (Read et al, 2005). In an expression analysis of endogenous Ret by reverse transcriptase PCR (RT-PCR), we detected high levels of Ret mRNA in larvae and pupae, and lower levels in the adult thorax and IFMs (Supplementary Fig S1). To achieve robust overexpression of activated Ret specifically in muscles, we used the UAS-GAL4 system and the Myocyte enhancer factor-2 (Mef2) GAL4 driver, which is active in all muscle tissues from the early embryo throughout larval and pupal stages and in the adult fly. Mef2>RetMEN2B overexpression caused lethality at 25°C, but at 18°C, viable progeny eclosed with lower frequency. Surviving transgenic flies displayed mild muscle abnormalities, including deposits of actin dispersed over the muscle tissue, and some abnormally thick and irregular myofibrils (Fig 1C, G, J). A recent RNAi screen for modifiers of muscle development (Schnorrer et al, 2010) identified a large number of lines with a highly reminiscent phenotypic class and designated this “actin blobs”, we therefore refer to this by the same term. When RetMEN2B was overexpressed in the background of Pink1 mutants, the majority of flies showed significantly improved muscle morphology, with only 12% of flies displaying degenerated myofibrils (Fig 1D and L). The frequency of flies with actin blobs also decreased markedly compared to RetMEN2B expressing controls, suggesting that Pink1 function may be required for this phenotype. However, in contrast to Pink1 mutants, park mutants overexpressing RetMEN2B showed no improvement as the frequency of degenerated myofibrils remained unchanged (Fig 1H and L). Expression of the RetMEN2B protein was examined by Western Blot of thorax homogenates and levels were similar between the Pink1 and park mutants, indicating that differences in transgene expression were not a likely cause of the differential response (Fig 1M). To determine if Ret protein expression or Ret signaling was required for the phenotypic rescue, we overexpressed wild-type (WT) Ret using the same GAL4 driver. We found that RetWT was unable to modify the phenotype probably because the putative Ret ligand was not present in the IFMs at significant levels at this stage (Supplementary Fig S2). Moreover, the effects of Ret on IFM morphology appeared rather specific, since overexpression of a constitutively active fibroblast growth factor receptor (FGFR), UAS-htlλ, caused a dramatic change in IFM fate (data not shown). Figure 1. RetMEN2B overexpression rescues Pink1 but not park mutant muscle degeneration A–K. Drosophila hemi-thoraces stained with phalloidin at low magnification (upper panels) showing overall indirect flight muscle (IFM) morphology, and at higher magnification (lower panels). High-magnification images of WT sarcomeres (I), sarcomeres with ‘actin blobs’ (J), and degenerated sarcomeres (K). Heterozygous controls (A, E) display normal IFM layout (upper panels), myofibril morphology (lower panels) and sarcomeres (I). Pink1 (B) and park mutants (F) display abnormal morphologies with truncated muscles (yellow arrow heads, upper panels) and disorganized myofibrils (lower panels) with degenerated sarcomere structure (K). Animals overexpressing RetMEN2B (C, G) display normal IFM layout (upper panels), fairly normal myofibril morphology with occasional deposits of mislocated actin filaments, and “actin blobs”, (red arrow heads, lower panels and J). RetMEN2B overexpression in Pink1 mutants largely rescues the mutant phenotypes, as the majority of animals display normal IFM morphology (D), while park mutants are not rescued (H). L. Percentage of flies with phenotype “wild type” (blue), “actin blobs” (green), “degenerated” (red) or “actin blobs and degenerated” (yellow). M. Western blot analysis of Ret expression in thorax homogenates from w1118 controls, and Pink1, or park mutants overexpressing RetMEN2B, indicating similar levels of Ret overexpression between the two mutant backgrounds. Tissue from three animals per sample. Tubulin was used as a loading control. N–U. Overexpression of UAS-RetMEN2B under control of Mhc-GAL4 and Tub-GAL80ts, pupae were shifted from 18 to 30°C at pupal stage 11, activating expression after muscle formation is completed. Heterozygous controls (N, R) and RetMEN2B late overexpressing animals display normal muscle and myofibril morphologies (N, P, R, S, T). Pink1 (O) and park mutants (S) display abnormal morphologies with truncated muscles and disorganized myofibrils with degenerated sarcomere structure (lower panels). Late RetMEN2B overexpression in Pink1 mutants (Q) largely rescues the mutant phenotypes, while park mutants (U) are not rescued. V. Percentage of flies with phenotype “wild type” (blue) or “degenerated” (red). Number of animals per genotype as depicted in figure. Data information: Scale bars: upper panels, 100 μm; lower panels, 10 μm. Source data are available online for this figure Source Data for Figure 1M [embj201284290-SourceData-Figure1M.pdf] Download figure Download PowerPoint Rescue of Pink1 mutants is not developmental The partial embryonic lethality and appearance of actin blobs by Mef2>RetMEN2B overexpression indicated that high levels of Ret signaling interfered with normal muscle development. Other receptor tyrosine kinases such as epidermal growth factor receptor (EGFR) and FGFR are known to regulate embryonic myoblast specification via Ras/Erk signaling (Carmena et al, 1998; Halfon et al, 2000), and the insulin receptor controls muscle size (Demontis & Perrimon, 2009). Therefore, it is plausible that active RetMEN2B affects these, or similar developmental processes. To verify that the rescue of the Pink1 mutants is not a developmental interaction, we utilized the GAL80ts system which permits transgene expression in a defined time window regulated by temperature. To drive RetMEN2B expression, we chose the GAL4 driver, Myosin heavy chain (Mhc) GAL4, which expresses only in differentiated muscles, not in myoblasts, in difference to Mef-GAL4 and generates higher expression. Unlike Mef2-GAL4, it causes complete lethality when driving RetMEN2B from embryonic stages. Flies were crossed at 18°C (non-permissive temperature), after which pupae were shifted to 30°C (permissive temperature) at pharate adult stage P11 ± 3 h (equivalent of 75 h APF at 25°C) (Flybase FBdv:00005349), a time well after completion of IFM development, but before the onset of apoptotic degeneration in Pink1 and park mutants (Greene et al, 2003; Clark et al, 2006). Analyses were again performed at 3–5 days post-eclosion. Using this protocol, Pink1 and park mutants showed degenerated myofibrils with a frequency of approximately 90% and 80% respectively as compared to controls (Fig 1N, O, R, S, V), the higher penetrance being likely due to the increased temperature. RetMEN2B-overexpressing flies eclosed with Mendelian frequencies and displayed fully normal muscle morphology, without the presence of actin blobs, confirming the hypothesis that the lethality and actin blob phenotypes have developmental origins (Fig 1P, T, V). When RetMEN2B was expressed in Pink1 mutants from this late pupal stage and onwards, it again largely rescued muscle degeneration, indicating that the rescue is not due to a developmental interaction, but a direct protective effect of Ret signaling on degenerating tissue (Fig 1Q and V). Interestingly, park mutants were again not rescued using this expression protocol (Fig 1U and V). Ret signaling rescues mitochondrial morphology in flight muscles One possibility is that RetMEN2B inhibits muscle degeneration without directly targeting the primary cause of the Pink1 phenotype: mitochondrial impairments (Clark et al, 2006). To test this possibility, we analyzed the ultrastructure of mitochondria using transmission electron microscopy. IFMs from control flies showed regular organization of myofibrils and densely packed mitochondria with intact cristae (Fig 2A, E, L, M). Pink1 and park mutants displayed a heterogeneous population of mitochondria with the majority having significantly enlarged sizes and mild or severe disruption of their cristae structure, when compared to control mitochondria (Fig 2B, F, I-M). Mef2>RetMEN2B overexpression in control flies did not alter normal mitochondria morphology (Fig 2C, G, L, M). However, in Pink1 mutants, RetMEN2B overexpression significantly reduced the fraction of severely impaired mitochondria and increased the fraction of mitochondria with WT-like cristae structure (Fig 2D and L). In contrast, park mutants showed no improvement of structural impairments when RetMEN2B was overexpressed (Fig 2H and M). These results demonstrate that RetMEN2B can rescue mitochondrial impairments of pink1 but not park mutants, suggesting that the mitochondrial deficiencies of the two mutant strains have partially different origins. Figure 2. RetMEN2B rescues mitochondrial cristae structure of Pink1 mutants A–K. Transmission electron microscopy images of indirect flight muscles. Heterozygous controls (A, E) and animals overexpressing RetMEN2B (B, F) display normal mitochondria of similar size with highly dense cristae structure. Pink1 and park mutants have enlarged mitochondria with broken cristae (C, G). Phenotype can vary from mild to severe. High-power images of mitochondria are shown for the categories “wild type” (I), “mild” (J), “severe” phenotype (K). RetMEN2B overexpression partially restores mitochondrial size and cristae structure in Pink1 (D), but not park mutants (H). Scale bar, 2 μm. L, M. Percentages of mitochondria of the indicated categories, 500–800 mitochondria per animal, averages of 6 animals per genotype. Download figure Download PowerPoint Ret rescues mitochondrial morphology in dopaminergic neurons To address whether RetMEN2B also rescues the morphology of mitochondria in dopaminergic neurons, we overexpressed RetMEN2B using TH-GAL4 together with the mitochondrial marker mitoGFP (Pilling et al, 2006). Pink1 and park mutants displayed severely enlarged mitochondria as compared to controls (Fig 3A, B, E, F, I, J). RetMEN2B overexpression in a control background did not significantly alter the normal mitochondrial background (Fig 3C, G, I, J). However, when overexpressed in Pink1 mutants, mitochondrial size was significantly rescued (Fig 3D and I). Quantification of mitochondrial volumes revealed that in the presence of RetMEN2B the abundance of normal mitochondria was increased, while the fraction of enlarged mitochondria decreased to levels similar to those of control flies. Merely, the 4% largest mitochondria were not rescued. In line with the analysis of mitochondria in muscle, mitochondrial morphology in neurons of park mutants was not rescued by RetMEN2B (Fig 3H and J). Figure 3. Rescue of Pink1 mutant dopamine neuron mitochondria by RetMEN2B A–H. Confocal maximum projections (left panels) and isosurface renderings (right panels) of dopamine neuron mitochondria in the PPL1 cluster of dopaminergic neurons, visualized by mitoGFP and immunostainings against GFP and TH. Genotypes: All flies contain TH-GAL4 and UAS-mitoGFP and Pink1, park mutant alleles, as well as UAS-RetMEN2B as indicated. Isosurface renderings are color-coded according to volume from 0 to 3 μm3. RetMEN2B-overexpressing control animals (C, G) display normal mitochondrial morphology as compared to non-transgenic controls (A, E). Pink1 mutants (B) and park mutants (F) display severely enlarged mitochondria, and RetMEN2B partially rescues mitochondrial size in Pink1 mutants (D), but not in park mutants (H). Scale bar, 5 μm. I, J. Mitochondrial volume distributions of (A–D) and (E–H) in categories as indicated. Due to differences in staining and imaging conditions, data between the Pink1 and park datasets cannot be directly compared. n = 8–20 animals per genotype. Download figure Download PowerPoint GDNF/Ret signaling rescues mitochondrial defects in mammalian cells In order to assess whether signaling from endogenous Ret can also rescue mitochondrial impairments caused by loss of PINK1 function, we used the human dopaminergic neuroblastoma cell line SH-SY5Y, which expresses endogenous Ret. Acute knock-down of PINK1 in this cell line was previously shown to cause fragmentation of the mitochondrial network (Lutz et al, 2009) (Fig 4A, B, D). Stimulation of Ret by GDNF and soluble GFRα-1 rescued mitochondrial fragmentation, demonstrating that endogenous mammalian Ret can rescue mitochondrial impairments (Fig 4C and D). A semi-quantitative RT-PCR analysis of PINK1 mRNA controlled that GDNF/GFRα-1 stimulation did not upregulate PINK1 levels (Fig 4E). Figure 4. Activation of Ret signaling mammalian cells rescues PINK1 deficiency, but has no effect on mitophagy A–C. SH-SY5Y cells expressing endogenous Ret, transfected with scrambled control siRNA (A) display normal tubular mitochondrial morphology, visualized by immunostaining for TOM20 (white); DAPI (blue) indicates nuclei. Cells silenced for PINK1 expression display increased mitochondrial fragmentation (B). Stimulation of Ret signaling by treatment of cells with GDNF together with soluble GFRα-1 rescues mitochondrial fragmentation after PINK1 knockdown (C). D. Quantification of cells with either tubular (gray) or fragmented (blue) mitochondria. E. Quantification of PINK1 mRNA by quantitative RT-PCR indicates that GDNF/GFRα-1 treatment has no effect on PINK1 expression. F–M. SH-SY5Y cells were treated with CCCP for 2 or 24 h to depolarize mitochondria, and then stained for HSP60 (red), DAPI (blue) and Parkin or Ret (green) as indicated. Cells with endogenous Parkin expression display low levels of mitophagy (F) and no cells fully cleared of mitochondria were detected 24 h after CCCP treatment. Cells overexpressing Parkin display translocation of Parkin to mitochondria 2 h after CCCP treatment and complete clearance of mitochondria by 24 h after adding CCCP (G). White arrowheads indicate cells without detectable mitochondria. Silencing of PINK1 by siRNA largely inhibits Parkin translocation and mitophagy (H), whereas silencing of Ret has no effect on mitophagy alone (I) or in cells overexpressing Parkin (J). Overexpression of constitutively active RetMEN2A does not activate mitophagy in control or PINK1-silenced cells (K, L), and does not modulate Parkin translocation or mitophagy in Parkin-overexpressing cells (M). N. Quantification of the experiments described in (F–M). O. Quantification of mRNA after PINK1 or Ret silencing by quantitative RT-PCR. Download figure Download PowerPoint Ret rescues mitochondrial morphology independently of Parkin-induced mitophagy Although the data so far suggested that Ret rescues Pink1-deficient mitochondria independently of Parkin, we cannot exclude that Ret signaling activates Parkin translocation to mitochondria, thus promoting their clearance through mitophagy. To test this hypothesis, we treated SH-SY5Y cells overexpressing Parkin with carbonyl cyanide m-chlorophenyl hydrazone (CCCP) to depolarize mitochondria. CCCP treatment induced recruitment of Parkin to mitochondria (detected 2 h after adding CCCP) followed by the removal of depolarized mitochondria in about 50% of Parkin-expressing SH-SY5Y cells (monitored 24 h later) (Fig 4G and N). Parkin-induced mitophagy required the presence of PINK1, as described previously (Geisler et al, 2010; Narendra et al, 2010; Vives-Bauza et al, 2010), but was not impaired in cells silenced for Ret expression (Fig 4H, I, J, N, O). Moreover, the overexpression of constitutively active RetMEN2A did not induce Parkin translocation or mitophagy under any condition, including PINK1 knock-down with or without Parkin overexpression (Fig 4K, L, M, N). Similar results were obtained when GDNF and soluble GFRα-1 was used to activate signaling via endogenous Ret (Fig 4N). Furthermore, GDNF/GFRα-1 treatment also rescued mitochondrial fragmentation induced by PINK1 silencing HeLa cells, a cell type which does not express endogenous Parkin (Denison et al, 2003; Pawlyk et al, 2003), further indicating that Ret signaling rescues PINK1 loss-of-function phenotypes independently of Parkin (Supplementary Fig S3). Ret signaling rescues impaired bioenergetics of Pink1-deficient cells It has been reported previously that PINK1 deficiency impairs mitochondrial respiration (Gautier et al, 2008, 2012; Gandhi et al, 2009;" @default.
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• W2146081034 title "Ret rescues mitochondrial morphology and muscle degeneration of Drosophila Pink1 mutants" @default.
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• W2146081034 doi "https://doi.org/10.1002/embj.201284290" @default.
• W2146081034 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/4000093" @default.
• W2146081034 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/24473149" @default.
• W2146081034 hasPublicationYear "2014" @default.
• W2146081034 type Work @default.

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