CIN85 regulates dopamine receptor endocytosis and governs behaviour in mice
2010; Springer Nature; Volume: 29; Issue: 14 Linguagem: Inglês
10.1038/emboj.2010.120
ISSN1460-2075
AutoresNoriaki Shimokawa, Kaisa Haglund, Sabine M. Hölter, Caroline Grabbe, Vladimir Kirkin, Noriyuki Koibuchi, Christian Schultz, Jan Rozman, Daniela Hoeller, Chun‐Hong Qiu, Marina Londoño, Jun Ikezawa, Peter Jedlička, Birgit Stein, Stephan W. Schwarzacher, David P Wolfer, Nicole Ehrhardt, Rainer Heuchel, Ioannis P. Nezis, Andreas Brech, Mirko H. H. Schmidt, Helmut Fuchs, Valérie Gailus‐Durner, Martin Klingenspor, Oliver Bögler, Wolfgang Wurst, Thomas Deller, Martin Hrabé de Angelis, Ivan Đikić,
Tópico(s)Pancreatic function and diabetes
ResumoArticle15 June 2010free access CIN85 regulates dopamine receptor endocytosis and governs behaviour in mice Noriaki Shimokawa Noriaki Shimokawa Institute of Biochemistry II and Cluster of Excellence Macromolecular Complexes, Goethe University, Frankfurt (Main), Germany Department of Integrative Physiology, Gunma University Graduate School of Medicine, Gunma, Japan Search for more papers by this author Kaisa Haglund Kaisa Haglund Institute of Biochemistry II and Cluster of Excellence Macromolecular Complexes, Goethe University, Frankfurt (Main), Germany Department of Biochemistry, Institute for Cancer Research, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway Centre for Cancer Biomedicine, Faculty of Medicine, University of Oslo, Oslo, Norway Search for more papers by this author Sabine M Hölter Sabine M Hölter Institute of Developmental Genetics, Helmholtz Zentrum München, German Research Center for Environmental Health, Neuherberg, Germany Technische Universität München, Germany Search for more papers by this author Caroline Grabbe Caroline Grabbe Institute of Biochemistry II and Cluster of Excellence Macromolecular Complexes, Goethe University, Frankfurt (Main), Germany Search for more papers by this author Vladimir Kirkin Vladimir Kirkin Institute of Biochemistry II and Cluster of Excellence Macromolecular Complexes, Goethe University, Frankfurt (Main), Germany Search for more papers by this author Noriyuki Koibuchi Noriyuki Koibuchi Department of Integrative Physiology, Gunma University Graduate School of Medicine, Gunma, Japan Search for more papers by this author Christian Schultz Christian Schultz Institute of Clinical Neuroanatomy, Dr. Senckenberg Anatomy, Goethe University, Frankfurt (Main), Germany Medizinische Fakultät Mannheim der Universität Heidelberg, Zentrum für Biomedizin und Medizintechnik, Mannheim, Germany Search for more papers by this author Jan Rozman Jan Rozman Molecular Nutritional Medicine, Else-Kröner-Fresenius Center, Technische Universität München, Freising-Weihenstephan, Germany Institute of Experimental Genetics and German Mouse Clinic, Helmholtz Zentrum Muenchen, German Research Center for Environmental Health, Neuherberg, Germany Technische Universität München, Germany Search for more papers by this author Daniela Hoeller Daniela Hoeller Institute of Biochemistry II and Cluster of Excellence Macromolecular Complexes, Goethe University, Frankfurt (Main), Germany Search for more papers by this author Chun-Hong Qiu Chun-Hong Qiu Department of Integrative Physiology, Gunma University Graduate School of Medicine, Gunma, Japan Search for more papers by this author Marina B Londoño Marina B Londoño Department of Integrative Physiology, Gunma University Graduate School of Medicine, Gunma, Japan Search for more papers by this author Jun Ikezawa Jun Ikezawa Department of Integrative Physiology, Gunma University Graduate School of Medicine, Gunma, Japan Search for more papers by this author Peter Jedlicka Peter Jedlicka Institute of Clinical Neuroanatomy, Dr. Senckenberg Anatomy, Goethe University, Frankfurt (Main), Germany Search for more papers by this author Birgit Stein Birgit Stein Institute of Biochemistry II and Cluster of Excellence Macromolecular Complexes, Goethe University, Frankfurt (Main), Germany Search for more papers by this author Stephan W Schwarzacher Stephan W Schwarzacher Institute of Clinical Neuroanatomy, Dr. Senckenberg Anatomy, Goethe University, Frankfurt (Main), Germany Search for more papers by this author David P Wolfer David P Wolfer Institute of Anatomy, University of Zurich, and Institute of Human Movement Sciences and Sport, ETH Zurich, Zurich, Switzerland Search for more papers by this author Nicole Ehrhardt Nicole Ehrhardt Molecular Nutritional Medicine, Else-Kröner-Fresenius Center, Technische Universität München, Freising-Weihenstephan, Germany Institute of Experimental Genetics and German Mouse Clinic, Helmholtz Zentrum Muenchen, German Research Center for Environmental Health, Neuherberg, Germany Technische Universität München, Germany Search for more papers by this author Rainer Heuchel Rainer Heuchel Ludwig Institute for Cancer Research, Uppsala University, Uppsala, Sweden Karolinska Hospital, KFC, Clinical Research Center, Huddinge, Sweden Search for more papers by this author Ioannis Nezis Ioannis Nezis Department of Biochemistry, Institute for Cancer Research, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway Centre for Cancer Biomedicine, Faculty of Medicine, University of Oslo, Oslo, Norway Search for more papers by this author Andreas Brech Andreas Brech Department of Biochemistry, Institute for Cancer Research, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway Centre for Cancer Biomedicine, Faculty of Medicine, University of Oslo, Oslo, Norway Search for more papers by this author Mirko H H Schmidt Mirko H H Schmidt Institute of Biochemistry II and Cluster of Excellence Macromolecular Complexes, Goethe University, Frankfurt (Main), Germany Search for more papers by this author Helmut Fuchs Helmut Fuchs Institute of Experimental Genetics and German Mouse Clinic, Helmholtz Zentrum Muenchen, German Research Center for Environmental Health, Neuherberg, Germany Technische Universität München, Germany Search for more papers by this author Valerie Gailus-Durner Valerie Gailus-Durner Institute of Experimental Genetics and German Mouse Clinic, Helmholtz Zentrum Muenchen, German Research Center for Environmental Health, Neuherberg, Germany Technische Universität München, Germany Search for more papers by this author Martin Klingenspor Martin Klingenspor Molecular Nutritional Medicine, Else-Kröner-Fresenius Center, Technische Universität München, Freising-Weihenstephan, Germany Search for more papers by this author Oliver Bogler Oliver Bogler Departments of Neurosurgery, Neuro-Oncology and Brain Tumor Center, University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Wolfgang Wurst Wolfgang Wurst Institute of Developmental Genetics, Helmholtz Zentrum München, German Research Center for Environmental Health, Neuherberg, Germany Technische Universität München, Germany Search for more papers by this author Thomas Deller Thomas Deller Institute of Clinical Neuroanatomy, Dr. Senckenberg Anatomy, Goethe University, Frankfurt (Main), Germany Search for more papers by this author Martin Hrabé de Angelis Martin Hrabé de Angelis Institute of Experimental Genetics and German Mouse Clinic, Helmholtz Zentrum Muenchen, German Research Center for Environmental Health, Neuherberg, Germany Technische Universität München, Germany Search for more papers by this author Ivan Dikic Corresponding Author Ivan Dikic Institute of Biochemistry II and Cluster of Excellence Macromolecular Complexes, Goethe University, Frankfurt (Main), Germany Tumor Biology Program, Mediterranean Institute for Life Sciences, Mestrovicevo Setaliste, Split, Croatia Search for more papers by this author Noriaki Shimokawa Noriaki Shimokawa Institute of Biochemistry II and Cluster of Excellence Macromolecular Complexes, Goethe University, Frankfurt (Main), Germany Department of Integrative Physiology, Gunma University Graduate School of Medicine, Gunma, Japan Search for more papers by this author Kaisa Haglund Kaisa Haglund Institute of Biochemistry II and Cluster of Excellence Macromolecular Complexes, Goethe University, Frankfurt (Main), Germany Department of Biochemistry, Institute for Cancer Research, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway Centre for Cancer Biomedicine, Faculty of Medicine, University of Oslo, Oslo, Norway Search for more papers by this author Sabine M Hölter Sabine M Hölter Institute of Developmental Genetics, Helmholtz Zentrum München, German Research Center for Environmental Health, Neuherberg, Germany Technische Universität München, Germany Search for more papers by this author Caroline Grabbe Caroline Grabbe Institute of Biochemistry II and Cluster of Excellence Macromolecular Complexes, Goethe University, Frankfurt (Main), Germany Search for more papers by this author Vladimir Kirkin Vladimir Kirkin Institute of Biochemistry II and Cluster of Excellence Macromolecular Complexes, Goethe University, Frankfurt (Main), Germany Search for more papers by this author Noriyuki Koibuchi Noriyuki Koibuchi Department of Integrative Physiology, Gunma University Graduate School of Medicine, Gunma, Japan Search for more papers by this author Christian Schultz Christian Schultz Institute of Clinical Neuroanatomy, Dr. Senckenberg Anatomy, Goethe University, Frankfurt (Main), Germany Medizinische Fakultät Mannheim der Universität Heidelberg, Zentrum für Biomedizin und Medizintechnik, Mannheim, Germany Search for more papers by this author Jan Rozman Jan Rozman Molecular Nutritional Medicine, Else-Kröner-Fresenius Center, Technische Universität München, Freising-Weihenstephan, Germany Institute of Experimental Genetics and German Mouse Clinic, Helmholtz Zentrum Muenchen, German Research Center for Environmental Health, Neuherberg, Germany Technische Universität München, Germany Search for more papers by this author Daniela Hoeller Daniela Hoeller Institute of Biochemistry II and Cluster of Excellence Macromolecular Complexes, Goethe University, Frankfurt (Main), Germany Search for more papers by this author Chun-Hong Qiu Chun-Hong Qiu Department of Integrative Physiology, Gunma University Graduate School of Medicine, Gunma, Japan Search for more papers by this author Marina B Londoño Marina B Londoño Department of Integrative Physiology, Gunma University Graduate School of Medicine, Gunma, Japan Search for more papers by this author Jun Ikezawa Jun Ikezawa Department of Integrative Physiology, Gunma University Graduate School of Medicine, Gunma, Japan Search for more papers by this author Peter Jedlicka Peter Jedlicka Institute of Clinical Neuroanatomy, Dr. Senckenberg Anatomy, Goethe University, Frankfurt (Main), Germany Search for more papers by this author Birgit Stein Birgit Stein Institute of Biochemistry II and Cluster of Excellence Macromolecular Complexes, Goethe University, Frankfurt (Main), Germany Search for more papers by this author Stephan W Schwarzacher Stephan W Schwarzacher Institute of Clinical Neuroanatomy, Dr. Senckenberg Anatomy, Goethe University, Frankfurt (Main), Germany Search for more papers by this author David P Wolfer David P Wolfer Institute of Anatomy, University of Zurich, and Institute of Human Movement Sciences and Sport, ETH Zurich, Zurich, Switzerland Search for more papers by this author Nicole Ehrhardt Nicole Ehrhardt Molecular Nutritional Medicine, Else-Kröner-Fresenius Center, Technische Universität München, Freising-Weihenstephan, Germany Institute of Experimental Genetics and German Mouse Clinic, Helmholtz Zentrum Muenchen, German Research Center for Environmental Health, Neuherberg, Germany Technische Universität München, Germany Search for more papers by this author Rainer Heuchel Rainer Heuchel Ludwig Institute for Cancer Research, Uppsala University, Uppsala, Sweden Karolinska Hospital, KFC, Clinical Research Center, Huddinge, Sweden Search for more papers by this author Ioannis Nezis Ioannis Nezis Department of Biochemistry, Institute for Cancer Research, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway Centre for Cancer Biomedicine, Faculty of Medicine, University of Oslo, Oslo, Norway Search for more papers by this author Andreas Brech Andreas Brech Department of Biochemistry, Institute for Cancer Research, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway Centre for Cancer Biomedicine, Faculty of Medicine, University of Oslo, Oslo, Norway Search for more papers by this author Mirko H H Schmidt Mirko H H Schmidt Institute of Biochemistry II and Cluster of Excellence Macromolecular Complexes, Goethe University, Frankfurt (Main), Germany Search for more papers by this author Helmut Fuchs Helmut Fuchs Institute of Experimental Genetics and German Mouse Clinic, Helmholtz Zentrum Muenchen, German Research Center for Environmental Health, Neuherberg, Germany Technische Universität München, Germany Search for more papers by this author Valerie Gailus-Durner Valerie Gailus-Durner Institute of Experimental Genetics and German Mouse Clinic, Helmholtz Zentrum Muenchen, German Research Center for Environmental Health, Neuherberg, Germany Technische Universität München, Germany Search for more papers by this author Martin Klingenspor Martin Klingenspor Molecular Nutritional Medicine, Else-Kröner-Fresenius Center, Technische Universität München, Freising-Weihenstephan, Germany Search for more papers by this author Oliver Bogler Oliver Bogler Departments of Neurosurgery, Neuro-Oncology and Brain Tumor Center, University of Texas MD Anderson Cancer Center, Houston, TX, USA Search for more papers by this author Wolfgang Wurst Wolfgang Wurst Institute of Developmental Genetics, Helmholtz Zentrum München, German Research Center for Environmental Health, Neuherberg, Germany Technische Universität München, Germany Search for more papers by this author Thomas Deller Thomas Deller Institute of Clinical Neuroanatomy, Dr. Senckenberg Anatomy, Goethe University, Frankfurt (Main), Germany Search for more papers by this author Martin Hrabé de Angelis Martin Hrabé de Angelis Institute of Experimental Genetics and German Mouse Clinic, Helmholtz Zentrum Muenchen, German Research Center for Environmental Health, Neuherberg, Germany Technische Universität München, Germany Search for more papers by this author Ivan Dikic Corresponding Author Ivan Dikic Institute of Biochemistry II and Cluster of Excellence Macromolecular Complexes, Goethe University, Frankfurt (Main), Germany Tumor Biology Program, Mediterranean Institute for Life Sciences, Mestrovicevo Setaliste, Split, Croatia Search for more papers by this author Author Information Noriaki Shimokawa1,2,‡, Kaisa Haglund1,3,4,‡, Sabine M Hölter5,6, Caroline Grabbe1, Vladimir Kirkin1, Noriyuki Koibuchi2, Christian Schultz7,8, Jan Rozman9,10,11, Daniela Hoeller1, Chun-Hong Qiu2, Marina B Londoño2, Jun Ikezawa2, Peter Jedlicka7, Birgit Stein1, Stephan W Schwarzacher7, David P Wolfer12, Nicole Ehrhardt9,10,11, Rainer Heuchel13,14, Ioannis Nezis3,4, Andreas Brech3,4, Mirko H H Schmidt1, Helmut Fuchs10,11, Valerie Gailus-Durner10,11, Martin Klingenspor9, Oliver Bogler15, Wolfgang Wurst5,6, Thomas Deller7, Martin Hrabé de Angelis10,11 and Ivan Dikic 1,16 1Institute of Biochemistry II and Cluster of Excellence Macromolecular Complexes, Goethe University, Frankfurt (Main), Germany 2Department of Integrative Physiology, Gunma University Graduate School of Medicine, Gunma, Japan 3Department of Biochemistry, Institute for Cancer Research, The Norwegian Radium Hospital, Oslo University Hospital, Oslo, Norway 4Centre for Cancer Biomedicine, Faculty of Medicine, University of Oslo, Oslo, Norway 5Institute of Developmental Genetics, Helmholtz Zentrum München, German Research Center for Environmental Health, Neuherberg, Germany 6Technische Universität München, Germany 7Institute of Clinical Neuroanatomy, Dr. Senckenberg Anatomy, Goethe University, Frankfurt (Main), Germany 8Medizinische Fakultät Mannheim der Universität Heidelberg, Zentrum für Biomedizin und Medizintechnik, Mannheim, Germany 9Molecular Nutritional Medicine, Else-Kröner-Fresenius Center, Technische Universität München, Freising-Weihenstephan, Germany 10Institute of Experimental Genetics and German Mouse Clinic, Helmholtz Zentrum Muenchen, German Research Center for Environmental Health, Neuherberg, Germany 11Technische Universität München, Germany 12Institute of Anatomy, University of Zurich, and Institute of Human Movement Sciences and Sport, ETH Zurich, Zurich, Switzerland 13Ludwig Institute for Cancer Research, Uppsala University, Uppsala, Sweden 14Karolinska Hospital, KFC, Clinical Research Center, Huddinge, Sweden 15Departments of Neurosurgery, Neuro-Oncology and Brain Tumor Center, University of Texas MD Anderson Cancer Center, Houston, TX, USA 16Tumor Biology Program, Mediterranean Institute for Life Sciences, Mestrovicevo Setaliste, Split, Croatia ‡These authors contributed equally to this work *Corresponding author. Institute of Biochemistry II, Goethe University Medical School, Theodor-Stern-Kai 7, Frankfurt 60596, Germany. Tel.: +49 69 6301 83647; Fax: +49 69 6301 5577; E-mail: [email protected] The EMBO Journal (2010)29:2421-2432https://doi.org/10.1038/emboj.2010.120 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 Despite extensive investigations of Cbl-interacting protein of 85 kDa (CIN85) in receptor trafficking and cytoskeletal dynamics, little is known about its functions in vivo. Here, we report the study of a mouse deficient of the two CIN85 isoforms expressed in the central nervous system, exposing a function of CIN85 in dopamine receptor endocytosis. Mice lacking CIN85 exon 2 (CIN85Δex2) show hyperactivity phenotypes, characterized by increased physical activity and exploratory behaviour. Interestingly, CIN85Δex2 animals display abnormally high levels of dopamine and D2 dopamine receptors (D2DRs) in the striatum, an important centre for the coordination of animal behaviour. Importantly, CIN85 localizes to the post-synaptic compartment of striatal neurons in which it co-clusters with D2DRs. Moreover, it interacts with endocytic regulators such as dynamin and endophilins in the striatum. Absence of striatal CIN85 causes insufficient complex formation of endophilins with D2DRs in the striatum and ultimately decreased D2DR endocytosis in striatal neurons in response to dopamine stimulation. These findings indicate an important function of CIN85 in the regulation of dopamine receptor functions and provide a molecular explanation for the hyperactive behaviour of CIN85Δex2 mice. Introduction Cbl-interacting protein of 85 kDa (CIN85)/SH3-domain kinase binding protein 1 (SH3KBP1) forms, together with CMS (p130Cas ligand with multiple SH3 domains)/CD2-associated protein (CD2AP), a family of adaptor molecules with established functions in coordinating the spatial and temporal assembly of protein complexes during receptor endocytosis, formation of kidney glomeruli and organization of the immunological synapse in T cells (Dikic, 2002). Common to both CIN85/SH3KBP1 (alias CIN85) and CMS/CD2AP is a core structural organization comprising three N-terminal SH3 domains, followed by a centrally located proline-rich region and a C-terminal coiled coil (Dikic, 2002). In addition, in a functional perspective, both family members share certain characteristics, given that their SH3 domains specifically recognize a consensus Px(P/A)xxR motif, present in Cbl (Petrelli et al, 2002; Soubeyran et al, 2002; Kurakin et al, 2003; Kobayashi et al, 2004; Jozic et al, 2005; Moncalian et al, 2006) and other proteins involved in endocytosis, including synaptojanin, Huntingtin-interacting protein 1-related, Alix and Dab2 (Chen et al, 2000; Kowanetz et al, 2004), as well as regulators of the actin cytoskeleton, such as cortactin and the actin-capping protein CAPZ (Kirsch et al, 2001; Welsch et al, 2001; Cormont et al, 2003; Hutchings et al, 2003; Lynch et al, 2003). Indeed, a function of CIN85 and CMS/CD2AP in the regulation of actin dynamics is emphasized by their ability to directly interact with F-actin and induce bundling of actin filaments (Gaidos et al, 2007). The multitude of verified interaction partners has placed CIN85 as a central adaptor molecule involved in the recruitment of the endocytic machinery required for the internalization of a variety of cell surface receptors, including growth factor receptors (such as EGFR, Met and VEGFR) (Petrelli et al, 2002; Soubeyran et al, 2002; Kobayashi et al, 2004), immunoglobulin IgE receptors in mast cells (Molfetta et al, 2005), as well as during the infectious internalization of the bacterial pathogen Listeria monocytogenes (Veiga and Cossart, 2005). Despite sharing many overlapping molecular functions in cultured cells, investigations in mice deficient of CD2AP have highlighted that CIN85 and CD2AP may have distinct functions in vivo. In agreement with its abundance in the kidney, mice lacking CD2AP suffer from a nephrotic syndrome caused by defective podocyte functionality during the formation of the glomerular slit diaphragm, eventually resulting in lethality at 6–8 weeks of age (Shih et al, 1999). An important function for CD2AP in spermatocyte production has furthermore been established, given that podocyte-specific reintroduction of transgenic CD2AP in knockout mice is sufficient to rescue animals into adulthood, but renders male mice infertile (Grunkemeyer et al, 2005). Interestingly, the podocyte and the cells of the basal seminiferous tubule are both cell types in which CIN85 is poorly expressed. As a result of alternative splicing and differential promoter usage, the mouse CIN85 genomic locus gives rise to multiple isoforms, including CIN85-xl, CIN85-l (CIN85), CIN85-ΔA, CIN85-m, CIN85-s, CIN85-t and CIN85-h (Supplementary Figure S1) (Buchman et al, 2002; Finniss et al, 2004). The different isoforms display specific patterns of expression, among which CIN85-xl and CIN85-l are most abundant in brain and CIN85-l and CIN85-ΔA in thymus and spleen (Figure 2D; Supplementary Figure S1; Buchman et al, 2002). Lower levels of various isoforms can also be detected in kidney, muscle, skin, heart, lung and testis (Buchman et al, 2002 and unpublished observations). CD2AP is on the other hand predominantly expressed in spleen, thymus, heart, kidney, lung, muscle and liver, but seems to be lacking in neuronal cells (Dustin et al, 1998; Shih et al, 1999; Li et al, 2000). In this study, we report a novel function of CIN85 in the regulation of post-synaptic dopamine receptor endocytosis in striatal neurons. In wild-type mice, CIN85 resides post-synaptically and associates with endocytic regulators, such as dynamin and endophilins. In mouse striatal neurons, absence of brain-specific CIN85 expression results in inefficient complex formation between dopamine receptors and endophilins, as well as reduced internalization of stimulated dopamine receptors. Mice deficient of brain-specific CIN85 expression show hyperactive phenotypes, which in many ways resemble the behavioural aberrations displayed in human beings affected by attention deficit hyperactivity disorder (ADHD), a disorder strongly associated with abnormal dopamine signalling. Results Specific isoforms of CIN85 localize post-synaptically in neurons As CIN85 is highly expressed in the brain (Buchman et al, 2002), we were interested to investigate the function of CIN85 in the central nervous system (CNS). We initially analysed the expression pattern of CIN85 in various brain regions prepared from both mouse and rat (Figure 1A; Supplementary Figure S2 and data not shown) and found both of the major isoforms expressed in brain, CIN85-xl and CIN85-l, to be abundant in most brain regions tested, in line with earlier reported observations (Bian et al, 2008). To refine our survey of CIN85 localization on a sub-cellular level, we subsequently performed immunohistochemical analysis of endogenous CIN85 in cultured primary rat hippocampal neurons with mature synapses. In agreement with earlier reported observations (Kawata et al, 2006), and as shown in Figure 1B, we found high levels of CIN85 in the somatodendritic compartment, in which it frequently clustered in dendritic shafts, as well as within dendritic spines. Dendritic spines are small protrusions extending from the surface of dendrites, which are believed to be the main sites of excitatory synapses and thus vital centres for synaptic transmission in the brain (Segal, 2005). Figure 1.CIN85 is highly expressed in neurons in which it localizes to post-synaptic sites. (A) Western blot showing CIN85 expression in different mouse brain regions. Lysates (15 μg protein/lane) of the indicated brain regions from wild-type (+/+) and CIN85Δex2 knockout (−/−) mice were separated by 7% SDS–PAGE and immunoblotted with antibodies against CIN85 (CT). Equal protein loading was confirmed by re-blotting with anti-β-actin antibodies. (B) In primary hippocampal neurons derived from wild-type rat, CIN85 (SETA antibody, green) is localized to dendritic spines, in which it is accumulated in post-synaptic compartments, co-localizing with F-actin (red, upper panel) and PSD-95 (red, middle panel), closely juxtaposing the pre-synaptic synaptophysin protein (red, lower panel). Scale bars: 10 μm. (C) CIN85 (green) localizes to actin-positive (red), spine-like structures in dendrites of primary rat striatal neurons. Cells were fixed and stained with antibodies against CIN85 (SETA antibody, green) and with rhodamine phalloidin (red). Scale bars: 10 μm. (D) CIN85 is present in post-synaptic compartments in mouse brains. Synaptosome (SNS) fractions were isolated using the Percoll-step-gradient method from whole brain lysates as described in the Supplementary data. The western blot shows that CIN85, PSD-95 and synaptophysin are present in SNS fractions and that extraction with Triton X-100 solubilizes pre-synaptic synaptophysin into supernatant 1 (S1), whereas CIN85 and PSD-95 are present in post-synaptic fractions (pellet 2, P2, after a second Triton X-100 extraction). Supernatant 2 (S2) is the supernatant after the second Triton X-100 extraction. (E) CIN85 localizes to post-synaptic compartments in the striatum. Synaptosomes from mouse striata were prepared using a sucrose density-gradient method as described in the Supplementary data and immunoblotted with antibodies against CIN85 and PSD-95. WSL, whole striatal lysates; SNS, synaptosomes; PSD-s, supernatant of PSD fraction; PSD-p, pellet of PSD fraction. Each lane contains 15 μg of total protein. Download figure Download PowerPoint We confirmed the localization of CIN85 in dendritic spines by showing a co-localization of CIN85 with F-actin, which is abundant in these structures (Figure 1B, upper panel). In addition, by counter-staining primary hippocampal neurons for CIN85 together with markers for pre-synaptic (synaptophysin) and post-synaptic (PSD-95) compartments, we found CIN85 to co-localize with PSD-95 at post-synaptic sites, closely juxtaposing synaptophysin (Figure 1B, middle and lower panel, respectively). In addition, in cultured primary striatal neurons, CIN85 was enriched in spine-like structures in dendrites (Figure 1C). Aiming at further characterizing the localization of CIN85 in neurons, we subsequently isolated synaptosomes, which are enriched in pre- and post-synaptic structures (Booth and Clark, 1978), from whole mouse brain preparations, as well as from isolated mouse striata, using conventional Percoll step-gradient or sucrose density-gradient centrifugations, respectively. Whereas CIN85, PSD-95 and synaptophysin were all present in crude synaptosomal isolates from whole mouse brains, further extraction with Triton X-100, which is known to specifically solubilize pre-synaptic compartments and thus enrich post-synaptic components, depicted an enrichment of CIN85 together with PSD-95 in the post-synaptic fractions (Figure 1D). Similar results were obtained in isolated mouse striata in which CIN85 co-fractionated with PSD-95 in post-synaptic density preparations (Figure 1E). Mice lacking brain-specific CIN85 isoforms are hyperactive To investigate the function of CIN85 in the CNS, we next set out to generate mice deficient of the two major CIN85 isoforms expressed in the brain (CIN85-xl and CIN85-l). Using homologous recombination, we targeted exon 2 of the CIN85 genomic locus for deletion (Figure 2A) and could by a combinatorial approach based on Southern blot (Figure 2B) and PCR (Figure 2C; Supplementary Figure S3) confirm a successful integration of the targeting vector and absence of CIN85 exon 2 (CIN85Δex2). In agreement with the observed loss of CIN85Δex2 on the DNA level, we could furthermore confirm the absence of CIN85-xl and CIN85-l isoforms in brain, as well as CIN85-l in thymus and spleen, in CIN85Δex2 knockout mice by western blot analysis (Figure 2D). As expected, whereas all CIN85 protein variants encoded by transcripts initiated from promoter #1 (CIN85-xl, CIN85-l and the shorter CIN85-ΔCP) were abolished in CIN85Δex2 mice, none of the transcripts using alternative, downstream promoters, such as that of CIN85-ΔA (Figure 2D; Supplementary Figure S1; Buchman et al, 2002), were affected by the induced recombination event. Importantly, the neo-cassette, which as a result of the homologous recombination event replaces exon 2 in CIN85Δex2 animals, has been designed with a terminal translational stop codon, which should make ribosomal read-through and re-initiation highly unlikely. Moreover, given that exons 1 and 3 are out of frame, we anticipate that splicing events skipping the introduced LacZ/neo-cassette should result in inappropriate and prematurely terminated translation. Figure 2.Generation of CIN85Δex2 knockout mice. (A) Gene targeting of the CIN85 locus by removal of exon 2 by homologous recombination. The targeting vector consisted of a 6.0 kb 5′ homology region, the pGNA backbone containing a LacZ/neomycin cassette followed by a 3.0 kb 3′ homology region. The wild-type and targeted loci with their respective restriction sites are indicated. The arrows (1, 2, 3, 4) represent the primers used for genotypin
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