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Related ESCRT-III Subunit CHMP2B

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Related ESCRT-III Subunit CHMP2B The Journal of Neuroscience, February 18, 2015 • 35(7):3155–3173 • 3155 Cellular/Molecular Regulation of Postsynaptic Function by the Dementia- Related ESCRT-III Subunit CHMP2B X Romain Chassefeyre,1,2* Jose´ Martínez-Herna´ndez,1,2* Federica Bertaso,3,4,5 Nathalie Bouquier,3,4,5 Be´atrice Blot,1,2 Marine Laporte,1,2 Sandrine Fraboulet,1,2 Yohann Coute´,6,7 Anny Devoy,8 Adrian M. Isaacs,8 Karin Pernet-Gallay,1,2 Re´my Sadoul,1,2 XLaurent Fagni,3,4,5 and Yves Goldberg1,2,9 1Institut National de la Sante´ et de la Recherche Me´dicale (INSERM), Unite´ 836, F-38042 Grenoble, France, 2Universite´ Grenoble Alpes, Grenoble Institut des Neurosciences (GIN), F-38042 Grenoble, France, 3CNRS, UMR-5203, Institut de Ge´nomique Fonctionnelle, F-34094 Montpellier, France, 4Universite´s de Montpellier 1 & 2, UMR-5203, F-34094 Montpellier, France, 5INSERM, Unite´ 661, F-34094 Montpellier, France, 6INSERM, Unite´ 1038, F-38054 Grenoble, France, 7Commissariat a` l’Energie Atomique (CEA), Institut de Recherches en Technologies et Sciences pour le Vivant (iRTSV), Laboratoire de Biologie a` Grande Echelle, F-38054 Grenoble, France, 8Department of Neurodegenerative Disease, University College London Institute of Neurology, London WC1N 3BG, United Kingdom, and 9CEA, iRTSV, Groupe Physiopathologie du Cytosquelette (GPC), F-38054 Grenoble, France The charged multivesicular body proteins (Chmp1–7) are an evolutionarily conserved family of cytosolic proteins that transiently assembles into helical polymers that change the curvature of cellular membrane domains. Mutations in human CHMP2B cause fronto- temporal dementia, suggesting that this protein may normally control some neuron-specific process. Here, we examined the function, localization, and interactions of neuronal Chmp2b. The protein was highly expressed in mouse brain and could be readily detected in neuronal dendrites and spines. Depletion of endogenous Chmp2b reduced dendritic branching of cultured hippocampal neurons, de- creased excitatory synapse density in vitro and in vivo, and abolished activity-induced spine enlargement and synaptic potentiation. To understand the synaptic effects of Chmp2b, we determined its ultrastructural distribution by quantitative immuno-electron microscopy and its biochemical interactions by coimmunoprecipitation and mass spectrometry. In the hippocampus in situ, a subset of neuronal Chmp2b was shown to concentrate beneath the perisynaptic membrane of dendritic spines. In synaptoneurosome lysates, Chmp2b was stably bound to a large complex containing other members of the Chmp family, as well as postsynaptic scaffolds. The supramolecular Chmp assembly detected here corresponds to a stable form of the endosomal sorting complex required for transport-III (ESCRT-III), a ubiquitous cytoplasmic protein complex known to play a central role in remodeling of lipid membranes. We conclude that Chmp2b- containing ESCRT-III complexes are also present at dendritic spines, where they regulate synaptic plasticity. We propose that synaptic ESCRT-III filaments may function as a novel element of the submembrane cytoskeleton of spines. Key words: ESCRT filaments; frontotemporal dementia; postsynaptic scaffold; spinoskeleton; structural plasticity Introduction cellular membrane remodeling during inward vesiculation of The endosomal sorting complex required for transport-III multivesicular endosomes, mitotic cell abscission, and viral bud- (ESCRT-III) is a ubiquitous protein complex that is crucial to ding (for review, see Henne et al., 2013; McCullough et al., 2013). The core subunits of ESCRT-III are a family of evolutionarily conserved proteins, called charged multivesicular body proteins Received Feb. 10, 2014; revised Dec. 23, 2014; accepted Jan. 6, 2015. Author contributions: R.S., L.F., and Y.G. designed research; R.C., J.M.-H., F.B., N.B., B.B., M.L., S.F., Y.C., A.D., (Chmp1-7). Dominant mutations in subunit CHMP2B cause A.M.I., K.P.-G., and Y.G. performed research; Y.C. and A.M.I. contributed unpublished reagents/analytic tools; R.C., frontotemporal dementia (FTD; Skibinski et al., 2005; Isaacs et J.M.-H., F.B., N.B., Y.C., K.P.-G., R.S., L.F., and Y.G. analyzed data; R.C., J.M.-H., A.M.I., R.S., L.F., and Y.G. wrote the al., 2011), raising the question whether some neuron-specific paper. process is controlled by this protein. Chmp2b and other ESCRT- This work was supported by a grant from the Agence Nationale de la Recherche (ANR), MALZ program, III subunits exist in the cytosol in a monomeric, autoinhibited France. R.C. and M.L. were supported by the Ministe`re de l’Enseignement Supe´rieur et de la Recherche, France. J.M.-H. and N.B. were supported by ANR. A.M.I. was supported by the United Kingdom Medical state. After release of intramolecular inhibition, individual sub- Research Council. We thank Y. Saoudi for help with microscopy and N. Jaafari, A. Petiot, and W. Weissenhorn units polymerize into circular or helical filaments (20–200 nm for critically reading this manuscript. wide, subject to subunit species) that adhere to lipid membranes *R.C. and J.M.-H. contributed equally to this work. and change their curvature (Shim et al., 2007; Hanson et al., 2008; The authors declare no competing financial interests. This article is freely available online through the J Neurosci Author Open Choice option. Lata et al., 2008; Bajorek et al., 2009; Henne et al., 2012; Buchk- Correspondence should be addressed to either Yves Goldberg or Rémy Sadoul,, Institut des Neurosciences, UJF ovich et al., 2013). Site Sante´, Chemin Ferrini, 38042 Grenoble cedex 9, France. E-mail: [email protected] or [email protected]. R. Chassefeyre’s present address: The Scripps Research Institute, La Jolla, CA 92037. This is an Open Access article distributed under the terms of the Creative Commons Attribution License DOI:10.1523/JNEUROSCI.0586-14.2015 (http://creativecommons.org/licenses/by/3.0),whichpermitsunrestricteduse,distributionandreproductioninany Copyright © 2015 Chassefeyre et al. medium provided that the original work is properly attributed. 3156 • J. Neurosci., February 18, 2015 • 35(7):3155–3173 Chassefeyre, Martínez-Herna´ndez et al. • ESCRT-III Function at Synapses Table 1. Plasmids used in this study Plasmid name Cloning technique Cloning sites Feature Tags Source pIRES2-EGFP Starting vector Clontech pIRES2-EGFP-CHMP2B WT In-Fusion (Clontech) XhoI, XhoI RNAi res. C-terminal FLAG This study pBI-CMV3 Starting vector Clontech pBI-CMV3-CHMP2B WT In-Fusion (Clontech) BamHI, HindIII RNAi res. This study pBI-CMV3-CHMP2B R19/22/26A In-Fusion (Clontech) BamHI, HindIII RNAi res. This study pBI-CMV3-CHMP2B L4D/F5D In-Fusion (Clontech) BamHI, HindIII RNAi res. This study pBI-CMV3-CHMP2B L207D/L210D In-Fusion (Clontech) BamHI, HindIII RNAi res. This study pSuper-mCherry Starting vector Belly et al., 2010 pSuper-mCherry shRNA vs CHMP2B Standard ligation HindIII, BglII Belly et al., 2010 pSuper-mCherry shRNA vs GFP Standard ligation HindIII, BglII This study pCDNA3.1-CHMP2B WT Topo (Invitrogen) C-terminal HA This study pCDNA3.1-CHMP2B R19/22/26A Topo (Invitrogen) C-terminal HA This study pCDNA3.1-CHMP4B Topo (Invitrogen) C-terminal FLAG Bodon et al., 2011 The cloning technique used to construct each plasmid and, if applicable, the associated kit supplier are indicated. RNAi res. Indicates the encoded mRNA is resistant to RNAi by the Chmp2b shRNA. We have previously shown that Chmp2b preferentially po- pSuper-mCherry. cDNAs encoding wild-type (WT) or mutant ver- lymerizes under the plasma membrane and, when overexpressed, sions of the RNAi-resistant Chmp2b (Belly et al., 2010) were cloned in organizes into helices that deform patches of the cell surface into the pBI-CMV3 vector (Clontech), yielding pBI-CMV3-Chmp2b WT, R19/22/26A L4D/F5D long protrusions (Bodon et al., 2011). Holomeric ESCRT-III pBI-CMV3-Chmp2b , and pBI-CMV3-Chmp2b .In consists of heteropolymeric Chmp filaments assembled at sites of these vectors, Chmp2b is coexpressed with fluorescent protein ZsGreen endosomal or plasma membrane deformation, after recruitment by from a bidirectional promoter. cDNA encoding wild-type, RNAi- resistant Chmp2b (carrying a single C-terminal Flag tag; Bodon et al., the upstream-acting complexes ESCRT-I and -II and/or by the 2011) was inserted upstream of the IRES sequence of the pIRES2-EGFP Chmp4b-binding protein Alix. ESCRT-III assemblies at membranes vector (Clontech), yielding pIRES2-EGFP-Chmp2b (used in Fig. 6B). In are thought to be transient, being dissociated after interaction with this vector, Chmp2b is coexpressed with EGFP from a bicistronic tran- the depolymerizing ATPase Vps4. This depolymerization step is script. All constructs were verified by sequencing (Beckman Coulter). coupled to scission of membrane necks modeled by ESCRT-III fila- Antibodies. See Table 2 for details of antibodies and dilutions. ments (Henne et al., 2013). Animals. Animals were handled and killed in conformity with Euro- FTD-associated CHMP2B mutants are deregulated and form pean law and internal regulations of INSERM. Pregnant Oncins France aberrant polymeric structures in transfected cells (Skibinski et al., souche A (OFA; a substrain of Sprague Dawley) rats (Charles River) were 2005). Strong expression of such a CHMP2B mutant in cultured used for primary neuron cultures, and 2-month-old OFA rats of both cortical neurons is rapidly lethal (Lee et al., 2007), and transgenic sexes were used for biochemical analysis of brain tissue. The rats were mice expressing the same mutant throughout their brain display deeply anesthetized by isoflurane inhalation before decapitation, quickly progressive neurodegenerative changes (Ghazi-Noori et al., followed by cesarean section or brain dissection. Three-month-old male 2012). These effects have been linked to defects in autophagic and C57BL/6 mice and 1.3-month-old female Chmp2b KO mice (here called KD mice since knock-out is incomplete; Ghazi-Noori et al., 2012)or endosomal degradation pathways (Filimonenko et al., 2007; Lee female WT littermates (on C57BL/6 background) were used for immu- et al., 2007; Urwin et al., 2010).
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