The Susd2 protein regulates neurite growth and excitatory synaptic density in hippocampal cultures Yann Nadjar 1 , Antoine Triller, Jean-Louis Bessereau 2 , Andrea Dumoulin ⁎ Ecole Normale Supérieure, IBENS, INSERM U1024, 75005 Paris, France abstract article info Article history: Received 16 April 2014 Revised 12 December 2014 Accepted 12 February 2015 Available online 25 February 2015 Keywords: CCP domain Sushi Adhesion molecule cleavage Axon elongation Dendritic outgrowth Synapse regulation Complement control protein (CCP) domains have adhesion properties and are commonly found in proteins that control the complement immune system. However, an increasing number of proteins containing CCP domains have been reported to display neuronal functions. Susd2 is a transmembrane protein containing one CCP domain. It was previously identified as a tumor-reversing protein, but has no characterized function in the CNS. The present study investigates the expression and function of Susd2 in the rat hippocampus. Characterization of Susd2 during development showed a peak in mRNA expression two weeks after birth. In hippocampal neuronal cultures, the same expression profile was observed at 15 days in vitro for both mRNA and protein, a time consistent with synaptogenesis in our model. At the subcellular level, Susd2 was located on the soma, axons and dendrites, and appeared to associate preferentially with excitatory synapses. Inhibition of Susd2 by shRNAs led to decreased numbers of excitatory synaptic profiles, exclusively. Also, morphological parameters were studied on young (5DIV) developing neurons. After Susd2 inhibition, an increase in dendritic tree length but a decrease in axon elongation were observed, suggesting changes in adhesion properties. Our results demonstrate a dual role for Susd2 at different developmental stages, and raise the question whether Susd2 and other CCP-containing proteins expressed in the CNS could be function-related. © 2015 Elsevier Inc. All rights reserved. 1. Introduction In the nervous system, adhesion molecules participate in neuronal migration, neurite outgrowth, synapse formation, maintenance and plasticity, their diversity and patterns of expression allowing the elabo- ration of complex neuronal networks (Togashi et al., 2009). Altered functions of such proteins may lead to neurological developmental disorders (Ye et al., 2010; Redies et al., 2012), reflecting sometimes subtle defects in neuronal circuitry. Adhesion molecules are often part of large protein families, characterized by a specific protein domain with adhesive properties. This is the case for proteins of the immuno- globulin superfamily, for integrins and N-cadherins, and for the newly identified leucin rich repeat (LRR) superfamily (Linhoff et al., 2009; Nam et al., 2011). Recently, the proteins containing the complement control protein (CCP) domain have also been shown to be involved in several aspects of neuronal development in invertebrates and verte- brates (Nakayama and Hama, 2011). CCP domains, also named Sushi or SCR (for a short consensus repeat), contain approximately 60 amino acids and are characterized by a consensus sequence that includes four invariant cysteines partici- pating in an ovoid ternary structure (Schmidt et al., 2008). The CCP domains support protein–protein and protein–carbohydrate interac- tions. They were first identified in a number of plasmatic proteins of the immune system, in particular proteins regulating activation of the complement system (Sjöberg et al., 2009; Trouw and Daha, 2011). In Drosophila, the Hikaru genki gene codes a secreted protein with five CCP domains, identified in a screen for mutants exhibiting reduced locomotion (Hoshino et al., 1996). Location in the synaptic cleft and specific requirement of protein expression during development for correct behavioral phenotype suggested a role in synapse formation. In Caenorhabditis elegans, LEV-9, a secreted protein containing eight CCP domains, was shown to localize at the neuromuscular junction and to be necessary for the clustering of nicotinic acetylcholine receptors (Gendrel et al., 2009). In humans, three genes coding CCP- containing proteins were found to be related to clinical conditions: CSMD1/2, Srpx2, and Sez6. CSMD1/2 proteins are expressed in the nervous system and were identified as genetic susceptibility factors in schizophrenia (Håvik et al., 2011). Srpx2 was found to be mutated in two different families suffering from rolandic seizures, mental Molecular and Cellular Neuroscience 65 (2015) 82–91 Abbreviations: aa, amino acid(s); AMOP, adhesion-associated domain in Muc-4 and other proteins; CCP, complement control protein; DIV, days in vitro; HA, human influenza hemagglutinin; HEK, human embryonic kidney; HRP, horseradish peroxidase; LRR, leucine rich repeat; SCR, short consensus repeat; SO, somatomedin B-like domain; VWD, von Willebrand factor type D domain. ⁎ Corresponding author at: INSERM U1024, IBENS, 46 rue d'Ulm, Ecole Normale Supérieure, 75005 Paris, France. E-mail address: andrea.dumoulin@ens.fr (A. Dumoulin). 1 Present address: Département de Neurologie, Hôpital Pitié Salpétrière, Boulevard de l'Hôpital, 75013 Paris, France. 2 Present address: University Claude Bernard Lyon1, CGphiMC UMR CNRS 5534, 69622 Villeurbanne, France. http://dx.doi.org/10.1016/j.mcn.2015.02.007 1044-7431/© 2015 Elsevier Inc. All rights reserved. Contents lists available at ScienceDirect Molecular and Cellular Neuroscience journal homepage: www.elsevier.com/locate/ymcne