Asterisks (*) indicate IgG heavy or light chains. D. subject to Ponesimod distinct regulatory mechanisms. Keywords:LRRTM, neuroligin, neurexin, synaptogenesis, excitatory synapse formation == INTRODUCTION == Synapse assembly, maturation, Rabbit Polyclonal to HEXIM1 specification, and maintenance are likely driven by a multitude of trans-synaptic cell-adhesion molecules. Multiple synaptic cell-adhesion molecules may contribute to these processes, including but not limited to neurexins and neuroligins (Ushkaryov et al., 1992;Ichtchenko et al., 1995), ephrins and Eph receptors (Torres et al., 1998), SynCAMs (Biederer et al., 2002), and netrin G-ligands (Kim et al., 2006). A key technical advance in studying synaptic cell-adhesion molecules was the discovery that expression of such Ponesimod proteins in non-neuronal cells can potently enhance formation of synapses onto these cells (i.e., induce presynaptic differentiation of axons), when these cells are co-cultured with neurons (Scheiffele et al., 2000;Biederer et al., 2002;Graf et al., 2004;Kim et al., 2006). In this assay, referred to as the artificial synapse-formation assay, SynCAMs, neuroligins/neurexins, and NGLs are active (see references cited above). Most recently, a family of neuronal leucine-rich repeat proteins called LRRTMs was also identified as postsynaptic proteins that are active in this assay (Linhoff et al., 2009; Brose, 2009). LRRTMs comprise a family of four homologous leucine-rich repeat proteins that are selectively expressed in neurons with a differential distribution in brain (Lauren et al., 2003). LRRTM1 is usually a maternally suppressed gene that is associated paternally with handedness and schizophrenia (Francks et al., 2007;Ludwig et al., 2009). All LRRTMs induce presynaptic differentiation in artificial synapse-formation assays, and LRRTM2 is usually localized to excitatory synapses (Linhoff et al., 2009). Moreover, deletion of LRRTM1 in mice causes an increase in the immunoreactivity for the vesicular glutamate transporter VGLUT1 (Linhoff et al., 2009), a morphological change similar Ponesimod to that observed in neuroligin-3 R451C knockin mice (Tabuchi et al., 2007). Together, these data indicate that LRRTMs may be postsynaptic cell-adhesion molecules similar to neuroligins. However, these data raise important new questions, for example whether LRRTMs also alter synapse numbers in neurons, and more significantly, which presynaptic molecules they might interact with. Here, we examined the role of LRRTMs in neurons, focusing on LRRTM2 because or its well-documented localization to synapses (Linhoff et al., 2009). We demonstrate that LRRTM2 selectively induces excitatory synapse formation in the artificial synapse-formation assay, and increases excitatory synapse density in transfected neurons. Moreover, we identify neurexins as the presynaptic receptors for LRRTM2, and demonstrate that neurexin-binding to LRRTM2 is usually tightly regulated by alternative splicing of neurexins at splice site #4 (SS#4). Our data expand the trans-synaptic conversation network mediating synaptic cell adhesion, and suggest that neurexins generally nucleate trans-synaptic signaling. == RESULTS == == LRRTM2 Induces Excitatory Presynaptic Specializations in the Artificial Synapse-Formation Assay == We transfected COS cells with plasmids encoding only mVenus (control), or mVenus-fusion proteins of LRRTM2 or neuroligin-1, and co-cultured the transfected COS cells with cultured hippocampal neurons. After two days of co-culture, samples were fixed, immunolabeled for mVenus and synaptic markers, and analyzed by quantitative fluorescence microscopy (Figures 1A1B). == Physique 1. LRRTM2 Expression in COS cells and in Cultured Hippocampal Neurons Increases Excitatory Synapse Density. == A. LRRTM2 selectively promotes formation of excitatory synapses in the artificial synapse-formation assay. Hippocampal neurons were co-cultured for two days with COS cells expressing mVenus alone (control), an LRRTM2-mVenus fusion protein (LRRTM2), or an mVenus fusion protein of neuroligin-1 lacking inserts in splice sites A and B (NL1AB). Panels show representative immunofluorescence images of the co-cultures stained with antibodies to mVenus (green; GFP) and to various pre- Ponesimod and postsynaptic markers (red; VGLUT1, vesicular glutamate transporter 1; VGAT, vesicular GABA transporter). Coincident green and red signals are shown in yellow (scale bar = 25 m; applies to all images). B. Quantitation of the artificial synapse formation activity of LRRTM2 and neuroligin-1. Experiments as described in A were quantified by measuring the ratio of the synaptic marker staining to mVenus fluorescence (for absolute red and green fluorescence values, seeFigure S1). C..