We performed our screen with low (150mM) and high (500mM) salt washes, and under both conditions we found no Neurexin peptides in the Fc negative control indicating a high level specificity in our assay (Table S1)

We performed our screen with low (150mM) and high (500mM) salt washes, and under both conditions we found no Neurexin peptides in the Fc negative control indicating a high level specificity in our assay (Table S1). CNS synapses is limited. Synaptic cell adhesion molecules are key players in organizing synapse formation between appropriate synaptic partners. Hetero- or homophilic adhesive interactions across a nascent synapse allow target cell acknowledgement and subsequent bidirectional differentiation of pre- and postsynaptic elements. Previous work has identified several synaptic cell adhesion molecules that can mediatetrans-synaptic conversation to induce pre- and postsynaptic differentiation. These synaptogenic adhesion molecules include the neuroligins (Ichtchenko et al., 1995;Scheiffele et al., 2000;Track et al., 1999), synCAM (Biederer et al., 2002), NGL-2 and NGL-3 (Kim et al., 2006;Woo et al., 2009) and EphB2 (Kayser et al., 2006). To identify novel transmembrane proteins that might regulate the development and function of synapses, we carried out anin silicoscreen for genes that showed subregion-specific expression in the adult hippocampus as reported in the Allen Brain Atlas (Lein et al., 2007) and encoded proteins with extracellular protein-protein conversation domains. Candidate genes were subsequently tested for putative synaptogenic properties in heterologous cell-neuron coculture assays (Biederer and Scheiffele, 2007;Scheiffele et al., 2000). This approach led to the identification of Leucine-Rich Repeat Transmembrane (LRRTM) proteins as proteins that could induce presynaptic differentiation. During the course (±)-ANAP of the study Linhoff and colleagues reported that LRRTM proteins can induce synapse formation in hippocampal neurons (Linhoff et al., (±)-ANAP 2009), but several major questions remained unanswered. Importantly, whether endogenous LRRTMs contribute to synaptic function in vivo was not known, and the receptors that mediate the effects of LRRTM had not been identified. Here we provide evidence that endogenous LRRTM2 regulates excitatory synapse development and function in vitro and in vivo, and identify Neurexins as functional receptors for LRRTM2. == Results == == LRRTM2 Regulates Excitatory Synapse Formation in Hippocampal Neurons == TheLRRTMgene family consists of four genes, LRRTM1-4. Analysis of theLRRTMgene expression pattern by in situ hybridization showed that these genes are differentially expressed in the developing cortex and hippocampus (Physique S1). To determine the subcellular localization of LRRTM proteins, we transfected 293T cells with tagged constructs, and examined their distribution by immunofluorescence. Myc-tagged LRRTM2-4 and LRRTM4L all localized to the cell membrane in transfected 293T cells (Physique S1). LRRTM1 however remained largely intracellular, despite the presence of a predicted transmembrane domain name (±)-ANAP (Laurn et al., 2003), suggesting that LRRTM1 may require additional proteins for proper membrane targeting. In a heterologous synapse induction assay, in which LRRTM proteins expressed in 293T cells were co-cultured with hippocampal neurons, we found that LRRTM2 (±)-ANAP was more effective than the other LRRTM genes in inducing presynaptic differentiation (Physique S2). We therefore decided to investigate whether endogenous LRRTM2 contributed to synapse formation. To determine if LRRTM2 was targeted to excitatory synapses, hippocampal neurons were cotransfected with myc-tagged LRRTM2 and GFP, and immunostained for synaptic markers. Myc-LRRTM2 clustered in the heads of dendritic spines, where it colocalized with the excitatory postsynaptic marker PSD-95, but not with the inhibitory postsynaptic marker gephyrin (Physique 1A). The quality of immunofluorescence with currently available LRRTM2 Cbll1 antibodies is not suitable to draw definitive conclusion about the localization of the endogenous protein, but the localization of the tagged proteins suggests that LRRTM2 primarily localizes to the postsynaptic density of excitatory synapses. == Physique 1. == Knockdown of LRRTM2 Decreases Excitatory Synapse Density in Hippocampal Neurons (A) Hippocampal neurons were cotransfected with myc-LRRTM2 and GFP at DIV 10 and immunostained for postsynaptic markers and GFP at DIV 17. Top panels, myc-LRRTM2 colocalizes with the excitatory postsynaptic marker PSD-95 in dendritic spines (arrowheads). Bottom panels, myc-LRRTM2 does not colocalize with the inhibitory postsynaptic marker gephyrin. GFP fluorescence shown in blue for better visualization. (B) 293T cells were cotransfected with mouse myc-LRRTM2 and the vacant pSUPER vector (sh-vector) or pSUPER made up of a shRNA against mouse and rat LRRTM2 (sh-LRRTM2) for 48 hr. The LRRTM2 shRNA reduces expression of mouse LRRTM2 by 90%, but human myc-LRRTM2 is not affected. Samples were probed with a -actin antibody to verify equivalent loading. (C) Knockdown of endogenous LRRTM2 in hippocampal neurons. Neurons were infected with control or sh-LRRTM2 made up of lentiviral vectors (LV) at DIV 6 and analyzed at DIV.