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• W2024301757 abstract "Delineating the molecular basis of synapse development is crucial for understanding brain function. Cocultures of neurons with transfected fibroblasts have demonstrated the synapse-promoting activity of candidate molecules. Here, we performed an unbiased expression screen for synaptogenic proteins in the coculture assay using custom-made cDNA libraries. Reisolation of NGL-3/LRRC4B and neuroligin-2 accounts for a minority of positive clones, indicating that current understanding of mammalian synaptogenic proteins is incomplete. We identify LRRTM1 as a transmembrane protein that induces presynaptic differentiation in contacting axons. All four LRRTM family members exhibit synaptogenic activity, LRRTMs localize to excitatory synapses, and artificially induced clustering of LRRTMs mediates postsynaptic differentiation. We generate LRRTM1–/– mice and reveal altered distribution of the vesicular glutamate transporter VGLUT1, confirming an in vivo synaptic function. These results suggest a prevalence of LRR domain proteins in trans-synaptic signaling and provide a cellular basis for the reported linkage of LRRTM1 to handedness and schizophrenia. Delineating the molecular basis of synapse development is crucial for understanding brain function. Cocultures of neurons with transfected fibroblasts have demonstrated the synapse-promoting activity of candidate molecules. Here, we performed an unbiased expression screen for synaptogenic proteins in the coculture assay using custom-made cDNA libraries. Reisolation of NGL-3/LRRC4B and neuroligin-2 accounts for a minority of positive clones, indicating that current understanding of mammalian synaptogenic proteins is incomplete. We identify LRRTM1 as a transmembrane protein that induces presynaptic differentiation in contacting axons. All four LRRTM family members exhibit synaptogenic activity, LRRTMs localize to excitatory synapses, and artificially induced clustering of LRRTMs mediates postsynaptic differentiation. We generate LRRTM1–/– mice and reveal altered distribution of the vesicular glutamate transporter VGLUT1, confirming an in vivo synaptic function. These results suggest a prevalence of LRR domain proteins in trans-synaptic signaling and provide a cellular basis for the reported linkage of LRRTM1 to handedness and schizophrenia. Chemical synapses represent the principal means of communication between neurons. While significant progress has been made in determining the developmental signals that guide axons to their targets, the molecular mechanisms that determine the final establishment of circuits remain incompletely understood. Synapses are asymmetric cellular junctions composed of a presynaptic vesicle release site, a synaptic cleft, and a specialized postsynaptic receptive apparatus. A major challenge in elucidating the mechanisms that govern synapse formation and maturation is the enormous variety of synapse types in the mammalian brain. It is likely that this diversity requires contribution from a large number of molecular signals. In addition to neuronal transmembrane proteins, secreted factors such as FGFs and neuronal pentraxins, and glial-derived factors such as thrombospondin and complement cascade proteins shape synapse development (Stevens et al., 2007Stevens B. Allen N.J. Vazquez L.E. Howell G.R. Christopherson K.S. Nouri N. Micheva K.D. Mehalow A.K. Huberman A.D. Stafford B. et al.The classical complement cascade mediates CNS synapse elimination.Cell. 2007; 131: 1164-1178Abstract Full Text Full Text PDF PubMed Scopus (1652) Google Scholar, Waites et al., 2005Waites C.L. Craig A.M. Garner C.C. Mechanisms of vertebrate synaptogenesis.Annu. Rev. Neurosci. 2005; 28: 251-274Crossref PubMed Scopus (355) Google Scholar). Significant attention has focused on the role of synaptic cell adhesion molecules (CAMs) in synapse development (Ackley and Jin, 2004Ackley B.D. Jin Y. Genetic analysis of synaptic target recognition and assembly.Trends Neurosci. 2004; 27: 540-547Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, Dalva et al., 2007Dalva M.B. McClelland A.C. Kayser M.S. Cell adhesion molecules: signalling functions at the synapse.Nat. Rev. Neurosci. 2007; 8: 206-220Crossref PubMed Scopus (401) Google Scholar, Ko and Kim, 2007Ko J. Kim E. Leucine-rich repeat proteins of synapses.J. Neurosci. Res. 2007; 85: 2824-2832Crossref PubMed Scopus (39) Google Scholar, Yamagata et al., 2003Yamagata M. Sanes J.R. Weiner J.A. Synaptic adhesion molecules.Curr. Opin. Cell Biol. 2003; 15: 621-632Crossref PubMed Scopus (275) Google Scholar). Given the potential for a trans-synaptic interaction that could bridge the presynaptic release apparatus with the postsynaptic density, Scheiffele and colleagues tested whether neuroligin expressed in transfected HEK cells could assemble presynaptic terminals when cocultured with axons from pontine explants. Indeed, expression of neuroligin in HEK cells induced the clustering of synaptic vesicles in contacting axons (Scheiffele et al., 2000Scheiffele P. Fan J. Choih J. Fetter R. Serafini T. Neuroligin expressed in nonneuronal cells triggers presynaptic development in contacting axons.Cell. 2000; 101: 657-669Abstract Full Text Full Text PDF PubMed Scopus (910) Google Scholar), via direct interaction with neurexins. In a parallel fashion, expression of β-neurexin in COS cells induces postsynaptic differentiation in contacting dendrites (Graf et al., 2004Graf E.R. Zhang X. Jin S.X. Linhoff M.W. Craig A.M. Neurexins induce differentiation of GABA and glutamate postsynaptic specializations via neuroligins.Cell. 2004; 119: 1013-1026Abstract Full Text Full Text PDF PubMed Scopus (706) Google Scholar). These results show that a single molecular interaction can organize many aspects of presynaptic and postsynaptic assembly. Further studies indicate selective roles of different neuroligin and neurexin splice isoforms at excitatory versus inhibitory synapses (Chubykin et al., 2007Chubykin A.A. Atasoy D. Etherton M.R. Brose N. Kavalali E.T. Gibson J.R. Sudhof T.C. Activity-dependent validation of excitatory versus inhibitory synapses by neuroligin-1 versus neuroligin-2.Neuron. 2007; 54: 919-931Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar, Craig and Kang, 2007Craig A.M. Kang Y. Neurexin-neuroligin signaling in synapse development.Curr. Opin. Neurobiol. 2007; 17: 43-52Crossref PubMed Scopus (403) Google Scholar). Moreover, analyses of knockout mice indicate an essential role for neurexins and neuroligins in synapse development (Missler et al., 2003Missler M. Zhang W. Rohlmann A. Kattenstroth G. Hammer R.E. Gottmann K. Sudhof T.C. Alpha-neurexins couple Ca2+ channels to synaptic vesicle exocytosis.Nature. 2003; 424: 939-948Crossref Scopus (475) Google Scholar, Varoqueaux et al., 2006Varoqueaux F. Aramuni G. Rawson R.L. Mohrmann R. Missler M. Gottmann K. Zhang W. Sudhof T.C. Brose N. Neuroligins determine synapse maturation and function.Neuron. 2006; 51: 741-754Abstract Full Text Full Text PDF PubMed Scopus (552) Google Scholar). Neuroligin-1, -2, -3 triple-knockout mice die at birth due to defects in excitatory and inhibitory transmission, although single-neuroligin knockouts have only subtle phenotypes (Chubykin et al., 2007Chubykin A.A. Atasoy D. Etherton M.R. Brose N. Kavalali E.T. Gibson J.R. Sudhof T.C. Activity-dependent validation of excitatory versus inhibitory synapses by neuroligin-1 versus neuroligin-2.Neuron. 2007; 54: 919-931Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar, Jamain et al., 2008Jamain S. Radyushkin K. Hammerschmidt K. Granon S. Boretius S. Varoqueaux F. Ramanantsoa N. Gallego J. Ronnenberg A. Winter D. et al.Reduced social interaction and ultrasonic communication in a mouse model of monogenic heritable autism.Proc. Natl. Acad. Sci. USA. 2008; 105: 1710-1715Crossref PubMed Scopus (390) Google Scholar, Varoqueaux et al., 2006Varoqueaux F. Aramuni G. Rawson R.L. Mohrmann R. Missler M. Gottmann K. Zhang W. Sudhof T.C. Brose N. Neuroligins determine synapse maturation and function.Neuron. 2006; 51: 741-754Abstract Full Text Full Text PDF PubMed Scopus (552) Google Scholar). The fibroblast-neuron coculture assay has proven to be a useful tool in testing candidate proteins for a role in synaptic development (Biederer and Scheiffele, 2007Biederer T. Scheiffele P. Mixed-culture assays for analyzing neuronal synapse formation.Nat. Protocols. 2007; 2: 670-676Crossref PubMed Scopus (108) Google Scholar, Craig et al., 2006Craig A.M. Graf E.R. Linhoff M.W. How to build a central synapse: clues from cell culture.Trends Neurosci. 2006; 29: 8-20Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). Coculture assays have revealed synaptogenic activity for four families of neuronal CAMs, and in some cases their binding partners: neuroligins and partner neurexins, SynCAMs/Necls (Biederer et al., 2002Biederer T. Sara Y. Mozhayeva M. Atasoy D. Liu X. Kavalali E.T. Sudhof T.C. SynCAM, a synaptic adhesion molecule that drives synapse assembly.Science. 2002; 297: 1525-1531Crossref PubMed Scopus (603) Google Scholar), EphBs and partner ephrinBs (Aoto et al., 2007Aoto J. Ting P. Maghsoodi B. Xu N. Henkemeyer M. Chen L. Postsynaptic ephrinB3 promotes shaft glutamatergic synapse formation.J. Neurosci. 2007; 27: 7508-7519Crossref PubMed Scopus (69) Google Scholar, Kayser et al., 2006Kayser M.S. McClelland A.C. Hughes E.G. Dalva M.B. Intracellular and trans-synaptic regulation of glutamatergic synaptogenesis by EphB receptors.J. Neurosci. 2006; 26: 12152-12164Crossref PubMed Scopus (163) Google Scholar), and netrin G ligands (NGLs/LRRC4s) (Kim et al., 2006Kim S. Burette A. Chung H.S. Kwon S.K. Woo J. Lee H.W. Kim K. Kim H. Weinberg R.J. Kim E. NGL family PSD-95-interacting adhesion molecules regulate excitatory synapse formation.Nat. Neurosci. 2006; 9: 1294-1301Crossref PubMed Scopus (188) Google Scholar). Knockout mice studies have validated roles for EphBs/ephrinBs (Aoto et al., 2007Aoto J. Ting P. Maghsoodi B. Xu N. Henkemeyer M. Chen L. Postsynaptic ephrinB3 promotes shaft glutamatergic synapse formation.J. Neurosci. 2007; 27: 7508-7519Crossref PubMed Scopus (69) Google Scholar, Henderson et al., 2001Henderson J.T. Georgiou J. Jia Z. Robertson J. Elowe S. Roder J.C. Pawson T. The receptor tyrosine kinase EphB2 regulates NMDA-dependent synaptic function.Neuron. 2001; 32: 1041-1056Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar, Henkemeyer et al., 2003Henkemeyer M. Itkis O.S. Ngo M. Hickmott P.W. Ethell I.M. Multiple EphB receptor tyrosine kinases shape dendritic spines in the hippocampus.J. Cell Biol. 2003; 163: 1313-1326Crossref PubMed Scopus (228) Google Scholar, Kayser et al., 2006Kayser M.S. McClelland A.C. Hughes E.G. Dalva M.B. Intracellular and trans-synaptic regulation of glutamatergic synaptogenesis by EphB receptors.J. Neurosci. 2006; 26: 12152-12164Crossref PubMed Scopus (163) Google Scholar) as well as neurexins/neuroligins in synapse development. Evidence is accumulating that proteins testing positive for synaptogenic activity in this assay may not be essential for initiating the formation of or maintaining the integrity of synaptic junctions. Rather, these factors may serve a role in the maturation of the synapse, recruiting components necessary for synaptic function. Here we used the fibroblast-neuron coculture assay to search for novel synaptogenic proteins. We created and screened a set of full-length size-selected cDNA expression libraries from developing rat brain. Screening to date reveals a prevalence of leucine-rich repeat (LRR) synaptogenic proteins arising from this unbiased approach. We identify the leucine rich repeat transmembrane neuronal (LRRTM) protein family as able to instruct excitatory presynaptic differentiation and to mediate postsynaptic differentiation. Furthermore, we generate an LRRTM1 knockout mouse and reveal a modest synaptic phenotype, suggesting a synaptic basis for the recently reported linkage of LRRTM1 to handedness and schizophrenia (Francks et al., 2007Francks C. Maegawa S. Lauren J. Abrahams B.S. Velayos-Baeza A. Medland S.E. Colella S. Groszer M. McAuley E.Z. Caffrey T.M. et al.LRRTM1 on chromosome 2p12 is a maternally suppressed gene that is associated paternally with handedness and schizophrenia.Mol. Psychiatry. 2007; 12 (1057): 1129-1139Crossref PubMed Scopus (248) Google Scholar). Parts of this work have previously been published in doctoral dissertations (Linhoff, 2008Linhoff M.W. Expression Screening for Proteins Involved in CNS Presynaptic Differentiation and Initial Characterization of the Synaptogenic LRRTM Protein Family. Washington University in St. Louis, St. Louis2008Google Scholar, Lauren, 2007Lauren J. Characterization of LRRTM and NGR Gene Families: Expression and Functions. Helsinki University Printing House, Helsinki2007Google Scholar). To test the feasibility of converting the fibroblast-neuron coculture assay into an expression screen, neuroligin-1 and neuroligin-2 cDNAs were diluted up to 1:1000 with either a soluble CFP expression plasmid or a pool of inactive cDNAs. Neuroligin-2 could be diluted 500-fold while still consistently yielding a positive synaptogenic signal on a single neuron-COS cell coculture coverslip, while neuroligin-1 could only be diluted 100-fold (data not shown). Thus, a pool size of ∼250 clones would be likely to yield novel clones with synaptogenic activity of similar potency as neuroligins. This pool size of 250 is considerably smaller than typical pool sizes of 1,000 - 10,000 used for expression screens involving more sensitive detection technologies (e.g., Fournier et al., 2001Fournier A.E. GrandPre T. Strittmatter S.M. Identification of a receptor mediating Nogo-66 inhibition of axonal regeneration.Nature. 2001; 409: 341-346Crossref PubMed Scopus (916) Google Scholar). Furthermore, since we are expecting active proteins to be transmembrane or secreted, positive clones would have to be full-length to contain the signal peptide essential for proper trafficking. Thus, the first step in the screen was to generate an unamplified cDNA expression library with a high percentage of full-length clones (Figure 1A). We isolated mRNA from rat forebrain at P11, the peak of synaptogenesis (Micheva and Beaulieu, 1996Micheva K.D. Beaulieu C. Quantitative aspects of synaptogenesis in the rat barrel field cortex with special reference to GABA circuitry.J. Comp. Neurol. 1996; 373: 340-354Crossref PubMed Scopus (215) Google Scholar). To ensure representation of full-length molecules, we used the biotinylated cap-trapper method that had been developed for large-scale sequencing projects (Figures S1A and S1B) (Carninci et al., 1996Carninci P. Kvam C. Kitamura A. Ohsumi T. Okazaki Y. Itoh M. Kamiya M. Shibata K. Sasaki N. Izawa M. et al.High-efficiency full-length cDNA cloning by biotinylated CAP trapper.Genomics. 1996; 37: 327-336Crossref PubMed Scopus (243) Google Scholar). The resultant cap-trapped cDNA was subjected to rigorous size fractionation, and individual unamplified libraries were maintained. Mean insert size of the resultant libraries was confirmed by restriction digestion (Figure S1C), and sequencing of over 50 clones indicated that all contained the 5′ ATG. We were successful in creating high-quality expression libraries for inserts up to 5 kb using pcDNA3.1 as the host expression vector (Figure 1A). Figure 1B illustrates the protocol that we used for the screen to identify cDNA pools that contained synaptogenic activity. Synaptogenic activity was assayed by the immunocytochemical detection of experimentally induced hemisynapses. The cocultures were immunostained with synapsin I antibody to detect synaptic vesicle clustering at contact sites between COS cells and axons. Coimmunostaining for the postsynaptic markers PSD-95 family and gephyrin ensured that synapsin immunoreactivity associated with bona fide interneuronal synapses was not counted as a false positive. A cDNA pool was considered positive if it generated any COS cell with significant associated synapsin clusters unapposed to PSD-95 family or gephyrin. We screened positive cDNA pools using PCR to detect known synaptogenic factors. The first positive pool, PD026, was found in the 4–5 kb library, tested positive for neuroligin-2 using PCR, and was not fractionated to isolate the single responsible synaptogenic cDNA. Further screening within the 3–4 kb library resulted in another positive pool, PC064, which tested PCR negative for neuroligins. We subdivided the PC064 cDNA pool to isolate the synaptogenic clone. Generation of arrayed clones and DNA from a positive subpool aided in identifying the active clone. The isolated clone, PC064-89-29-40 (pool PC064, subpool 89, subpool 29, clone 40), was identified as leucine-rich repeat transmembrane protein LRRTM1. The LRRTM protein family consists of four members, each possessing ten extracellular leucine-rich repeats, a single transmembrane domain, and an ∼70 amino acid cytoplasmic domain (Figures 1B and S2) (Lauren et al., 2003Lauren J. Airaksinen M.S. Saarma M. Timmusk T. A novel gene family encoding leucine-rich repeat transmembrane proteins differentially expressed in the nervous system.Genomics. 2003; 81: 411-421Crossref PubMed Scopus (112) Google Scholar). We cloned each of the LRRTM family members from rat cDNA, fused CFP to the C terminus, and tested each protein for synaptogenic activity in the coculture assay. N-cadherin has been shown previously to not induce presynaptic clustering (Scheiffele et al., 2000Scheiffele P. Fan J. Choih J. Fetter R. Serafini T. Neuroligin expressed in nonneuronal cells triggers presynaptic development in contacting axons.Cell. 2000; 101: 657-669Abstract Full Text Full Text PDF PubMed Scopus (910) Google Scholar); we confirmed this (see below) and used N-cadherin as a negative control. As an additional control, we used the LRR protein, AMIGO, which has been shown to interact with the axons of hippocampal neurons and induce neurite outgrowth and fasciculation (Kuja-Panula et al., 2003Kuja-Panula J. Kiiltomaki M. Yamashiro T. Rouhiainen A. Rauvala H. AMIGO, a transmembrane protein implicated in axon tract development, defines a novel protein family with leucine-rich repeats.J. Cell Biol. 2003; 160: 963-973Crossref PubMed Scopus (105) Google Scholar). To obtain a quantitative measure of each protein's ability to instruct presynaptic differentiation, we measured the amount of synapsin clustering associated with transfected COS cells and not associated with MAP2-positive dendrites to exclude interneuronal synapses. Robust synaptogenic activity was observed for LRRTM1-CFP, LRRTM2-CFP, LRRTM4-CFP, and neuroligin-2-CFP (Figures 2A, 2B, and S3B). In some instances clustering of the CFP fusion protein could be observed apposed to synapsin puncta (Figures 2A and 2B, insets). LRRTM3-CFP consistently yielded limited activity in the coculture assay (Figure S3A). In contrast to the LRRTMs, synaptogenic activity was not observed for N-cadherin-CFP or the LRR-containing AMIGO-CFP (see Figure 2C for images of AMIGO-CFP). LRRTM1-CFP, LRRTM2-CFP, LRRTM4-CFP, and neuroligin-2-CFP each induced a >14-fold increase in synapsin clustering when compared to results obtained from N-cadherin-CFP or AMIGO-CFP-transfected cells (Figure 2D, n = 20 transfected COS cells per construct; ANOVA p < 0.0001; t test versus N-cadherin-CFP and AMIGO-CFP p < 0.005). In contrast, synapsin clustering associated with LRRTM3-CFP was 1.6- to 3.3-fold above that associated with N-cadherin-CFP or AMIGO-CFP (t test versus N-cadherin and AMIGO p < 0.05). In separate experiments to confirm the lack of presynaptic inducing activity of N-cadherin compared with no surface protein expression, we compared the percentage of cells exhibiting any clusters of synapsin not associated with PSD-95 or gephyrin for COS cells expressing N-cadherin-CFP (1.6% ± 0.3%) or expressing CFP (1.8% ± 0.5%; p > 0.1). Axonal contact area determined from dephospho-tau immunoreactivity was also higher for LRRTM2-CFP, LRRTM4-CFP, and neuroligin-2-CFP than for N-cadherin-CFP or AMIGO-CFP (Figure S3C; ANOVA p < 0.0001), suggesting an effect on axon adhesion as well as presynaptic differentiation. The less than 2-fold increase in axon contact area for LRRTM2-CFP or LRRTM4-CFP was not sufficient to explain the >14-fold increase in synapsin clustering, indicating a direct presynaptic effect. Thus, measures of synapsin area normalized per axon contact area also revealed significant induction of presynaptic differentiation by LRRTMs (Figure 2E). Although the most synaptogenic factors tended to have increased axon contact area, the same phenomenon did not hold true for LRRTM1-CFP. This result is likely due to the reduced surface expression of LRRTM1 relative to the other family members. We observed that LRRTM1 accumulates extensively in the endoplasmic reticulum of transfected nonneuronal cells (data not shown), and this result has been observed previously (Francks et al., 2007Francks C. Maegawa S. Lauren J. Abrahams B.S. Velayos-Baeza A. Medland S.E. Colella S. Groszer M. McAuley E.Z. Caffrey T.M. et al.LRRTM1 on chromosome 2p12 is a maternally suppressed gene that is associated paternally with handedness and schizophrenia.Mol. Psychiatry. 2007; 12 (1057): 1129-1139Crossref PubMed Scopus (248) Google Scholar). Since synapsin is a vesicle-associated protein, we also tested for other markers of presynaptic differentiation. LRRTMs induced clustering of the active zone cytomatrix protein bassoon (Figures S4A–S4D), as well as the integral synaptic vesicle membrane protein synaptophysin (Figures S4E–S4H). We tested whether the synaptogenic activity was restricted to excitatory or inhibitory presynaptic differentiation. The CFP fusion proteins were scored blind for significant clustering of the presynaptic vesicular neurotransmitter transporters, VGLUT1 and VGAT, to identify excitatory or inhibitory presynaptic differentiation, respectively. Significant clustering of VGLUT1 unapposed to PSD-95 family was observed for LRRTM1-CFP, LRRTM2-CFP, LRRTM3-CFP, LRRTM4-CFP, and neuroligin-2-CFP (Figures 2F–2J; ANOVA p < 0.0001; t test versus N-cadherin-CFP and AMIGO-CFP p < 0.0005). In contrast, only LRRTM2-CFP and neuroligin-2 showed robust clustering of VGAT unapposed to gephyrin (Figures S3D–S3F; ANOVA p < 0.0001). Since LRRTM1 and LRRTM2 demonstrated the most potent synaptogenic activity of the four family members, we focused further efforts on these two family members. To test whether the induced presynaptic clusters were functional for release, we used whole-cell patch voltage clamp to record currents from HEK293T cells cotransfected with LRRTM2-CFP and NMDA receptor subunits YFP-NR1 and NR2A and cocultured with hippocampal neurons. A similar assay was used to show that neuroligin-1 coexpressed with NMDA receptors could induce excitatory postsynaptic current (EPSC)-like events in HEK cells cocultured with cerebellar granule neurons (Fu et al., 2003Fu Z. Washbourne P. Ortinski P. Vicini S. Functional excitatory synapses in HEK293 cells expressing neuroligin and glutamate receptors.J. Neurophysiol. 2003; 90: 3950-3957Crossref PubMed Scopus (83) Google Scholar). Here, bursts of spontaneous activity much like NMDA receptor currents typical of cultured neurons were recorded from the HEK293T cells expressing LRRTM2-CFP and NMDA receptors (Figure 3A). These bursts of high-amplitude events were abolished by tetrodotoxin (TTX) and reduced to miniature EPSC-like events, indicating that the bursts arise from action-potential-dependent neuronal network activity that drives glutamate release onto the transfected cell. In contrast, HEK293T cells cotransfected with YFP-NR1, NR2A, and N-cadherin-CFP as a negative control exhibited only rare spontaneous events and lacked the bursting activity observed for LRRTM2-CFP-expressing cells (Figure 3B). These rare synaptic-like events may correspond to fusion of immature synaptic vesicles or may arise from orphan release sites of axons (Krueger et al., 2003Krueger S.R. Kolar A. Fitzsimonds R.M. The presynaptic release apparatus is functional in the absence of dendritic contact and highly mobile within isolated axons.Neuron. 2003; 40: 945-957Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). The frequency and amplitude of events onto N-cadherin-expressing cells was not significantly altered by TTX. Amplitude of events onto LRRTM2-CFP-expressing cells was reduced 4.6-fold by TTX (n = 5 cells with frequency >0.1 Hz), indicating the formation of multiple functional release sites. Quantitative analysis confirmed a greater than 20-fold overall increase in frequency of spontaneous EPSC-like events and 6.6-fold increase in frequency of miniature EPSC-like events in HEK293T cells expressing LRRTM2-CFP compared with N-cadherin-CFP-expressing cells (Figure 3C; n ≥ 10, p < 0.005 t test). The amplitude of spontaneous but not miniature events was also significantly increased for LRRTM2-CFP versus N-cadherin-CFP-expressing cells (Figure 3D; p < 0.05 t test comparison of mean cell values). Mean amplitude of miniature events was 89 and 99 pA for N-cadherin or LRRTM2-CFP-expressing cells, respectively, suggesting that sufficient levels of diffuse surface NMDA receptors were obtained in the HEK cells to provide a sensitive assay of release. All synaptic-like events were abolished by NMDA receptor antagonist APV (data not shown). Thus, the presynaptic specializations induced by LRRTM2 expression are functional with respect to evoked and spontaneous glutamatergic synaptic vesicle exocytosis. To determine if the LRR domain was necessary for synaptogenic activity, a deletion mutant lacking the LRR domain, ΔLRR-LRRTM2-CFP, was tested in the coculture assay. The deletion mutant was expressed on the cell surface, but presynaptic differentiation was not observed in axons that contacted the surface of transfected COS cells (Figure S5). To determine whether the LRR domain of LRRTM2 is sufficient to instruct presynaptic differentiation, a fusion protein of the LRR domain of LRRTM2 with a myc-epitope tagged placental alkaline phosphatase (AP), LRRTM2 LRR-AP, was linked via biotinylated anti-myc antibody onto neutravidin beads. Contact of these LRR-AP-coated beads with isolated axons of hippocampal neurons induced clustering of synapsin or VGLUT1 at contact sites (Figures 4A and 4B, right panels). Beads coated with the control AP protein did not display synaptogenic activity (Figures 4A and 4B, left panels). Random counts revealed synapsin clustering at 34.1% of LRR-AP bead contacts and only 2.1% of control AP bead contacts (n = 50 fields). The mean synapsin intensity under all LRR-AP beads was 6.3-fold higher than that under control AP beads (p < 0.0001). Clustering of the active zone marker bassoon could also be detected at LRR-AP bead-axon contact sites. In fact, there appeared to be separation between the bassoon-labeled active zone and the VGLUT1-positive vesicle pool, with the active zone most closely apposed to the bead surface (Figure 4B, right panel, inset). The LRR-AP beads did not appear to cluster VGAT at contact sites with GAD-positive axons (data not shown). These results show that the LRR domain of LRRTM2 is necessary and sufficient to induce excitatory presynaptic differentiation without any contribution from other factors. To begin to assess subcellular localization, we created extracellular YFP fusions of LRRTM1 and LRRTM2 and transfected hippocampal neurons in culture. YFP-LRRTM-1 or -2 expressed at low level in hippocampal neurons traffic to dendrites and assume a punctate, synaptic-like pattern (Figures 5A–5C). YFP-LRRTM-1 or -2 colocalized with PSD-95 family proteins opposite VGLUT1 puncta. YFP-LRRTM-1 and -2 appeared to be exclusively colocalized with the excitatory postsynaptic scaffolding proteins of the PSD-95 family but not with the inhibitory postsynaptic scaffolding protein gephyrin. Quantitation of thresholded puncta revealed 72.6% overlap of YFP-LRRTM2 puncta with PSD-95 family, compared with 4.5% overlap of mirror images as control, and 8.0% overlap with gephyrin. These results suggest that the function of LRRTMs may be restricted to glutamatergic and not GABAergic hippocampal synapses, although more definitive tests and more diverse contexts are required. The C terminus of all four LRRTM members ends in a pattern of residues (-E-C-E-V) that resembles the X-S/T-X-V Class I PDZ domain ligand pattern (Sheng and Sala, 2001Sheng M. Sala C. PDZ domains and the organization of supramolecular complexes.Annu. Rev. Neurosci. 2001; 24: 1-29Crossref PubMed Scopus (1008) Google Scholar). Furthermore, a glutamate at the −3 position is preferred for PDZ domains 1 and 2 of PSD-95, for which several natural ligands terminate in -E-S/T-D-V (Sheng and Sala, 2001Sheng M. Sala C. PDZ domains and the organization of supramolecular complexes.Annu. Rev. Neurosci. 2001; 24: 1-29Crossref PubMed Scopus (1008) Google Scholar). Although a cysteine has not been reported in the −2 position for PDZ domain ligands, the similarity prompted us to test whether LRRTMs might bind PSD-95. We tested whether YFP-LRRTM2 could colocalize with PSD-95 in COS cells. YFP-LRRTM2 frequently forms small clusters in transfected COS cells. When PSD-95-mRFP was cotransfected with YFP-LRRTM2, the two proteins colocalized precisely in larger discrete aggregates (Figure 5D). Similar coclustering has been observed for coexpressed PSD-95 and other ligands and is thought to reflect the formation of large complexes via multimerization of PSD-95 and binding to ligand. Deletion of the C-terminal E-C-E-V residues of YFP-LRRTM2 disrupted the colocalization with PSD-95 (Figure 5E). We also confirmed the interaction by coimmunoprecipitation of myc-tagged PSD-95 with YFP-LRRTM2 coexpressed in COS cells (Figure 5F). Again, delet" @default.
• W2024301757 created "2016-06-24" @default.
• W2024301757 creator A5004495969 @default.
• W2024301757 creator A5005787962 @default.
• W2024301757 creator A5008833193 @default.
• W2024301757 creator A5011027783 @default.
• W2024301757 creator A5011071667 @default.
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• W2024301757 creator A5026147594 @default.
• W2024301757 creator A5062212424 @default.
• W2024301757 creator A5063001738 @default.
• W2024301757 date "2009-03-01" @default.
• W2024301757 modified "2023-10-18" @default.
• W2024301757 title "An Unbiased Expression Screen for Synaptogenic Proteins Identifies the LRRTM Protein Family as Synaptic Organizers" @default.
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