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Multiple trans-synaptic complexes organize synapse development, yet their roles in the mature brain and cooperation remain unclear. We analyzed the postsynaptic adhesion protein LRRTM1 in the prefrontal cortex (PFC), a region relevant to cognition and disorders. LRRTM1 knockout (KO) mice had fewer synapses, and we asked whether other synapse organizers counteract further loss. This determined that the immunoglobulin family member SynCAM 1 controls synapse number in PFC and was upregulated upon LRRTM1 loss. Combined LRRTM1 and SynCAM 1 deletion substantially lowered dendritic spine number in PFC, but not hippocampus, more than the sum of single KO impairments. Their cooperation extended presynaptically, and puncta of Neurexins, LRRTM1 partners, were less abundant in double KO (DKO) PFC. Electrophysiology and fMRI demonstrated aberrant neuronal activity in DKO mice. Further, DKO mice were impaired in social interactions and cognitive tasks. Our results reveal concerted roles of LRRTM1 and SynCAM 1 across synaptic, network, and behavioral domains., (© 2023. The Author(s).)

Neurexins are key organizer molecules that regulate synaptic function and are implicated in autism and schizophrenia. β-neurexins interact with numerous cell adhesion and receptor molecules, but their neuronal localization remains elusive. Using single-molecule tracking and high-resolution microscopy to detect neurexin1β and neurexin3β in primary hippocampal neurons from knockin mice, we demonstrate that endogenous β-neurexins are present in fewer than half of excitatory and inhibitory synapses. Moreover, we observe a large extrasynaptic pool of β-neurexins on axons and show that axonal β-neurexins diffuse with higher surface mobility than those transiently confined within synapses. Stimulation of neuronal activity further increases the mobility of synaptic and axonal β-neurexins, whereas inhibition causes the opposite. Blocking ectodomain cleavage by metalloproteases also reduces β-neurexin mobility and enhances glutamate release. These findings suggest that the surface mobility of endogenous β-neurexins inside and outside of synapses is dynamically regulated and linked to neuronal activity., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

C1ql3 is a secreted neuronal protein that binds to BAI3, an adhesion-class GPCR. C1ql3 is homologous to other gC1q-domain proteins that control synapse numbers, but a role for C1ql3 in regulating synapse density has not been demonstrated. We show in cultured neurons that C1ql3 expression is activity dependent and supports excitatory synapse density. Using newly generated conditional and constitutive C1ql3 knockout mice, we found that C1ql3-deficient mice exhibited fewer excitatory synapses and diverse behavioral abnormalities, including marked impairments in fear memories. Using circuit-tracing tools and conditional ablation of C1ql3 targeted to specific brain regions, we demonstrate that C1ql3-expressing neurons in the basolateral amygdala project to the medial prefrontal cortex, that these efferents contribute to fear memory behavior, and that C1ql3 is required for formation and/or maintenance of these synapses. Our results suggest that C1ql3 is a signaling protein essential for subsets of synaptic projections and the behaviors controlled by these projections., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Neurotransmission at different synapses is highly variable, and cell-adhesion molecules like α-neurexins (α-Nrxn) and their extracellular binding partners determine synapse function. Although α-Nrxn affect transmission at excitatory and inhibitory synapses, the contribution of neurexophilin-1 (Nxph1), an α-Nrxn ligand with restricted expression in subpopulations of inhibitory neurons, is unclear. To reveal its role, we investigated mice that either lack or overexpress Nxph1. We found that genetic deletion of Nxph1 impaired GABAB receptor (GABA(B)R)-dependent short-term depression of inhibitory synapses in the nucleus reticularis thalami, a region where Nxph1 is normally expressed at high levels. To test the conclusion that Nxph1 supports presynaptic GABA(B)R, we expressed Nxph1 ectopically at excitatory terminals in the neocortex, which normally do not contain this molecule but can be modulated by GABA(B)R. We generated Nxph1-GFP transgenic mice under control of the Thy1.2 promoter and observed a reduced short-term facilitation at these excitatory synapses, representing an inverse phenotype to the knockout. Consistently, the diminished facilitation could be reversed by pharmacologically blocking GABA(B)R with CGP-55845. Moreover, a complete rescue was achieved by additional blocking of postsynaptic GABA(A)R with intracellular picrotoxin or gabazine, suggesting that Nxph1 is able to recruit or stabilize both presynaptic GABA(B)R and postsynaptic GABA(A)R. In support, immunoelectron microscopy validated the localization of ectopic Nxph1 at the synaptic cleft of excitatory synapses in transgenic mice and revealed an enrichment of GABA(A)R and GABA(B)R subunits compared with wild-type animals. Thus, our data propose that Nxph1 plays an instructive role in synaptic short-term plasticity and the configuration with GABA receptors.

The development of neuronal networks in the brain requires the differentiation of functional synapses. Neurobeachin (Nbea) was identified as a putative regulator of membrane protein trafficking associated with tubulovesicular endomembranes and postsynaptic plasma membranes. Nbea is essential for evoked transmission at neuromuscular junctions, but its role in the central nervous system has not been characterized. Here, we have studied central synapses of a newly generated gene-trap knockout (KO) mouse line at embryonic day 18, because null-mutant mice are paralysed and die perinatally. Although the overall brain architecture was normal, we identified major abnormalities of synaptic function in mutant animals. In acute slices from the brainstem, both spontaneous excitatory and inhibitory postsynaptic currents were clearly reduced and failure rates of evoked inhibitory responses were markedly increased. In addition, the frequency of miniature excitatory and both the frequency and amplitudes of miniature inhibitory postsynaptic currents were severely diminished in KO mice, indicating a perturbation of both action potential-dependent and -independent transmitter release. Moreover, Nbea appears to be important for the formation and composition of central synapses because the area density of mature asymmetric contacts in the fetal brainstem was reduced to 30% of wild-type levels, and the expression levels of a subset of synaptic marker proteins were smaller than in littermate controls. Our data demonstrate for the first time a function of Nbea at central synapses that may be based on its presumed role in targeting membrane proteins to synaptic contacts, and are consistent with the 'excitatory-inhibitory imbalance' model of autism where Nbea gene rearrangements have been detected in some patients.

Two families of cell-adhesion molecules, predominantly presynaptic neurexins and postsynaptic neuroligins, are important for the formation and functioning of synapses in the brain, and mutations in several genes encoding these transmembrane proteins have been found in autism patients. However, very little is known about how neurexins are targeted to synapses and which mechanisms regulate this process. Using various epitope-tagged neurexins in primary hippocampal neurons of wild-type and knock-out mice in vitro and in transgenic animals in vivo, we show that neurexins are trafficked throughout neurons via transport vesicles and the plasma membrane insertion of neurexins occurs preferentially in the axonal/synaptic compartment. We also observed that exit of neurexins from the ER/Golgi and correct targeting require their PDZ-binding motif at the C terminus, whereas two presumptive ER retention signals are inactive. The ubiquitous presence of neurexin-positive transport vesicles and absence of bassoon colabeling demonstrate that these carriers are not active zone precursor vesicles, but colocalization with CASK, RIM1alpha, and calcium channels suggests that they may carry additional components of the exocytotic machinery. Our data indicate that neurexins are delivered to synapses by a polarized and regulated targeting process that involves PDZ-domain mediated interactions, suggesting a novel pathway for the distribution of neurexins and other synaptic proteins.

Alpha-neurexins are synaptic cell-surface molecules that are required for Ca(2+)-triggered exocytosis. Mice lacking all three alpha-neurexins show drastically reduced neurotransmitter release at excitatory and inhibitory synapses and die early postnatally. Although previous histological analysis of newborn alpha-neurexin triple mutants revealed only a moderate reduction in the density of type II synapses in the brainstem, cell culture studies proposed that neurexins are prominently involved in synapse formation. To assess the contribution of alpha-neurexins to the formation and structural properties of synapses in vivo, we performed a detailed morphological analysis of the brains from surviving adult double knockout mice lacking two of the three alpha-neurexins. Despite their impaired neurotransmission, we did not observe any gross anatomical defects or changes in the distribution of synaptic proteins in adult mutants. Only mild structural alterations were found: a approximately 20% reduction of neuropil area in many brain regions, resulting predominantly from shortened distal dendritic branches and fewer spines, as demonstrated by Golgi impregnation of pyramidal neurons. Quantitative electron microscopy revealed ultrastructurally normal type I and II terminals and a approximately 30% decrease in the density of type II synapses in the neocortex. To exclude errors in pathfinding, we investigated axonal projections in the olfactory bulb of newborn knockouts and did not observe any changes. Therefore, alpha-neurexins are not essential for the formation of the vast majority of synapses in vivo but rather regulate the function of these synapses.

The LDL receptor-related protein 1 (LRP1) is a multifunctional cell surface receptor that is highly expressed on neurons. Neuronal LRP1 in vitro can mediate ligand endocytosis, as well as modulate signal transduction processes. However, little is known about its role in the intact nervous system. Here, we report that mice that lack LRP1 selectively in differentiated neurons develop severe behavioral and motor abnormalities, including hyperactivity, tremor, and dystonia. Since their central nervous systems appear histoanatomically normal, we suggest that this phenotype is likely attributable to abnormal neurotransmission. This conclusion is supported by studies of primary cultured neurons that show that LRP1 is present in close proximity to the N-methyl-D-aspartate (NMDA) receptor in dendritic synapses and can be coprecipitated with NMDA receptor subunits and the postsynaptic density protein PSD-95 from neuronal cell lysates. Moreover, treatment with NMDA, but not dopamine, reduces the interaction of LRP1 with PSD-95, indicating that LRP1 participates in transmitter-dependent postsynaptic responses. Together, these findings suggest that LRP1, like other ApoE receptors, can modulate synaptic transmission in the brain.

Synapses are specialized intercellular junctions in which cell adhesion molecules connect the presynaptic machinery for neurotransmitter release to the postsynaptic machinery for receptor signalling. Neurotransmitter release requires the presynaptic co-assembly of Ca2+ channels with the secretory apparatus, but little is known about how synaptic components are organized. Alpha-neurexins, a family of >1,000 presynaptic cell-surface proteins encoded by three genes, link the pre- and postsynaptic compartments of synapses by binding extracellularly to postsynaptic cell adhesion molecules and intracellularly to presynaptic PDZ domain proteins. Using triple-knockout mice, we show that alpha-neurexins are not required for synapse formation, but are essential for Ca2+-triggered neurotransmitter release. Neurotransmitter release is impaired because synaptic Ca2+ channel function is markedly reduced, although the number of cell-surface Ca2+ channels appears normal. These data suggest that alpha-neurexins organize presynaptic terminals by functionally coupling Ca2+ channels to the presynaptic machinery.

Neurexins constitute a large family of highly variable cell-surface molecules that may function in synaptic transmission and/or synapse formation. Each of the three known neurexin genes encodes two major neurexin variants, α- and β-neurexins, that are composed of distinct extracellular domains linked to identical intracellular sequences. Deletions of one, two, or all three α-neurexins in mice recently demonstrated their essential role at synapses. In multiple α-neurexin knock-outs, neurotransmitter release from excitatory and inhibitory synapses was severely reduced, primarily probably because voltage-dependent Ca2+ channels were impaired. It remained unclear, however, which neurexin variants actually influence exocytosis and Ca2+ channels, which domain of neurexins is required for this function, and which Ca2+-channel subtypes are regulated. Here, we show by electrophysiological recordings that transgenic neurexin 1α rescues the release and Ca2+-current phenotypes, whereas transgenic neurexin 1β has no effect, indicating the importance of the extracellular sequences for the function of neurexins. Because neurexin 1α rescued the knock-out phenotype independent of the α-neurexin gene deleted, these data are consistent with a redundant function among different α-neurexins. In both knock-out and transgenically rescued mice, α-neurexins selectively affected the component of neurotransmitter release that depended on activation of N- and P/Q-type Ca2+ channels, but left L-type Ca2+ channels unscathed. Our findings indicate that α-neurexins represent organizer molecules in neurotransmission that regulate N- and P/Q-type Ca2+ channels, constituting an essential role at synapses that critically involves the extracellular domains of neurexins. [ABSTRACT FROM AUTHOR]

Synapses are specialized intercellular junctions in which cell adhesion molecules connect the presynaptic machinery for neurotransmitter release to the postsynaptic machinery for receptor signalling. Neurotransmitter release requires the presynaptic co-assembly of Ca2+ channels with the secretory apparatus, but little is known about how synaptic components are organized. a-Neurexins, a family of >1,000 presynaptic cell-surface proteins encoded by three genes, link the pre- and postsynaptic compartments of synapses by binding extracellularly to postsynaptic cell adhesion molecules and intracellularly to presynaptic PDZ domain proteins. Using triple-knockout mice, we show that a-neurexins are not required for synapse formation, but are essential for Ca2+-triggered neurotransmitter release. Neurotransmitter release is impaired because synaptic Ca2+ channel function is markedly reduced, although the number of cell-surface Ca2+ channels appears normal. These data suggest that a-neurexins organize presynaptic terminals by functionally coupling Ca2+ channels to the presynaptic machinery. [ABSTRACT FROM AUTHOR]

Neurexophilin 3 (Nxph3) is a specific ligand of synaptic α-neurexins that are essential for efficient neurotransmitter release. Previous biochemical work demonstrated that Nxph3 interacts with an extracellular domain of α-neurexins in a tight complex; however, no information is available on the localization or functional role of Nxph3 in the brain. Here, we generated lacZ reporter gene knock-in mice to investigate the distribution of Nxph3 at the single-cell level and Nxph3 knockout mice to examine its functional importance. Nxph3 expression was restricted mostly to subplate-derived neurons in cortical layer 6b, granule cells in the vestibulocerebellum, and Cajal-Retzius cells during development. Colabeling experiments demonstrated that neurons expressing Nxph3 do not belong to a uniform cell type. Morphological analyses and systematic behavioral testing of knockout mice revealed no anatomical defects but uncovered remarkable functional abnormalities in sensory information processing and motor coordination, evident by increased startle response, reduced prepulse inhibition, and poor rotarod performance. Since Nxph3-deficient mice behaved normally while performing a number of other tasks, our data suggest an important role for Nxph3 as a locally and temporally regulated neuropeptide-like molecule, presumably acting in a complex with α-neurexins in select neuronal circuits. [ABSTRACT FROM AUTHOR]