Distinct Alterations in Dendritic Spine Morphology in the Absence of ß-Neurexins.

Dendritic spines are essential for synaptic function because they constitute the postsynaptic compartment of the neurons that receives the most excitatory input. The extracellularly shorter variant of the presynaptic cell adhesion molecules neurexins, ß-neurexin, has been implicated in various aspects of synaptic function, including neurotransmitter release. However, its role in developing or stabilizing dendritic spines as fundamental computational units of excitatory synapses has remained unclear. Here, we show through morphological analysis that the deletion of ß-neurexins in hippocampal neurons in vitro and in hippocampal tissue in vivo affects presynaptic dense-core vesicles, as hypothesized earlier, and, unexpectedly, alters the postsynaptic spine structure. Specifically, we observed that the absence of ß-neurexins led to an increase in filopodial-like protrusions in vitro and more mature mushroom-type spines in the CA1 region of adult knockout mice. In addition, the deletion of ß-neurexins caused alterations in the spine head dimension and an increase in spines with perforations of their postsynaptic density but no changes in the overall number of spines or synapses. Our results indicate that presynaptic ß-neurexins play a role across the synaptic cleft, possibly by aligning with postsynaptic binding partners and glutamate receptors via transsynaptic columns.

PI(4,5)P2 controls slit diaphragm formation and endocytosis in Drosophila nephrocytes.

Drosophila nephrocytes are an emerging model system for mammalian podocytes and proximal tubules as well as for the investigation of kidney diseases. Like podocytes, nephrocytes exhibit characteristics of epithelial cells, but the role of phospholipids in polarization of these cells is yet unclear. In epithelia, phosphatidylinositol(4,5)bisphosphate (PI(4,5)P2) and phosphatidylinositol(3,4,5)-trisphosphate (PI(3,4,5)P3) are asymmetrically distributed in the plasma membrane and determine apical-basal polarity. Here, we demonstrate that both phospholipids are present in the plasma membrane of nephrocytes, but only PI(4,5)P2 accumulates at slit diaphragms. Knockdown of Skittles, a phosphatidylinositol(4)phosphate 5-kinase, which produces PI(4,5)P2, abolished slit diaphragm formation and led to strongly reduced endocytosis. Notably, reduction in PI(3,4,5)P3 by overexpression of PTEN or expression of a dominant-negative phosphatidylinositol-3-kinase did not affect nephrocyte function, whereas enhanced formation of PI(3,4,5)P3 by constitutively active phosphatidylinositol-3-kinase resulted in strong slit diaphragm and endocytosis defects by ectopic activation of the Akt/mTOR pathway. Thus, PI(4,5)P2 but not PI(3,4,5)P3 is essential for slit diaphragm formation and nephrocyte function. However, PI(3,4,5)P3 has to be tightly controlled to ensure nephrocyte development.

Apical-basal polarity regulators are essential for slit diaphragm assembly and endocytosis in Drosophila nephrocytes.

Apical-basal polarity is a key feature of most epithelial cells and it is regulated by highly conserved protein complexes. In mammalian podocytes, which emerge from columnar epithelial cells, this polarity is preserved and the tight junctions are converted to the slit diaphragms, establishing the filtration barrier. In Drosophila, nephrocytes show several structural and functional similarities with mammalian podocytes and proximal tubular cells. However, in contrast to podocytes, little is known about the role of apical-basal polarity regulators in these cells. In this study, we used expansion microscopy and found the apical polarity determinants of the PAR/aPKC and Crb-complexes to be predominantly targeted to the cell cortex in proximity to the nephrocyte diaphragm, whereas basolateral regulators also accumulate intracellularly. Knockdown of PAR-complex proteins results in severe endocytosis and nephrocyte diaphragm defects, which is due to impaired aPKC recruitment to the plasma membrane. Similar, downregulation of most basolateral polarity regulators disrupts Nephrin localization but had surprisingly divergent effects on endocytosis. Our findings suggest that morphology and slit diaphragm assembly/maintenance of nephrocytes is regulated by classical apical-basal polarity regulators, which have distinct functions in endocytosis.

Auxiliary α2δ1 and α2δ3 Subunits of Calcium Channels Drive Excitatory and Inhibitory Neuronal Network Development.

VGCCs are multisubunit complexes that play a crucial role in neuronal signaling. Auxiliary α2δ subunits of VGCCs modulate trafficking and biophysical properties of the pore-forming α1 subunit and trigger excitatory synaptogenesis. Alterations in the expression level of α2δ subunits were implicated in several syndromes and diseases, including chronic neuropathic pain, autism, and epilepsy. However, the contribution of distinct α2δ subunits to excitatory/inhibitory imbalance and aberrant network connectivity characteristic for these pathologic conditions remains unclear. Here, we show that α2δ1 overexpression enhances spontaneous neuronal network activity in developing and mature cultures of hippocampal neurons. In contrast, overexpression, but not downregulation, of α2δ3 enhances neuronal firing in immature cultures, whereas later in development it suppresses neuronal activity. We found that α2δ1 overexpression increases excitatory synaptic density and selectively enhances presynaptic glutamate release, which is impaired on α2δ1 knockdown. Overexpression of α2δ3 increases the excitatory synaptic density as well but also facilitates spontaneous GABA release and triggers an increase in the density of inhibitory synapses, which is accompanied by enhanced axonaloutgrowth in immature interneurons. Together, our findings demonstrate that α2δ1 and α2δ3 subunits play distinct but complementary roles in driving formation of structural and functional network connectivity during early development. An alteration in α2δ surface expression during critical developmental windows can therefore play a causal role and have a profound impact on the excitatory-to-inhibitory balance and network connectivity.SIGNIFICANCE STATEMENT The computational capacity of neuronal networks is determined by their connectivity. Chemical synapses are the main interface for transfer of information between individual neurons. The initial formation of network connectivity requires spontaneous electrical activity and the calcium channel-mediated signaling. We found that, in early development, auxiliary α2δ3 subunits of calcium channels foster presynaptic release of GABA, trigger formation of inhibitory synapses, and promote axonal outgrowth in inhibitory interneurons. In contrast, later in development, α2δ1 subunits promote the glutamatergic neurotransmission and synaptogenesis, as well as strongly enhance neuronal network activity. We propose that formation of connectivity in neuronal networks is associated with a concerted interplay of α2δ1 and α2δ3 subunits of calcium channels.

Human CCR5high effector memory cells perform CNS parenchymal immune surveillance via GZMK-mediated transendothelial diapedesis.

Although the CNS is immune privileged, continuous search for pathogens and tumours by immune cells within the CNS is indispensable. Thus, distinct immune-cell populations also cross the blood-brain barrier independently of inflammation/under homeostatic conditions. It was previously shown that effector memory T cells populate healthy CNS parenchyma in humans and, independently, that CCR5-expressing lymphocytes as well as CCR5 ligands are enriched in the CNS of patients with multiple sclerosis. Apart from the recently described CD8+ CNS tissue-resident memory T cells, we identified a population of CD4+CCR5high effector memory cells as brain parenchyma-surveilling cells. These cells used their high levels of VLA-4 to arrest on scattered VCAM1, their open-conformation LFA-1 to crawl preferentially against the flow in search for sites permissive for extravasation, and their stored granzyme K (GZMK) to induce local ICAM1 aggregation and perform trans-, rather than paracellular diapedesis through unstimulated primary brain microvascular endothelial cells. This study included peripheral blood mononuclear cell samples from 175 healthy donors, 29 patients infected with HIV, with neurological symptoms in terms of cognitive impairment, 73 patients with relapsing-remitting multiple sclerosis in remission, either 1-4 weeks before (n = 29), or 18-60 months after the initiation of natalizumab therapy (n = 44), as well as white matter brain tissue of three patients suffering from epilepsy. We here provide ex vivo evidence that CCR5highGZMK+CD4+ effector memory T cells are involved in CNS immune surveillance during homeostasis, but could also play a role in CNS pathology. Among CD4+ T cells, this subset was found to dominate the CNS of patients without neurological inflammation ex vivo. The reduction in peripheral blood of HIV-positive patients with neurological symptoms correlated to their CD4 count as a measure of disease progression. Their peripheral enrichment in multiple sclerosis patients and specific peripheral entrapment through the CNS infiltration inhibiting drug natalizumab additionally suggests a contribution to CNS autoimmune pathology. Our transcriptome analysis revealed a migratory phenotype sharing many features with tissue-resident memory and Th17.1 cells, most notably the transcription factor eomesodermin. Knowledge on this cell subset should enable future studies to find ways to strengthen the host defence against CNS-resident pathogens and brain tumours or to prevent CNS autoimmunity.

Protein mutated in paroxysmal dyskinesia interacts with the active zone protein RIM and suppresses synaptic vesicle exocytosis.

Paroxysmal nonkinesigenic dyskinesia (PNKD) is an autosomal dominant episodic movement disorder precipitated by coffee, alcohol, and stress. We previously identified the causative gene but the function of the encoded protein remains unknown. We also generated a PNKD mouse model that revealed dysregulated dopamine signaling in vivo. Here, we show that PNKD interacts with synaptic active zone proteins Rab3-interacting molecule (RIM)1 and RIM2, localizes to synapses, and modulates neurotransmitter release. Overexpressed PNKD protein suppresses release, and mutant PNKD protein is less effective than wild-type at inhibiting exocytosis. In PNKD KO mice, RIM1/2 protein levels are reduced and synaptic strength is impaired. Thus, PNKD is a novel synaptic protein with a regulatory role in neurotransmitter release.

Modulation of synaptic function through the α-neurexin-specific ligand neurexophilin-1.

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.

Promoter-like sequences regulating transcriptional activity in neurexin and neuroligin genes.

Synapse function requires the cell-adhesion molecules neurexins (Nrxn) and neuroligins (Nlgn). Although these molecules are essential for neurotransmission and prefer distinct isoform combinations for interaction, little is known about their transcriptional regulation. Here, we started to explore this important aspect because expression of Nrxn1-3 and Nlgn1-3 genes is altered in mice lacking the transcriptional regulator methyl-CpG-binding protein2 (MeCP2). Since MeCP2 can bind to methylated CpG-dinucleotides and Nrxn/Nlgn contain CpG-islands, we tested genomic sequences for transcriptional activity in reporter gene assays. We found that their influence on transcription are differentially activating or inhibiting. As we observed an activity difference between heterologous and neuronal cell lines for distinct Nrxn1 and Nlgn2 sequences, we dissected their putative promoter regions. In both genes, we identify regions in exon1 that can induce transcription, in addition to the alternative transcriptional start points in exon2. While the 5'-regions of Nrxn1 and Nlgn2 contain two CpG-rich elements that show distinct methylation frequency and binding to MeCP2, other regions may act independently of this transcriptional regulator. These data provide first insights into regulatory sequences of Nrxn and Nlgn genes that may represent an important aspect of their function at synapses in health and disease.

Concerted roles of LRRTM1 and SynCAM 1 in organizing prefrontal cortex synapses and cognitive functions.

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.

Deletion of ß-Neurexins in Mice Alters the Distribution of Dense-Core Vesicles in Presynapses of Hippocampal and Cerebellar Neurons.

Communication between neurons through synapses includes the release of neurotransmitter-containing synaptic vesicles (SVs) and of neuromodulator-containing dense-core vesicles (DCVs). Neurexins (Nrxns), a polymorphic family of cell surface molecules encoded by three genes in vertebrates (Nrxn1-3), have been proposed as essential presynaptic organizers and as candidates for cell type-specific or even synapse-specific regulation of synaptic vesicle exocytosis. However, it remains unknown whether Nrxns also regulate DCVs. Here, we report that at least ß-neurexins (ß-Nrxns), an extracellularly smaller Nrxn variant, are involved in the distribution of presynaptic DCVs. We found that conditional deletion of all three ß-Nrxn isoforms in mice by lentivirus-mediated Cre recombinase expression in primary hippocampal neurons reduces the number of ultrastructurally identified DCVs in presynaptic boutons. Consistently, colabeling against marker proteins revealed a diminished population of chromogranin A- (ChrgA-) positive DCVs in synapses and axons of ß-Nrxn-deficient neurons. Moreover, we validated the impaired DCV distribution in cerebellar brain tissue from constitutive ß-Nrxn knockout (ß-TKO) mice, where DCVs are normally abundant and ß-Nrxn isoforms are prominently expressed. Finally, we observed that the ultrastructure and marker proteins of the Golgi apparatus, responsible for packaging neuropeptides into DCVs, seem unchanged. In conclusion, based on the validation from the two deletion strategies in conditional and constitutive KO mice, two neuronal populations from the hippocampus and cerebellum, and two experimental protocols in cultured neurons and in the brain tissue, this study presented morphological evidence that the number of DCVs at synapses is altered in the absence of ß-Nrxns. Our results therefore point to an unexpected contribution of ß-Nrxns to the organization of neuropeptide and neuromodulator function, in addition to their more established role in synaptic vesicle release.

Endogenous ß-neurexins on axons and within synapses show regulated dynamic behavior.

Neurexins are key organizer molecules that regulate synaptic function and are implicated in autism and schizophrenia. ß-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 neurexin1ß and neurexin3ß in primary hippocampal neurons from knockin mice, we demonstrate that endogenous ß-neurexins are present in fewer than half of excitatory and inhibitory synapses. Moreover, we observe a large extrasynaptic pool of ß-neurexins on axons and show that axonal ß-neurexins diffuse with higher surface mobility than those transiently confined within synapses. Stimulation of neuronal activity further increases the mobility of synaptic and axonal ß-neurexins, whereas inhibition causes the opposite. Blocking ectodomain cleavage by metalloproteases also reduces ß-neurexin mobility and enhances glutamate release. These findings suggest that the surface mobility of endogenous ß-neurexins inside and outside of synapses is dynamically regulated and linked to neuronal activity.

An extracellular vesicle-related gene expression signature identifies high-risk patients in medulloblastoma.

BACKGROUND: Medulloblastoma (MB) is a malignant brain tumor in childhood. It comprises 4 subgroups with different clinical behaviors. The aim of this study was to characterize the transcriptomic landscape of MB, both at the level of individual tumors as well as in large patient cohorts. METHODS: We used a combination of single-cell transcriptomics, cell culture models and biophysical methods such as nanoparticle tracking analysis and electron microscopy to investigate intercellular communication in the MB tumor niche. RESULTS: Tumor cells of the sonic hedgehog (SHH)-MB subgroup show a differentiation blockade. These cells undergo extensive metabolic reprogramming. The gene expression profiles of individual tumor cells show a partial convergence with those of tumor-associated glial and immune cells. One possible cause is the transfer of extracellular vesicles (EVs) between cells in the tumor niche. We were able to detect EVs in co-culture models of MB tumor cells and oligodendrocytes. We also identified a gene expression signature, EVS, which shows overlap with the proteome profile of large oncosomes from prostate cancer cells. This signature is also present in MB patient samples. A high EVS expression is one common characteristic of tumors that occur in high-risk patients from different MB subgroups or subtypes. CONCLUSIONS: With EVS, our study uncovered a novel gene expression signature that has a high prognostic significance across MB subgroups.

Alpha-neurexins couple Ca2+ channels to synaptic vesicle exocytosis.

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.

Enhanced LTP of population spikes in the dentate gyrus of mice haploinsufficient for neurobeachin.

Deletion of the autism candidate molecule neurobeachin (Nbea), a large PH-BEACH-domain containing neuronal protein, has been shown to affect synaptic function by interfering with neurotransmitter receptor targeting and dendritic spine formation. Previous analysis of mice lacking one allele of the Nbea gene identified impaired spatial learning and memory in addition to altered autism-related behaviours. However, no functional data from living heterozygous Nbea mice (Nbea+/-) are available to corroborate the behavioural phenotype. Here, we explored the consequences of Nbea haploinsufficiency on excitation/inhibition balance and synaptic plasticity in the intact hippocampal dentate gyrus of Nbea+/- animals in vivo by electrophysiological recordings. Based on field potential recordings, we show that Nbea+/- mice display enhanced LTP of the granule cell population spike, but no differences in basal synaptic transmission, synapse numbers, short-term plasticity, or network inhibition. These data indicate that Nbea haploinsufficiency causes remarkably specific alterations to granule cell excitability in vivo, which may contribute to the behavioural abnormalities in Nbea+/- mice and to related symptoms in patients.

Polarized targeting of neurexins to synapses is regulated by their C-terminal sequences.

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.

Neurobeachin, a protein implicated in membrane protein traffic and autism, is required for the formation and functioning of central synapses.

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.

Neurexophilin 3 is highly localized in cortical and cerebellar regions and is functionally important for sensorimotor gating and motor coordination.

Neurexophilin 3 (Nxph3) is a specific ligand of synaptic alpha-neurexins that are essential for efficient neurotransmitter release. Previous biochemical work demonstrated that Nxph3 interacts with an extracellular domain of alpha-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 alpha-neurexins in select neuronal circuits.

Deletion of alpha-neurexins does not cause a major impairment of axonal pathfinding or synapse formation.

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.

Neuronal LRP1 functionally associates with postsynaptic proteins and is required for normal motor function in mice.

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.

Extracellular domains of alpha-neurexins participate in regulating synaptic transmission by selectively affecting N- and P/Q-type Ca2+ channels.

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, alpha- and beta-neurexins, that are composed of distinct extracellular domains linked to identical intracellular sequences. Deletions of one, two, or all three alpha-neurexins in mice recently demonstrated their essential role at synapses. In multiple alpha-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 1alpha rescues the release and Ca2+-current phenotypes, whereas transgenic neurexin 1beta has no effect, indicating the importance of the extracellular sequences for the function of neurexins. Because neurexin 1alpha rescued the knock-out phenotype independent of the alpha-neurexin gene deleted, these data are consistent with a redundant function among different alpha-neurexins. In both knock-out and transgenically rescued mice, alpha-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 alpha-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.