Presynaptic nerve terminals release neurotransmitters repeatedly, often at high frequency, and in relative isolation from neuronal cell bodies. Repeated release requires cycles of SNARE-complex assembly and disassembly, with continuous generation of reactive SNARE-protein intermediates. Although many forms of neurodegeneration initiate presynaptically, only few pathogenic mechanisms are known, and the functions of presynaptic proteins linked to neurodegeneration, such as α-synuclein, remain unclear. Here, we find that maintenance of continuous presynaptic SNARE-complex assembly required a non-classical chaperone activity mediated by synucleins. Specifically, α-synuclein directly bound to the SNARE-protein synaptobrevin-2/VAMP2, and promoted SNARE-complex assembly. Moreover, triple knockout mice lacking synucleins developed age-dependent neurological impairments, exhibited decreased SNARE-complex assembly, and perished prematurely. Thus, synucleins may function to sustain normal SNAREcomplex assembly in a presynaptic terminal during aging.In presynaptic terminals, neurotransmitter release requires a tightly coordinated membrane fusion machinery whose central components are soluble NSF attachment protein receptor (SNARE) and Sec1/Munc18-like proteins (1-3). Terminals release neurotransmitters thousands of times per minute; during each release reaction, SNARE-complex assembly and disassembly generates highly reactive unfolded SNARE protein intermediates, rendering the terminals potentially vulnerable to activity-dependent degeneration. Indeed, much evidence points to presynaptic terminals as an initiation site for neurodegeneration (4-6), and knockout (KO) of at least one presynaptic chaperone protein, cysteine string protein-α (CSPα), causes fulminant neurodegeneration in mice (7). Synucleins are abundant presynaptic proteins that are expressed from three genes (α-, β-and γ-synuclein; 8). α-Synuclein is involved in neurodegeneration (9-11), and γ-synuclein may contribute to progression of many types of cancer (12). Synucleins may modify neurotransmitter release (13,14), but their physiological functions remain unknown. Strikingly, transgenic expression of α-synuclein abolishes the lethal neurodegeneration induced by KO of CSPα, whereas deletion of endogenous synucleins accelerates this neurodegeneration (15). CSPα KO mice exhibit decreased levels of the SNARE protein SNAP-25 and impaired SNARE-complex assembly, suggesting a link between SNARE-complex assembly and neurodegeneration. α-* To whom correspondence should be addressed: tcs1@stanford.edu. HHMI Author Manuscript HHMI Author Manuscript HHMI Author ManuscriptSynuclein rescues SNARE-complex assembly but not SNAP-25 levels (15). This result indicates that α-synuclein may enhance SNARE-protein function, and thereby compensate for the CSPα deletion. To address this hypothesis, we here examine the role of α-synuclein in SNARE-complex assembly and in the maintenance of continuous SNARE-cycling in presynaptic terminals over the lifetime of an animal.We imm...

Autism spectrum disorders (ASDs) are characterized by impairments in social behaviors that are sometimes coupled to specialized cognitive abilities. A small percentage of ASD patients carry mutations in genes encoding neuroligins, which are postsynaptic cell-adhesion molecules. We introduced one of these mutations into mice: the Arg451-->Cys451 (R451C) substitution in neuroligin-3. R451C mutant mice showed impaired social interactions but enhanced spatial learning abilities. Unexpectedly, these behavioral changes were accompanied by an increase in inhibitory synaptic transmission with no apparent effect on excitatory synapses. Deletion of neuroligin-3, in contrast, did not cause such changes, indicating that the R451C substitution represents a gain-of-function mutation. These data suggest that increased inhibitory synaptic transmission may contribute to human ASDs and that the R451C knockin mice may be a useful model for studying autism-related behaviors.

Neuroligins enhance synapse formation in vitro, but surprisingly are not required for the generation of synapses in vivo. We now show that in cultured neurons, neuroligin-1 overexpression increases excitatory, but not inhibitory, synaptic responses, and potentiates synaptic NMDAR/AMPAR ratios. In contrast, neuroligin-2 overexpression increases inhibitory, but not excitatory, synaptic responses. Accordingly, deletion of neuroligin-1 in knockout mice selectively decreases the NMDAR/AMPAR ratio, whereas deletion of neuroligin-2 selectively decreases inhibitory synaptic responses. Strikingly, chronic inhibition of NMDARs or CaM-Kinase II, which signals downstream of NMDARs, suppresses the synapse-boosting activity of neuroligin-1, whereas chronic inhibition of general synaptic activity suppresses the synapse-boosting activity of neuroligin-2. Taken together, these data indicate that neuroligins do not establish, but specify and validate, synapses via an activity-dependent mechanism, with different neuroligins acting on distinct types of synapses. This hypothesis reconciles the overexpression and knockout phenotypes and suggests that neuroligins contribute to the use-dependent formation of neural circuits.

Deletions in the neurexin-1␣ gene were identified in large-scale unbiased screens for copy-number variations in patients with autism or schizophrenia. To explore the underlying biology, we studied the electrophysiological and behavioral phenotype of mice lacking neurexin-1␣. Hippocampal slice physiology uncovered a defect in excitatory synaptic strength in neurexin-1␣ deficient mice, as revealed by a decrease in miniature excitatory postsynaptic current (EPSC) frequency and in the input-output relation of evoked postsynaptic potentials. This defect was specific for excitatory synaptic transmission, because no change in inhibitory synaptic transmission was observed in the hippocampus. Behavioral studies revealed that, compared with littermate control mice, neurexin-1␣ deficient mice displayed a decrease in prepulse inhibition, an increase in grooming behaviors, an impairment in nestbuilding activity, and an improvement in motor learning. However, neurexin-1␣ deficient mice did not exhibit any obvious changes in social behaviors or in spatial learning. Together, these data indicate that the neurexin-1␣ deficiency induces a discrete neural phenotype whose extent correlates, at least in part, with impairments observed in human patients.autism ͉ neuroligin ͉ schizophrenia ͉ synaptic cell-adhesion ͉ synapse N eurexins are neuronal cell-surface proteins that were identified as receptors for ␣-latrotoxin, a presynaptic toxin that triggers massive neurotransmitter release (1-3). Neurexins are largely presynaptic proteins that form a transsynaptic celladhesion complex with postsynaptic neuroligins (4, 5). Vertebrates express three neurexin genes, each of which includes two promoters that direct the synthesis of the longer ␣-neurexins and the shorter ␤-neurexins (6, 7). Neurexins are expressed in all neurons, and are subject to extensive alternative splicing, generating Ͼ1,000 splice variants, some of which exhibit highly regulated developmental and spatial expression patterns (8).The properties of neurexins suggested that they function as synaptic recognition molecules (1), and mediate transsynaptic interactions via binding to neuroligins (4). In support of this overall concept, ␣-neurexin triple KO mice exhibit major impairments in synaptic transmission that manifest largely, but not exclusively, as presynaptic changes (9-13). Importantly, ␣-neurexin KO mice do not display a major decrease in the number of excitatory synapses, and only a moderate decrease in inhibitory synapses (9, 13). Thus, neurexins appear to be essential components of synaptic function whose roles extend to several different components of synapses.In recent years, enormous progress in human genetics has led to the identification of multiple genes that are linked to autism spectrum disorders (ASDs) and schizophrenia. Interestingly, copy-number variations in the neurexin-1␣ gene (but not the neurexin-1␤ gene) were repeatedly observed in patients with ASDs (14 -21) and schizophrenia (22-25). Because 0.5% of all ASD cases appear to harbor neurexin-1␣ gen...

Neuroligins (NLs) are a family of neural cell-adhesion molecules that are involved in excitatory/inhibitory synapse specification. Multiple members of the NL family (including NL1) and their binding partners have been linked to cases of human autism and mental retardation. We have now characterized NL1-deficient mice in autism-and mental retardation-relevant behavioral tasks. NL1 knock-out (KO) mice display deficits in spatial learning and memory that correlate with impaired hippocampal long-term potentiation. In addition, NL1 KO mice exhibit a dramatic increase in repetitive, stereotyped grooming behavior, a potential autism-relevant abnormality. This repetitive grooming abnormality in NL1 KO mice is associated with a reduced NMDA/AMPA ratio at corticostriatal synapses. Interestingly, we further demonstrate that the increased repetitive grooming phenotype can be rescued in adult mice by administration of the NMDA receptor partial coagonist D-cycloserine. Broadly, these data are consistent with a role of synaptic cell-adhesion molecules in general, and NL1 in particular, in autism and implicate reduced excitatory synaptic transmission as a potential mechanism and treatment target for repetitive behavioral abnormalities.

Multiple independent mutations in neuroligin genes were identified in patients with familial autism, including the R451C substitution in neuroligin-3 (NL3). Previous studies showed that NL3 R451C knock-in mice exhibited modestly impaired social behaviors, enhanced water maze learning abilities, and increased synaptic inhibition in the somatosensory cortex, and they suggested that the behavioral changes in these mice may be caused by a general shift of synaptic transmission to inhibition. Here, we confirm that NL3 R451C mutant mice behaviorally exhibit social interaction deficits and electrophysiologically display increased synaptic inhibition in the somatosensory cortex. Unexpectedly, however, we find that the NL3 R451C mutation produced a strikingly different phenotype in the hippocampus. Specifically, in the hippocampal CA1 region, the NL3 R451C mutation caused an ∼1.5-fold increase in AMPA receptor-mediated excitatory synaptic transmission, dramatically altered the kinetics of NMDA receptor-mediated synaptic responses, induced an approximately twofold up-regulation of NMDA receptors containing NR2B subunits, and enhanced longterm potentiation almost twofold. NL3 KO mice did not exhibit any of these changes. Quantitative light microscopy and EM revealed that the NL3 R451C mutation increased dendritic branching and altered the structure of synapses in the stratum radiatum of the hippocampus. Thus, in NL3 R451C mutant mice, a single point mutation in a synaptic cell adhesion molecule causes context-dependent changes in synaptic transmission; these changes are consistent with the broad impact of this mutation on murine and human behaviors, suggesting that NL3 controls excitatory and inhibitory synapse properties in a region-and circuit-specific manner.synapse formation A utism spectrum disorders (ASDs) constitute a heterogeneous group of neurodevelopmental diseases with a strong genetic component (1-3). The identification of multiple ASD candidate genes that encode synaptic proteins suggested that ASDs may involve impairments in synaptic transmission (4-12). In particular, numerous mutations in neuroligins, a family of postsynaptic cell adhesion molecules, have been identified in 10,12). Consistent with the hypothesis that synaptic dysfunction contributes to ASD pathogenesis (13, 14), mouse models of two ASD-associated neuroligin mutations exhibit functional changes in synaptic transmission (15, 16).Neuroligins are not critical for the initial establishment of synapses but are required for normal synapse function (17)(18)(19)(20). Deletion of neuroligin-1 or -2 selectively impairs excitatory or inhibitory synaptic transmission, respectively (17,18,21,22), whereas at least in the somatosensory cortex, deletion of neuroligin-3 (NL3) causes no major synaptic phenotype (15). Three ASD-relevant neuroligin mutations were characterized in mouse models: the R451C substitution in NL3 (15, 23), a loss of function mutation of NL4 (24), and a point mutation that was identified in the cytoplasmic tail of NL4 in an ASD patient...

Neuroligins are evolutionarily conserved postsynaptic cell-adhesion molecules that function, at least in part, by forming trans-synaptic complexes with presynaptic neurexins. Different neuroligin isoforms perform diverse functions and exhibit distinct intracellular localizations, but contain similar cytoplasmic sequences whose role remains largely unknown. Here, we analysed the effect of a single amino-acid substitution (R704C) that targets a conserved arginine residue in the cytoplasmic sequence of all neuroligins, and that was associated with autism in neuroligin-4. We introduced the R704C mutation into mouse neuroligin-3 by homologous recombination, and examined its effect on synapses in vitro and in vivo. Electrophysiological and morphological studies revealed that the neuroligin-3 R704C mutation did not significantly alter synapse formation, but dramatically impaired synapse function. Specifically, the R704C mutation caused a major and selective decrease in AMPA receptor-mediated synaptic transmission in pyramidal neurons of the hippocampus, without similarly changing NMDA or GABA receptor-mediated synaptic transmission, and without detectably altering presynaptic neurotransmitter release. Our results suggest that the cytoplasmic tail of neuroligin-3 has a central role in synaptic transmission by modulating the recruitment of AMPA receptors to postsynaptic sites at excitatory synapses.

Objective:The objectives of this study were to measure the global impact of the pandemic on the volumes for intravenous thrombolysis (IVT), IVT transfers, and stroke hospitalizations over 4 months at the height of the pandemic (March 1 to June 30, 2020) compared with two control 4-month periods.Methods:We conducted a cross-sectional, observational, retrospective study across 6 continents, 70 countries, and 457 stroke centers. Diagnoses were identified by their ICD-10 codes and/or classifications in stroke databases.Results:There were 91,373 stroke admissions in the 4 months immediately before compared to 80,894 admissions during the pandemic months, representing an 11.5% (95%CI, -11.7 to - 11.3, p<0.0001) decline. There were 13,334 IVT therapies in the 4 months preceding compared to 11,570 procedures during the pandemic, representing a 13.2% (95%CI, -13.8 to -12.7, p<0.0001) drop. Interfacility IVT transfers decreased from 1,337 to 1,178, or an 11.9% decrease (95%CI, -13.7 to -10.3, p=0.001). Recovery of stroke hospitalization volume (9.5%, 95%CI 9.2-9.8, p<0.0001) was noted over the two later (May, June) versus the two earlier (March, April) pandemic months. There was a 1.48% stroke rate across 119,967 COVID-19 hospitalizations. SARS-CoV-2 infection was noted in 3.3% (1,722/52,026) of all stroke admissions.Conclusions:The COVID-19 pandemic was associated with a global decline in the volume of stroke hospitalizations, IVT, and interfacility IVT transfers. Primary stroke centers and centers with higher COVID19 inpatient volumes experienced steeper declines. Recovery of stroke hospitalization was noted in the later pandemic months.