Markus Missler

Neuroligins Determine Synapse Maturation and Function

Synaptogenesis, the generation and maturation of functional synapses between nerve cells, is an essential step in the development of neuronal networks in the brain. It is thought to be triggered by members of the neuroligin family of postsynaptic cell adhesion proteins, which may form transsynaptic contacts with presynaptic alpha- and beta-neurexins and have been implicated in the etiology of autism. We show that deletion mutant mice lacking neuroligin expression die shortly after birth due to respiratory failure. This respiratory failure is a consequence of reduced GABAergic/glycinergic and glutamatergic synaptic transmission and network activity in brainstem centers that control respiration. However, the density of synaptic contacts is not altered in neuroligin-deficient brains and cultured neurons. Our data show that neuroligins are required for proper synapse maturation and brain function, but not for the initial formation of synaptic contacts.

Neuroligin 1: A splice site-specific ligand for β-neurexins

Neurexins are neuronal cell surface proteins with hundreds of isoforms generated by alternative splicing. Here we describe neuroligin 1, a neuronal cell surface protein that is enriched in synaptic plasma membranes and acts as a splice site-specific ligand for beta-neurexins. Neuroligin 1 binds to beta-neurexins only if they lack an insert in the alternatively spliced sequence of the G domain, but not if they contain an insert. The extracellular sequence of neuroligin 1 is composed of a catalytically inactive esterase domain homologous to acetylcholinesterase. In situ hybridization reveals that alternative splicing of neurexins at the site recognized by neuroligin 1 is highly regulated. These findings support a model whereby alternative splicing of neurexins creates a family of cell surface receptors that confers interactive specificity onto their resident neurons.

α-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. α-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 α-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 α-neurexins organize presynaptic terminals by functionally coupling Ca2+ channels to the presynaptic machinery.

COMPLEXINS : CYTOSOLIC PROTEINS THAT REGULATE SNAP RECEPTOR FUNCTION

A family of proteins called complexins was discovered that compete with alpha-SNAP, but not synaptotagmin, for SNAP receptor binding. Complexins I and II are highly homologous hydrophilic proteins that are tightly conserved, with 100% identity among mouse, rat, and human complexin II. They are enriched in neurons where they colocalize with syntaxin and SNAP-25; in addition, complexin II is expressed ubiquitously at low levels. Complexins bind weakly to syntaxin alone and not at all to synaptobrevin and SNAP-25, but strongly to the SNAP receptor-core complex composed of these three molecules. They compete with alpha-SNAP for binding to the core complex but not with other interacting molecules, including synaptotagmin I, suggesting that the complexins regulate the sequential interactions of alpha-SNAP and synaptotagmins with the SNAP receptor during exocytosis.

Neurexins: Three genes and 1001 products

The human brain has approximately 10(12) neurons, three orders of magnitude more than there are basepairs in the human genome. Each neuron is connected to other neurons by thousands of synapses, creating a dense network of communicating neurons. Cell-recognition events between neurons at, and outside of synapses, are likely to guide the development and maintenance of the complex network formed by neurons. However, little is known about which proteins are important for neuronal cell recognition. Neurexins, a family of polymorphic cell-surface proteins, might mediate some of these cell recognition events. Thousands of neurexin isoforms are generated from three genes by usage of alternative promoters and alternative splicing. These isoforms are displayed on the neuronal cell surface, with different classes of neurons expressing distinct combinations of isoforms. Neurexins probably have a multitude of ligands, some of which interact only with subsets of neurexin isoforms. This review describes the properties of the neurexin protein family and their potential roles in neuronal cell adhesion and intercellular signaling.

SV2A and SV2B Function as Redundant Ca2+ Regulators in Neurotransmitter Release

SV2 proteins are abundant synaptic vesicle proteins expressed in two major (SV2A and SV2B) and one minor isoform (SV2C) that resemble transporter proteins. We now show that SV2B knockout mice are phenotypically normal while SV2A- and SV2A/SV2B double knockout mice exhibit severe seizures and die postnatally. In electrophysiological recordings from cultured hippocampal neurons, SV2A- or SV2B-deficient cells exhibited no detectable abnormalities. Neurons lacking both SV2 isoforms, however, experienced sustained increases in Ca2+-dependent synaptic transmission when two or more action potentials were triggered in succession. These increases could be reversed by EGTA-AM. Our data suggest that without SV2 proteins, presynaptic Ca2+ accumulation during consecutive action potentials causes abnormal increases in neurotransmitter release that destabilize synaptic circuits and induce epilepsy.

The Making of Neurexins

Abstract: Neurexins are neuronal cell‐surface proteins with up to thousands of isoforms. These isoforms are generated by alternative splicing of transcripts from six promoters in three genes. The structure of neurexins resembles cell‐surface receptors with a modular architecture suggestive of a sequential assembly during evolution. Neurexins probably perform multiple functions in the brain. They participate in intercellular junctions in which β‐neurexins tightly bind to a second class of neuronal cell‐surface receptors called neuroligins. Intracellularly, the neurexin/neuroligin junction is bound by CASK on the neurexin side and PSD95 on the neuroligin side. CASK and PSD95 are homologous membrane‐associated guanylate kinases that bind to the neurexin/neuroligin junction via PDZ domains, creating an asymmetric junction (neurexin/neuroligin) with similar intracellular binding partners. In addition to a function as cell‐adhesion molecules, neurexins may also serve as a signalling receptor, because a class of ligands for α‐neurexins called neurexophilins is similar to peptide hormones. Finally, at least one neurexin isoform, neurexin Iα, represents a high‐affinity receptor for α‐latrotoxin, which is a potent excitatory neurotoxin. Thus, neurexins constitute a large family of neuronal receptors that may be involved in multiple interactive functions between neurons.

Synaptic Cell Adhesion

Chemical synapses are asymmetric intercellular junctions that mediate synaptic transmission. Synaptic junctions are organized by trans-synaptic cell adhesion molecules bridging the synaptic cleft. Synaptic cell adhesion molecules not only connect pre- and postsynaptic compartments, but also mediate trans-synaptic recognition and signaling processes that are essential for the establishment, specification, and plasticity of synapses. A growing number of synaptic cell adhesion molecules that include neurexins and neuroligins, Ig-domain proteins such as SynCAMs, receptor phosphotyrosine kinases and phosphatases, and several leucine-rich repeat proteins have been identified. These synaptic cell adhesion molecules use characteristic extracellular domains to perform complementary roles in organizing synaptic junctions that are only now being revealed. The importance of synaptic cell adhesion molecules for brain function is highlighted by recent findings implicating several such molecules, notably neurexins and neuroligins, in schizophrenia and autism.

Extracellular Domains of α-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, 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.

Neurexophilin Binding to α-Neurexins A SINGLE LNS DOMAIN FUNCTIONS AS AN INDEPENDENTLY FOLDING LIGAND-BINDING UNIT

α-Neurexins (Iα, IIα, and IIIα) are receptor-like proteins expressed in hundreds of isoforms on the neuronal cell surface. The extracellular domains of α-neurexins are composed of six LNS repeats, named after homologous sequences in theLaminin A G domain, Neurexins, andSex hormone-binding globulin, with three interspersed epidermal growth factor-like domains. Purification of neurexin Iα revealed that it is tightly complexed to a secreted glycoprotein called neurexophilin 1. Neurexophilin 1 is a member of a family of at least four genes and resembles a neuropeptide, suggesting a function as an endogenous ligand for α-neurexins. We have now used recombinant proteins and knockout mice to investigate which isoforms and domains of different neurexins and neurexophilins interact with each other. We show that neurexophilins 1 and 3 but not 4 (neurexophilin 2 is not expressed in rodents) bind to a single individual LNS domain, the second overall LNS domain in all three α-neurexins. Although this domain is alternatively spliced, all splice variants bind, suggesting that alternative splicing does not regulate binding. Using homologous recombination to disrupt the neurexophilin 1 gene, we generated mutant mice that do not express detectable neurexophilin 1 mRNA. Mice lacking neurexophilin 1 are viable with no obvious morbidity or mortality. However, homozygous mutant mice exhibit male sterility, probably because homologous recombination resulted in the co-insertion into the neurexophilin gene of herpes simplex virus thymidine kinase, which is known to cause male sterility. In the neurexophilin 1 knockout mice, neurexin Iα is complexed with neurexophilin 3 but not neurexophilin 4, suggesting that neurexophilin 1 is redundant with neurexophilin 3 and that neurexophilins 1 and 3 but not 4 bind to neurexins. This hypothesis was confirmed using expression experiments. Our data reveal that the six LNS and three epidermal growth factor domains of neurexins are independently folding ligand-binding domains that may interact with distinct targets. The results support the notion that neurexophilins represent a family of extracellular signaling molecules that interact with multiple receptors including all three α-neurexins.