© BioMed Central Ltd 2013
Published: 30 September 2013
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© BioMed Central Ltd 2013
Published: 30 September 2013
The neurexin family of cell adhesion proteins consists of three members in vertebrates and has homologs in several invertebrate species. In mammals, each neurexin gene encodes an α-neurexin in which the extracellular portion is long, and a β-neurexin in which the extracellular portion is short. As a result of alternative splicing, both major isoforms can be transcribed in many variants, contributing to distinct structural domains and variability. Neurexins act predominantly at the presynaptic terminal in neurons and play essential roles in neurotransmission and differentiation of synapses. Some of these functions require the formation of trans-synaptic complexes with postsynaptic proteins such as neuroligins, LRRTM proteins or cerebellin. In addition, rare mutations and copy-number variations of human neurexin genes have been linked to autism and schizophrenia, indicating that impairments of synaptic function sustained by neurexins and their binding partners may be relevant to the pathomechanism of these debilitating diseases.
Neurexins are transmembrane proteins that function primarily at the cell surface of neurons [1–3]. Neurexin variants are essential for Ca2+-dependent transmission at diverse types of excitatory and inhibitory synapses from the central and peripheral nervous system [4–8], and play additional roles in their formation and differentiation [9–14]. One of the most intensely studied features of neurexins is their ability to bind extracellularly to proteins of other synaptically connected neurons. The first and prototypical interaction partner discovered was postsynaptic neuroligin [15, 16]. However, a number of additional molecules associated with the synaptic cleft have been identified as binding partners, including neurexophilin [17–19], dystroglycan , LRRTM proteins [21, 22] and cerebellin [23, 24]. Neurexin isoforms bound to neuroligins, for example, can form trans-synaptic complexes at excitatory and inhibitory synapses that are involved in synapse specification, establishment, maturation and plasticity. Important from a medical point of view, impairments caused by mutations in the neurexin-neuroligin complex  lead to an imbalance of excitatory to inhibitory activity in neuronal circuits, which has been implicated in the pathomechanisms of autism spectrum disorders  and schizophrenia .
Comparison of human and murine neurexin genes
In addition to the two major α-neurexin and β-neurexin variants, vertebrate neurexin genes contain five conserved alternative splice sites in the α-neurexin coding sequence (SS#1 to SS#5) and two in β-neurexin (SS#4 and SS#5) that by permutation allow for about 3,908 possible neurexin variants. For example, the SS#1 of neurexin 1 consists of four mini-exons (2, 3, 4 and 5; Figure 2a) that can be inserted in 24 permutations . In addition, some of the splice events may lead to soluble isoforms lacking the membrane-bound carboxy-terminal part of the protein . Alternative splicing is a hallmark of all neurexin genes [1, 30–32, 34, 35], and has received considerable attention because binding to postsynaptic partners was found to depend on splicing events, at least partially. Some alternatively spliced exons in neurexins are more conserved than exons that are constitutively expressed , supporting the idea that long introns with weak splice sites and rare splice events result in higher conservation of the entire inserted DNA, often indicating functionally important protein sequences . In particular, the inserted protein sequences at SS#2 and SS#4 are highly conserved and all known α-neurexin interacting proteins bind to the domains where SS#2 and SS#4 are located (see below).
α-Neurexins contain six LNS (laminin-neurexin-sex hormone binding globulin) domains with three epidermal growth factor-like (EGF) domains interspersed (Figure 1, upper panel). The shorter β-neurexins are identical to the carboxyl terminus of α-neurexins starting from αLNS6 but have a unique amino-terminal stretch of 37 histidine-rich residues (Figure 1, lower panel). All neurexins are N-glycosylated and the sequence between αLNS6 and the transmembrane region is characterized by O-glycosylation . The cytosolic domains have a potential endoplasmatic retention signal, a cytoskeleton integrating protein 4.1, and a PDZ-binding motif that is required for trafficking of neurexins .
Interaction partners of neurexins
Binding partners specific for α-Nrxn:
Shared by α-Nrxn and β-Nrxn:
αLNS2, αLNS6, βLNS
The binding of some of these proteins to αLNS2 or αLNS6 can be modified by alternative splicing that occurs in a hypervariable region in the vicinity of the Ca2+-binding site (Figure 4). While neurexophilin binds αLNS2 independently of alternative splicing , dystroglycan and LRRTM require a splice insert-free LNS domain [20, 48] and cerebellin binds presumably directly to the insert in SS#4 of αLNS6/βLNS [23, 24]. Splice insert dependency of neurexin/neuroligin complex formation is more complicated because neuroligins also have two splice sites, termed A and B. While all neurexins share the five splice sites, the neuroligins differ: neuroligin 1 contains splice sites A and B , neuroligin 2 and neuroligin 3 have only splice site A  and neuroligin 4 is not alternatively spliced . Co-crystal data exist for the binding interface of neurexin 1αLNS6/βLNS without insert in SS#4 to neuroligin 1 and 4 [40–42], and neuroligin 3 is predicted to form similar complexes [40–42]. In contrast, the proposed binding interface of neuroligin 2 to αLNS6 differs structurally with a G500Q change from neuroligin 1 to 2, which raises the possibility that neuroligin 2 uses an alternative binding epitope [42, 51].
Affinity purification of neuroligin with the extracellular domain of β-neurexin originally suggested that only β-neurexin without an insert in SS#4 (−SS#4) binds neuroligin 1 . This apparent splice insert dependency of neurexin binding to neuroligin then led to the generalized idea of a splice code that classifies specific pairings in the neurexin/neuroligin complex (for neurexins: ±SS#4; for neuroligins: ±A, ±B) according to specific roles at excitatory and inhibitory synapses [13, 15, 16, 52, 53]. Subsequently, it has been shown that also α-neurexins, even with insert in SS#4, bind to neuroligin 1(−B)  and neuroligins 2 and 3, albeit with lower affinity than β-neurexins [54, 55]. Biochemical experiments have now established that, with one exception discussed below, any neurexin can bind any neuroligin [54, 56] and that neurexins + SS#4 yield considerable amounts of protein complexes with neuroligins if only the incubation time is long enough . This behavior can be explained by recent crystal structures of β-neurexin + SS#4 that show a remarkable displacement of the inserts at SS#4 [54, 57].
Finally, the conservation of the splice insert sequence in SS#4 is in accordance with the conformational switch  that (i) increases affinity for Ca2+ binding by positioning an additional Ca2+ coordinating residue , and (ii) requires a match to the sequence of β10 that is replaced by the SS#4 insert. However, the reason for the conservation may be different: since the insert sequence at SS#4 itself binds exclusively to cerebellin [23, 24] and cerebellin constitutes an ancestral protein, it can be hypothesized that the interaction of neurexins + SS#4 with cerebellin may be responsible for the evolutionary pressure on the splice insert conservation, rather than the interaction of neurexin with neuroligins that is reduced by the alternative splicing at SS#4.
The discovery of neurexins as a receptor for α-latrotoxin , a neurotoxin that causes massive neurotransmitter vesicle release from terminals, has argued in favor of a presynaptic localization. This location has been confirmed by the finding of a prominent presynaptic release phenotype in α-neurexin knockout (KO) mice [6, 65]. Nevertheless, additional postsynaptic defects and localization of transgenically expressed variants may indicate that a small population of postsynaptic neurexins exists [5, 66]. Due to the lack of isoform-specific antibodies for high-resolution morphology, endogenous neurexin proteins have not been mapped systematically to subpopulations of neurons and/or synapses by immunolabeling. Localization patterns have been obtained mostly from mRNA studies [1, 67–69] and by subcellular fractionation [65, 69]. In situ hybridization data reveal that neurexins 1/2 and neurexin 3 may be expressed initially in distinct cell populations , whereas in the mature central nervous system the α-neurexin and β-neurexin isoforms are distributed in a partially overlapping, partially differential pattern [1, 67]. In particular, the three β-isoforms show a more unique distribution, in which, for example, neurexin 1β is restricted to cortical layers 2 and 3, thalamus and parts of the hippocampus [1, 67]. Using the regulation by alternative splicing, juvenile neurons in chicken express insert-negative neurexin variants . With progressing neuronal and synaptic development, the number of insert-positive variants increases . Since insert-negative neurexins have the highest potential to bind to known interaction partners (Table 2), these data suggest that maturation is accompanied by reduced binding capacities for neuroligins, LRRTM and dystroglycan. Instead, insert-positive variants at SS#4 favor the binding to cerebellin [24, 70]. Interestingly, in the cerebellum where the cerebellin/GluRδ2 complex is abundantly expressed , much higher levels of neurexins lacking all inserts have been found compared with the rest of the brain . These results are consistent with an activity-controlled expression of neurexin + SS#4 and, thereby, a regulated interaction with cerebellin/GluRδ2. Supporting this idea of an activity-dependent ‘splice-code’ that changes the profile of neurexins for binding partners, the generation of different splice variants was shown to be coupled to synaptic activity via the Ca2+/calmodulin-dependent kinase pathway and involves RNA-binding protein SAM68 [71, 72]. For example, it has been shown that the inclusion of a splice insert at SS#3 in neurexin 2 depends on depolarization and Ca2+ influx . Furthermore, the expression of + SS#3/+SS#4-containing variants follows closely the activity rhythm in autonomous oscillating cells of the suprachiasmatic nucleus , and + SS#4 expression is reduced in α-neurexin isoforms after applying a learning and memory paradigm . Unfortunately, expression results from different species and different experimental paradigms are sometimes contradictory [68, 75], suggesting that more research is needed to establish the regulated variability of splice variants and to determine which variants are actually realized under which conditions.
KO studies in mice established the importance of α-neurexins as essential because they are required for Ca2+-dependent exocytosis at neuronal synapses [4–7, 11, 37]. For β-neurexins, in contrast, no results from KO studies have been published yet.
The deletion of two or three α-neurexin isoforms resulted in severely impaired spontaneous and evoked neurotransmitter release at excitatory and inhibitory synapses in brainstem and neocortex [5, 6]. Even the deletion of a single isoform, neurexin 1α, resulted in a reduction of spontaneous release from excitatory synapses in hippocampal pyramidal neurons , emphasizing the importance of every neurexin for synaptic homeostasis . In addition, the loss of one or more α-neurexin isoforms reduced Ca2+ currents and caused unresponsiveness to specific blockers , suggesting that an impaired Ca2+-channel function is part of the process. It remains unclear, however, how the deletion of α-neurexins uncouples N-type and P/Q-type Ca2+ channels from the neurotransmitter release machinery [37, 76]. A direct interaction of the extracellular domains of α-neurexins and the pore-forming subunits of the Ca2+ channels appears unlikely as neurexins are not required for normal Ca2+ currents per se, and the surface expression and number of Ca2+ channels were also unchanged in KO neurons .
Any mechanistic explanation of the effect of α-neurexins on Ca2+ channels also needs to consider the observation that the carboxyl terminus binds to PDZ-domain proteins such as CASK  and Mints . Both, CASK and Mints interact with the β-subunit of N-type Ca2+ channels, while Mints also interact with P/Q-type Ca2+ channels . This complex, in turn, could be coupled to synaptic vesicles by the interaction of α-neurexin with synaptotagmin  and/or Mints to Munc18 . Although this molecular pathway provides a possible link between neurexins, Ca2+ channels and the release machinery, the comparatively moderate effect of genetic deletion of CASK and Mint on synaptic transmission [81, 82] does not support a crucial contribution of these molecules. More work needs to be done to integrate α-neurexins into the current view of Ca2+-channel tethering or positioning by synaptotagmins, RIMs, liprins and CAST/ERC/ELKS, which also appears independent of Mint or CASK . In addition, recent advances on the function of Ca2+-channel α2δ subunits as important modulators of synaptic transmission  suggest alternative routes to influence Ca2+-channel activity and mobility . This includes the possibility, albeit speculative, of direct or indirect interference with extracellular domains of α-neurexins that could explain why β-neurexins do not rescue the α-neurexin KO phenotype .
Studies using co-cultures between primary neurons and non-neuronal cells transfected with neurexins or neuroligins have uncovered their ability to stimulate the de novo formation of functional synapses by clustering presynaptic or postsynaptic proteins [12, 14]. Surface expression of neurexins induces clusters of PSD95 and gephyrin at excitatory and inhibitory postsynapses of contacting dendrites [10, 13]. Expression of neuroligins, in turn, induces clustering of presynaptic marker proteins on contacting axons  and different neuroligin isoforms appear to trigger differentiation of excitatory versus inhibitory terminals [9, 53, 86]. Interestingly, this strong synaptogenic effect of overexpressed neurexins and neuroligins observed in these cell culture assays has not been matched by prominently reduced numbers of excitatory and inhibitory synapses in loss-of-function mouse models [6, 11, 87, 88]. For example, the multiple KO of α-neurexins leads to a moderate reduction of symmetric, presumably inhibitory, synapses and leaves excitatory synapse density unscathed that at the same time displays a severely impaired neurotransmitter release [5, 6, 11]. For neuroligins that have served as the prototypical synaptogenic molecule in vitro, there are no visible effects on synapse numbers in multiple or single KO mice [87, 88]. Overexpression versus deletion strategies cannot be the sole reason for these differences because lentiviral-mediated expression of neurexins has failed to elevate synapse numbers  and transgenic overexpression of neurexin in mice does not increase mini frequencies above wild-type levels . Since RNAi-mediated knock-down of neurexins, in turn, can lower the numbers of excitatory and inhibitory synapses in cultured neurons , it is clear that more research is needed to define the role of the neurexin/neuroligin complex in synapse formation.
Synapse formation assays have also been used to decipher the putative splice code for preferred binding between neurexins and neuroligins, and to other partners. Most studies using neurexins have been performed with overexpressed β-neurexin variants that represent the best binding partner for all neuroligin isoforms regardless of alternative splice inserts in either protein [15, 46, 54, 89], as also discussed above (Structural features and the splice-code hypothesis). Accordingly, β-neurexin instantly reaches the maximal synaptogenetic effect , and optimizing binding to neuroligin by deglycosylation or removal of the B insert does not significantly increase clustering of synaptic proteins . In contrast, only a few cell culture studies have been performed with α-neurexins [12, 75, 89]. These were limited to α-neurexin + SS#4 variants that bind reliably only to neuroligins without insert B  but do not reach the complex forming capacity of β-neurexin + SS#4 to neuroligin 1-B . Since neuroligin 1-B was shown to cluster and bind α-neurexins, it is not surprising that most synaptogenic effects of overexpressed α-neurexins have been observed at inhibitory synapses [12, 75]. This is because inhibitory synapses contain mostly neuroligin 2 [12, 91], which has similar biochemical binding properties to neuroligin 1-B . As α-neurexins look more diffusibly distributed on the axonal surface  but are clustered by neuroligin 2/neuroligin 1-B , it can be hypothesized that α-neurexins are the more potent variants for dynamic adaptations that may be particularly relevant for inhibitory synapses.
The outcome of the autism genome-wide association study projects surprisingly revealed only weak correlations for ASD to common genetic variants, but identified genes with rare single nucleotide polymorphisms (SNPs) or copy number variations that have a considerable impact . Such rare mutations have been found in the α-neurexin coding region of nrxn1[97–99], nrxn3 and the signal peptide of β-neurexins . An excess of mutations in these genes is found in patients with ASD [27, 102], schizophrenia [103, 104] and substance abuse and impulsive behavior . Historically, the neuroligin 3 single mutation R451C has been the first SNP of a protein gene associated with ASD  but other molecules such as nrxn1, nrxn3, nlgn3, nlgn4, shank2, shank3 and genomic regions at 1q21.1 and 16p11.2 are now accepted as bona fide ASD risk loci . Some of the single site mutations found in patients have been introduced in mouse models, such as neuroligin 3 R451C [93, 107] and neuroligin 4 R704C . Interestingly, analysis of mutations in mice also demonstrates converging phenotypes of different risk loci . As might be expected, the mouse models recapitulate some but not all aspects of the diseases: for example, repetitive grooming as stereotype behavior in neurexin 1α KO, but not the social disabilities . When tested in cell culture or biochemical assays, most mutations cause a complete loss of expression or largely reduced trafficking of the defective protein to synapses [109–111]. These observations highlight the central role of neurexins and neuroligins at the synapse and have prompted new research into the protein interaction network across the synaptic cleft that may provide insights into higher cognitive functions at the molecular level.
Invertebrate models have already proven excellent systems to study multiple mutations in neurexin and neuroligin genes that are impossible to obtain in mice  or to follow effects on synaptic cell adhesion by imaging in live animals . Due to the sequence conservation of neurexin and neuroligin throughout the animal kingdom, identification of mutations and binding partners in one species facilitates the finding of orthologs, and allows the description of a canonical protein network. For example, binding to neuroligin is blocked in all species investigated by a synthetic aspartate to alanine mutation in the neurexin αLNS6 domain that corresponds to the essential Ca2+-binding residue D137 of β-neurexin [41, 46, 114]. In addition, mutations Y189H, L319SSM and L849Q, which inhibit neuroligin function in Drosophila, can be readily localized on the mammalian neuroligin crystal structure  and are likely to destabilize the fold of the extracellular (Y85, L235) or the transmembrane domain (L712). This could explain the reduced level of neuroligin reaching the postsynapse , similar to other ASD mutations in mammals [97–101]. Finally, the fact that a synthetic D356R mutation in Drosophila neuroligin 1 rescues the KO phenotype  suggests neurexin-independent functions of neuroligin, as the corresponding mutation D271R in rat neuroligin 1 was found to inhibit neurexin binding .
Unlike these structural similarities, any functional comparisons need to keep in mind that mostly presynaptic α-neurexins interact with postsynaptic neuroligin in vertebrates, as discussed above. In C. elegans, in contrast, neurexin and also neuroligin are expressed presynaptically and postsynaptically [33, 113] and retrograde trans-synaptic signaling from the postsynapse to the presynapse in the worm is modulated by an interaction in trans and cis simultaneously . It is also important to realize that while C. elegans expresses a β-neurexin with a yet unresolved function , flies rely on a single α-neurexin alone [35, 117]. It is therefore not surprising that the functional phenotypes in vertebrate and invertebrate neurexin mutant animals share similarities but can also differ considerably (reviewed in detail in ). For example, analyses of Drosophila loss-of-function mutants of α-neurexins have described effects on synapse ultrastructure [35, 117] that are absent from the mouse KOs [6, 11], whereas both model systems suffer from impaired neurotransmission. These limitations notwithstanding, the recent finding of a triple complex of α-neurexin/syd-1/liprin-α at the active zone of neuromuscular junctions in flies , for another example, will encourage the search for a similar complex in mammals that might help to solve the question why and how α-neurexins couple Ca2+ channels to release sites.
In addition to synapses of the central nervous system, neurexin isoforms have been reported to act in smooth muscle cells [116, 120, 121], pancreatic β-islet cells [122–124], melanotrophs of the hypophysis  and endothelial cells . For example, α-neurexins and neuroligins modulate Ca2+-triggered exocytosis from melanotrophs in the hypophysis  and from insulin-secreting β cells in the endocrine pancreas’s islets of Langerhans . In β cells, the cytosolic domain of α-neurexins is essential for insulin granule docking through an indirect interaction with granuphilin, which lines vesicles to the cell surface membrane that are ready for fusion . In this process, the number of release-ready vesicles is homeostatically regulated by neurexin or granuphilin, while the reduction of either protein increases glucose-sensitive fusion. Interestingly, granuphilin is selectively expressed in β cells and melanotrophs, which might explain why α-neurexins function in both cell types. Since the granuphilin homolog Rab3A plays a similar role in the docking of synaptic vesicles in neurons, canonical protein complexes consisting of α-neurexins-CASK-Mint1/2-Rab3a/Granuphilin-Munc18 have been suggested .
The neurexin/neuroligin pair most likely represents one of the best characterized protein complexes at the neuronal synapse. Its modulation due to alternative splicing and isoform pairings is remarkable and its roles in synaptic function and differentiation are essential. However, important issues remain to be addressed.
First, it is incompletely understood if α-neurexins and β-neurexins have overlapping  or different functions at the synapse. Rescue experiments have suggested that their functions are non-redundant , but analysis of multiple β-neurexin KOs and comparative knock-down studies will be necessary to address this issue directly.
Second, the apparent preference of α-neurexins for GABAergic synapses as observed in some assays [10, 12, 13, 75] needs to be reconciled with the KO mouse phenotype that is characterized by a dramatic release impairment that affects both excitatory and inhibitory synapses [4, 6].
Third, neurexins act at the synapse but only little is known about how they are transported to the presynaptic terminal during intracellular trafficking. It has been shown that neurexin targeting requires a PDZ-binding motif interaction in mouse neurons  and a Syd-1/RhoGAP100F-dependent delivery in Drosophila. However, the characteristics of the vesicular pathways responsible and the dynamics of the transport are unclear.
Fourth, most known interacting proteins of neurexins bind to the last LNS domain of α-neurexin/the single LNS domain of β-neurexin, and only neurexophilin and dystroglycan are known to bind to αLNS2 (Table 2). It needs to be studied if the additional domains in α-neurexin simply act as spacers or if they provide additional sites for binding partners that have yet to be discovered.
Fifth, the early expression and the preference of juvenile neurons for neurexins without splice inserts [67, 68] suggest an additional role of some neurexin variants in developmental processes such as neurite growth [11, 127] that needs to be explored in more detail.
Finally, human genetic work and mouse models have linked the neurexin/neuroligin complex to synapse-related neuropsychiatric disorders such as autism and schizophrenia . It will be one of the most challenging tasks ahead of us to unravel the underlying cellular mechanisms that explain, for example, why mutations in the same molecules lead to diverse symptoms, a prerequisite to develop more causative therapeutic strategies.
Autism spectrum disorder
Epidermal growth factor-like
Laminin-neurexin-sex hormone binding globulin
Single nucleotide polymorphism.
This work is supported by Deutsche Forschungsgemeinschaft grant number SFB629, TPB11 (MM).