Interactive Fly, Drosophila

Neurexins: cloning and general biology

In the nervous system, glial cells greatly outnumber neurons but the full extent of their role in determining neural activity remains unknown. The axotactin (axo) gene of Drosophila is shown to encode a member of the neurexin protein superfamily secreted by glia and subsequently localized to axonal tracts. The axo gene was isolated in a screen for temperature-sensitive paralytic mutations. Null mutations of axo cause temperature-sensitive paralysis and a corresponding blockade of axonal conduction. At 37 degrees C., compound action potentials in mutant nerves are either lost or are reduced by more than 90%, in contrast with wild-type nerves, in which no failure is seen. Thus, the AXO protein appears to be a component of a glial-neuronal signaling mechanism that helps to determine the membrane electrical properties of target axons. It is hypothesized that an axo-dependent signal is required for the normal expression, localization, or clustering of some set of ion channels (Yuan, 1999).

The Axo protein contains several motifs, including an amino-terminal hydrophobic signal sequence followed by a consensus cleavage site, three cystein-rich epidermal growth factor repeats, five laminin G repeats, and a fibrinogen beta/gamma-like segment. Several features distinguish Axo from other members of the neurexin superfamily: a Kunitz-like domain; the presence of both a Laminin G domain (characteristic of neurexins) and a fibrinogen domain adjacent to the second EGF repeat; and the absence of a transmembrane domain, which suggests that Axo is secreted rather than membrane associated. Beginning at embryonic stage 13, axo transcripts are detected in the differentiating nervous system in a segmentally repeated pattern of discrete spots directly overlaying the longitudinal axonal tracts. This pattern corresponds with the location of a subset of glial cells, including longitudinal glia and segmental boundary cells. Axo protein distribution differs from that of the axo transcript. The protein coincides with axonal tracts in the ventral cord, brain and parts of the peripheral nervous system. Thus, Axo appears to be secreted from glia and subsequently localized to axonal tracts (Yuan, 1999).

In Caenorhabditis elegans, vulval induction is mediated by the let-23 receptor tyrosine kinase (RTK)/ Ras signaling pathway. The precise localization of let-23 RTK at epithelial junctions is essential for the vulval induction, and requires three genes, including lin-2, -7, and -10. The mammalian homolog of lin-2 has been identified as CASK, a protein interacting with neurexin, a neuronal adhesion molecule. CASK has recently been reported to interact with syndecans and an actin-binding protein, band 4.1, at epithelial and synaptic junctions, and to play central roles in the formation of cell-cell junctions. The product of C. elegans lin-7 directly interacts with let-23 RTK and localizes let-23 RTK at epithelial junctions. Three rat homologs of lin-7 are ubiquitously expressed in various tissues. These homologs accumulate at the junctional complex region in cultured Madin-Darby canine kidney cells, and are also localized at the synaptic junctions in neurons. The mammalian homologs of lin-7 may be implicated in the formation of cell-cell junctions (Irie, 1999).

Neurexins are highly variable transmembrane proteins hypothesized to be nerve terminal-specific cell adhesion molecules. As a test of the hypothesis that neurexin is restricted to the nerve terminal, neurexins were examined in the electric organ of the elasmobranch electric fish. Specific antibodies generated against the intracellular domain of electric fish neurexin were used in immunocytochemical and Western blot analyses of the electromotor neurons that innervate the electric organ. Neurexin is not expressed at electric organ nerve terminals, as would be expected by the neurexin hypothesis. Instead, neurexin is expressed by electromotor neurons and on myelinated axons. This neurexin has a molecular weight of 140 kDa, consistent with an alpha-neurexin. In addition, perineurial cells of the electromotor nerve also express a neurexin. These cells surround bundles of axons to form a diffusion barrier and are thought to be a special form of fibroblast. The results of the study argue against a universal role for neurexins as nerve terminal-specific proteins but suggest that neurexins are involved in axon-Schwann cell and perineurial cell interactions (Russell, 1997).

Neurexins, a family of cell surface proteins specific to brain, are transcribed from two promoters in three genes, resulting in three alpha- and three beta-neurexins. There are differential but overlapping distributions of neurexin isoforms in different classes of neurons. Alpha-neurexins are alternatively spliced at five canonical positions; beta-neurexins at two. Characterization of many independent bovine neurexin I alpha cDNAs suggests that different splice sites are used independently. This creates the potential to express more than 1000 distinct neurexin proteins in brain. The splicing pattern is conserved in rat and cow. Thus, in addition to somatic gene rearrangement (immunoglobulins and T cell receptors) and large gene families (odorant receptors), alternative splicing potentially represents a third mechanism for creating a large number of cell surface receptors that are expressed by specific subsets of cells (Ullrich, 1995).

Synaptotagmin I and neurexin I mRNAs, coding for proteins involved in neurotransmitter secretion, become detectable in primary sympathetic ganglia shortly after initial induction of the noradrenergic transmitter phenotype. To test whether the induction of these more general neuronal genes is mediated by signals known to initiate noradrenergic differentiation in a neuronal subpopulation, their expression was examined in noradrenergic neurons induced by ectopic overexpression of growth and transcription factors. Overexpression of BMP4 or Phox2a in vivo results in synaptotagmin I and neurexin I expression in ectopically located noradrenergic cells. In vitro, BMP4 initiates synaptotagmin I and neurexin I expression in addition to tyrosine hydroxylase induction. Thus, the induction of synaptotagmin I and neurexin I, which are expressed in a large number of different neuron populations, can be accomplished by growth and transcription factors available only to a subset of neurons. These findings suggest that the initial expression of proteins involved in neurotransmitter secretion is regulated by different signals in different neuron populations (Patzke, 2001).

Expression of the major isoforms of three neurexin genes was analyzed in the developing embryonic nervous system of mice. Transcripts of all three genes are detected as early as embryonic day 10 (E10) and increased with maturation of the nervous system. RNAs of the major neurexin isoforms (alpha and beta) are found throughout the central nervous system exclusively in postmitotic neurons and at least 1 d before synapses are formed. In contrast, in the PNS, the alpha- and beta-isoforms display differential expression patterns. Neurexin III mRNA shows a more restricted regional expression than neurexin I and II transcripts. These expression profiles are consistent with the hypothesis that the neurexins have a function in early neuronal differentiation and axogenesis (Puschel, 1995).

To explore the possibility that overproduction of neuronal acetylcholinesterase (AChE) confers changes in both cholinergic and morphogenic intercellular interactions, developmental responses to neuronal AChE overexpression were studied in motoneurons and neuromuscular junctions of AChE-transgenic mice. Perikarya of spinal cord motoneurons are consistently enlarged from embryonic through adult stages in AChE-transgenic mice. Atypical motoneuron development is accompanied by premature enhancement in the embryonic spinal cord expression of choline acetyltransferase mRNA, encoding the acetylcholine-synthesizing enzyme choline acetyltransferase. In contrast, the mRNA encoding for neurexin-Ibeta, the heterophilic ligand of the AChE-homologous neuronal cell surface protein neuroligin, is drastically lower in embryonic transgenic spinal cord than in controls. Postnatal cessation of these dual transcriptional responses is followed by late-onset deterioration in neuromotor performance that is associated with gross aberrations in neuromuscular ultrastructure and with pronounced amyotrophy. These findings demonstrate embryonic feedback mechanisms to neuronal AChE overexpression that are attributable to both cholinergic and cell-cell interaction pathways, suggesting that embryonic neurexin Ibeta expression is concerted in vivo with AChE levels and indicating that postnatal changes in neuronal AChE-associated proteins may be involved in late-onset neuromotor pathologies (Andres, 1997).

In yeast two-hybrid screens for intracellular molecules interacting with different neurexins, a single interacting protein called CASK has been identified. CASK is composed of an N-terminal Ca2+, calmodulin-dependent protein kinase sequence and a C-terminal region that is similar to the intercellular junction proteins dlg-A, PSD95/SAP90, SAP97, Z01, and Z02 (Drosophila homolog: Discs large). The C-terminal region also contains DHR-, SH3-, and guanylate kinase domains. CASK is enriched in the synaptic plasma membranes of the brain, but is also detectable at low levels in all tissues tested. The cytoplasmic domains of all three neurexins bind CASK in a salt-labile interaction. In neurexin I, this interaction is dependent on the C-terminal three residues. Thus, CASK is a membrane-associated protein that combines domains found in Ca(2+)-activated protein kinases and in proteins specific for intercellular junctions, suggesting that it may be a signaling molecule operating at the plasma membrane, possibly in conjunction with neurexins (Hata, 1996).

A novel multivalent PDZ domain protein, CIPP (for channel-interacting PDZ domain protein) is expressed exclusively in brain and kidney. Within the brain, the highest CIPP mRNA levels are found in neurons of the cerebellum, inferior colliculus, vestibular nucleus, facial nucleus, and thalamus. Furthermore, the inward rectifier K+ (Kir) channel, Kir4.1 (also called Kir1.2), has been identified as a cellular CIPP ligand. Among several other Kir channels tested, only the closely related Kir4.2 (or Kir1.3) also interacts with CIPP. In addition, specific PDZ domains within CIPP associate selectively with the C-termini of N-methyl-D-aspartate subtypes of glutamate receptors, as well as neurexins and neuroligins, cell surface molecules enriched in synaptic membranes. Thus, CIPP may serve as a scaffold that brings structurally diverse but functionally connected proteins into close proximity at the synapse. The functional consequences of CIPP expression on Kir4.1 channels were studied using whole-cell voltage clamp techniques in Kir4.1 transfected COS-7 cells. On average, Kir4.1 current densities are doubled by cotransfection with CIPP (Kurschner, 1998).

Synaptotagmin, a major intrinsic membrane protein of synaptic vesicles that binds Ca2+, was purified from bovine brain and immobilized onto Sepharose 4B. Affinity chromatography of brain membrane proteins on immobilized synaptotagmin reveals binding of alpha- and beta-neurexins to synaptotagmin in a Ca(2+)-independent manner. Synaptotagmin specifically interacts with the cytoplasmic domains of neurexins but not of control proteins. This interaction is dependent on a highly conserved, 40 amino acid sequence that makes up most of the cytoplasmic tails of the neurexins. These data suggest a direct interaction between the cytoplasmic domains of a plasma membrane protein (the neurexins) and a protein specific for a subcellular organelle (synaptotagmin). Such an interaction could have an important role in the docking and targeting of synaptic vesicles in the nerve terminal (Hata, 1993).

The interaction of the synaptic vesicle protein, synaptotagmin, and the presynaptic alpha-latrotoxin receptor, a neurexin, is thought to be involved in docking of synaptic vesicles at active sites or the modulation of neurotransmitter release. Pieces of synaptotagmin containing the carboxyl terminus are capable of purifying neurexins from solubilized brain homogenates. Pieces as small as a synthesized peptide corresponding to the COOH-terminal 34 amino acids are capable of enriching neurexins 100-fold. The binding of neurexins to synaptotagmin is calcium-independent and of moderate affinity. This COOH-terminal segment of synaptotagmin is conserved in all species characterized to date. Reflective of this, a synthetic peptide corresponding to the carboxyl terminus of Drosophila synaptotagmin is capable of purification of rat neurexins, suggesting the possibility that this interaction may also exist in Drosophila. It is proposed that the carboxyl terminus of synaptotagmin binds to the carboxyl terminus of the neurexins and that this interaction may mediate docking of synaptic vesicles or modulation of neurotransmitter release (Perin, 1994).

Neurexin functions at nodes

Ranvier nodes are flanked by paranodal regions where oligodendrocytes or Schwann cells interact closely with axons. Paranodes play a critical role in the physiological properties of myelinated nerve fibers. Paranodin, a prominent 180 kDa transmembrane neuronal glycoprotein, was purified and cloned from adult rat brain, and found to be highly concentrated in axonal membranes at their junction with myelinating glial cells, in paranodes of central and peripheral nerve fibers. The large extracellular domain of paranodin is related to neurexins, and its short intracellular tail binds protein 4.1, a cytoskeleton-anchoring protein. Paranodin may be a critical component of the macromolecular complex involved in the tight interactions between axons and myelinating glial cells characteristic of the paranodal region (Menegoz, 1997).

Mice incapable of synthesizing the abundant galactolipids of myelin exhibit disrupted paranodal axo-glial interactions in the central and peripheral nervous systems. Using these mutants, the role that axo-glial interactions play in the establishment of axonal protein distribution in the region of the node of Ranvier has been analyzed. Whereas the clustering of the nodal proteins, sodium channels, ankyrin(G), and neurofascin is only slightly affected, the distribution of potassium channels and paranodin, proteins that are normally concentrated in the regions juxtaposed to the node, is dramatically altered. The potassium channels, which are normally concentrated in the paranode/juxtaparanode, are not restricted to this region but are detected throughout the internode in the galactolipid-deficient mice. Paranodin/contactin-associated protein (Caspr), a paranodal protein that is a potential neuronal mediator of axon-myelin binding, is not concentrated in the paranodal regions but is diffusely distributed along the internodal regions. Collectively, these findings suggest that the myelin galactolipids are essential for the proper formation of axo-glial interactions and demonstrate that a disruption in these interactions results in profound abnormalities in the molecular organization of the paranodal axolemma (Dupree, 1999).

The axons of myelinated nerves in the adult nervous system are subdivided into several distinct structural and functional domains that each differ in their molecular composition. The generation of these specialized subcellular structures is essential for the efficient and rapid propagation of action potential via saltatory conduction. Several distinct subdomains can be found in the axonal membrane, the nodes of Ranvier, the paranodes, and the juxtaparanodes. The nodes of Ranvier are characterized by a high concentration of voltage-gated Na+ channels, which enable the regeneration of the action potential. Several other proteins are localized to the axolemma at the nodes, including Na+/K+ ATPases, the spectrin-binding protein ankyrin, and the cell adhesion proteins NrCAM and Neurofascin. Junctions that are formed between the axon and the myelinating cell (called the paranodal junctions) border the nodes of Ranvier. In this region, the compact myelin lamellae open up into a chain of cytoplasmic loops that form a series of septate-like junctions with the axon. The paranodal region is thought to serve several functions: to anchor the myelin to the axon; to form a partial barrier that isolates the periaxonal space under the myelin from the electrical activity at the nodes, and to physically demarcate boundaries that limit the lateral diffusion of membrane components, thereby confining them to the node. A third specialized region in myelinated axons is defined as the juxtaparanode. This region, often referred to as the paranodal main segment, is located in a short zone just beyond the innermost paranodal junctions, separating them from the internodes that lie beneath the compact myelin sheaths. In large fibers in the PNS, this region contributes to the axon-Schwann cell network, a structure of thin axonal processes that are enclosed by protrusions of the Schwann cell cytoplasmic layer. This complex network is implicated in axonal transport and in the lysosyme-mediated degradation of transported material. Although no prominent network is present in the CNS, some invasion of the axoplasm by the inner cytoplasmic loop occurs at this site. The juxtaparanodal region is characterized by the presence of heteromultimers of the Shaker-like K+ channel alpha subunits Kv1.1 and Kv1.2, and the cytoplasmic Kvbeta2 subunit. The precise localization of voltage-activated K+ channels to this region may stabilize conduction and help to maintain the internodal resting potential (Poliak, 1999 and references therein).

Contactin-associated protein (Caspr; also known as Paranodin) is a member of the neurexin superfamily, a group of transmembrane proteins that mediate cell-cell interactions in the nervous system. It was originally identified in a complex with contactin that binds to the receptor protein tyrosine phosphatase beta (RPTPbeta) and as a major lectin-binding protein from rat brain. Caspr is concentrated at the paranode of Ranvier in the septate-like junctions that are formed between axons and the terminal loops of oligodendrocytes and myelinating Schwann cells. Both the localization of Caspr and the development of the septate junctions occur with the maturation of the myelinated fiber, suggesting that Caspr may be an essential component of the paranodal junction. The notion that Caspr plays a role in the generation and integrity of the paranodal junctions is also supported by studies in Drosophila demonstrating that the Caspr-related Neurexin IV (Nrx-IV) is an essential component of the ectodermally derived pleated septate junctions (Baumgartner, 1996 ). Nrx-IV mutants are devoid of septate junctions between glial cells, resulting in the breakdown of the blood-nerve barrier. These mutants lack the transverse septate that is characteristic of these junctions, which are structurally similar to the paranodal junctions (Baumgartner, 1996 ). Although Caspr and the related Drosophila Nrx-IV have a similar primary structure, they differ in their cytoplasmic tail. Caspr contains a proline-rich sequence, while Drosophila Nrx-IV has a shorter cytoplasmic region containing a binding site for PDZ domains (Bellen, 1998). This structural difference suggests the existence of other members of this group in vertebrates. Caspr2, a new member of this family, is a component of the juxtaparanodal region in myelinated axons (Poliak, 1999).

Rapid conduction in myelinated axons depends on the generation of specialized subcellular domains to which different sets of ion channels are localized. Caspr2, a mammalian homolog of Drosophila Nrx-IV and the closely related molecule Caspr/Paranodin demarcate distinct subdomains in myelinated axons. An alignment of human Caspr proteins with the amino acid sequence of Drosophila Nrx-IV reveals that Caspr2 is more related (34% identity) to Nrx-IV than is Caspr (29% identity). Like Nrx-IV, Caspr2 lacks the proline-glycine-tyrosine repeats that are found near the transmembrane domain of Caspr. The major difference between Caspr2 and Caspr is found in the cytoplasmic domain. While both share a protein 4.1 binding sequence at the juxtamembrane region, they diverge thereafter. The intracellular region of Caspr2 is more similar to glycophorin C (65% identity) and Nrx-IV (37% identity) than to Caspr (29% identity). Caspr2, Nrx-IV, and all the neurexins each contain a short amino acid sequence at their C terminus, which serves as a binding site for type II PDZ domains. This consensus sequence is not found in Caspr. Altogether, these sequence similarities indicate that Caspr2 is most likely the mammalian ortholog of Drosophila Nrx-IV. While contactin-associated protein (Caspr) is present at the paranodal junctions, Caspr2 is precisely colocalized with Shaker-like K+ channels in the juxtaparanodal region. Caspr2 specifically associates with Kv1.1, Kv1.2, and their Kvbeta2 subunit. This association involves the C-terminal sequence of Caspr2, which contains the putative PDZ binding site. These results suggest a role for Caspr family members in the local differentiation of the axon into distinct functional subdomains (Poliak, 1999).

The localization of the Caspr proteins to distinct domains could be controlled by interactions with extracellular ligands, as well as with intracellular proteins within the axon. Candidate axonal proteins that may be involved in targeting and localization of Caspr2 include members of the protein 4.1 family and proteins containing PDZ domains. Caspr2 and Nrx-IV share in their cytoplasmic tail a high sequence homology with the erythrocyte transmembrane protein glycophorin C. This protein is found in a ternary complex also containing protein 4.1 and the membrane-associated guanylate kinase protein p55. The cytoplasmic domain of Caspr has been shown to interact with protein 4.1 from rat brain, and Nrx-IV associates directly with the N-terminal region of Coracle, a Drosophila protein 4.1 homolog. Genetic analyses in Drosophila indicate interdependence between protein 4.1/Coracle and Nrx-IV for correct localization. In Nrx-IV mutants, protein 4.1/Coracle is mislocalized and is not found in septate junctions. In coracle null flies, Nrx-IV reaches the lateral membrane but is not subsequently maintained at the septate junctions. Another protein that is required for the proper localization of Nrx-IV is Disc lost. This protein contains four PDZ domains, two of which interact with the C-terminal tail of Nrx-IV. By analogy with Nrx-IV and glycophorin C, it is likely that the localization of Caspr2 to the juxtaparanodal region may also involve a complex interaction with cytoplasmic proteins, such as members of the protein 4.1 family and PDZ domain-containing proteins (Poliak, 1999 and references therein).

Neurexins and synapses

Neurexins are a large family of proteins that act as neuronal cell-surface receptors. The function and localization of the various neurexins, however, have not yet been clarified. Beta-neurexins are candidate receptors for neuroligin-1, a postsynaptic membrane protein that can trigger synapse formation at axon contacts. Studies in mammalian cell culture reveal that neurexins are concentrated at synapses and purified neuroligin is sufficient to cluster neurexin and to induce presynaptic differentiation. Oligomerization of neuroligin is required for its function, and beta-neurexin clustering is found to be sufficient to trigger the recruitment of synaptic vesicles through interactions that require the cytoplasmic domain of neurexin. A two-step model is proposed in which postsynaptic neuroligin multimers initially cluster axonal neurexins. In response to this clustering, neurexins nucleate the assembly of a cytoplasmic scaffold to which the exocytotic apparatus is recruited (Dean, 2003).

Analysis of the molecular mechanism of neuroligin-induced synapse formation shows that that overexpression of neuroligin stimulates pre- and post-synaptic differentiation in cultured hippocampal neurons, suggesting that neuroligin is a limiting component of the postsynaptic machinery involved in synapse formation. Neuroligin activity depends on its interaction with neurexins. It was found that (1) endogenous neurexins are concentrated in synaptic terminals, (2) postsynaptic multimers of neuroligin-1 are sufficient to trigger the recruitment of neurexin to newly forming synaptic sites and (3) clustering of neurexin induces recruitment of synaptic vesicles (Dean, 2003).

The neurexin family of proteins was first identified as high-affinity receptors for the venom alpha-latrotoxin. Although an involvement of neurexins in the latrotoxin response of neuronal cells is now well documented, the subcellular localization and normal function of the neurexins are still unknown. Using a newly generated neurexin antibody that recognizes most neurexin isoforms, it has been shown that neurexins are concentrated at synapses. Neurexin immunoreactivity is not completely restricted to synapses, so it cannot be determined whether the additional non-synaptic neurexin pool consists of specific non-synaptic isoforms, or whether most neurexins are localized to both synapses and non-synaptic regions of the plasma membrane. In either case, the data show that at least a subset of neurexin family members is concentrated at CNS synapses.

Neuroligin was originally isolated as a splice-specific ligand of beta-neurexins by affinity chromatography. Using a functional in vitro assay, it has been demonstrated that neuroligins have a synaptogenic activity, but it remained unclear whether this activity of neuroligin is mediated through a neurexin. Strong evidence has now been provided that neurexin functions as a neuroligin receptor in synapse formation: overexpression of neuroligin in neurons induces recruitment of neurexins to newly forming terminals and promotes the formation of synaptic specializations. This activity of neuroligin-expressing cells can be mimicked by purified neuroligin presented to axons in a fluid lipid bilayer on a bead. Mutations in neuroligin that inhibit neurexin binding or adhesion with neurexin-expressing cells reduce the synapse-promoting activity of the protein (Dean, 2003).

Moreover, the results indicate that lateral clustering has a critical role in neuroligin-neurexin signaling. Clustering of beta-neurexin in axons with multimerized antibodies is sufficient to trigger the accumulation of synaptic vesicles, whereas crosslinking of neurexin with monomeric antibodies is ineffective. This suggests a highly cooperative process in which a minimum number of beta-neurexins (at least four) must be clustered to result in productive signaling. These findings support a striking mechanistic analogy between the induction of synaptic differentiation in CNS neurons and synapse formation in the immune system where receptor clustering represents a crucial event in T-cell activation (Dean, 2003).

How can the clustering of beta-neurexin trigger the assembly of presynaptic terminals? For beta-neurexin to be active it must have an intact cytoplasmic C-terminal tail, which contains a PDZ-binding motif. This domain is known to interact with the cytoplasmic scaffolding molecules CIPP, CASK/Lin-2 and Mint/Lin-10/X11alpha and may interact with other proteins that have not yet been identified. The clustering of beta-neurexin monomers in the axon by neuroligin tetramers may either recruit specific downstream signaling components to the beta-neurexin at the forming contacts or activate signaling through components that are 'pre-bound' to beta-neurexin. In either case, the small neurexin clusters would provide the minimal nucleation site for assembling a presynaptic protein scaffold and, subsequently, the secretory apparatus. Most of the cytoplasmic neurexin binding proteins are multivalent and can generate a scaffold with additional neurexin binding sites. Thus, the initial complexes could be enlarged by recruitment of more beta-neurexins, and, subsequently, additional neuroligins, forming an expanding cell-cell contact. Such a model might also account for the fact that clustering of ephrinB by EphB receptors induces the recruitment of adapters and signaling molecules resulting in a retrograde signal transmitted into the ephrinB-expressing cell (Dean, 2003).

The model predicts that a beta-neurexin is localized to the presynaptic terminal. With the immunological reagents currently available, direct evidence cannot be provided for a presynaptic localization of beta-neurexin. Several lines of evidence are provided, however, that support such a localization: (1) a pan-neurexin antibody that recognizes alpha- and beta-neurexins shows that neurexin isoforms are concentrated at synapses and in axonal growth cones; (2) clustering of endogenous neurexins with neuroligin-coated beads induces presynaptic specializations in the absence of postsynaptic elements and (3) clustering of epitope-tagged beta-neurexin in axons with antibodies triggers the recruitment of synaptic vesicles. It is possible that individual neurexin isoforms show differential distribution over pre- and postsynaptic plasma membrane domains. To address this question, beta-neurexin-specific antibodies are currently being generated, and ultrastructural studies with pan-neurexin antibodies are underway (Dean, 2003).

Whereas neuroligin-neurexin signaling is sufficient to promote synaptic differentiation, during development both molecules function in concert with several additional trans-synaptic signaling factors such as WNTs, cadherins, Ig-domain proteins and Eph receptors. One factor whose function at least partially overlaps with that of neuroligin is the Ig-domain protein SynCAM. Whereas neuroligin-neurexin interactions are heterophilic and therefore provide the synapse-induction process with directionality, SynCAM apparently functions through homophilic interactions. It is noteworthy that neurexin and SynCAM contain binding sites for the same cytoplasmic scaffolding proteins, indicating that they may converge on common downstream effectors. A common cytoplasmic scaffold may thus integrate adhesive and signaling cues from several extracellular pathways and allow for a stepwise assembly of trans-synaptic complexes. Future work should elucidate whether cross-talk between different adhesion and signaling system exists and, if so, how it contributes to the regulation of synapse formation and synaptic specificity in the CNS (Dean, 2003).

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 post-synaptic compartments of synapses by binding both extracellularly to postsynaptic cell adhesion molecules and intracellularly to presynaptic PDZ domain proteins. Using triple-knockout mice, it has been shown 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 appear normal. These data suggest that alpha-neurexins organize presynaptic terminals by functionally coupling Ca²⁺ channels to the presynaptic machinery (Missler, 2003).

Marked advances have been made in the understanding of postsynaptic mechanisms, for example, the discovery of stargazins and the molecular description of glutamate receptor recycling. Furthermore, neuroligins and SynCAMs have been identified as postsynaptic molecules that can induce differentiation of presynaptic nerve terminals. However, little is known about the presynaptic mechanisms that assemble the secretory machinery for neurotransmitter release in nerve terminals precisely opposite to the postsynaptic density, and little is known about the mechanisms that control the activity and location of Ca²⁺ channels. In the present study, alpha-neurexins, presynaptic cell-adhesion molecules that are coupled to intracellular PDZ domain proteins, are shown to be required for normal neurotransmitter release, and deletion of alpha-neurexins impairs the function of synaptic Ca²⁺ channels. These results reveal a link between two previously unconnected processes -- synaptic cell adhesion and voltage-gated Ca²⁺-signalling -- and suggest the hypothesis that alpha-neurexins couple synaptic cell adhesion to presynaptic Ca²⁺-channels and other parts of the presynaptic secretory machinery (Missler, 2003).

Deletion of alpha-neurexins impairs both spontaneous and evoked neurotransmitter release as measured in excitatory and inhibitory synapses in the brainstem and neocortex. The decrease in mini frequency, the decrease in evoked responses, the increase in failure rates, and the lack of noticeable changes in postsynaptic receptor activity demonstrate a primary presynaptic impairment. This impairment is not due to developmental abnormalities. The only structural change found in alpha-neurexin KO mice is a selective decline in the number of inhibitory synapses that can not account for the much larger decrease in inhibitory synaptic transmission. This decline could be due to the special role of GABAergic synaptic transmission in driving early development, which may lead to enhanced activity-dependent elimination of inhibitory synapses in alpha-neurexin KO mice (Missler, 2003).

It is proposed that alpha-neurexins function as presynaptic cell adhesion and/or cell recognition molecules that couple extracellular synaptic interactions to the intracellular organization of the presynaptic secretory apparatus and Ca²⁺ channels. alpha-neurexins are clearly not subunits of Ca²⁺ channels because the function of Ca²⁺ channels is normal in many cells lacking alpha-neurexins (such as chromaffin or muscle cells). alpha-neurexins are not 'chaperones' for Ca²⁺ channels because at least N-type channels (which contributed predominantly to the Ca²⁺ current reduction) are transported to the cell-surface in alpha-neurexin KO mice and exhibit a normal function. One model to explain how alpha-neurexins determine the synaptic activity of Ca²⁺ channels is that Ca²⁺ channel activity is negatively regulated in mature neurons, and that synaptic alpha-neurexins function to selectively empower Ca²⁺ channels at the synapse. Such a mechanism would concentrate Ca²⁺ influx to synapses, and imply a neurexin-dependent pathway by which postsynaptic neurons could control presynaptic Ca²⁺ channel activity. According to this proposal, alpha-neurexins act as trans-synaptic cell-adhesion molecules that participate in a modular organization of presynaptic terminals by mediating the localized activation of Ca²⁺ channels (Missler, 2003).

Formation of synaptic connections requires alignment of neurotransmitter receptors on postsynaptic dendrites opposite matching transmitter release sites on presynaptic axons. ß-neurexins and neuroligins form a trans-synaptic link at glutamate synapses. Neurexin alone is sufficient to induce glutamate postsynaptic differentiation in contacting dendrites. Surprisingly, neurexin also induces GABA postsynaptic differentiation. Conversely, neuroligins induce presynaptic differentiation in both glutamate and GABA axons. Whereas neuroligins-1, -3, and -4 localize to glutamate postsynaptic sites, neuroligin-2 localizes primarily to GABA synapses. Direct aggregation of neuroligins reveals a linkage of neuroligin-2 to GABA and glutamate postsynaptic proteins, but the other neuroligins link only to glutamate postsynaptic proteins. Furthermore, mislocalized expression of neuroligin-2 disperses postsynaptic proteins and disrupts synaptic transmission. These findings indicate that the neurexin-neuroligin link is a core component mediating both GABAergic and glutamatergic synaptogenesis, and differences in isoform localization and binding affinities may contribute to appropriate differentiation and specificity (Graf, 2004).

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. This study shows 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. These 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 (Zhang, 2005).

Alpha latrotoxin binds neurexin

Continued: Neurexin Evolutionary homologs part 2/3 | part 3/3

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