Alternative splicing controls selective trans-synaptic interactions of the neuroligin-neurexin complex.

Formation of synapses requires specific cellular interactions that organize pre- and postsynaptic compartments. The neuroligin-neurexin complex mediates heterophilic adhesion and can trigger assembly of glutamatergic and GABAergic synapses in cultured hippocampal neurons. Both neuroligins and neurexins are encoded by multiple genes. Alternative splicing generates large numbers of isoforms, which may engage in selective axo-dendritic interactions. We explored whether alternative splicing of the postsynaptic neuroligins modifies their activity toward glutamatergic and GABAergic axons. We find that small extracellular splice insertions restrict the function of neuroligin-1 and -2 to glutamatergic and GABAergic contacts and alter interaction with presynaptic neurexins. The neuroligin isoforms associated with GABAergic contacts bind to neurexin-1alpha and a subset of neurexin-1betas. In turn, these neurexin isoforms induce GABAergic but not glutamatergic postsynaptic differentiation. Our findings suggest that alternative splicing plays a central role in regulating selective extracellular interactions through the neuroligin-neurexin complex at glutamatergic and GABAergic synapses.

Caspr regulates the processing of contactin and inhibits its binding to neurofascin.

Three cell adhesion molecules are present at the axoglial junctions that form between the axon and myelinating glia on either side of nodes of Ranvier. These include an axonal complex of contacin-associated protein (Caspr) and contactin, which was proposed to bind NF155, an isoform of neurofascin located on the glial paranodal loops. Here, we show that NF155 binds directly to contactin and that surprisingly, coexpression of Caspr inhibits this interaction. This inhibition reflects the association of Caspr with contactin during biosynthesis and the resulting expression of a low molecular weight (LMw), endoglycosidase H-sensitive isoform of contactin at the cell membrane, which remains associated with Caspr but is unable to bind NF155. Accordingly, deletion of Caspr in mice by gene targeting results in a shift from the LMw- to a HMw-contactin glycoform. These results demonstrate that Caspr regulates the intracellular processing and transport of contactin to the cell surface, thereby affecting its ability to interact with other cell adhesion molecules.

Retention of a cell adhesion complex at the paranodal junction requires the cytoplasmic region of Caspr.

An axonal complex of cell adhesion molecules consisting of Caspr and contactin has been found to be essential for the generation of the paranodal axo-glial junctions flanking the nodes of Ranvier. Here we report that although the extracellular region of Caspr was sufficient for directing it to the paranodes in transgenic mice, retention of the Caspr-contactin complex at the junction depended on the presence of an intact cytoplasmic domain of Caspr. Using immunoelectron microscopy, we found that a Caspr mutant lacking its intracellular domain was often found within the axon instead of the junctional axolemma. We further show that a short sequence in the cytoplasmic domain of Caspr mediated its binding to the cytoskeleton-associated protein 4.1B. Clustering of contactin on the cell surface induced coclustering of Caspr and immobilized protein 4.1B at the plasma membrane. Furthermore, deletion of the protein 4.1B binding site accelerated the internalization of a Caspr-contactin chimera from the cell surface. These results suggest that Caspr serves as a "transmembrane scaffold" that stabilizes the Caspr/contactin adhesion complex at the paranodal junction by connecting it to cytoskeletal components within the axon.

Silencing of neuroligin function by postsynaptic neurexins.

The formation of neuronal circuits during development involves a combination of synapse stabilization and elimination events. Synaptic adhesion molecules are thought to play an important role in synaptogenesis, and several trans-synaptic adhesion systems that promote the formation and maturation of synapses have been identified. The neuroligin-neurexin complex is a heterophilic adhesion system that promotes assembly and maturation of synapses through bidirectional signaling. In this protein complex, postsynaptic neuroligins are thought to interact trans-synaptically with presynaptic neurexins. However, the subcellular localization of neurexins has not been determined. Using immunoelectron microscopy, we found that endogenous neurexins and epitope-tagged neurexin-1beta are localized to axons and presynaptic terminals in vivo. Unexpectedly, neurexins are also abundant in the postsynaptic density. cis-expression of neurexin-1beta with neuroligin-1 inhibits trans-binding to recombinant neurexins, blocks the synaptogenic activity of neuroligin-1, and reduces the density of presynaptic terminals in cultured hippocampal neurons. Our results demonstrate that the function of neurexin proteins is more diverse than previously anticipated and suggest that postsynaptic cis-interactions might provide a novel mechanism for silencing the activity of a synaptic adhesion complex.