The specificity of interactions between neurons is believed to be mediated by diverse cell adhesion molecules, including members of the cadherin superfamily. Whereas mechanisms of classical cadherin adhesion have been studied extensively, much less is known about the related protocadherins (Pcdhs), which together make up the majority of the superfamily. Here we use quantitative cell aggregation assays and biochemical analyses to characterize cis and trans interactions among the 22-member γ-Pcdh family, which have been shown to be critical for the control of synaptogenesis and neuronal survival. We show that γ-Pcdh isoforms engage in trans interactions that are strictly homophilic. In contrast to classical cadherins, γ-Pcdh interactions are only partially Ca 2+ -dependent, and their specificity is mediated through the second and third extracellular cadherin (EC) domains (EC2 and EC3), rather than through EC1. The γ-Pcdhs also interact both covalently and noncovalently in the cis-orientation to form multimers both in vitro and in vivo. In contrast to γ-Pcdh trans interactions, cis interactions are highly promiscuous, with no isoform specificity. We present data supporting a model in which γ-Pcdh cistetramers represent the unit of their adhesive trans interactions. Unrestricted tetramerization in cis, coupled with strictly homophilic interactions in trans, predicts that the 22 γ-Pcdhs could form 234,256 distinct adhesive interfaces. Given the demonstrated role of the γ-Pcdhs in synaptogenesis, our data have important implications for the molecular control of neuronal specificity.cadherin | calcium-dependent | synaptogenesis | recognition | trafficking

SUMMARY The 22 γ-protocadherins (γ-Pcdhs) potentially specify thousands of distinct homophilic adhesive interactions in the brain. Neonatal lethality of mice lacking the Pcdh-γ gene cluster has, however, precluded analysis of many brain regions. Here, we use a conditional Pcdh-γ allele to restrict mutation to the cerebral cortex and find that, in contrast to other central nervous system phenotypes, loss of γ-Pcdhs in cortical neurons does not affect their survival or result in reduced synaptic density. Instead, mutant cortical neurons exhibit severely reduced dendritic arborization. Mutant cortices have aberrantly high levels of protein kinase C (PKC) activity and of phosphorylated (inactive) myristoylated alanine-rich C-kinase substrate, a PKC target that promotes arborization. Dendrite complexity can be rescued in Pcdh-γ mutant neurons by inhibiting PKC, its upstream activator phospholipase C, or the γ-Pcdh binding partner focal adhesion kinase. Our results reveal a distinct role for the γ-Pcdhs in cortical development and identify a signaling pathway through which they play this role.

Molecular diversity of surface receptors has been hypothesized to provide a mechanism for selective synaptic connectivity. Neurexins are highly diversified receptors that drive the morphological and functional differentiation of synapses. Using a single cDNA sequencing approach, we detected 1,364 unique neurexin-α and 37 neurexin-β mRNAs produced by alternative splicing of neurexin pre-mRNAs. This molecular diversity results from near-exhaustive combinatorial use of alternative splice insertions in Nrxn1α and Nrxn2α. By contrast, Nrxn3α exhibits several highly stereotyped exon selections that incorporate novel elements for posttranscriptional regulation of a subset of transcripts. Complexity of Nrxn1α repertoires correlates with the cellular complexity of neuronal tissues, and a specific subset of isoforms is enriched in a purified cell type. Our analysis defines the molecular diversity of a critical synaptic receptor and provides evidence that neurexin diversity is linked to cellular diversity in the nervous system.

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Chadman, K. K. et al. Minimal aberrant behavioral phenotypes of neuroligin-3 R451C knockin mice.

The unique anatomical and functional features of principal and interneuron populations are critical for the appropriate function of neuronal circuits. Cell type-specific properties are encoded by selective gene expression programs that shape molecular repertoires and synaptic protein complexes. However, the nature of such programs, particularly for post-transcriptional regulation at the level of alternative splicing is only beginning to emerge. We here demonstrate that transcripts encoding the synaptic adhesion molecules neurexin-1,2,3 are commonly expressed in principal cells and interneurons of the mouse hippocampus but undergo highly differential, cell type-specific alternative splicing. Principal cell-specific neurexin splice isoforms depend on the RNA-binding protein Slm2. By contrast, most parvalbumin-positive (PV+) interneurons lack Slm2, express a different neurexin splice isoform and co-express the corresponding splice isoform-specific neurexin ligand Cbln4. Conditional ablation of Nrxn alternative splice insertions selectively in PV+ cells results in elevated hippocampal network activity and impairment in a learning task. Thus, PV-cell-specific alternative splicing of neurexins is critical for neuronal circuit functionDOI: http://dx.doi.org/10.7554/eLife.22757.001

SUMMARY The 22 γ-Protocadherin (γ-Pcdh) cell adhesion molecules are critical for the elaboration of complex dendritic arbors in the cerebral cortex. Here, we provide evidence that the γ-Pcdhs negatively regulate synapse development by inhibiting the postsynaptic cell adhesion molecule neuroligin-1 (Nlg1). Mice lacking all γ-Pcdhs in the forebrain exhibit significantly increased dendritic spine density in vivo, while spine density is significantly decreased in mice overexpressing one of the 22 γ-Pcdh isoforms. Co-expression of γ-Pcdhs inhibits the ability of Nlg1 to increase spine density and to induce presynaptic differentiation in hippocampal neurons in vitro. The γ-Pcdhs physically interact in cis with Nlg1 both in vitro and in vivo, and we present evidence that this disrupts Nlg1 binding to its presynaptic partner neurexin1β. Together with prior work, these data identify a mechanism through which γ-Pcdhs could coordinate dendrite arbor growth and complexity with spine maturation in the developing brain.

Background: ␥-Protocadherin adhesion molecules regulate dendrite arborization by inhibiting focal adhesion kinase (FAK). Results: Protein kinase C (PKC) phosphorylation of a C-terminal serine disrupts ␥-protocadherin inhibition of FAK and reduces dendrite arborization. Conclusion:The ability of the ␥-protocadherins to promote dendrite arborization is regulated by phosphorylation. Significance: These data provide much-needed mechanistic insight into the regulation of a critical neurodevelopmental adhesion molecule.