REVIEWS Specification of synaptic connectivity by cell surface interactions Joris de Wit1 and Anirvan Ghosh2 Abstract | The molecular diversification of cell surface molecules has long been postulated to impart specific surface identities on neuronal cell types. The existence of unique cell surface identities would allow neurons to distinguish one another and connect with their appropriate target cells. Although progress has been made in identifying cell type-specific surface molecule repertoires and in characterizing their extracellular interactions, determining how this molecular diversity contributes to the precise wiring of neural circuitry has proven challenging. Here, we review the role of the cadherin, neurexin, immunoglobulin and leucine-rich repeat protein superfamilies in the specification of connectivity. The emerging evidence suggests that the concerted actions of these proteins may critically contribute to the assembly of neural circuits. Axon initial segment A hallmark of CNS organization is the highly precise pat- The molecular mechanisms that regulate wiring A specialized subcellular tern of connectivity between neurons. The location of a specificity and synaptic diversity in the vertebrate brain compartment in the first part synapse on a target cell and its distinctive structural and are only beginning to be understood. Classic work from of the axon that contains high 2 3 concentrations of channels, functional properties are key factors in determining the Langley and Sperry on the regeneration of nerve fibres scaffolding proteins and flow of information in a neural circuit. in the mature nervous system indicated that neurons adhesion molecules and that The nervous system uses many mechanisms to can rewire with remarkable specificity. Their work sug- initiates action potentials. accomplish the formidable task of connecting neurons gested the presence of “individual identification tags” with their appropriate target cells (reviewed in REF. 1). on cells and fibres that would allow neurons to distin- The organization of many brain regions into distinct guish one another and selectively connect to target cells anatomical layers (laminae) is one key structural feature by “specific chemical affinities” (REF. 3). In recent years, that aids wiring specificity. The targeting of processes to substantial progress has been made in identifying and specific laminae, which we refer to as laminar specific- characterizing molecularly diverse cell surface protein ity, limits the range of target cells or surfaces available families, which may to some extent act as the surface for synapse formation. Within a particular lamina, pro- tags envisioned by Langley and Sperry. Advances in cesses can synapse with a specific cell type that resides genomics and proteomics are enabling the analysis of in that layer, such as a principal cell or an interneuron cell type-specific repertoires of surface proteins and the (cellular specificity). Neurons can also form synapses systematic, large-scale mapping of their extracellular onto a specific subcellular domain of a target cell, such interaction networks. Despite these advances, deter- 1VIB Center for the Biology as the axon initial segment or dendritic compartment mining how this molecular diversity contributes to the of Disease and Center (subcellular specificity). In the final step of assembling encoding of wiring specificity and synaptic diversity for Human Genetics, KU Leuven, Herestraat 49, functionally connected circuits, synapses between dif- remains a considerable challenge. 3000 Leuven, Belgium. ferent types of neurons are differentiated into structur- Several requirements can be envisioned for cell 2Neuroscience Discovery, ally and functionally distinct synapse types (synaptic surface proteins that regulate wiring specificity and Roche Innovation Center diversity). These different levels of specificity are closely synaptic diversity. Such proteins should be expressed Basel, F. Hoffman‑La Roche, Grenzacherstrasse 124, related. For example, dendrites from principal neurons in distinct populations of neurons, be capable of inter- 4070 Basel, Switzerland. in brain regions such as the hippocampus or the cortex acting in trans with their binding partners, and provide Correspondence to J.d.W. span multiple laminae and form a different type of syn- enough molecular diversity to confer cell type- and joris.dewit@ apse with a distinct presynaptic partner in each lamina. synapse type-specific identities. In this Review, we cme.vib-kuleuven.be Depending on the viewpoint, this could be considered as start by introducing several superfamilies of cell sur- doi:10.1038/nrn.2015.3 an example of laminar specificity, subcellular specificity face proteins that meet these requirements: the cadher- Published online 10 Dec 2015 or synaptic diversity. ins, neurexins, leucine-rich repeat (LRR) proteins and 22 | JANUARY 2016 | VOLUME 17 www.nature.com/nrn © 2016 Macmillan Publishers Limited. All rights reserved REVIEWS immunoglobulin (Ig) proteins. The molecular diversity domain that contains catenin-binding sites (FIG. 1A). of these protein families arises either through the large Approximately 20 classic cadherins exist, and these are size of the underlying gene family or through the alter- further classified as type I or type II cadherins based on native splicing of a more-limited number of genes. We amino acid sequence identity in their first EC repeat11. review the evidence for cell type-specific expression of Classic cadherin trans interactions are mediated by the these proteins and discuss to what extent current in vivo EC1 domain (FIG. 1A) and are preferentially homophilic, evidence supports a role for them in wiring specific- but they can also be heterophilic within each class; thus, ity and synaptic diversity. Other cell surface proteins type I cadherins can interact with other type I cad- exhibiting cell type-specific expression patterns have herins, and type II cadherins can interact with other been implicated in the specification of connectivity, type II cadherins14–18. and a brief, albeit incomplete, overview of additional The protocadherins were identified based on their molecular players in this process is provided in BOX 1. structural similarity to cadherins19 and are subdivided Note that, owing to space limitations, we do not discuss into clustered and non-clustered protocadherins. The the cellular and molecular mechanisms that guide axons role of non-clustered protocadherins in connectivity towards their target cells, nor do we provide an over- is not well understood20 and is not discussed here. view of all the cellular and molecular mechanisms that The clustered protocadherins comprise the α‑, β‑ and are known to contribute to synaptic specificity, as these γ‑clusters, which are arranged in three tandem arrays have been extensively reviewed elsewhere1,4–6. Finally, on a single chromosome that encodes a total of 58 iso- this Review focuses on the rodent CNS, and we refer the forms in mice21–23. The α- and γ‑protocadherin clusters reader to several excellent recent reviews that discuss contain variable exons encoding complete extracellular, synaptic specificity in invertebrate systems7–9. transmembrane and proximal intracellular domains. Each exon contains its own promoter and is spliced to Cell surface protein families three constant exons that encode the common remain- Classic cadherins and protocadherins. The cadherin der of the cytoplasmic domain24,25. The β‑cluster lacks superfamily comprises more than 100 transmem- constant exons and instead contains exons that seem brane glycoproteins that can be grouped into several to encode complete proteins. Five phylogenetically subfamilies, of which the classic cadherins and pro- divergent members from the α- and γ‑clusters, which tocadherins have been the most studied in relation to differ from their respective cluster members and are connectivity10–13. Classic cadherins contain five extracel- more similar to each other, have been grouped into a lular cadherin (EC) repeats and a conserved intracellular separate C-cluster23. Although structurally similar, clustered protocad- herins differ from classic cadherins in several aspects, Box 1 | Other cell surface proteins that contribute to wiring specificity with direct implications for their role in connectivity. Many cell surface proteins other than those focused on in this Review have been Protocadherins contain six EC repeats instead of five, and implicated in the specification of connectivity in the vertebrate nervous system, and we their cytoplasmic tails lack canonical catenin‑binding­­­­ highlight a few of them here. The semaphorins (SEMAs) and their plexin signalling sites (FIG. 1B). Protocadherin isoforms from the α‑, β‑ receptors are best known for their roles as repulsive axon-guidance cues132, but they and γ‑gene clusters engage in highly specific homophilic also contribute to laminar and cellular specificity. In the retina, the transmembrane trans interactions that are mediated by the EC2 and EC3 protein SEMA6A and its receptor plexin A4 are localized in specific sublaminae of the domains, instead of by the EC1 domain26. Protocadherins inner plexiform layer (IPL). Loss of SEMA6A or plexin A4 severely disrupts can associate in cis and form multimers (FIG. 1B), which lamina-specific arborization of amacrine and retinal ganglion cells133. A complex interplay between SEMA6s and plexin receptors also contributes to lamina-specific have different trans binding specificities than individual