The roles of cadherins in controlling organization and function of the synapse

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1 The roles of cadherins in controlling organization and function of the synapse Contact information: Seung-Hye Lee: Louis F. Reichardt: (415) (phone) (415) (fax) Seung-Hye Lee and Louis F. Reichardt Neuroscience Program, Department of Physiology and Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA Keywords: cadherin, α-catenin, β-catenin, cell adhesion molecule, δ-catenin, Fat cadherin, Flamingo, N- cadherin, p120ctn, PDZ domain, protocadherin, synapse

2 Synopsis Cadherins are a large superfamily of cell adhesion molecules defined by the presence of multiple extracellular copies of the cadherin motif. The classical, seven-pass transmembrane (Flamingo), protocadherin, and Fat cadherins are expressed in synapses and control synapse formation, function and plasticity. The classical cadherins regulate axon-target interactions as well as pre and postsynaptic differentiation. Flamingo cadherins appear to act at early stages of synapse development. Protocadherins regulate synaptic density and seem likely to regulate synaptic specificity. Fat cadherins control organization of the F-actin cytoskeleton and are highly expressed in the brain, but their synaptic roles remain to be studied.

3 Introduction Cadherins constitute a large superfamily of cell-to-cell adhesion molecules defined by the presence of multiple copies of a unique domain termed the cadherin motif in their extracellular domains. Cadherins mediate the formation of many types of cell-cell junctions, including adherens junctions and desmosomes in epithelia, adherens junctions and complexus adherens in endothelia, intercalated disks in cardiac myocytes, and synaptic junctions between axons and their target cells. The vast majority of the cadherins are transmembrane proteins with functionally important interactions mediated by both extracellular and cytoplasmic domains. Some cadherins also have functionally significant cis-interactions that control their oligomerization and the strength of cell-to-cell adhesion. Most of the more than 80 mammalian cadherins are expressed in the nervous system and many have been shown to be present at synapses and in some instances to regulate synapse development or function. Synapses are formed through a series of reciprocal inductive events in which contact between axons and their targets (muscle at NMJ, dendrites in CNS) is followed by assembly of pre- and postsynaptic constituents. Many cadherin members function in synapse formation and plasticity. The classical cadherins were identified initially as Ca 2+ -dependent cell adhesion molecules that mediated primarily homophilic adhesion. One of the cadherins motifs present in a cadherin contain hydrophobic residues including one or two tryptophan residues mediating interactions with the same cadherin present on other cells. Gene sequence analysis has enlarged the cadherin superfamily membership, which can be subdivided into several subfamilies based upon homology, structure and function. The classical cadherins have been divided into Type I and Type II subfamilies, distinguished by the presence of conserved amino acid residues in the most

4 N-terminal cadherin motif which results in differences in the structures of their interaction interfaces. All classical cadherins in vertebrates are single pass transmembrane proteins that contain five tandem repeats of the cadherin motif in their extracellular domains and a conserved cytoplasmic domain that mediates through linker proteins named catenins association with the F- actin cytoskeleton. Invertebrates contain similar cadherins that also mediate interactions through the catenins with the F-actin cytoskeleton, but have different numbers of extracellular cadherin motifs. The desmosomal cadherins, the desmogleins and desmocollins, constitute a distinct family of cadherins that mediate through their cytoplasmic domains interactions through the plakophilins and desmoplakins with intermediate filament networks. As they are not found at synapses, they will not be discussed further in this article. Additional cadherin subgroups include the 7-pass transmembrane or flamingo cadherins, and the protocadherins. In addition, there are a number of proteins containing the cadherin motif that do not fit into these subfamilies, including the receptor tyrosine kinase c-ret, Fat, Ksp-cadherin, and LI-cadherin. c-ret is stimulated by the GDNF-family of neurotrophic factors and is not discussed further in this contribution. Atypical glycosyl phosphatidylinositol (GPI)-linked cadherin, T-cadherin is expressed in neurons and regulates neurite outgrowth. Classical cadherins Each member of the classical cadherins mediates primarily homophilic interactions with the same cadherin on neighboring cells. Of particular interest, several members have been shown to be associated with networks of interconnected neurons within the brain and to be present at synapses, suggesting a role in establishing specific synaptic connections. Type I cadherins like

5 N-, E-, P-, R-cadherins and many different members of type II cadherins, for example, MNcadherin, cadherin-6, 8, 11, are observed at the synapses. At synapses, cadherins are typically found in puncta adherens-like structures within or surrounding synapses, suggesting they play a role in organizing synaptic membrane domains similar to their roles in organizing adherens junctions and surrounding membrane domains in epithelial cells. In epithelial cells, cadherins typically collaborate to form adherens junctions with a subfamily of the immunoglobulin superfamily, named nectins. Nectins promote cell adhesion, but exhibit strongest interactions with related, but not identical family members. In contrast to epithelia, synapses are not symmetric and it is not clear how cadherins functioning alone can ensure that axons form synapses with dendrites as opposed to other axons. Recent work has demonstrated that differential transport of different nectins to axonal vs. dendritic compartments promotes the preferential interactions observed by axons with dendrites. In this manner, the nectins may help ensure that only appropriate synaptic contacts are formed or stabilized within the CNS. Mutations of several cadherin genes or perturbation of cadherin function impair normal synapse development and function in vertebrates and invertebrates. Cadherins appear to play roles in several stages of synapse development. Synaptic adhesion molecules, including the cadherins, are important in directing the localization and specificity of interactions between presynaptic axons with postsynaptic targets. In many instances, cadherins are required for this target selection process. In the Xenopus retina, for example, impairment of N-cadherin function slows axon growth, thereby preventing axons from reaching their normal targets in the optic tectum. In elegant recent experiments analyzing the pattern of synaptic connections formed by photoreceptor axons in Drosophila, N-cadherin appears to mediate homophilic and attractive interactions between the growth cones of photoreceptors and their

6 targets that precede synaptic partner selection. Inhibition of N-cadherin function disrupts laminar-specific connectivity in chick optic tectum and results in overshooting of thalamic afferents beyond layer IV to as far as the pial surface in cortical-thalamic slice cultures. Cadherins are also required for synapse maturation. For presynaptic maturation, active zone proteins and synaptic vesicles are concentrated at synaptic contacts. During CNS development, presynaptic compartments are known to be assembled from preassembled packets of active zone proteins, synaptic vesicles and associated synaptic proteins and they are trapped at the sites of cell-cell contacts. The N-cadherin complex plays an important role in localizing synaptic vesicles at nascent synapses in vitro. In the CNS, many excitatory synapses are made onto dendritic spines, actin filament-based protrusions. N-cadherin-mediated contact stabilization and modulation of the actin cytoskeleton through interacting signaling molecules are essential for proper spine maturation in vitro. Finally, cadherins have a role in synaptic efficacy in vivo. The deletion of cadherin-11 alters mouse behavior and synaptic plasticity. Taken together, these results indicate that cadherins have roles in synaptic development, specificity and function. It seems almost certain that mutations in the many additional genes encoding classical cadherins will also prove to have interesting synaptic and behavioral phenotypes, especially when mutations are combined to reduce functional redundancy. The large number of neuronallyexpressed classic cadherins complicates studies on their neural functions because of the potential for compensation. In considering the roles of cadherins in directing synaptic development and function, it is important to consider the proteins that are associated with cadherins. Through these proteins, cadherin ligation regulates several distinct signaling pathways which impinge on neuronal development and synapse development and function. Two major classes of proteins associate

7 directly with the classic cadherins: members of the β-catenin (β-catenin and plakoglobin) and p120catenin (p120ctn) ( p120ctn, δ-catenin, p0071, and ARVCF) families. Members of each family contain a central domain consisting of several armadillo (arm) repeats and flanking N-and C-terminal domains. The cadherin cytoplasmic domains bind to the arm repeats of these proteins. β-catenin controls cell adhesion and cytoskeletal organization through interactions with α- catenin, and with Rho family GTPase regulators, such as IQGAP and RICS. Some of these interactions are known to be competitive with cadherin binding. In addition the presence of β- catenin is required for activation of the classical Wnt signaling pathway and the role of β-catenin in this pathway is mediated through interactions with transcription factors, such as the TCFs, negative regulators, such as ICAT (inhibitor of beta-catenin and TCF), and regulators of protein stability, such as APC, axin and GSK3β. Phosphorylation of cadherins and β-catenin regulate their interactions, functions and turnover and many protein kinases and phosphatases have been shown to control the stability of the cadherin-β-catenin complex following extracellular stimuli. In the synapse, for example, neural activity induces dephosphorylation and redistribution of β- catenin into spines, where it interacts with cadherins to influence synaptic size and strength. Recently, BDNF has been shown to regulate tyrosine phosphorylation and interaction of β- catenin with cadherin, thereby modulating synaptic vesicle localization and synapse density in vitro. Within cadherin-centered junctions a major role of β-catenin is to provide a link from cadherins to α-catenin, which in turn promotes interactions with the F-actin cytoskeleton. The presence of β-catenin and α-catenin is required to promote strong adhesion between cells. The association between these two catenins is mediated through the N-terminal region of β-catenin, a domain containing multiple serine/threonine phosphorylation sites that regulate separately the

8 association with α-catenin and β-catenin degradation. Dendritic spines on αn-catenin-deficient neurons are unstable and highly mobile. Loss of αn-catenin reduces the stability and size of synaptic contacts, possibly by disruption of the cadherin-f-actin linkage or perturbation of cadherin-mediated regulation of F-actin organization. β-catenin and most p120ctn family proteins include C-terminal PDZ-domain interaction motifs. Through these cadherins can associate indirectly with the PDZ-domain-containing scaffold proteins know to be crucial for synapse organization. The β-catenin C-terminus binds the Cask- Mint-Veli complex, the neuronal isoform of MAGI (MAGUKS (Membrane-Associated Guanylate Kinases) with Inverted domain structure), S-SCAM (synaptic scaffold molecule), and additional PDZ-domain containing proteins, some of which modulate β-catenin s transcriptional functions. Through its C-terminus β-catenin functions as a scaffold that promotes promote localization of the Cask-Mint-Veli complex and synaptic vesicles to synapses. β-catenin has also been shown to promote dendrite growth and arborization through pathways independent of Wnt signaling. Through its interaction with MAGI-1b, β-catenin has been shown in non-neuronal cells to recruit the phosphatase PTEN to adhesion sites where it promotes their stability through localized suppression of Src-induced invasiveness in cancer cells. Src suppression is likely to promote adhesion site stability through control of Rho family GEFs and possibly Rho family effectors such as myosin II. Through these interactions PTEN may also regulate synapse stability. Another MAGUK family member which interacts with β-catenin, lp-dlg/kiaa0583, provides a potential alternative link through vinexin and vinculin to the F-actin cytoskeleton. Finally, there are poorly understood intramolecular interactions between the N-and C-terminal and arm domains in β-catenin that regulate the interactions of these domains with other proteins.

9 β-catenin is also an essential intermediate in the classical signaling pathway activated by Wnt proteins, but largely separate pools of β-catenin appear to be involved in its cadherin-associated and Wnt-pathway mediated functions. Wnt signaling results in stabilization of β-catenin and then it interacts with TCF family and other transcription factors to regulate gene expression. As a result of its essential role in this major Wnt signaling pathway, β-catenin has many additional roles within the nervous system, including regulation of neuroepithelial precursor proliferation, forebrain patterning, and sensory neuron specification. Wnt7a/Dishevelled-1 signaling promotes presynaptic assembly and neurotransmitter release in mossy fiber-granular cell synapse in the cerebellum, but it is not clear whether this is mediated by β-catenin-dependent canonical or noncanonical signaling pathway. Finally, plakoglobin, which is primarily expressed in epithelia, is a homologue of β-catenin that functions similarly in promoting cell-cell adhesion. Plakoglobin, though, cannot replace β-catenin in the Wnt signaling pathway. Recently, interest in plakoglobin has been increased by the observation that it is enriched in some synapses. Members of the p120ctn family interact with the membrane-proximal portion of the cytoplasmic domains of the classical cadherins. All members of this protein family have 10 putative arm repeats with high sequence identity (more than 45%) among members and flanking N- and C-terminal polypeptides with less homology. Except for p120ctn, these proteins include PDZ-domain interaction motifs at their C-terminals capable of interacting with several PDZproteins including synaptic scaffold proteins. p120ctn and δ-catenin are strongly expressed in the nervous system. Mice lacking δ-catenin are viable, but have many behavioral deficits that have been attributed to perturbations of synaptic function. In the hippocampus of mutant animals, short-term plasticity, as assessed by paired-pulse facilitation, is impaired, while long-term plasticity, as assessed by measurements of

10 long-term potentiation, is elevated following high frequency stimulation, but reduced following low frequency stimulation. Long-term depression is also impaired at hippocampal synapses in these animals. The absence of p120ctn results in early mouse embryo lethality. Mice in which p120ctn is eliminated solely in the forebrain are viable and have severely perturbed neuronal morphologies. In particular, the dendrites of neurons form with few spines and the density of synapses is severely reduced. This phenotype can be recapitulated in cell culture and rescue experiments indicate that p120ctn enhances spine density through inhibition of Rho activation while it promotes spine maturation through interactions with cadherins, acting either upstream or downstream of the cadherins. p120ctn family members have been shown to regulate cadherin turnover, intracellular signaling, and cytoskeletal organization in several cell types. First, the presence of p120ctn family members is important for stabilizing cadherins on the cell surface in vitro and in vivo. In their absence, cadherins are transported normally to the surface, but are rapidly endocytosed and degraded. p120ctn family members also play important roles in the signaling functions of adherent junctions. Cadherin-mediated adhesive junctions recruit and activate numerous signaling proteins, including PI3 kinase, Rac, Rho, and Arp2/3. The p120ctn family members promote assembly of these signaling centers through recruitment of the tyrosine kinases Fer, Fyn and Yes and phosphatases including SHP-1, PTP-µ, and DEP-1 and have been shown to substrates of several kinases including receptor tyrosine kinases and Src family kinases. In the nervous system δ-catenin also interacts with neurotransmitter receptors such as mglur1a and NR2A, and PDZ-containing scaffold proteins like S-SCAM. p120ctn family members regulate actin filament organization through regulation of Rho GTPases with p120ctn promoting activation of Rac and inhibition of Rho following cell adhesion. The C-terminal region of δ-

11 catenin also interacts in a phosphorylation-dependent manner with cortactin, an activator of the Arp2/3 complex, thereby providing a second pathway for regulation of the F-actin cytoskeleton. In addition, p120ctn family members also interact with the microtubule network directly as well as indirectly through kinesin. Somewhat analogously to the involvement of β-catenin in Wnt signaling, the arm repeats of p120ctn associate with the transcriptional repressor Kaiso and thereby control expression of Wnt11 and Xenopus morphogenesis. Finally, deletion of p120ctn from keratinocytes results in elevated Rho activity, which in turn promotes activation of NF-κB and a proinflammatory cascade. Deletion in acinar epithelial cells prevents normal gland development and results in delamination of epithelial cells, almost certainly as a consequence of decreased surface E- cadherin and/or NF-κB. In summary, the classical cadherins have been shown to regulate synapse formation, function and plasticity. Many of the functions of the cadherins are controlled through binding to two families of catenins, which in turn control cadherin complex signaling, association with the cytoskeleton, and association with molecules important in synaptic function, including transmitter receptors and synaptic vesicles. In the future, it will be important to understand how these interactions are regulated by signaling pathways activated through activity and neurotransmitter receptors because these seem likely to control alterations in synaptic efficiency, in part, through regulation of synaptic cell adhesion molecule function. Seven-pass (Flamingo-like) transmembrane cadherins The prototypic member of the seven-pass transmembrane cadherin family, Flamingo, was identified as a protein that mediates interactions between epithelial cells that control planar cell

12 polarity. In the planar cell polarity pathway, Flamingo acts through modulation of Wnt/frizzled signaling. Flamingo has nine cadherin repeats, two laminin G domains, five EGF-like domains and seven transmembrane domains and has been shown to promote cell aggregation in vitro. Celsr1, Celsr2 and Celsr3 are the mammalian orthologues of Flamingo and are expressed in brain and epithelial cells, exhibiting distinct expression patterns. Flamingo has several actions that influence early steps in neuronal differentiation that precede synapse formation. First in Drosophila sensory neurons, it acts cell autonomously to promote axon growth and prevent premature axon branching. In addition, flamingo controls the timing and length of dorsal dendrites of these neurons. Interestingly, it functions to delay their initiation and thereby limit their extent of growth. Flamingo is expressed on both the axons and dendrites of these neurons and some of the mutants that affect its function also affect its localization, suggesting that proper localization is required for its function. It has been suggested that Flamingo engages in both homophilic and heterophilic interactions, the latter with unknown ligands, to control the extent and patterning of dendrite growth. Flamingo is also expressed in the Drosophila visual system where its presence is required for normal targeting of R1-R6 photoreceptor axons to appropriate cartridges in the lamina and of R8 axons in the medulla. Flamingo is expressed on the surfaces of these growth cones during their invasion of the brain. Intriguingly, other genes known to be required in the planar cell polarity pathway are not required for normal target selection by Drosophila photoreceptor axons, so flamingo appears to be acting through a distinct pathway to regulate axon guidance. However, the genetic pathway through which Celsr3 controls axon tract development in mouse brain development seems likely to involve the planar cell polarity pathway because mice mutants in Celsr3 and Fzd-3, a member of Frizzled family, have similar phenotypes.

13 Protocadherins Protocadherins constitute a very large family of proteins containing up to seven cadherin-like motifs that are distinct from those present in classical cadherins, a single transmembrane domain, and distinct cytoplasmic domains. In mammals, many of these proteins are highly expressed in the nervous system where several have been localized to synapses. Their expression patterns in the brain frequently correlate with functional CNS subdivisions and it has been proposed that they have important roles in establishing and maintaining synaptic specificity. Four clusters (α, β, γ and δ) of genes encoding these proteins have been identified. Products of the protocadherinα cluster are also named the CNRs (cadherin-like neuronal receptors). In the α, β, and γ clusters, single exons, each of which is coupled to a separate promoter, encode the variable extracellular domains. In the α and γ clusters, the mrna product derived from each exon is spliced to an exon encoding the transmembrane and cytoplasmic domain, resulting in expression of several distinct extracellular domains, each fused to a constant cytoplasmic domain. β cluster expressed transcripts contains variable extracellular domains together with a transmembrane and cytoplasmic domain lack the shared, constant exon which is observed in the α and γ clusters. Genes in the fourth cluster, the δ-protocadherin genes, are distributed on different chromosomes and exhibit similar alternative splicing patterns to that observed in the α and γ clusters. Recent work has shown that, in addition to biallelic expression, there is monoallelic, but combinatorial expression of variable exons of the protocadherin-α and γ cluster in single Purkinje cells, i.e. only one of the two chromosomes can be used as a template for transcription of a single variable exon, but more than one variable exon can be expressed within a single neuron. In principle, this is a powerful mechanism to generate neuronal diversity and identity.

14 In contrast to the classical cadherins, however, there is comparatively little evidence that these proteins function through homophilic adhesion. Some reports suggest that hey may interact with each other, recognize RGD-binding β1 integrins, and interact with reelin. It has also been suggested that protocadherins control homophilic binding by classical cadherins. There is little information on the cytoplasmic signaling pathways regulated by the protocadherins. Fyn is reported to interact with CNR cytoplasmic domain. Pcdh18 has been shown to associate with Dab1, which has been shown to mediate reeling signaling upon phosphorylation by a Src family kinase like Fyn. Members of the α- and γ-protocadherin proteins have been localized to synapses and have been proposed to control synapse formation, specificity or maintenance. Analysis of mice in which the entire protocadherin-γ has been deleted results in early lethality with specific loss through apoptosis of interneurons in the spinal cord. In addition, the spinal cords of these animals had a lower density of synapses. Analysis of this mutant in a Bax gene mutant background, which prevents apoptosis, revealed a striking deficit in the density of both excitatory and inhibitory synapses. Synaptic function was also compromised in neurons cultured from these animals. The results provide direct evidence that this family of protocadherins promotes synaptic development and/or maturation. At present there is no evidence that they control synaptic specificity, but future studies on this and other protocadherins seem likely to be informative. Mechanisms through which these proteins control synapse development remain mysterious. Fat-related and other members of cadherin superfamily The Fat cadherins are single transmembrane pass proteins that typically contain in their extracellular domains 34 tandemly arrayed cadherin repeats, plus in the membrane proximal

15 region one or two laminin A subunit G domains and several EGF motifs. Involvement of Fat cadherins in synapse formation or function is purely speculative at this point. There are two and four Fat cadherin genes in Drosophila and mice, respectively. Mouse Fat2 mediates homophilic binding and is detected solely in cerebellar granule cells where it is localized to the axons of these cells, the parallel fibers. Its expression level is elevated during the time period of synapse formation by these cells and remains high at later times. Its localization suggests that it helps organize the parallel fibers, which form synapses primarily on the dendritic spines of Purkinje cells. Fat3 expression is more broadly expressed in the embryonic CNS, including the spinal cord, but there is no information on its CNS functions. Cadherin 23 is a novel single pass transmembrane cadherin with 27 extracellular tandemly organized cadherin repeats that is expressed in both hair cells in the inner ear and photoreceptors in the retina. Cadherin 23 is a component of the tip link in hair cell stereocilia and forms a complex with each of the other proteins whose genes are mutated in Usher Syndrome, a syndrome that results in deafness and blindness resulting from stereoclia disorganization. Through a C-terminal PDZ interaction motif cadherin 23 has been shown to associate with the PDZ-domain containing scaffold protein harmonin. Harmonin is an F-actin bundling protein that also associates protocadherin-15, Myosin VIIa, and a scaffold protein named Sans which contains several ankyrin repeats plus a SAM domain. In addition several transmembrane proteins, including usherin, a putative cell adhesion molecule, USH2B, a putative ion cotransporter, the very large G-protein coupled receptor USH2C and the 4-pass transmembrane protein clarin also associate with this complex. Mutations in each of the genes encoding these proteins also results in characteristic forms of Usher syndrome. In addition to their roles in

16 organization of stereocilia, several of these proteins are expressed at photoreceptor synapses, suggesting that they have additional functions at these synapses. The cytoplasmic domain of mammalian Fat1 has been shown to bind Ena/Vasp proteins and to regulate actin dynamics at cell-cell contacts. In Drosophila, absence of Fat, but not of Fat-like results in hyperproliferation of cells in imaginal disks. Fat also is involved in regulation of planar cell polarity (PCP) where it appears to modulate the strength of Wnt-Frizzled signaling although the mechanisms through which it interacts with the planar polarity pathway are not understood. It seems likely that future studies will reveal diverse functions for these proteins, including roles at the synapse, but at present these are not clear. Concluding remarks Here we have summarized present knowledge on the regulation and function of cadherin superfamily proteins known to be expressed at synapses or involved in synapse formation. Many of the mechanistic studies on have been performed in non-neuronal cell types and it will be fascinating determine whether these mechanisms govern their synaptic functions. Future studies are also expected to reveal the functions of individual genes and proteins in different types of synapse at different stages in their development.

17 Further reading Adato, A., Michel, V., Kikkawa, Y., Reiners, J., Alagramam, K.N., Weil, D., Yonekawa, H., Wolfrum, U., El-Amraoui, A., and Petit, C. (2005). Interactions in the network of Usher syndrome type 1 proteins. Hum Mol Genet 14, Bamji, S.X. (2005). Cadherins: actin with the cytoskeleton to form synapses. Neuron 47, Elia, L.P., Yamamoto, M., Zang, K., and Reichardt, L.F. (2006). p120 Catenin Regulates Dendritic Spine and Synapse Development through Rho-Family GTPases and Cadherins. Neuron 51, Hirayama, T., and Yagi, T. (2006). The role and expression of the protocadherin-alpha clusters in the CNS. Curr Opin Neurobiol 16, Inoue, A., and Sanes, J.R. (1997). Lamina-specific connectivity in the brain: regulation by N- cadherin, neurotrophins, and glycoconjugates. Science 276, Junghans, D., Haas, I.G., and Kemler, R. (2005). Mammalian cadherins and protocadherins: about cell death, synapses and processing. Curr Opin Cell Biol 17, Kimura, H., Usui, T., Tsubouchi, A., and Uemura, T. (2006). Potential dual molecular interaction of the Drosophila 7-pass transmembrane cadherin Flamingo in dendritic morphogenesis. J Cell Sci 119, Leckband, D., and Prakasam, A. (2006). Mechanism and dynamics of cadherin adhesion. Annu Rev Biomed Eng 8, Lee, C.H., Herman, T., Clandinin, T.R., Lee, R., and Zipursky, S.L. (2001). N-cadherin regulates target specificity in the Drosophila visual system. Neuron 30,

18 Lee, R.C., Clandinin, T.R., Lee, C.H., Chen, P.L., Meinertzhagen, I.A., and Zipursky, S.L. (2003). The protocadherin Flamingo is required for axon target selection in the Drosophila visual system. Nat Neurosci 6, Lilien, J., and Balsamo, J. (2005). The regulation of cadherin-mediated adhesion by tyrosine phosphorylation/dephosphorylation of beta-catenin. Curr Opin Cell Biol 17, Prakash, S., Caldwell, J.C., Eberl, D.F., and Clandinin, T.R. (2005). Drosophila N-cadherin mediates an attractive interaction between photoreceptor axons and their targets. Nat Neurosci 8, Reiners, J., Nagel-Wolfrum, K., Jurgens, K., Marker, T., and Wolfrum, U. (2006). Molecular basis of human Usher syndrome: deciphering the meshes of the Usher protein network provides insights into the pathomechanisms of the Usher disease. Exp Eye Res 83, Salinas, P.C., and Price, S.R. (2005). Cadherins and catenins in synapse development. Curr Opin Neurobiol 15, Takeichi, M., and Abe, K. (2005). Synaptic contact dynamics controlled by cadherin and catenins. Trends Cell Biol 15, Tanoue, T., and Takeichi, M. (2005). New insights into Fat cadherins. J Cell Sci 118, Togashi, H., Abe, K., Mizoguchi, A., Takaoka, K., Chisaka, O., and Takeichi, M. (2002). Cadherin regulates dendritic spine morphogenesis. Neuron 35, Weiner, J.A., Wang, X., Tapia, J.C., and Sanes, J.R. (2005). Gamma protocadherins are required for synaptic development in the spinal cord. Proc Natl Acad Sci U S A. 102, Wheelock, M.J., and Johnson, K.R. (2003). Cadherins as modulators of cellular phenotype. Annu Rev Cell Dev Biol 19,

19 Yagi, T., and Takeichi, M. (2000). Cadherin superfamily genes: functions, genomic organization, and neurologic diversity. Genes Dev 14, Yu, X., and Malenka, R. C. (2003). _β-catenin is critical for dendritic morphogenesis. Nature Neurosci 6,

20 Table 1. The synaptic phenotypes upon perturbation and suggested function of main cadherin components Class Molecules System Mechanism of Inhibition and Synaptic phenotypes Function Classical cadherin N-Cadherin Cadherin-11 β-catenin αn-catenin p120 catenin δ-catenin Chick optic tectum in vivo Thalamic slice culture Drosophila visual system Cultured hippocampal neurons Mouse hippocampus Hippocampal pyramidal neurons in vivo and in vitro Cultured hippocampal neurons Cultured hippocampal neurons Hippocampal pyramidal neurons in vivo and in vitro Hippocampal pyramidal neurons in vivo Ab Inhibition: overshooting of retinal axons that fail to stop in normal target layers Ab Inhibition: overshooting of thalamic afferent beyond layer IV to the pial surface Genetic mutation: Disruption of target selection by R1-R8 cell axons in the lamina and medulla Dominant negative inhibition: longer dendritic protrusions; smaller spine head width; decreased presynaptic puncta; inhibition of late phase LTP Genetic mutation: increased LTP in CA1 region,; altered fearand anxiety-related responses Genetic mutation: disruption in the localization of synaptic vesicles and synaptic transmission Phosphorylation mutant (Y654F): increased spine β-catenin levels; increased PSD size and mepsc frequency Genetic mutation: increased dendritic spine length and decreased spine stability Overexpression: increased spine density and stabilization of spines Genetic mutation: reduced dendritic spine and synapse densities in vivo and in vitro; misregulated Rho family GTPase activity Genetic mutation: cognitive defect; impaired short-term and long-term plasticity Protocadherin Protocadherin-γ Mouse spinal cord Genetic mutation: decreased spinal cord synapse density, reduced synaptic current amplitude, sensori-motor defect Seven-pass transmembrane cadherin Flamingo Drosophila visual system Genetic mutation: inappropriate target selection by R1-R6 cell axons in the lamina; disruption of local pattern of synaptic terminals in the medulla Target recognition Target recognition Target recognition Synaptic maturation Synaptic plasticity Maturation of synapse Synaptic plasticity? Maturation of synapse Maturation of synapse Maturation of synapse; synaptic plasticity Induction or maturation of synapse Target recognition

21 A A C C D D B B E E

22 ? CASK/Mint /Veli Synaptic vesicles α-catenin β-catenin Presynaptic membrane p120 catenins β-catenin S-SCAM GluR6 Cdk5 LAR RICS IQGAP Dynein PI3 kinase PTP-κ SHP-2 α-catenin Afadin Formin α-actinin Spectrin Vinculin ZO-1 Zyxin Receptors Rac Cadherin Presenilin-1 AMPAR PTP-µ PTP1B Cortactin p120 catenin Fer Fyn Yes Kinesin PTP-µ Rho δ-catenin S-SCAM Densin-180 Presenilin-1 mglur1a NR2A Abl Erbin Cortactin Postsynaptic membrane

23 Figure 1. Importance of cadherin complex in synapse development. Examples of synaptic phenotypes resulting from perturbations of cadherin and catenin functions are shown. (A, A ) N-cadherin regulates lamina-specific connectivity in the brain. Injection of N-cadherin monoclonal antibody-producing hybridoma into chick optic tectum results in extension of some retinal axons past their normal target in laminae B and D in the optic tectum in vivo. Retinal axons were visualized by DiI injection. Reproduced by permission from Inoue, A., and Sanes, J. R., (B, B ) In Drosophila N-cadherin is required for R cell axon target connectivity. In cadherin mutant animals, the normal projection patterning by R1-R6 axons is disrupted and R cell growth cones fail to extend to their normal targets. DiI was injected into single ommatidia. Data are kindly provided by Thomas R. Clandinin. (C, C ) β-catenin plays a role in synaptic vesicle localization and presynaptic assembly. Deletion of β-catenin results in a reduction of vesicle number per synapse in the stratum radiatum of the CA1 hippocampus. (D. D ) p120catenin regulates dendritic spine development in vivo. Pyramidal neurons from the CA1 hippocampus which lacks p120 catenin exhibit reduced spine number compare to control neurons. Pyramidal neurons were visualized by Golgi-staining. (E, E ) N-cadherin regulates dendritic spine morphogenesis of hippocampal pyramidal neurons. Neurons expressing cn390δ, an N-cadherin mutant which lacks its extracellular domain, form immature and elongated filopodial-like spines and spines with bifurcated heads. Cells were stained with F-actin (green). Transfected cells were labeled in red. Reproduced by permission from Togashi, H. et al., Figure 2. Proteins constituents and interactions in cadherin adhesion complexes. Classical cadherins interact with catenins. Both cadherins and catenins also interact with diverse additional proteins, thereby regulating many intracellular signaling pathways. Reported

24 interacting partners of cadherin, β-catenin, α-catenin, p120 catenin and δ-catenin are listed. The proteins which have been shown to interact with cadherin components in the synapse are shown in blue. Cadherin signaling regulates the actin cytoskeleton through p120 catenin familymediated regulation of Rho family GTPases and cortactin. Through association with cadherins mediated through β-catenin, α-catenin provides a physical link to actin-binding proteins and the F-actin cytoskeleton.

25 Suggested cross-references to other articles MS 335 Extracellular matrix (laminin, fibronectin, cadherin, integrin) MS 347 Dendritic development (Activity, Wnt, & Rho GTPases) MS 348 Developmental synaptic plasticity: LTP, LTD, and synapse formation and elimination MS 349 Presynaptic development & active zones MS 351 Retrograde transsynaptic influences MS355 Synaptic adhesion molecules MS 356 β-catenin: localization of synaptic vesicles MS 357 Postsynaptic development, muscle (agrin, MuSK, rapsyn, neuregulin) MS 360 Postsynaptic development, neuronal (molecular scaffolds) MS1788 Assembly of postsynaptic specialization