- Email: [email protected]
Cadherin-mediated adhesion at the interneuronal synapse
593
Cadherin-mediated adhesion at the interneuronal synapse Juan L Brusés One of the recent advances in the molecular definition of a synapse has been the identification of cadherins as major structural components. The presence of classic (N- and E-) cadherins in the synaptic complex is not surprising considering the ultrastructural similarities between interneuronal synapses and the adhesive junctions formed between epithelial cells. However, the role of these adhesion molecules and their junctions in this context is likely to encompass both developmental and physiological phenomena that are unique to the synapse. Moreover, the recent finding that a much broader family of cadherin-related receptors is also located at the synaptic complex has fuelled speculation that cadherins have a role in generation of specificity in synaptic connectivity as well as structure. Addresses Cellular Biochemistry and Biophysics Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, New York 10021, USA; e-mail: [email protected] Current Opinion in Cell Biology 2000, 12:593–597 0955-0674/00/$ — see front matter © 2000 Elsevier Science Ltd. All rights reserved. Abbreviations AJ adherens junction apCAM Aplysia cell adhesion molecule CNS central nervous system CNRs cadherin-related neuronal receptors E-cadherin epithelial cadherin EM electron microscopy LTP long-term potentiation N-cadherin neural cadherin NCAM neural cell adhesion molecule NMDA N-methyl-D-aspartic acid PA puncta adherentia Pcadh protocadherin SJ synaptic junction
Introduction The interneuronal synaptic complex consists of the synaptic junction (SJ), which includes pre-and postsynaptic elements for neurotransmission, and, in many cases, a second adhesive structure called the puncta adherentia (PA) (Figure 1) [1]. SJs and PAs are easily distinguishable at the ultrastructural level. PAs are symmetrical structures similar to the adherens junction (AJ) formed between epithelial cells, whereas SJs are asymmetrical junctions with a large thickening of the postsynaptic membrane. In addition, neurotransmitter vesicles are found in the proximity of the presynaptic membrane of SJs (Figure 1). Both light and electron microscopy (EM) studies have indicated that not all synapses are associated with PAs and that PAs exist at many sites other than synapses [1–3]. PAs are much more abundant in developing tissue than in adult neural tissues [2,4], and their level of association with the synaptic complex varies between different regions of the
nervous system [3,5]. Thus, it appears that the presence or absence of PAs at a synaptic complex reflects the functional or developmental state of the synapse. It remains unclear, however, whether the presence of PAs next to the SJ affects synaptic transmission.
Cadherin-mediated synaptic adhesion: ‘o-rings’ or ‘spot welds’ The classic cadherins and their associated proteins (called catenins) are highly concentrated at AJ in epithelia and other tissues, where together they serve as a major adhesive component [6–9]. Both cadherins and their junctional partners catenins have been detected within the synaptic complex by confocal fluorescence microscopy and by immunogold EM [2,10], indicating that PAs in the nervous system are cadherin-mediated junctions similar, if not identical, to AJs. Two morphologically distinct forms of cadherin-based adhesive structures have been described at the synaptic complex: a spot-shaped structure or macula, which corresponds to the PA, and a belt-like structure surrounding the SJ, which resembles the zonula adherens (ZA) of epithelial cells. Utilizing confocal microscopic analysis, Fannon and Colman [10] detected N- and E-cadherin near the area of transmitter release in the en marron and glomerular synapses of the mouse cerebellum and proposed a model in which the synaptic active zone is surrounded by an adhesion belt formed by cadherins [10]. In agreement with this model, immunogold EM studies of synapse cross-sections localized αN- and β-catenins at both sides of the active zone in the adult mouse central nervous system (CNS) [2]. However, belt-shaped AJs are not ubiquitous in the CNS. Confocal observations of synapses with labeled synaptophysin have shown a punctuate distribution of catenins within the synaptic area [2], and these cadherin-positive spots are frequently located next to the transmitter release zone [10]. Morphological studies utilizing three-dimensional EM reconstruction of axo-spinous hippocampal synapses have determined that their PAs almost invariably have a macular shape [3,5,11]. Although these studies do not establish the presence of cadherins, their morphology and location closely resembles cadherin-mediated junctions. In summary, AJs may be belt-shaped in certain types of synapses but are more commonly present as macular PAs. This distinction is important in that it suggests that cadherin-based adhesion may serve to stabilize the synaptic complex but not necessarily to seal its borders against molecular diffusion. Finally, it should be noted that cadherins that are not clustered into junctions can still mediate adhesion [8] and the SJ itself has adhesive properties, thus the presence or absence of PAs alone should not be taken as a reflection of the adhesive stability of the synaptic complex.
594
Cell-to-cell contact and extracellular matrix
of the mutually exclusive distribution of N- and E-cadherins in excitatory and inhibitory synaptic complexes, it was proposed that cadherins may establish specific neuronal connectivity [10,12–14]. Similarly, the distribution of several classic cadherins in distinct structures, layers, and circuits in the nervous system indicates that these molecules could play an important role in the formation and interconnection of brain structures [15–27,28•]. In any case, the relatively small family of classic cadherins can only account for a limited number of the molecular codes that are required for establishing complex neuronal connectivity. Thus, attention has recently turned to the protocadherins (Pcadhs), which constitute a much larger family of neural receptors [13,29].
Figure 1
(a)
Protocadherins: candidates for neural specificity Protocadherins are cadherin-related transmembrane receptors that constitute a diverse family with a number of subgroups [30–34]. A distinct Pcadh subfamily found in mice, called the cadherin-related neuronal receptors (CNRs), has been localized by immunogold EM to SJs in the mouse neocortex [35]. CNRs were isolated by their ability to bind to the tyrosine kinase Fyn and are exclusively expressed in the nervous system of both developing and adults animals. More than 20 members of the family have been isolated, and different neurons express distinct combinations of CNRs, indicating that they might participate in the creation of specific neuronal properties [35]. Although Pcadhs possess the features of adhesion molecules [30], their adhesive properties and presence at the synaptic complex have not been well established, with the exception of certain members of the CNR subfamily [35].
(b)
Pre m
v
PSD SJ
PA
Post Current Opinion in Cell Biology
Cellular components of the synaptic complex. (a) A microphotograph of an interneuronal chemical synapse in the chick ciliary ganglion. This photograph illustrates the components of the synaptic complex which are labelled in the drawing shown in (b). The presynaptic terminal (Pre) contains a high number of synaptic vesicles (v) filled with neurotransmitters, and receptors are concentrated on the surface of the postsynaptic elements (Post), which also contain the machinery for signal transduction. The area within the larger bracket corresponds to the synaptic junction (SJ) and active zone where neurotransmission is believed to take place. This structure is easily identifiable by the presence of synaptic vesicles in the proximity of the presynaptic membrane and the larger thickening of the postsynaptic membrane (PSD). The puncta adherentia (PA) is a distinct adhesive structure associated with the synaptic complex with a parallel and symmetrical disposition of both membranes. (b) The major components of the synaptic complex and their characteristic features from the photograph in (a) are outlined in this illustration. Pre, presynaptic terminal; Post, Post-synaptic element; v, synaptic vesicles; PSD, postsynaptic density; m, mitochondria; SJ, synaptic junction; PA, puncta adherentia. Scale bar, 250 nm.
Classic cadherins and neuronal connectivity The change in surface distribution of cadherins — from a diffuse to a punctuate pattern — during the in vitro formation of synaptic contacts suggests that cadherins might participate in the formation of the synapse [12]. Furthermore, on the basis
The proposed role of Pcadh in the formation and maintenance of specific neuronal connections has been further supported by the recent discovery of 52 novel human Pcadh genes [36••]. The Pcadh ectodomain possesses characteristics of the classic cadherins in terms of domain structure and conserved amino acids at relevant positions, however, both families of molecules have distinct structural features. For example, Pcadhs contain six or seven cadherin domains within the extracellular region, whereas classic cadherin ectodomains are composed of five cadherin domains [30]. Also, the His–Ala–Val (HAV) tripetide and flanking regions that are well conserved among classic cadherins are replaced in Pcadhs by a similar but not identical sequence [36••]. Finally, classic cadherins have a highly conserved cytoplasmic domain, whereas Pcadhs possess a divergent cytoplasmic tail [30]. Remarkably, Pcadh genes have a genomic organization that could potentially provide significant molecular diversity. In contrast to the genomic sequence of the classic cadherin extracellular domain, which is encoded by multiple exons interrupted by several introns, each Pcadh ectodomain is encoded by a single large exon. The Pcadhs genes are located in the same region of the genome, and they are clustered in three groups namely Pcadh-α, -β, and -δ, which contain at least 15, 15, and 22 genes respectively [36••,37]. Each
Cadherin-mediated adhesion at the interneuronal synapse Brusés
gene family is composed of multiple exons, each of which encodes a similar but nonidentical ectodomain. Downstream of this region are three exons that encode the cytoplasmic domain of the protein, which varies among Pcadhs subfamilies, indicating that the cytoplasmic-binding domains are the same within a particular subfamily, but different between the three groups. A combination of transcriptional mechanisms, including a single promoter for each group of exons or a single promoter for each exon both followed by an alternative splicing step, could in principle operate over this genomic architecture and provide a diversity of proteins with varying extracellular domains and constant cytoplasmic tails [36••]. The regulatory mechanisms that control the rearrangement and expression of Pcadhs is as yet not understood, nevertheless, the generation of a variety of similar cadherin-like molecules that differ in their binding properties while transducing the same intracellular signal in distinct cells, could translate into a large repertoire of cell–cell interactions. The ability of the neuron to recognize its neighbors and identify its potential targets is a key process in the formation of neuronal connections. Thus, the potential diversity of surface receptors provided by the Pcadh family may contribute to the establishment of neuronal connectivity [36••,38•].
Modification of adhesion at synaptic contacts In addition to synaptogenesis, synaptic changes in the expression level and functional state of adhesion molecules are believed to be required for the structural and functional modifications associated with plasticity of the mature synapse [39]. In Aplysia, long-term facilitation of synaptic efficacy induced by neuronal activity is accompanied by a downregulation of the surface expression of apCAM, the Aplysia homologue of NCAM [40,41]. In agreement with the hypothesis that an activity-dependent reduction in surface adhesion is required for synaptic plasticity, a number of studies have shown that function-blocking antibodies or peptides against various adhesion molecules (e.g. NCAM, L1, integrins, and cadherins) can block or affect long-term potentiation [29,39,42,43]. Several different mechanisms have been proposed for the regulation of cadherin-mediated adhesion [44]. In a recent study, Tanaka and co-workers [45••] addressed at the molecular level the effect of neuronal activity on the conformational and functional state of N-cadherin in cultured hippocampal neurons. Interestingly, it was found that depolarization caused by NMDA either induces dimerization or chemically stabilizes any N-cadherin dimers already present. This renders the molecule resistant to enzymatic proteolytic degradation. N-cadherin dimer formation [46,47] and the clustering of multiple molecules [48] are both known to stabilize homophilic interactions and strengthen surface adhesion. The consequences of these changes for synaptic function are not yet known but imply that neuronal activity further stabilizes the pre- and postsynaptic membrane.
595
From the studies of apCAM, it would seem illogical that an increase in synaptic adhesion enhances synaptic plasticity. However, if N-cadherin is localized at restricted areas of the synaptic complex, such as the PA, the stabilization of this adhesive structure associated with the SJ might allow for structural changes at the synaptic active zone without the loss of the synaptic contact. Stimulation protocols that lead to long-term potentiation cause significant structural changes in dendritic spines [49–52], and excessive activation can cause the loss of spines [53]. The strengthening of the synaptic complex via a cadherin-mediated PA might therefore help to maintain synaptic contact between the bouton and the highly dynamic dendritic spines during this period of intense activity and structural remodeling.
Conclusions and future directions From the studies discussed here, it seems that classic cadherins and Pcadhs both play important roles in the formation and function of synaptic connections. Although classic cadherins are likely to participate in the establishment of the synaptic junction and remain as an adhesive mechanism between the pre- and postsynaptic membranes, Pcadh may be better suited to specification of neuronal connectivity. This specification may be brought about by the larger diversity of receptors generated from the Pcadh family. Both systems might influence remodeling of the adult nervous system. Further studies are needed to define the mode and extent to which cadherins participate in the formation and maintenance of a synaptic contact. Unfortunately, targeted gene disruption has not provided much information about the synaptic role of cadherins because of embryo lethality at early developmental stages. The generation of transgenic animals that express mutated molecules with dominantnegative effects under the control of inducible promoters might allow manipulation of cadherin function at particular developmental stages. This would allow its role in synapse assembly to be assessed. The use of in vitro models of synapse formation using genetically modified cells might provide an alternative approach. Finally, characterizing the expression patterns of individual Pcadh subfamily members, their adhesive capabilities and understanding the role of their signaling pathways will be necessary to determine whether these molecules provide the molecular codes required for specific neuronal connectivity.
Acknowledgements I thank Urs Rutishauser and Barry Gumbiner for their critical review of this manuscript.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest •• of outstanding interest 1.
Peters A, Palay SL, Webster HD: The Fine Structure of the Nervous System. New York: Oxford University Press; 1991.
596
Cell-to-cell contact and extracellular matrix
2.
Uchida N, Honjo Y, Johnson KR, Wheelock MJ, Takeichi M: The catenin/cadherin adhesion system is localized in synaptic junctions bordering transmitter release zones. J Cell Biol 1996, 135:767-779.
3.
Spacek J, Harris KM: Three-dimensional organization of cell adhesion junctions at synapses and dendritic spines in area CA1 of the rat hippocampus. J Comp Neurol 1998, 393:58-68.
4.
Vaughn JE: Fine structure of synaptogenesis in the vertebrate central nervous system. Synapse 1989, 3:255-285.
5.
Spacek J: Relationships between synaptic junctions, puncta adhaerentia and the spine apparatus at neocortical axo-spinous synapses. A serial section study. Anat Embryol (Berl) 1985, 173:129-135.
6.
Takeichi M: Morphogenetic roles of classic cadherins. Curr Opin Cell Biol 1995, 7:619-627.
7.
Gumbiner BM: Cell adhesion: the molecular basis of tissue architecture and morphogenesis. Cell 1996, 84:345-357.
8.
Yap AS, Brieher WM, Gumbiner BM: Molecular and functional analysis of cadherin-based adherens junctions. Annu Rev Cell Dev Biol 1997, 13:119-146.
9.
Gottardi C, Niessen CM, Gumbiner BM: The Adherens Junction. Edited by Beckerle M. Oxford: Oxford University Press; 2000.
10. Fannon AM, Colman DR: A model for central synaptic junctional complex formation based on the differential adhesive specificities of the cadherins. Neuron 1996, 17:423-434. 11. Szumowski KM, Harris KM: Pictures in cell biology. Threedimensional reconstruction of synaptic ultrastructures. Trends Cell Biol 1999, 9:205. 12. Benson DL, Tanaka H: N-cadherin redistribution during synaptogenesis in hippocampal neurons. J Neurosci 1998, 18:6892-6904. 13. Uemura T: The cadherin superfamily at the synapse: more members, more missions. Cell 1998, 93:1095-1098. 14. Shapiro L, Colman DR: The diversity of cadherins and implications for a synaptic adhesive code in the CNS. Neuron 1999, 23:427-430. 15. Matsunami H, Takeichi M: Fetal brain subdivisions defined by R- and E-cadherin expressions: evidence for the role of cadherin activity in region-specific, cell-cell adhesion. Dev Biol 1995, 172:466-478. 16. Redies C, Takeichi M: Cadherins in the developing central nervous system: an adhesive code for segmental and functional subdivisions. Dev Biol 1996, 180:413-423. 17.
Takeichi M, Uemura T, Iwai Y, Uchida N, Inoue T, Tanaka T, Suzuki SC: Cadherins in brain patterning and neural network formation. Cold Spring Harb Symp Quant Biol 1997, 62:505-510.
18. Takeichi M, Matsunami H, Inoue T, Kimura Y, Suzuki S, Tanaka T: Roles of cadherins in patterning of the developing brain. Dev Neurosci 1997, 19:86-87. 19. Suzuki SC, Inoue T, Kimura Y, Tanaka T, Takeichi M: Neuronal circuits are subdivided by differential expression of type-II classic cadherins in postnatal mouse brains. Mol Cell Neurosci 1997, 9:433-447. 20. Sanes JR, Yamagata M: Formation of lamina-specific synaptic connections. Curr Opin Neurobiol 1999, 9:79-87. 21. Inoue T, Tanaka T, Suzuki SC, Takeichi M: Cadherin-6 in the developing mouse brain: expression along restricted connection systems and synaptic localization suggest a potential role in neuronal circuitry. Dev Dyn 1998, 211:338-351. 22. Arndt K, Redies C: Development of cadherin-defined parasagittal subdivisions in the embryonic chicken cerebellum. J Comp Neurol 1998, 401:367-381. 23. Arndt K, Nakagawa S, Takeichi M, Redies C: Cadherin-defined segments and parasagittal cell ribbons in the developing chicken cerebellum. Mol Cell Neurosci 1998, 10:211-228. 24. Wohrn JC, Puelles L, Nakagawa S, Takeichi M, Redies C: Cadherin expression in the retina and retinofugal pathways of the chicken embryo. J Comp Neurol 1998, 396:20-38.
25. Miskevich F, Zhu Y, Ranscht B, Sanes JR: Expression of multiple cadherins and catenins in the chick optic tectum. Mol Cell Neurosci 1998, 12:240-255. 26. Iwai Y, Usui T, Hirano S, Steward R, Takeichi M, Uemura T: Axon patterning requires DN-cadherin, a novel neuronal adhesion receptor, in the Drosophila embryonic CNS. Neuron 1997, 19:77-89. 27.
Huntley GW, Benson DL: Neural (N)-cadherin at developing thalamocortical synapses provides an adhesion mechanism for the formation of somatopically organized connections. J Comp Neurol 1999, 407:453-471.
28. Wohrn JC, Nakagawa S, Ast M, Takeichi M, Redies C: Combinatorial • expression of cadherins in the tectum and the sorting of neurites in the tectofugal pathways of the chicken embryo. Neuroscience 1999, 90:985-1000. This study describes the distribution and combinatorial localization of classic cadherins in the chick visual system. The authors characterize the cadherin expression in the tectal layers, retina, and their projections into the tectum. The results, although descriptive, support the view that different cadherins are players in the formation of particular brain structures. 29. Hagler DJ Jr, Goda Y: Synaptic adhesion: the building blocks of memory? Neuron 1998, 20:1059-1062. 30. Sano K, Tanihara H, Heimark RL, Obata S, Davidson M, St John T, Taketani S, Suzuki S: Protocadherins: a large family of cadherinrelated molecules in central nervous system. EMBO J 1993, 12:2249-2256. 31. Obata S, Sago H, Mori N, Rochelle JM, Seldin MF, Davidson M, St John T, Taketani S, Suzuki ST: Protocadherin Pcdh2 shows properties similar to, but distinct from, those of classical cadherins. J Cell Sci 1995, 108:3765-3773. 32. Obata S, Sago H, Mori N, Davidson M, St John T, Suzuki ST: A common protocadherin tail: multiple protocadherins share the same sequence in their cytoplasmic domains and are expressed in different regions of brain. Cell Adhes Commun 1998, 6:323-333. 33. Hirano S, Yan Q, Suzuki ST: Expression of a novel protocadherin, OL-protocadherin, in a subset of functional systems of the developing mouse brain. J Neurosci 1999, 19:995-1005. 34. Yagi T, Takeichi M: Cadherin superfamily genes: functions, genomic organization, and neurologic diversity. Genes Dev 2000, 14:1169-1180. 35. Kohmura N, Senzaki K, Hamada S, Kai N, Yasuda R, Watanabe M, Ishii H, Yasuda M, Mishina M, Yagi T: Diversity revealed by a novel family of cadherins expressed in neurons at a synaptic complex. Neuron 1998, 20:1137-1151. 36. Wu Q, Maniatis T: A striking organization of a large family of •• human neural cadherin-like cell adhesion genes. Cell 1999, 97:779-790. The identification of 52 novel human cadherin-like genes is reported in this study. Using an iterative BLAST search analysis and a computer-assisted sequence analysis of a portion of the human genome, the study described 52 Pcadh genes organized into three tandem gene clusters. Multiple variable single exons encoding the extracellular domain of the protein are located upstream of three constant exons that encode the cytoplasmic domain. This genomic organization indicates that a number of different molecular combinations can be achieved via gene rearrangement. This genomic structure further supports the hypothesis that Pcadh can establish a complex molecular code for neuronal connectivity. 37.
Sugino H, Hamada S, Yasuda R, Tuji A, Matsuda Y, Fujita M, Yagi T: Genomic organization of the family of CNR cadherin genes in mice and humans. Genomics 2000, 63:75-87.
38. Wu Q, Maniatis T: Large exons encoding multiple ectodomains are • a characteristic feature of protocadherin genes. Proc Natl Acad Sci USA 2000, 97:3124-3129. See annotation [36••]. 39. Murase S, Schuman EM: The role of cell adhesion molecules in synaptic plasticity and memory. Curr Opin Cell Biol 1999, 11:549-553. 40. Mayford M, Barzilai A, Keller F, Schacher S, Kandel ER: Modulation of an NCAM-related adhesion molecule with long-term synaptic plasticity in Aplysia. Science 1992, 256:638-644. 41. Zhu H, Wu F, Schacher S: Changes in expression and distribution of Aplysia cell adhesion molecules can influence synapse formation and elimination in vitro. J Neurosci 1995, 15:4173-4183.
Cadherin-mediated adhesion at the interneuronal synapse Brusés
597
42. Tang L, Hung CP, Schuman EM: A role for the cadherin family of cell adhesion molecules in hippocampal long-term potentiation. Neuron 1998, 20:1165-1175.
47.
43. Yamagata K, Andreasson KI, Sugiura H, Maru E, Dominique M, Irie Y, Mik N, Hayashi Y, Yoshioka M, Kaneko K et al.: Arcadlin is a neural activity-regulated cadherin involved in long term potentiation. J Biol Chem 1999, 274:19473-19479.
48. Yap AS, Brieher WM, Pruschy M, Gumbiner BM: Lateral clustering of the adhesive ectodomain: a fundamental determinant of cadherin function. Curr Biol 1997, 7:308-315.
44. Gumbiner BM: Regulation of cadherin adhesive activity. J Cell Biol 2000, 148:399-404.
49. Engert F, Bonhoeffer T: Dendritic spine changes associated with hippocampal long-term synaptic plasticity. Nature 1999, 399:66-70.
45. Tanaka H, Shan W, Phillips GR, Arndt K, Bozdagi O, Shapiro L, •• Huntley GW, Benson DL, Colman DR: Molecular modification of N-cadherin in response to synaptic activity. Neuron 2000, 25:93-107. Utilizing an in vitro system of synapses formed between hippocampal neurons, this study demonstrates that neuronal depolarization induced by NMDA causes N-cadherin dimerization at the synapse. The N-cadherin redistribution occurs in 30 min and returns to normal levels in 2 hours. This study indicates that electrical activity induces an increase in synaptic adhesion via changes in the conformational state of N-cadherin. 46. Brieher WM, Yap AS, Gumbiner BM: Lateral dimerization is required for the homophilic binding activity of C-cadherin. J Cell Biol 1996, 135:487-496.
Tamura K, Shan WS, Hendrickson WA, Colman DR, Shapiro L: Structure-function analysis of cell adhesion by neural (N-) cadherin. Neuron 1998, 20:1153-1163.
50. Maletic-Savatic M, Malinow R, Svoboda K: Rapid dendritic morphogenesis in CA1 hippocampal dendrites induced by synaptic activity. Science 1999, 283:1923-1927. 51. Toni N, Buchs PA, Nikonenko I, Bron CR, Muller D: LTP promotes formation of multiple spine synapses between a single axon terminal and a dendrite. Nature 1999, 402:421-425. 52. Wong WT, Wong RO: Rapid dendritic movements during synapse formation and rearrangement. Curr Opin Neurobiol 2000, 10:118-124. 53. Harris KM: Structure, development, and plasticity of dendritic spines. Curr Opin Neurobiol 1999, 9:343-348.
Cadherin-mediated adhesion at the interneuronal synapse Juan L Brusés One of the recent advances in the molecular definition of a synapse has been the identification of cadherins as major structural components. The presence of classic (N- and E-) cadherins in the synaptic complex is not surprising considering the ultrastructural similarities between interneuronal synapses and the adhesive junctions formed between epithelial cells. However, the role of these adhesion molecules and their junctions in this context is likely to encompass both developmental and physiological phenomena that are unique to the synapse. Moreover, the recent finding that a much broader family of cadherin-related receptors is also located at the synaptic complex has fuelled speculation that cadherins have a role in generation of specificity in synaptic connectivity as well as structure. Addresses Cellular Biochemistry and Biophysics Program, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, New York 10021, USA; e-mail: [email protected] Current Opinion in Cell Biology 2000, 12:593–597 0955-0674/00/$ — see front matter © 2000 Elsevier Science Ltd. All rights reserved. Abbreviations AJ adherens junction apCAM Aplysia cell adhesion molecule CNS central nervous system CNRs cadherin-related neuronal receptors E-cadherin epithelial cadherin EM electron microscopy LTP long-term potentiation N-cadherin neural cadherin NCAM neural cell adhesion molecule NMDA N-methyl-D-aspartic acid PA puncta adherentia Pcadh protocadherin SJ synaptic junction
Introduction The interneuronal synaptic complex consists of the synaptic junction (SJ), which includes pre-and postsynaptic elements for neurotransmission, and, in many cases, a second adhesive structure called the puncta adherentia (PA) (Figure 1) [1]. SJs and PAs are easily distinguishable at the ultrastructural level. PAs are symmetrical structures similar to the adherens junction (AJ) formed between epithelial cells, whereas SJs are asymmetrical junctions with a large thickening of the postsynaptic membrane. In addition, neurotransmitter vesicles are found in the proximity of the presynaptic membrane of SJs (Figure 1). Both light and electron microscopy (EM) studies have indicated that not all synapses are associated with PAs and that PAs exist at many sites other than synapses [1–3]. PAs are much more abundant in developing tissue than in adult neural tissues [2,4], and their level of association with the synaptic complex varies between different regions of the
nervous system [3,5]. Thus, it appears that the presence or absence of PAs at a synaptic complex reflects the functional or developmental state of the synapse. It remains unclear, however, whether the presence of PAs next to the SJ affects synaptic transmission.
Cadherin-mediated synaptic adhesion: ‘o-rings’ or ‘spot welds’ The classic cadherins and their associated proteins (called catenins) are highly concentrated at AJ in epithelia and other tissues, where together they serve as a major adhesive component [6–9]. Both cadherins and their junctional partners catenins have been detected within the synaptic complex by confocal fluorescence microscopy and by immunogold EM [2,10], indicating that PAs in the nervous system are cadherin-mediated junctions similar, if not identical, to AJs. Two morphologically distinct forms of cadherin-based adhesive structures have been described at the synaptic complex: a spot-shaped structure or macula, which corresponds to the PA, and a belt-like structure surrounding the SJ, which resembles the zonula adherens (ZA) of epithelial cells. Utilizing confocal microscopic analysis, Fannon and Colman [10] detected N- and E-cadherin near the area of transmitter release in the en marron and glomerular synapses of the mouse cerebellum and proposed a model in which the synaptic active zone is surrounded by an adhesion belt formed by cadherins [10]. In agreement with this model, immunogold EM studies of synapse cross-sections localized αN- and β-catenins at both sides of the active zone in the adult mouse central nervous system (CNS) [2]. However, belt-shaped AJs are not ubiquitous in the CNS. Confocal observations of synapses with labeled synaptophysin have shown a punctuate distribution of catenins within the synaptic area [2], and these cadherin-positive spots are frequently located next to the transmitter release zone [10]. Morphological studies utilizing three-dimensional EM reconstruction of axo-spinous hippocampal synapses have determined that their PAs almost invariably have a macular shape [3,5,11]. Although these studies do not establish the presence of cadherins, their morphology and location closely resembles cadherin-mediated junctions. In summary, AJs may be belt-shaped in certain types of synapses but are more commonly present as macular PAs. This distinction is important in that it suggests that cadherin-based adhesion may serve to stabilize the synaptic complex but not necessarily to seal its borders against molecular diffusion. Finally, it should be noted that cadherins that are not clustered into junctions can still mediate adhesion [8] and the SJ itself has adhesive properties, thus the presence or absence of PAs alone should not be taken as a reflection of the adhesive stability of the synaptic complex.
594
Cell-to-cell contact and extracellular matrix
of the mutually exclusive distribution of N- and E-cadherins in excitatory and inhibitory synaptic complexes, it was proposed that cadherins may establish specific neuronal connectivity [10,12–14]. Similarly, the distribution of several classic cadherins in distinct structures, layers, and circuits in the nervous system indicates that these molecules could play an important role in the formation and interconnection of brain structures [15–27,28•]. In any case, the relatively small family of classic cadherins can only account for a limited number of the molecular codes that are required for establishing complex neuronal connectivity. Thus, attention has recently turned to the protocadherins (Pcadhs), which constitute a much larger family of neural receptors [13,29].
Figure 1
(a)
Protocadherins: candidates for neural specificity Protocadherins are cadherin-related transmembrane receptors that constitute a diverse family with a number of subgroups [30–34]. A distinct Pcadh subfamily found in mice, called the cadherin-related neuronal receptors (CNRs), has been localized by immunogold EM to SJs in the mouse neocortex [35]. CNRs were isolated by their ability to bind to the tyrosine kinase Fyn and are exclusively expressed in the nervous system of both developing and adults animals. More than 20 members of the family have been isolated, and different neurons express distinct combinations of CNRs, indicating that they might participate in the creation of specific neuronal properties [35]. Although Pcadhs possess the features of adhesion molecules [30], their adhesive properties and presence at the synaptic complex have not been well established, with the exception of certain members of the CNR subfamily [35].
(b)
Pre m
v
PSD SJ
PA
Post Current Opinion in Cell Biology
Cellular components of the synaptic complex. (a) A microphotograph of an interneuronal chemical synapse in the chick ciliary ganglion. This photograph illustrates the components of the synaptic complex which are labelled in the drawing shown in (b). The presynaptic terminal (Pre) contains a high number of synaptic vesicles (v) filled with neurotransmitters, and receptors are concentrated on the surface of the postsynaptic elements (Post), which also contain the machinery for signal transduction. The area within the larger bracket corresponds to the synaptic junction (SJ) and active zone where neurotransmission is believed to take place. This structure is easily identifiable by the presence of synaptic vesicles in the proximity of the presynaptic membrane and the larger thickening of the postsynaptic membrane (PSD). The puncta adherentia (PA) is a distinct adhesive structure associated with the synaptic complex with a parallel and symmetrical disposition of both membranes. (b) The major components of the synaptic complex and their characteristic features from the photograph in (a) are outlined in this illustration. Pre, presynaptic terminal; Post, Post-synaptic element; v, synaptic vesicles; PSD, postsynaptic density; m, mitochondria; SJ, synaptic junction; PA, puncta adherentia. Scale bar, 250 nm.
Classic cadherins and neuronal connectivity The change in surface distribution of cadherins — from a diffuse to a punctuate pattern — during the in vitro formation of synaptic contacts suggests that cadherins might participate in the formation of the synapse [12]. Furthermore, on the basis
The proposed role of Pcadh in the formation and maintenance of specific neuronal connections has been further supported by the recent discovery of 52 novel human Pcadh genes [36••]. The Pcadh ectodomain possesses characteristics of the classic cadherins in terms of domain structure and conserved amino acids at relevant positions, however, both families of molecules have distinct structural features. For example, Pcadhs contain six or seven cadherin domains within the extracellular region, whereas classic cadherin ectodomains are composed of five cadherin domains [30]. Also, the His–Ala–Val (HAV) tripetide and flanking regions that are well conserved among classic cadherins are replaced in Pcadhs by a similar but not identical sequence [36••]. Finally, classic cadherins have a highly conserved cytoplasmic domain, whereas Pcadhs possess a divergent cytoplasmic tail [30]. Remarkably, Pcadh genes have a genomic organization that could potentially provide significant molecular diversity. In contrast to the genomic sequence of the classic cadherin extracellular domain, which is encoded by multiple exons interrupted by several introns, each Pcadh ectodomain is encoded by a single large exon. The Pcadhs genes are located in the same region of the genome, and they are clustered in three groups namely Pcadh-α, -β, and -δ, which contain at least 15, 15, and 22 genes respectively [36••,37]. Each
Cadherin-mediated adhesion at the interneuronal synapse Brusés
gene family is composed of multiple exons, each of which encodes a similar but nonidentical ectodomain. Downstream of this region are three exons that encode the cytoplasmic domain of the protein, which varies among Pcadhs subfamilies, indicating that the cytoplasmic-binding domains are the same within a particular subfamily, but different between the three groups. A combination of transcriptional mechanisms, including a single promoter for each group of exons or a single promoter for each exon both followed by an alternative splicing step, could in principle operate over this genomic architecture and provide a diversity of proteins with varying extracellular domains and constant cytoplasmic tails [36••]. The regulatory mechanisms that control the rearrangement and expression of Pcadhs is as yet not understood, nevertheless, the generation of a variety of similar cadherin-like molecules that differ in their binding properties while transducing the same intracellular signal in distinct cells, could translate into a large repertoire of cell–cell interactions. The ability of the neuron to recognize its neighbors and identify its potential targets is a key process in the formation of neuronal connections. Thus, the potential diversity of surface receptors provided by the Pcadh family may contribute to the establishment of neuronal connectivity [36••,38•].
Modification of adhesion at synaptic contacts In addition to synaptogenesis, synaptic changes in the expression level and functional state of adhesion molecules are believed to be required for the structural and functional modifications associated with plasticity of the mature synapse [39]. In Aplysia, long-term facilitation of synaptic efficacy induced by neuronal activity is accompanied by a downregulation of the surface expression of apCAM, the Aplysia homologue of NCAM [40,41]. In agreement with the hypothesis that an activity-dependent reduction in surface adhesion is required for synaptic plasticity, a number of studies have shown that function-blocking antibodies or peptides against various adhesion molecules (e.g. NCAM, L1, integrins, and cadherins) can block or affect long-term potentiation [29,39,42,43]. Several different mechanisms have been proposed for the regulation of cadherin-mediated adhesion [44]. In a recent study, Tanaka and co-workers [45••] addressed at the molecular level the effect of neuronal activity on the conformational and functional state of N-cadherin in cultured hippocampal neurons. Interestingly, it was found that depolarization caused by NMDA either induces dimerization or chemically stabilizes any N-cadherin dimers already present. This renders the molecule resistant to enzymatic proteolytic degradation. N-cadherin dimer formation [46,47] and the clustering of multiple molecules [48] are both known to stabilize homophilic interactions and strengthen surface adhesion. The consequences of these changes for synaptic function are not yet known but imply that neuronal activity further stabilizes the pre- and postsynaptic membrane.
595
From the studies of apCAM, it would seem illogical that an increase in synaptic adhesion enhances synaptic plasticity. However, if N-cadherin is localized at restricted areas of the synaptic complex, such as the PA, the stabilization of this adhesive structure associated with the SJ might allow for structural changes at the synaptic active zone without the loss of the synaptic contact. Stimulation protocols that lead to long-term potentiation cause significant structural changes in dendritic spines [49–52], and excessive activation can cause the loss of spines [53]. The strengthening of the synaptic complex via a cadherin-mediated PA might therefore help to maintain synaptic contact between the bouton and the highly dynamic dendritic spines during this period of intense activity and structural remodeling.
Conclusions and future directions From the studies discussed here, it seems that classic cadherins and Pcadhs both play important roles in the formation and function of synaptic connections. Although classic cadherins are likely to participate in the establishment of the synaptic junction and remain as an adhesive mechanism between the pre- and postsynaptic membranes, Pcadh may be better suited to specification of neuronal connectivity. This specification may be brought about by the larger diversity of receptors generated from the Pcadh family. Both systems might influence remodeling of the adult nervous system. Further studies are needed to define the mode and extent to which cadherins participate in the formation and maintenance of a synaptic contact. Unfortunately, targeted gene disruption has not provided much information about the synaptic role of cadherins because of embryo lethality at early developmental stages. The generation of transgenic animals that express mutated molecules with dominantnegative effects under the control of inducible promoters might allow manipulation of cadherin function at particular developmental stages. This would allow its role in synapse assembly to be assessed. The use of in vitro models of synapse formation using genetically modified cells might provide an alternative approach. Finally, characterizing the expression patterns of individual Pcadh subfamily members, their adhesive capabilities and understanding the role of their signaling pathways will be necessary to determine whether these molecules provide the molecular codes required for specific neuronal connectivity.
Acknowledgements I thank Urs Rutishauser and Barry Gumbiner for their critical review of this manuscript.
References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest •• of outstanding interest 1.
Peters A, Palay SL, Webster HD: The Fine Structure of the Nervous System. New York: Oxford University Press; 1991.
596
Cell-to-cell contact and extracellular matrix
2.
Uchida N, Honjo Y, Johnson KR, Wheelock MJ, Takeichi M: The catenin/cadherin adhesion system is localized in synaptic junctions bordering transmitter release zones. J Cell Biol 1996, 135:767-779.
3.
Spacek J, Harris KM: Three-dimensional organization of cell adhesion junctions at synapses and dendritic spines in area CA1 of the rat hippocampus. J Comp Neurol 1998, 393:58-68.
4.
Vaughn JE: Fine structure of synaptogenesis in the vertebrate central nervous system. Synapse 1989, 3:255-285.
5.
Spacek J: Relationships between synaptic junctions, puncta adhaerentia and the spine apparatus at neocortical axo-spinous synapses. A serial section study. Anat Embryol (Berl) 1985, 173:129-135.
6.
Takeichi M: Morphogenetic roles of classic cadherins. Curr Opin Cell Biol 1995, 7:619-627.
7.
Gumbiner BM: Cell adhesion: the molecular basis of tissue architecture and morphogenesis. Cell 1996, 84:345-357.
8.
Yap AS, Brieher WM, Gumbiner BM: Molecular and functional analysis of cadherin-based adherens junctions. Annu Rev Cell Dev Biol 1997, 13:119-146.
9.
Gottardi C, Niessen CM, Gumbiner BM: The Adherens Junction. Edited by Beckerle M. Oxford: Oxford University Press; 2000.
10. Fannon AM, Colman DR: A model for central synaptic junctional complex formation based on the differential adhesive specificities of the cadherins. Neuron 1996, 17:423-434. 11. Szumowski KM, Harris KM: Pictures in cell biology. Threedimensional reconstruction of synaptic ultrastructures. Trends Cell Biol 1999, 9:205. 12. Benson DL, Tanaka H: N-cadherin redistribution during synaptogenesis in hippocampal neurons. J Neurosci 1998, 18:6892-6904. 13. Uemura T: The cadherin superfamily at the synapse: more members, more missions. Cell 1998, 93:1095-1098. 14. Shapiro L, Colman DR: The diversity of cadherins and implications for a synaptic adhesive code in the CNS. Neuron 1999, 23:427-430. 15. Matsunami H, Takeichi M: Fetal brain subdivisions defined by R- and E-cadherin expressions: evidence for the role of cadherin activity in region-specific, cell-cell adhesion. Dev Biol 1995, 172:466-478. 16. Redies C, Takeichi M: Cadherins in the developing central nervous system: an adhesive code for segmental and functional subdivisions. Dev Biol 1996, 180:413-423. 17.
Takeichi M, Uemura T, Iwai Y, Uchida N, Inoue T, Tanaka T, Suzuki SC: Cadherins in brain patterning and neural network formation. Cold Spring Harb Symp Quant Biol 1997, 62:505-510.
18. Takeichi M, Matsunami H, Inoue T, Kimura Y, Suzuki S, Tanaka T: Roles of cadherins in patterning of the developing brain. Dev Neurosci 1997, 19:86-87. 19. Suzuki SC, Inoue T, Kimura Y, Tanaka T, Takeichi M: Neuronal circuits are subdivided by differential expression of type-II classic cadherins in postnatal mouse brains. Mol Cell Neurosci 1997, 9:433-447. 20. Sanes JR, Yamagata M: Formation of lamina-specific synaptic connections. Curr Opin Neurobiol 1999, 9:79-87. 21. Inoue T, Tanaka T, Suzuki SC, Takeichi M: Cadherin-6 in the developing mouse brain: expression along restricted connection systems and synaptic localization suggest a potential role in neuronal circuitry. Dev Dyn 1998, 211:338-351. 22. Arndt K, Redies C: Development of cadherin-defined parasagittal subdivisions in the embryonic chicken cerebellum. J Comp Neurol 1998, 401:367-381. 23. Arndt K, Nakagawa S, Takeichi M, Redies C: Cadherin-defined segments and parasagittal cell ribbons in the developing chicken cerebellum. Mol Cell Neurosci 1998, 10:211-228. 24. Wohrn JC, Puelles L, Nakagawa S, Takeichi M, Redies C: Cadherin expression in the retina and retinofugal pathways of the chicken embryo. J Comp Neurol 1998, 396:20-38.
25. Miskevich F, Zhu Y, Ranscht B, Sanes JR: Expression of multiple cadherins and catenins in the chick optic tectum. Mol Cell Neurosci 1998, 12:240-255. 26. Iwai Y, Usui T, Hirano S, Steward R, Takeichi M, Uemura T: Axon patterning requires DN-cadherin, a novel neuronal adhesion receptor, in the Drosophila embryonic CNS. Neuron 1997, 19:77-89. 27.
Huntley GW, Benson DL: Neural (N)-cadherin at developing thalamocortical synapses provides an adhesion mechanism for the formation of somatopically organized connections. J Comp Neurol 1999, 407:453-471.
28. Wohrn JC, Nakagawa S, Ast M, Takeichi M, Redies C: Combinatorial • expression of cadherins in the tectum and the sorting of neurites in the tectofugal pathways of the chicken embryo. Neuroscience 1999, 90:985-1000. This study describes the distribution and combinatorial localization of classic cadherins in the chick visual system. The authors characterize the cadherin expression in the tectal layers, retina, and their projections into the tectum. The results, although descriptive, support the view that different cadherins are players in the formation of particular brain structures. 29. Hagler DJ Jr, Goda Y: Synaptic adhesion: the building blocks of memory? Neuron 1998, 20:1059-1062. 30. Sano K, Tanihara H, Heimark RL, Obata S, Davidson M, St John T, Taketani S, Suzuki S: Protocadherins: a large family of cadherinrelated molecules in central nervous system. EMBO J 1993, 12:2249-2256. 31. Obata S, Sago H, Mori N, Rochelle JM, Seldin MF, Davidson M, St John T, Taketani S, Suzuki ST: Protocadherin Pcdh2 shows properties similar to, but distinct from, those of classical cadherins. J Cell Sci 1995, 108:3765-3773. 32. Obata S, Sago H, Mori N, Davidson M, St John T, Suzuki ST: A common protocadherin tail: multiple protocadherins share the same sequence in their cytoplasmic domains and are expressed in different regions of brain. Cell Adhes Commun 1998, 6:323-333. 33. Hirano S, Yan Q, Suzuki ST: Expression of a novel protocadherin, OL-protocadherin, in a subset of functional systems of the developing mouse brain. J Neurosci 1999, 19:995-1005. 34. Yagi T, Takeichi M: Cadherin superfamily genes: functions, genomic organization, and neurologic diversity. Genes Dev 2000, 14:1169-1180. 35. Kohmura N, Senzaki K, Hamada S, Kai N, Yasuda R, Watanabe M, Ishii H, Yasuda M, Mishina M, Yagi T: Diversity revealed by a novel family of cadherins expressed in neurons at a synaptic complex. Neuron 1998, 20:1137-1151. 36. Wu Q, Maniatis T: A striking organization of a large family of •• human neural cadherin-like cell adhesion genes. Cell 1999, 97:779-790. The identification of 52 novel human cadherin-like genes is reported in this study. Using an iterative BLAST search analysis and a computer-assisted sequence analysis of a portion of the human genome, the study described 52 Pcadh genes organized into three tandem gene clusters. Multiple variable single exons encoding the extracellular domain of the protein are located upstream of three constant exons that encode the cytoplasmic domain. This genomic organization indicates that a number of different molecular combinations can be achieved via gene rearrangement. This genomic structure further supports the hypothesis that Pcadh can establish a complex molecular code for neuronal connectivity. 37.
Sugino H, Hamada S, Yasuda R, Tuji A, Matsuda Y, Fujita M, Yagi T: Genomic organization of the family of CNR cadherin genes in mice and humans. Genomics 2000, 63:75-87.
38. Wu Q, Maniatis T: Large exons encoding multiple ectodomains are • a characteristic feature of protocadherin genes. Proc Natl Acad Sci USA 2000, 97:3124-3129. See annotation [36••]. 39. Murase S, Schuman EM: The role of cell adhesion molecules in synaptic plasticity and memory. Curr Opin Cell Biol 1999, 11:549-553. 40. Mayford M, Barzilai A, Keller F, Schacher S, Kandel ER: Modulation of an NCAM-related adhesion molecule with long-term synaptic plasticity in Aplysia. Science 1992, 256:638-644. 41. Zhu H, Wu F, Schacher S: Changes in expression and distribution of Aplysia cell adhesion molecules can influence synapse formation and elimination in vitro. J Neurosci 1995, 15:4173-4183.
Cadherin-mediated adhesion at the interneuronal synapse Brusés
597
42. Tang L, Hung CP, Schuman EM: A role for the cadherin family of cell adhesion molecules in hippocampal long-term potentiation. Neuron 1998, 20:1165-1175.
47.
43. Yamagata K, Andreasson KI, Sugiura H, Maru E, Dominique M, Irie Y, Mik N, Hayashi Y, Yoshioka M, Kaneko K et al.: Arcadlin is a neural activity-regulated cadherin involved in long term potentiation. J Biol Chem 1999, 274:19473-19479.
48. Yap AS, Brieher WM, Pruschy M, Gumbiner BM: Lateral clustering of the adhesive ectodomain: a fundamental determinant of cadherin function. Curr Biol 1997, 7:308-315.
44. Gumbiner BM: Regulation of cadherin adhesive activity. J Cell Biol 2000, 148:399-404.
49. Engert F, Bonhoeffer T: Dendritic spine changes associated with hippocampal long-term synaptic plasticity. Nature 1999, 399:66-70.
45. Tanaka H, Shan W, Phillips GR, Arndt K, Bozdagi O, Shapiro L, •• Huntley GW, Benson DL, Colman DR: Molecular modification of N-cadherin in response to synaptic activity. Neuron 2000, 25:93-107. Utilizing an in vitro system of synapses formed between hippocampal neurons, this study demonstrates that neuronal depolarization induced by NMDA causes N-cadherin dimerization at the synapse. The N-cadherin redistribution occurs in 30 min and returns to normal levels in 2 hours. This study indicates that electrical activity induces an increase in synaptic adhesion via changes in the conformational state of N-cadherin. 46. Brieher WM, Yap AS, Gumbiner BM: Lateral dimerization is required for the homophilic binding activity of C-cadherin. J Cell Biol 1996, 135:487-496.
Tamura K, Shan WS, Hendrickson WA, Colman DR, Shapiro L: Structure-function analysis of cell adhesion by neural (N-) cadherin. Neuron 1998, 20:1153-1163.
50. Maletic-Savatic M, Malinow R, Svoboda K: Rapid dendritic morphogenesis in CA1 hippocampal dendrites induced by synaptic activity. Science 1999, 283:1923-1927. 51. Toni N, Buchs PA, Nikonenko I, Bron CR, Muller D: LTP promotes formation of multiple spine synapses between a single axon terminal and a dendrite. Nature 1999, 402:421-425. 52. Wong WT, Wong RO: Rapid dendritic movements during synapse formation and rearrangement. Curr Opin Neurobiol 2000, 10:118-124. 53. Harris KM: Structure, development, and plasticity of dendritic spines. Curr Opin Neurobiol 1999, 9:343-348.