MOLECULAR MECHANISMS OF SYNAPSE FORMATION: ADHESION MOLECULES
Functional Regulation of Synaptic Adhesion Complexes by Alternative Splicing
Peter Scheiffele, Ph.D.
The formation of synapses during the development of the central nervous system requires the coordinate differentiation of presynaptic and postsynaptic membrane domains. Cell adhesion molecules have been proposed to play important roles in directing this differentiation process as well as the selectivity of synaptic cell-cell interactions. We have explored the role of the heterophilic neuroligin-neurexin adhesion complex in synapse assembly. Neuroligins and neurexins each constitute protein families, with multiple isoforms generated from multiple genes through alternative transcription start sites and alternative splicing. We observed that the alternative splicing of neuroligin and neurexin isoforms underlies their selective function at GABAergic and glutamatergic synapses in hippocampal neurons. Specific classes of neuroligin and neurexin isoforms exclusively promote the assembly of glutamatergic presynaptic and postsynaptic structures, whereas other classes function exclusively at GABAergic synapses. These data suggest that the highly diverse extracellular domains of neuroligin and neurexin isoforms encode selective transsynaptic interactions that contribute to the assembly or modification of different types of central synapses.
SynCAMs: From Synaptic Adhesion to Synapse Formation
Thomas Biederer, Ph.D.
Synaptogenesis is a defining process of neuronal network formation. Synapse formation is most intense in early postnatal development, but synapses continue to turn over and form anew throughout adulthood. Only few interactions in the mammalian central nervous system (CNS) are known to directly induce new synapses. The analysis of these interactions, mediated by SynCAM 1 and the neuroligin/neurexin membrane proteins, has shown that these synaptic adhesion proteins can drive the formation of synaptic specializations through transsynaptic interactions. SynCAM 1 drives neurons to form fully functional presynaptic terminals upon physical contact.
SynCAM 1 is a homophilic cell adhesion molecule widely expressed in the CNS. It is an N-glycosylated, single-spanning membrane protein and is expressed during the peak period of synaptogenesis. Importantly, SynCAM 1 is enriched in synaptic membranes and induces the formation of new, functional presynaptic terminals in a coculture assay of hippocampal neurons. This activity of SynCAM 1 is monitored using live-cell optical imaging, and we employ it to reconstitute synaptic transmission. Using this reconstitution approach, properties of evoked synaptic transmission can be observed and analyzed under defined conditions in vitro. Concurringly, the overexpression of SynCAM 1 in transfected neurons increases the frequency of minis. This activity of SynCAM 1 depends on its cytosolic sequence, and the SynCAM 1-induced facilitation of minifrequencies is specific for excitatory currents.
Do these adhesive interactions at synaptic sites specify key properties of synaptogenesis? To begin to address this question in detail, we study the SynCAM family, founded by SynCAM 1. It consists of four members. All SynCAM proteins have three extracellular immunoglobulin-like domains and a highly conserved cytosolic tail with a PDZ interaction motif. We demonstrate through real-time RT-PCR analysis and in situ hybridization that the four SynCAM proteins are neuronally expressed, present in the developing vertebrate brain, and exhibit distinct regional and developmental expression profiles. Our biochemical studies identify that three of the four SynCAM proteins, including SynCAM 1, exert homophilic adhesive interactions. In addition, specific heterophilic adhesive interactions can occur among particular family members. These studies indicate that particular SynCAM interactions may constitute an adhesive code during synaptogenesis. We currently determine to which extent the four members of the SynCAM family of neuronal adhesion molecules drive the formation of presynaptic terminals. It is our hypothesis that all SynCAM proteins can drive synapse formation and that their homophilic and heterophilic interactions contribute to synapse specification.
These studies aim to provide deeper insight into the molecular mechanisms directing the initial steps of synapse formation in the CNS. Future analyses have a goal to determine the roles of synaptogenesis in the modulation of neuronal circuits.
Regulation of Synapse Formation and Sprouting by Cadherin Adhesion Complexes
Shernaz X. Bamji, Ph.D.
Recent studies suggest commonalities between the development of addictive behaviors and traditional learning models. For example, synaptic plasticity, which has long been the molecular correlate of learning and memory, has been demonstrated in neural reward circuits and is believed to contribute to the learning of addictive behaviors. The rapid formation and elimination of synaptic sites occurs throughout life and represents one aspect of synaptic plasticity in which synaptic communication is modified in the long term. Synaptic adhesion proteins are of particular interest in this context because presynaptic to postsynaptic membrane adhesion is one of the initial events during synapse formation and remains a fundamental component of the maintenance of synapses in maturity. Our studies demonstrate a role for the cadherin adhesion complex in the localization of synaptic vesicles to developing presynaptic compartments. Despite the requirement for cadherin-based adhesion in some aspects of synapse formation, we show that the maintenance of strong cell-cell adhesion is detrimental to the formation of new synapses in the presence of the plasticity factor brain-derived neurotrophic factor (BDNF). We show, using time-lapse confocal analysis, that BDNF mobilizes synaptic vesicles at existing synapses, resulting in small clusters of synaptic vesicles “splitting” away from synaptic sites. BDNF’s ability to mobilize synaptic vesicle clusters depends on the dissociation of cadherin/β-catenin adhesion complexes that occurs following tyrosine phosphorylation of β-catenin. Artificially maintaining cadherin/β-catenin complexes in the presence of BDNF abolishes the BDNF-mediated enhancement of synaptic vesicle mobility and also abolishes the longer term BDNF-mediated increase in synapse number. Together these data demonstrate that the disruption of cadherin/β-catenin complexes following BDNF treatment is an important molecular event through which BDNF increases synapse density. We are currently exploring the hypothesis that enhanced synaptic vesicle mobility contributes to the formation of new synapses at adjacent regions of the axon and that the disruption of strong cadherin-based adhesion catalyzes this event. We believe that the molecular adhesive machinery required for synapse assembly in development plays an essential role in modulating synaptic architecture in the context of plasticity-related structural remodeling.
Regulation of Synaptic Connectivity in C. Elegans: From Cell Adhesion to Morphogenetic Gradient
Kang Shen, M.D., Ph.D.
[Slides not available]
The presynaptic regions of axons accumulate synaptic vesicles and active zone and periactive zone proteins. The rules for the orderly recruitment of presynaptic components are not well understood. We systematically examined the molecular mechanisms of presynaptic development in egg-laying synapses of C. elegans, demonstrating that two scaffolding molecules, SYD-1 and SYD-2, play key roles in presynaptic assembly. SYD-2/liprin was previously shown to regulate the size and shape of active zones. We now show that in syd-1 and syd-2 mutants, synaptic vesicles and numerous other presynaptic proteins fail to accumulate at presynaptic sites. SYD-1 and SYD-2 function cell autonomously at presynaptic terminals, downstream of the synaptic specificity molecule SYG-1. SYD-1 likely acts upstream of SYD-2 to positively regulate its activity. These data suggest a hierarchical organization of presynaptic assembly, in which transmembrane specificity molecules initiate synaptogenesis by recruiting a few key scaffolding proteins, which in turn assemble other presynaptic components.