Maki Deguchi-Tawarada

CAST: A novel protein of the cytomatrix at the active zone of synapses that forms a ternary complex with RIM1 and Munc13-1

The cytomatrix at the active zone (CAZ) has been implicated in defining the site of Ca2+-dependent exocytosis of neurotransmitter. We have identified here a novel CAZ protein of ∼120 kD from rat brain and named it CAST (CAZ-associated structural protein). CAST had no transmembrane segment, but had four coiled-coil domains and a putative COOH-terminal consensus motif for binding to PDZ domains. CAST was localized at the CAZ of conventional synapses of mouse brain. CAST bound directly RIM1 and indirectly Munc13-1, presumably through RIM1, forming a ternary complex. RIM1 and Munc13-1 are CAZ proteins implicated in Ca2+-dependent exocytosis of neurotansmitters. Bassoon, another CAZ protein, was also associated with this ternary complex. These results suggest that a network of protein–protein interactions among the CAZ proteins exists at the CAZ. At the early stages of synapse formation, CAST was expressed and partly colocalized with bassoon in the axon shaft and the growth cone. The vesicles immunoisolated by antibassoon antibody–coupled beads contained not only bassoon but also CAST and RIM1. These results suggest that these CAZ proteins are at least partly transported on the same vesicles during synapse formation.

Physical and functional interaction of the active zone proteins, CAST, RIM1, and Bassoon, in neurotransmitter release

We have recently isolated a novel cytomatrix at the active zone (CAZ)–associated protein, CAST, and found it directly binds another CAZ protein RIM1 and indirectly binds Munc13-1 through RIM1; RIM1 and Munc13-1 directly bind to each other and are implicated in priming of synaptic vesicles. Here, we show that all the CAZ proteins thus far known form a large molecular complex in the brain, including CAST, RIM1, Munc13-1, Bassoon, and Piccolo. RIM1 and Bassoon directly bind to the COOH terminus and central region of CAST, respectively, forming a ternary complex. Piccolo, which is structurally related to Bassoon, also binds to the Bassoon-binding region of CAST. Moreover, the microinjected RIM1- or Bassoon-binding region of CAST impairs synaptic transmission in cultured superior cervical ganglion neurons. Furthermore, the CAST-binding domain of RIM1 or Bassoon also impairs synaptic transmission in the cultured neurons. These results indicate that CAST serves as a key component of the CAZ structure and is involved in neurotransmitter release by binding these CAZ proteins.

Nectin-like molecule-1/TSLL1/SynCAM3 : a neural tissue-specific immunoglobulin-like cell-cell adhesion molecule localizing at non-junctional contact sites of presynaptic nerve terminals, axons and glia cell processes

Nectins are Ca2+-independent immunoglobulin-like cell-cell adhesion molecules and comprise a family of four members. At the mossy fiber terminals of hippocampus, nectin-1 and nectin-3 localize at the presynaptic and postsynaptic sides of synaptic junctions, respectively, and their trans-interactions play a role in formation of synapses in cooperation with N-cadherin. Nectins are associated with the actin cytoskeleton through afadin, a nectin- and actin-filament-binding protein. Five nectin-like molecules (Necls) which have domain structures similar to those of nectins have been identified and here we characterize Necl-1/TSLL1/SynCAM3, from now on referred to as Necl-1. Tissue distribution analysis showed that Necl-1 was specifically expressed in the neural tissue. Immunofluorescence and immunoelectron microscopy revealed that Necl-1 localized at the contact sites among axons, their terminals, and glia cell processes that cooperatively formed synapses, axon bundles and myelinated axons. Necl-1 showed Ca2+-independent homophilic cell-cell adhesion activity. It furthermore showed Ca2+-independent heterophilic cell-cell adhesion activity with Necl-2/IGSF4/RA175/SgIGSF/TSLC1/SynCAM1 from now on referred to as Necl-2, nectin-1 and nectin-3, but not with Necl-5 or nectin-2. The C-terminal cytoplasmic region of Necl-1 did not bind afadin but bound membrane-associated guanylate kinase subfamily members that contain the L27 domain, including Dlg3, Pals2 and CASK. These results indicate that Necl-1 is a neural-tissue-specific Ca2+-independent immunoglobulin-like cell-cell adhesion molecule which potentially has membrane-associated guanylate kinase subfamily member-binding activity and localizes at the non-junctional cell-cell contact sites.

Synaptic activity prompts γ-secretase–mediated cleavage of EphA4 and dendritic spine formation

Alzheimers disease is an age-dependent neurodegenerative disorder that is characterized by a progressive decline in cognitive function. γ-secretase dysfunction is evident in many cases of early onset familial Alzheimers disease. However, the mechanism by which γ-secretase dysfunction results in memory loss and neurodegeneration is not fully understood. Here, we demonstrate that γ-secretase is localized at synapses and regulates spine formation. We identify EphA4, one of the Ephrin receptor family members, as a substrate of γ-secretase, and find that EphA4 processing is enhanced by synaptic activity. Moreover, overexpression of EphA4 intracellular domain increases the number of dendritic spines by activating the Rac signaling pathway. These findings reveal a function for EphA4-mediated intracellular signaling in the morphogenesis of dendritic spines and suggest that the processing of EphA4 by γ-secretase affects the pathogenesis of Alzheimers disease.

SAD: A Presynaptic Kinase Associated with Synaptic Vesicles and the Active Zone Cytomatrix that Regulates Neurotransmitter Release

A serine/threonine kinase SAD-1 in C. elegans regulates synapse development. We report here the isolation and characterization of mammalian orthologs of SAD-1, named SAD-A and SAD-B, which are specifically expressed in the brain. SAD-B is associated with synaptic vesicles and, like the active zone proteins CAST and Bassoon, is tightly associated with the presynaptic cytomatrix in nerve terminals. A short conserved region (SCR) in the COOH-terminus is required for the synaptic localization of SAD-B. Overexpression of SAD-B in cultured rat hippocampal neurons significantly increases the frequency of miniature excitatory postsynaptic current but not its amplitude. Introduction of SCR into presynaptic superior cervical ganglion neurons in culture significantly inhibits evoked synaptic transmission. Moreover, SCR decreases the size of the readily releasable pool measured by applying hypertonic sucrose. Furthermore, SAD-B phosphorylates the active zone protein RIM1 but not Munc13-1. These results suggest that mammalian SAD kinase presynaptically regulates neurotransmitter release.

Identification of activity‐regulated proteins in the postsynaptic density fraction

Background: The postsynaptic density (PSD) at synapses is a specialized submembranous structure where neurotransmitter receptors are linked to cytoskeleton and signalling molecules. Activity‐dependent dynamic change in the components of the PSD is a mechanism of synaptic plasticity. Identification of the PSD proteins and examination of their modulations dependent on synaptic activity will be valuable for an understanding of the molecular basis of learning and memory.

CAST2: identification and characterization of a protein structurally related to the presynaptic cytomatrix protein CAST

The cytomatrix at the active zone (CAZ) is thought to define the site of Ca2+‐dependent exocytosis of neurotransmitters. We have recently identified a novel CAZ protein from rat brain which we have named CAST (CAZ‐associated structural protein). CAST forms a large molecular complex with other CAZ proteins such as Bassoon, RIM1 and Munc13‐1, at least through direct binding to RIM1. Here, we have identified a rat protein that is structurally related to CAST and named it CAST2. Subcellular fractionation analysis of rat brain shows that CAST2 is also tightly associated with the postsynaptic density fraction. Like CAST, CAST2 directly binds RIM1 and forms a hetero‐oligomer with CAST. In primary cultured rat hippocampal neurones, CAST2 co‐localizes with Bassoon at synapses. Furthermore, immunoelectron microscopy reveals that CAST2 localizes to the vicinity of the presynaptic membrane of synapses in mouse brain. Sequence analysis reveals that CAST2 is a rat orthologue of the human protein ELKS. ELKS has also recently been identified as Rab6IP2 and ERC1. Accordingly, the original CAST is tentatively re‐named CAST1. These results indicate that CAST2 is a new component of the CAZ and, together with CAST1, may be involved in the formation of the CAZ structure.

Differential distribution of release-related proteins in the hippocampal CA3 area as revealed by freeze-fracture replica labeling

Synaptic vesicle release occurs at a specialized membrane domain known as the presynaptic active zone (AZ). Several membrane proteins are involved in the vesicle release processes such as docking, priming, and exocytotic fusion. Cytomatrix at the active zone (CAZ) proteins are structural components of the AZ and are highly concentrated in it. Localization of other release‐related proteins including target soluble N‐ethylmaleimide‐sensitive‐factor attachment protein receptor (t‐SNARE) proteins, however, has not been well demonstrated in the AZ. Here, we used sodium dodecyl sulfate‐digested freeze‐fracture replica labeling (SDS‐FRL) to analyze quantitatively the distribution of CAZ and t‐SNARE proteins in the hippocampal CA3 area. The AZ in replicated membrane was identified by immunolabeling for CAZ proteins (CAZ‐associated structural protein [CAST] and Bassoon). Clusters of immunogold particles for these proteins were found on the P‐face of presynaptic terminals of the mossy fiber and associational/commissural (A/C) fiber. Co‐labeling with CAST revealed distribution of the t‐SNARE proteins syntaxin and synaptosomal‐associated protein of 25 kDa (SNAP‐25) in the AZ as well as in the extrasynaptic membrane surrounding the AZ (SZ). Quantitative analysis demonstrated that the density of immunoparticles for CAST in the AZ was more than 100 times higher than in the SZ, whereas that for syntaxin and SNAP‐25 was not significantly different between the AZ and SZ in both the A/C and mossy fiber terminals. These results support the involvement of the t‐SNARE proteins in exocytotic fusion in the AZ and the role of CAST in specialization of the membrane domain for the AZ. J. Comp. Neurol. 489:195–216, 2005.

Rabconnectin-3, a novel protein that binds both GDP/GTP exchange protein and GTPase-activating protein for Rab3 small G protein family

Rab3A, a member of the Rab3 small G protein family, regulates Ca2+-dependent exocytosis of neurotransmitter. The cyclical activation and inactivation of Rab3A are essential for the Rab3A action in exocytosis. GDP-Rab3A is activated to GTP-Rab3A by Rab3 GDP/GTP exchange protein (Rab3 GEP), and GTP-Rab3A is inactivated to GDP-Rab3A by Rab3 GTPase-activating protein (Rab3 GAP). It remains unknown how or in which step of the multiple exocytosis steps these regulators are activated and inactivated. We isolated here a novel protein that was co-immunoprecipitated with Rab3 GEP and GAP by their respective antibodies from the crude synaptic vesicle fraction of rat brain. The protein, named rabconnectin-3, bound both Rab3 GEP and GAP. The cDNA of rabconnectin-3 was cloned from a human cDNA library and its primary structure was determined. Human rabconnectin-3 consisted of 3,036 amino acids and showed a calculated M r of 339,753. It had 12 WD domains. Tissue and subcellular distribution analyses in rat indicated that rabconnectin-3 was abundantly expressed in the brain where it was enriched in the synaptic vesicle fraction. Immunofluorescence and immunoelectron microscopy revealed that rabconnectin-3 was concentrated on synaptic vesicles at synapses. These results indicate that rabconnectin-3 serves as a scaffold molecule for both Rab3 GEP and GAP on synaptic vesicles.

Nectin-dependent localization of ZO-1 at puncta adhaerentia junctions between the mossy fiber terminals and the dendrites of the pyramidal cells in the CA3 area of adult mouse hippocampus

Nectin and afadin constitute a novel intercellular adhesion system that organizes adherens junctions in cooperation with the cadherin‐catenin system in epithelial cells. Nectin is a Ca2+‐independent immunoglobulin‐like adhesion molecule and afadin is an actin filament (F‐actin)‐binding protein that connects nectin to the actin cytoskeleton. At the puncta adhaerentia junctions (PAs) between the mossy fiber terminals and the dendrites of the pyramidal cells in the CA3 area of the adult mouse hippocampus, the nectin‐afadin system also colocalizes with the cadherin‐catenin system and has a role in the formation of synapses. ZO‐1 is another F‐actin‐binding protein that localizes at tight junctions (TJs) and connects claudin to the actin cytoskeleton in epithelial cells. The nectin‐afadin system is able to recruit ZO‐1 to the nectin‐based cell–cell adhesion sites in nonepithelial cells that have no TJs. In the present study, we investigated the localization of ZO‐1 in the mouse hippocampus. Immunofluorescence and immunoelectron microscopy revealed that ZO‐1 also localized at the PAs between the mossy fiber terminals and the dendrites of the pyramidal cells in the CA3 area of the adult mouse hippocampus, as described for afadin. ZO‐1 colocalized with afadin during the development of synaptic junctions and PAs. Microbeads coated with the extracellular fragment of nectin, which interacts with cellular nectin, recruited both afadin and ZO‐1 to the bead–cell contact sites in cultured rat hippocampal neurons. These results indicate that ZO‐1 colocalizes with nectin and afadin at the PAs and that the nectin‐afadin system is involved in the localization of ZO‐1. J. Comp. Neurol. 460:514–524, 2003.