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Denise Walker, Fré dé rique Berton, specifically expressed in distinct patterns in neural and Cé cile Raymond, Masakazu Kataoka 1 , neuroendocrine tissues (Ullrich et al., 1994;Marquèze et al., Yoko Shoji-Kasai 1 , Masami Takahashi 1 , Berton et al., 1997). Synaptotagmin I, a major mem- 1992, 1994 Yoshida et al., 1992;El Far et al., 1995) and to vesicle fusion and exocytosis of neurotransmitters. MichelP/Q-type (Martin-Moutot et al., 1996), but not L-type calThe interaction of synaptotagmins with native P/Q-type cium channels (El Far et al., 1995). Furthermore, co-exprescalcium channels was studied in solubilized synaptosion of syntaxin with calcium channels induces a somes from rat cerebellum. Antibodies against synaptomodification of current gating properties displaying a tagmins I and II, but not IV co-immunoprecipitated similar specificity for N-or P/Q-type channels [ 125 I]ω-conotoxin MVIIC-labelled calcium channels. (Bezprozvanny et al., 1995). These findings are consistent Direct interactions were studied between in vitro-transwith observations that neurotransmitter release at many lated [ 35 S]synaptotagmin I and fusion proteins concentral synapses is blocked by antagonists of N-or P/Qtaining cytoplasmic loops of the α 1 A subunit (BI type calcium channels, but is insensitive to inhibitors of isoform). Gel overlay revealed the association of L-type channels (Takahashi and Momiyama, 1993; Wheeler synaptotagmin I with a single region (residues 780-969) et al., 1994). Neuronal calcium channels are heteromeric located in the intracellular loop connecting homologous proteins constituted by an α 1 subunit which forms the transdomains II and III. Saturable calcium-independent membrane pore, associated with auxiliary α 2 δ and β subbinding occurred with equilibrium dissociation conunits (Birnbaumer et al., 1994). Association with core stants of 70 nM and 340 nM at 4°C and pH 7.4, and complexes involves syntaxin and SNAP25 binding to α 1 subunits on the cytoplasmic loop that links homologous association was blocked by addition of excess recombindomains II and III (Sheng et al., 1994(Sheng et al., , 1996; Rettig et al., ant synaptotagmin I. Direct synaptotagmin binding to 1996). the pore-forming subunit of the P/Q-type channel may It has been suggested that interactions between synaptic optimally locate the calcium-binding sites that initiate protein complexes and calcium channels may optimally exocytosis within a zone of voltage-gated calcium entry.locate the calcium sensor synaptotagmin within domains

BackgroundSynaptosomal-associated protein, 25 kDa (SNAP-25) regulates the exocytosis of neurotransmitters. Growing evidence suggests that SNAP-25 is involved in neuropsychiatric disorders, such as schizophrenia, attention-deficit/hyperactivity disorder, and epilepsy. Recently, increases in anxiety-related behaviors and epilepsy have been observed in SNAP-25 knock-in (KI) mice, which have a single amino acid substitution of Ala for Ser187. However, the molecular and cellular mechanisms underlying the abnormalities in this mutant remain unknown.ResultsIn this study, we found that a significant number of dentate gyrus (DG) granule cells was histologically and electrophysiologically similar to immature DG neurons in the dentate gyrus of the adult mutants, a phenomenon termed the “immature DG” (iDG). SNAP-25 KI mice and other mice possessing the iDG phenotype, i.e., alpha-calcium/calmodulin-dependent protein kinase II heterozygous mice, Schnurri-2 knockout mice, and mice treated with the antidepressant fluoxetine, showed similar molecular expression patterns, with over 100 genes similarly altered. A working memory deficit was also identified in mutant mice during a spontaneous forced alternation task using a modified T-maze, a behavioral task known to be dependent on hippocampal function. Chronic treatments with the antiepileptic drug valproate abolished the iDG phenotype and the working memory deficit in mutants.ConclusionsThese findings suggest that the substitution of Ala for Ser187 in SNAP-25 induces the iDG phenotype, which can also be caused by epilepsy, and led to a severe working memory deficit. In addition, the iDG phenotype in adulthood is likely an endophenotype for at least a part of some common psychiatric disorders.

Synaptosomal-associated protein of 25 kDa (SNAP-25) is a presynaptic protein essential for neurotransmitter release. Previously, we demonstrate that protein kinase C (PKC) phosphorylates Ser187 of SNAP-25, and enhances neurotransmitter release by recruiting secretory vesicles near to the plasma membrane. As PKC is abundant in the brain and SNAP-25 is essential for synaptic transmission, SNAP-25 phosphorylation is likely to play a crucial role in the central nervous system. We therefore generated a mutant mouse, substituting Ser187 of SNAP-25 with Ala using “knock-in” technology. The most striking effect of the mutation was observed in their behavior. The homozygous mutant mice froze readily in response to environmental change, and showed strong anxiety-related behavior in general activity and light and dark preference tests. In addition, the mutant mice sometimes exhibited spontaneously occurring convulsive seizures. Microdialysis measurements revealed that serotonin and dopamine release were markedly reduced in amygdala. These results clearly indicate that PKC-dependent SNAP-25 phosphorylation plays a critical role in the regulation of emotional behavior as well as the suppression of epileptic seizures, and the lack of enhancement of monoamine release is one of the possible mechanisms underlying these defects.

Phorbol ester treatment induces the phosphorylation of SNAP-25 at Ser(187) and the potentiation of Ca(2+)-induced dopamine (DA) and acetylcholine (Ach) release from PC12 cells. In order to evaluate the functional consequences of phosphorylation, quantitative analysis was carried out using an anti-phosphopeptide antibody that specifically recognizes SNAP-25 phosphorylated at Ser(187). DA and ACh release, assayed in low-K(+) as well as high-K(+) solution, increased by treating the cells with phorbol-12-myristate-13-acetate (PMA); however, the stimulation of high-K(+)-dependent release occurred at lower concentrations and with shorter exposures to PMA than that of the basal release in low-K(+)-solution. The PMA-induced phosphorylation of SNAP-25 did not correlate with the potentiation of high-K(+)-dependent neurotransmitter release. The potentiation of high-K(+)-dependent DA release by phorbol 12,13-diacetate (PDA), a water soluble phorbol ester, almost completely disappeared within 1 min after washing PDA in the presence of okadaic acid, conditions under which the phosphorylation of SNAP-25 persisted for at least 15 min. PMA-induced phosphorylation of SNAP-25 was inhibited by staurosporine, however, the potentiation of high-K(+)-dependent DA release was suppressed only partially. These results indicate that protein kinase activation does not account for a large fraction of the phorbol ester-induced potentiation of depolarization-dependent neurotransmitter release from PC12 cells.