Tyrosine kinases are expressed in many tissues, particularly in the central nervous system, and regulate various cellular functions. We report here that a src family tyrosine kinase-specific inhibitor, PP2, enhances neurotransmitter release from PC12 cells and primary cultured neurons. PP2 enhances only Ca 2؉ -dependent release; it does not affect basal release. These effects result from an enhancement of vesicular exocytosis and not from the reuptake or refilling of neurotransmitters because Ca 2؉ -dependent secretion of an exogenously expressed reporter protein, the human growth hormone (hGH), is also enhanced by PP2. Overexpression of constitutive active v-src, but not of a kinase-inactive mutant, suppressed Ca 2؉ -dependent release. In PP2-treated cells, Pyk2, paxillin, and some other proteins showed a decrease in tyrosine phosphorylation, and the enhancement of tyrosine phosphorylation of these proteins in response to Ca 2؉ influx was also reduced. Electron and fluorescence microscopy showed that PP2 treatment induced morphological change and decreased phalloidin reactivity at the filopodium-like structures on the processes of PC12 cells. Interestingly, inhibition of actin polymerization with cytochalasin D and latrunculin A enhanced Ca 2؉ -dependent, but not basal, release. It is possible that a src family tyrosine kinase, through the regulation of actin dynamics, has an inhibitory function to regulate neurotransmitter release.N eurotransmitters are accumulated in synaptic vesicles in presynaptic nerve terminals. Ca 2ϩ influx through voltagegated Ca 2ϩ channels triggers the release of neurotransmitters into the synaptic cleft by means of an exocytosis of synaptic vesicles. The modulation of neurotransmitter release is one of the cellular mechanisms for the regulation of synaptic transmission, and the involvement of various kinases has been suggested (1). Tyrosine kinases are classified as receptor types or nonreceptor types, both of which are abundant in the central nervous system. The receptor-type tyrosine kinase generally acts as a receptor for growth and trophic factors and plays an important role in the regulation of synaptic transmission through the up-regulation of neurotransmitter release from synapses of developing and mature neurons (2). The nonreceptor-type tyrosine kinase is well documented to have postsynaptic functions and is shown to phosphorylate neurotransmitter receptors, including nicotinic acetylcholine receptor (nACh-R), N-methyl-D-aspartate receptor (NMDA-R), and ␥-aminobutyric acid type A receptor (GABA A -R) (3). src kinase, a nonreceptor tyrosine kinase expressed in the central nervous system, is accumulated in synaptic vesicles and accounts for the majority of synapticvesicle tyrosine kinase activity (4, 5). Ca 2ϩ influx, which triggers neurotransmitter release, activates src kinase and induces tyrosine phosphorylation of several proteins, including synaptophysin, a synaptic vesicle protein (6-8). On synaptic vesicles, c-src interacts with synapsin, a perimembrane protein on these...

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.

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Pathological examination of dementia with Lewy bodies patients identified the presence of abnormal ␣-synuclein (␣Syn) aggregates in the presynaptic terminals. ␣Syn is involved in the regulation of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex. Importantly, ␣Syn-transgenic mouse and postmortem examination of patients with Parkinson's disease have demonstrated the abnormal distribution of SNARE protein in presynaptic terminals. In this study, we investigated the effects of SNARE dysfunction on endogenous ␣Syn using Snap25 S187A/S187A mutant mice. These mice have homozygous knock-in gene encoding unphosphorylatable S187A-substituted synaptosomal-associated protein of 25 kDa (SNAP-25). The mice displayed a significant age-dependent change in the distribution of ␣Syn and its Ser 129 -phosphorylated form in abnormally hypertrophied glutamatergic nerve terminals in the striatum. Electron-microscopic analysis revealed the abnormally condensed synaptic vesicles with concomitant mislocalization of ␣Syn protein to the periactive zone in the glutamatergic nerve terminals. However, the Snap25 S187A/S187A mutant mouse harbored no abnormalities in the nigrostriatal dopaminergic neurons. Our present results suggest that SNARE dysfunction is the initial trigger of mislocalization and accumulation of ␣Syn, and probably is an important pathomechanism of ␣-synucleinopathies.

Soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP)-25 is a neuronal SNARE protein essential for neurotransmitter release from presynaptic terminals. Three palmitoylated SNAP-25 family proteins: SNAP-25a, SNAP-25b, and SNAP-23, are expressed in the brain, but little is known about their distributions and functions. In the present study, we generated specific antibodies to distinguish these three homologous proteins. Immunoblot and immunohistochemical analyses revealed that SNAP-25b was distributed in synapse-enriched regions throughout almost the entire brain, whereas SNAP-25a and SNAP-23 were expressed in relatively specific brain regions with partially complementary expression patterns. SNAP-25a and SNAP-25b, but not SNAP-23, were also present in the axoplasm of nerve fibers. The intracellular localization was also different, and although SNAP-25b and SNAP-23 were found primarily in membrane and lipid raft-enriched fractions of mouse brain homogenates, a substantial amount of SNAP-25a was recovered in soluble fractions. In PC12 cells, SNAP-25b was localized to the plasma membrane, but SNAP-25a and SNAP-23 were distributed throughout the cytoplasm. The expression and distribution of these three proteins were also differentially regulated in the early postnatal period. These results indicate that the three SNAP-25 family proteins display a differential distribution in the brain as well as in neuronal cells, and possibly play distinct roles.