Saori Yamamori

A Single Amino Acid Mutation in SNAP-25 Induces Anxiety-Related Behavior in Mouse

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.

Differential expression of SNAP-25 family proteins in the mouse brain.

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. J. Comp. Neurol. 519:916–932, 2011.

Stress-induced phosphorylation of SNAP-25.

Synaptosomal-associated protein of 25 kDa (SNAP-25), a t-SNARE protein, plays a crucial role in neurotransmitter release by exocytosis. Protein kinase C phosphorylates SNAP-25 at Ser(187), however the physiological significance of this phosphorylation event in brain function remains unclear. In the present study, we found that SNAP-25 phosphorylation increased rapidly in the mouse brain following cold-water restraint stress. Both basal and stress-induced phosphorylation of SNAP-25 were high in stress-related brain regions, including the cerebral cortex, hippocampus, and amygdala, and the extent of phosphorylation increased with increasing amounts of stress. Intravenous administration of adrenaline increased SNAP-25 phosphorylation, although stress-induced phosphorylation was still observed in adrenalectomized mice. These results indicate that SNAP-25 phosphorylation is regulated in a stress-dependent manner through both central and peripheral mechanisms.

Epileptogenesis and epileptic maturation in phosphorylation site-specific SNAP-25 mutant mice

Snap25(S187A/S187A) mouse is a knock-in mouse with a single amino acid substitution at a protein kinase C-dependent phosphorylation site of the synaptosomal-associated protein of 25 kDa (SNAP-25), which is a target-soluble NSF attachment protein receptor (t-SNARE) protein essential for neurotransmitter release. Snap25(S187A/S187A) mice exhibit several distinct phenotypes, including reductions in dopamine and serotonin release in the brain, anxiety-like behavior, and cognitive dysfunctions. Homozygous mice show spontaneous epileptic convulsions, and about 15% of the mice die around three weeks after birth. The remaining mice survive for almost two years and exhibit spontaneous recurrent seizures throughout their lifetime. Here, we conducted long-term continuous video electroencephalogram recording of the mice and analyzed the process of epileptogenesis and epileptic maturation in detail. Spikes and slow-wave discharges (SWDs) were observed in the cerebral cortex and thalamus before epileptic convulsions began. SWDs showed several properties similar to those observed in absence seizures including (1) lack of in the hippocampus, (2) movement arrest during SWDs, and (3) inhibition by ethosuximide. Multiple generalized seizures occurred in all homozygous mice around three weeks after birth. However, seizure generation stopped within several days, and a seizure-free latent period began. Following a spike-free quiet period, the number of spikes increased gradually, and epileptic seizures reappeared. Subsequently, spontaneous seizures occurred cyclically throughout the life of the mice, and several progressive changes in seizure frequency, seizure duration, seizure cycle interval, seizure waveform, and the number and waveform of epileptic discharges during slow-wave sleep occurred with different time courses over 10 weeks. Anxiety-related behaviors appeared suddenly within three days after epileptic seizures began and were delayed markedly by oral administration of valproic acid. These results showed that Snap25(S187A/S187A) mice exhibited a variety of epilepsy-related phenomena, and thus, they will be useful for understanding the mechanisms of epileptogenesis, epileptic maturation, and the actions of antiepileptic drugs.

Two distinct regulatory mechanisms of neurotransmitter release by phosphatidylinositol 3-kinase

Recent studies have indicated that various growth factors are involved in synaptic functions; however, the precise mechanisms remain unclear. In order to elucidate the molecular mechanisms of the growth factor‐mediated regulation of presynaptic functions, the effects of epidermal growth factor (EGF) and insulin‐like growth factor‐1 (IGF‐1) on neurotransmitter release were studied in rat PC12 cells. Brief treatment with EGF and IGF‐1 enhanced Ca2+‐dependent dopamine release in a concentration‐dependent manner. EGF activated both mitogen‐activated protein kinase (MAPK) and phosphatidylinositol 3‐kinase (PI3‐kinase) pathways, and the EGF‐dependent enhancement of DA release was suppressed by a MAPK kinase inhibitor as well as by PI3‐kinase inhibitors. In striking contrast, IGF‐1 activated the PI3‐kinase pathway but not the MAPK pathway, and IGF‐1‐dependent enhancement was suppressed by a PI3‐kinase inhibitor but not by a MAPK kinase inhibitor. The enhanced green fluorescent protein‐tagged pleckstrin homology (PH) domain of protein kinase B, which selectively binds to phosphatidylinositol 3,4‐bisphosphate and phosphatidylinositol 3,4,5‐triphosphate, was translocated to the plasma membrane after treatment with either EGF or NGF. By contrast, no significant redistribution was induced by IGF‐1. These results indicate that PI3‐kinase participates in the enhancement of neurotransmitter release by two distinct mechanisms: EGF and NGF activate PI3‐kinase in the plasma membrane, whereas IGF‐1 activates PI3‐kinase possibly in the intracellular membrane, leading to enhancement of neurotransmitter release in a MAPK‐dependent and ‐independent manner respectively.

Dual mechanisms of rapid expression of anxiety-related behavior in pilocarpine-treated epileptic mice.

A mouse model of epilepsy was generated by inducing status epilepticus (SE) for either 1.5 or 4.5h with pilocarpine to study anxiety-related behaviors, changes in the electroencephalogram of the cerebral cortex and hippocampus, and expression of hippocampal proteins. The viability and rate of success of SE induction were high in C57BL/6N mice but not in C57BL/6J mice. C57BL/6N mice were immotile during the first 2days after SE; however, by the third day, most mice were recovered and exhibited strong anxiety-related behaviors in response to the light/dark preference test and open field test. There was a striking difference in the temporal appearance of anxiety-related behavior between the two SE durations: 1.5h SE mice exhibited strong anxiety-related behavior 3days after SE that gradually attenuated over the next few weeks, whereas 4.5h SE mice exhibited strong anxiety-related behavior 3days after SE that persisted even at nearly 1year after SE. Mice receiving both SE durations exhibited generalized seizures (GS) after SE; however, there was a marked difference in the timing and duration of GS appearance. Mice in the 4.5h SE group exhibited spontaneous GS from 4days to at least 96days after SE. In contrast, mice in the 1.5h SE group exhibited GS only within the first several days after SE; however, epileptic spike clusters continuously appeared in the cerebral cortex and hippocampus for up to twelve days after SE. Among the hippocampal proteins tested, only brain derived-neurotrophic factor (BDNF) exhibited altered expression in parallel with anxiety-related behavior. These results showed the possibility that BDNF expression in the hippocampus might cause anxiety-related behavior in adulthood.

NMDA receptor-dependent recruitment of calnexin to the neuronal plasma membrane.

Calnexin is a molecular chaperone that resides in the endoplasmic reticulum and participates in the folding and assembly of nascent proteins. In the present study, calnexin was found in both synaptic and non-synaptic membrane components of rat brain tissue. Immunohistochemical staining of mouse hippocampal sections revealed the presence of calnexin in the neuronal cell soma, as well as dendrite-enriched regions. Staining of permeabilized cultured rat hippocampal neurons with anti-calnexin antibody produced intense staining throughout the cytoplasm of the cell body and dendrites. In non-permeabilized cells, calnexin was found on the surface of the cell body and dendrites. To further confirm the surface localization of calnexin, cell surface proteins were selectively labeled with a membrane-impermeable biotinylation reagent. Calnexin and other plasma membrane proteins including N-methyl-D-aspartate (NMDA) receptor and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor were biotinylated, and the amount of calnexin on the plasma membrane markedly increased after NMDA receptor activation. These results suggest that a significant fraction of calnexin localizes to the neuronal cell membrane, and that this recruitment is regulated in an NMDA receptor-dependent manner. Moreover, immunoisolation of vesicles revealed co-localization of the AMPA receptor subunit, GluA2, and calnexin in post-endoplasmic reticulum intracellular membrane components. These findings provide support for the hypothesis that calnexin may play a role in NMDA receptor-dependent neuronal functions.

Protein phosphatase 2A dephosphorylates SNAP-25 through two distinct mechanisms in mouse brain synaptosomes

Synaptosomal-associated protein 25 (SNAP-25) plays an essential role in exocytotic neurotransmitter release as a t-SNARE protein. SNAP-25 is phosphorylated at Ser(187) in a protein kinase C (PKC)-dependent manner, but the mechanism for dephosphorylation has yet to be clarified. We investigated SNAP-25 dephosphorylation by comparing it to growth associated protein 43 (GAP-43), another PKC-dependent presynaptic phosphoprotein, in crude mouse brain synaptosome preparations. Phosphorylation levels for both SNAP-25 and GAP-43 increased significantly after treatment with PKC activator phorbol 12, 13-dibutyrate (PDB), and ionomycin treatment induced a striking reduction in a time-dependent manner. This dephosphorylation occurred only in the presence of extracellular Ca(2+), indicating involvement of a Ca(2+)-dependent phosphatase. Ca(2+)-dependent dephosphorylation was not suppressed by calcineurin/PP2B inhibitors such as FK506 and cyclosporine A. SNAP-25 dephosphorylation, however, was suppressed by calyculin A, a non-selective inhibitor of PP1 and PP2A, and okadaic acid selective for PP2A, but not by tautomycin selective for PP1. In contrast, none of these inhibitors suppressed GAP-43 dephosphorylation. PDB-induced SNAP-25 phosphorylation was enhanced by okadaic acid in a concentration-dependent manner. These results suggest that PP2A participates in SNAP-25 dephosphorylation through Ca(2+)-dependent and Ca(2+)-independent mechanisms but is not involved in GAP-43 dephosphorylation.

SNAP-25 phosphorylation at Ser187 regulates synaptic facilitation and short-term plasticity in an age-dependent manner

Neurotransmitter release is mediated by the SNARE complex, but the role of its phosphorylation has scarcely been elucidated. Although PKC activators are known to facilitate synaptic transmission, there has been a heated debate on whether PKC mediates facilitation of neurotransmitter release through phosphorylation. One of the SNARE proteins, SNAP-25, is phosphorylated at the residue serine-187 by PKC, but its physiological significance has been unclear. To examine these issues, we analyzed mutant mice lacking the phosphorylation of SNAP-25 serine-187 and found that they exhibited reduced release probability and enhanced presynaptic short-term plasticity, suggesting that not only the release process, but also the dynamics of synaptic vesicles was regulated by the phosphorylation. Furthermore, it has been known that the release probability changes with development, but the precise mechanism has been unclear, and we found that developmental changes in release probability of neurotransmitters were regulated by the phosphorylation. These results indicate that SNAP-25 phosphorylation developmentally facilitates neurotransmitter release but strongly inhibits presynaptic short-term plasticity via modification of the dynamics of synaptic vesicles in presynaptic terminals.

PKC-dependent phosphorylation of SNAP-25 plays a crucial role in the suppression of epileptogenesis and anxiety-related behavior in postnatal period of mouse

P3-s02 Comparison between fMRI and direct cortical stimulation for clinical retinotopic mapping Akihiro Shimotake 1 , Riki Matsumoto 1, Masanori Kanazu 2, Hiroki Yamamoto 2, Masao Matsuhashi 3, Nobukatsu Sawamoto 3, Yukihiro Yamao 4, Nobuhiro Mikuni 5, Susumu Miyamoto 4, Hidenao Fukuyama 3, Ryosuke Takahashi 1, Akio Ikeda 1 1 Dept Neurology, Grad Sch of Med, Kyoto Univ, Kyoto, Japan 2 Grad Sch of Human and Environmental Studies, Kyoto Univ, Kyoto, Japan 3 HBRC, Grad Sch of Med, Kyoto Univ, Kyoto, Japan 4 Dept Neurosurgery, Grad Sch of Med, Kyoto Univ, Kyoto, Japan 5 Dept Neurosurgery, Sapporo Med Univ, Sapporo, Japan