Wakate Dojo

Wakate Dojo 神経ネットワーク、細胞内・細胞間情報伝達

1DJ1-1 Activation of perineuronal net-positive excitatory neurons during associative memory learning Shota Morikawa^1,2,5，Yuji Ikegaya^3,4，Minoru Narita⁵，Hideki Tamura² ¹Laboratory of Gene Regulation Research, Graduate School of Biological Sciences, Nara Institute of Science and Technology (NAIST)，²Life Science Tokyo Advanced Research Center (L-StaR), Hoshi University School of Pharmacy and Pharmaceutical Sciences，³Laboratory of Chemical Pharmacology, Graduate School of Pharmaceutical Sciences, University of Tokyo，⁴Center for Information and Neural Networks, National Institute of Information and Communications Technology，⁵Department of Pharmacology, Hoshi University School of Pharmacy and Pharmaceutical Sciences Some neurons are enwrapped by a specialized matrix structure called a perineuronal net (PNN), which acts as a diffusion barrier for ion channels and receptors, scaffold binding for signaling molecules, and an ionic buffer. Therefore, PNNs modulate synaptic transmission and neuronal activity. PNNs in the adult brain are expressed in a region- and cell-specific manner; in particular, they are expressed around parvalbumin (PV)-positive inhibitory neurons, although some glutamatergic neurons express PNNs. Recent studies using enzymes that degrade PNNs demonstrate that PNNs regulate synaptic plasticity, critical period closure, and fear memory. Despite these important functions, it is unclear what differentiates neurons with PNNs from those without. Here, we show that PNNs are mainly expressed around excitatory neurons in brain regions related to emotional learning and memory. We then compared the expression of c-Fos, a marker of neuronal activity, between neurons with and without PNNs after fear conditioning, which is a form of associative learning.		1DJ1-2 SENP1 promote de-sumoylation of amyloid precursor protein Takuma Maruyama¹，Yoichiro Abe²，Takako Niikura¹ ¹Sophia Univ，²Dept Pharmacol. Sch Med, Keio Univ Amyloid β, a major component of a senile plaque, plays the central role in the pathogenesis of Alzheimer’s Disease (AD). Amyloid β is produced from amyloid precursor protein (APP) by β- and γ- cleavages. APP is modified by SUMO at Lys587 and Lys595 in APP695. These SUMOylation sites locate near the β-cleavage site. SUMOylation of APP decreases amyloid β production, suggesting the SUMOylation-dependent regulation of amyloid metabolism. However, the regulatory mechanism is largely unknown. SUMOylation is a reversible post-translational modification targeting lysine residues in substrate proteins. In human brain, three SUMO proteins, SUMO-1, SUMO-2 and SUMO-3, are expressed. SUMOylation is mediated by E1, E2, and E3 enzymes and reversed by de-SUMOylation enzymes. The sentrin/SUMO-specific proteases (SENPs) are major de-SUMOylation enzymes, and four family members (SENP1, 2, 3 and 6) are expressed in human brain. In this study, to identify SENP proteins for APP de-SUMOylation, we co-expressed APP695, SUMO1/2 and SENP proteins in HEK293 cells. We found that the de-SUMOylation of APP was induced by SENP1 and SENP2, but not SENP3. SENP1 but not SENP2 was colocalized with APP in HEK293 cells, suggesting that SENP1 can directly regulate the de-SUMOylation of APP. Moreover, the quantitative PCR revealed that SENP1 level was increased in an age-dependent manner only in female mice. Multiple studies have demonstrated that female mice compared with males show more aggressive amyloid pathology in AD mouse models. Our results provide a clue to understanding the role of SUMOylation in the sex difference in AD pathogenesis.

1DJ1-3 The effect of 8-nitro-cGMP on SNARE complex assembly and memory Yusuke Kishimoto¹，Shuichi Yanai²，Shogo Endo²，Takaaki Akaike³，Hideshi Ihara¹ ¹Department of Biological Science, Graduate School of Science, Osaka Prefecture University，²Aging Neuroscience Research Team, Tokyo Metropolitan Institute of Gerontology，³Department of Environmental Health Science and Molecular Toxicology, Graduate School of Medicine, Tohoku University 【Introductions】8-Nitroguanosine 3’,5’-cyclic monophosphate (8-nitro-cGMP) is a second messenger that mediates nitric oxide (NO)/reactive oxygen species (ROS) redox signal. This electrophilic molecule covalently binds to nucleophilic thiol group in protein, which is called protein S-guanylation, and exerts its functions. Our group has revealed that synaptosomal-assosiated protein 25 (SNAP-25), a member of soluble N-ethyl maleimide sensitive factor attachment protein receptor (SNARE) proteins, is subject to S-guanylation. Here, we have revealed the effect of S-gusnylation of SNAP-25 on the SNARE complex assembly and affinity of the complex with complexin (cplx). Moreover, we have investigated the effect of 8-nitro-cGMP on the brain functions, especially on memory.【Materials & Methods】SH-SY5Y neuroblastoma cells transfected with FLAG-tagged SNAP-25 and/or V5-tagged cplx were treated with 8-nitro-cGMP. Protein interactions were analyzed by co-immunoprecipitation (co-IP) and blue native (BN)-PAGE. 8-Nitro-cGMP was injected to C57BL6 mice’s intracerebroventricular (icv). Open field test and fear conditioning task were performed to assay activity and anxiety levels and memory. Brain proteins were analyzed by BN-PAGE.【Results & Discussions】Co-IPed V5-cplx by anti-FLAG antibody was decreased by 8-nitro-cGMP. SNAP-25 detected at high molecular mass were increased, by contrast, V5-tagged cplx were decreased by 8-nitro-cGMP. Behavioral tests revealed that 8-nitro-cGMP decreased rearing and context-dependent long-term memory. BN-PAGE revealed that the bands pattern of SNAP-25 and cplx in hippocampus were changed by 8-nitro-cGMP treatment. Our data suggest that 8-nitro-cGMP, a NO/ROS redox signal mediator, regulates SNARE complex functions and memory.		1DJ1-4 Effects of diet restriction on cognitive impairments and decreased hippocampal GAT2 expression in a genetic mouse model of Alzheimer’s disease Kazuki Hirashima，Daisuke Ibi，Kahori Sumiya，Takayosi Mamiya，Masayuki Hiramatsu Dept. Chem. Pharmacol., Fac. Pharm., Meijo Univ. OBJECTIVE: Alzheimer's disease (AD) is a neurodegenerative disorder characterized by a progressive decline in the cognitive function associated with neuropathological hallmarks such as β-amyloid (Aβ) plaques and neurofibrillary tangles. Diet restriction (DR) in both clinical studies and preclinical animal models has been suggested to protect against AD. Although we have already reported that the intraventricular administration of amyloid β-peptide (25-35) impairs learning and memory, and changes GABA transporter 2 (GAT2) expression in the hippocampus of mice, it remains unclear whether DR prevents memory deficits in genetic animal models of AD. Here, we investigated the effects of DR on memory deficits and hippocampal GAT2 expression in a genetic mouse model of AD. METHODS: We used triple transgenic (3xTg) mice expressing mutated genes of APP, presenilin-1, and tau as an animal model of AD, and performed Y-maze and novel object recognition (NOR) tests at both 6 and 9 months old to investigate short-term and object recognition memories, respectively. Wild-type (WT) mice of the same age were used as a control. DR, restricted to 70% of the ad libitum food intake of control mice, was started at 6 months old, and continued for 3 months. Thereafter, GAT2 and Aβ expressions were analyzed by Western blotting.RESULTS: Memory deficits and the down-regulation of hippocampal GAT2 expression in 3xTg mice became evident at 9 months old compared with WT mice, which were prevented by DR for 3 months from 6 months old. In contrast, 3xTg mice showed an increased expression of Aβ in the hippocampus, but DR did not affect it.DISCUSSION: These results suggest that DR may have a preventive effect against learning and memory deficits in patients with AD mediated by GAT2 induction.

1DJ1-5 Effects of maternal immune activation on the mossy fiber density and Reelin expression in the hippocampal dentate gyrus in the offspring of mice Nayu Koide，Daisuke Ibi，Takayoshi Mamiya，Masayuki Hiramatsu Dept. Chem. Pharmacol., Fac. Pharm., Meijo Univ. OBJECTIVE: Maternal immune activation (MIA) following infection increases the risk of psychiatric disorders. We have already shown that MIA induced by polyinosinic-polycytidylic acid (polyI:C) treatment of pregnant mice causes behavioral abnormalities of the offspring in adulthood. Reelin, a secretory protein from GABAergic interneurons in the hippocampal dentate gyrus (DG), has been reported to play a key role in mossy fiber development. A clinical study indicated that Reelin expression is decreased in the brains of patients with schizophrenia: however, the involvement of Reelin in brain dysfunction triggered by MIA remains unknown. Here, we investigated the effect of MIA on the hippocampal Reelin expression in the offspring of polyI:C-treated pregnant mice.METHODS: Pregnant C57BL/6J mice were intraperitoneally treated with polyI:C (20 mg/kg) on gestation day 9. The expressions of Reelin, somatostatin (a GABAergic interneuron marker), and synaptoporin (a mossy fiber marker) in the hippocampus of the offspring in adulthood were analyzed by immunostaining.RESULTS: MIA decreased the number of Reelin-positive cells, and the ratio of Reelin-positive cells colocalized with somatostatin in the hippocampal DG. We then analyzed synaptoporin expression to investigate whether the down-regulation of Reelin expression affects the mossy fiber density. The synaptoporin signal intensity was also decreased in the hippocampal DG.DISCUSSION: The results suggest that the reduction of Reelin expression on somatostatin-positive neurons in the hippocampal DG may down-regulate the density of mossy fibers, leading to behavioral abnormalities. Here, we also show the effect of recombinant Reelin microinjection into the DG on the mossy fiber density and behavioral abnormalities in the offspring.		1DJ1-6 Effect of chronic betaine intake on learning and memory impairments and hippocampal GAT2 expression in genetic mouse model of Alzheimer’s disease Kahori Sumiya，Daisuke Ibi，Kazuki Hirashima，Takayoshi Mamiya，Masayuki Hiramatsu Dept.Chem.Pharmacol., Fac.Pharm., Meijo Univ. OBJECTIVE: GABA transporter 2 (GAT2) transports not only GABA but also betaine, an amino acid derived from natural plants. Although we have already reported that acute treatment with betaine ameliorates learning and memory impairments in mice receiving the intracerebroventricular injection of amyloid-β (Aβ), it remains unclear whether chronic betaine treatment prevents brain dysfunction in genetic animal models of Alzheimer’s disease (AD). In this study, we investigated the effect of chronic betaine intake on learning and memory impairments and hippocampal GAT2 expression in a genetic mouse model of AD.METHODS: We used triple transgenic mice expressing mutated genes of APP , presenilin-1, and tau as an animal model of AD (3xTg-AD mice) and performed Y-maze and novel object recognition (NOR) tests at both 6 and 9 months old to investigate short-term and object recognition memories, respectively. Wild-type (WT) mice of the same age were used as a control. Betaine was added at a concentration of 10 g/L to drinking water at 6 months old, and continued for 3 months. GAT2 expression levels in the hippocampus were analyzed by Western blotting at 9 months old.RESULTS: Learning and memory impairments in 3xTg-AD mice were not observed at 6 months old, but they became evident 3 months later, which was prevented by chronic betaine intake for 3 months from 6 months old. GAT2 expression levels in the hippocampus of 3xTg-AD mice at 9 months old were decreased compared with WT mice. DISCUSSION: These results suggest that betaine may become a preventive medicine/supplement for AD.