神経化学教育口演

神経化学教育口演 Neurological Disorders

1G3-01 ATP supplementation therapy for ALS with SIGMAR1 mutation Shinoda Yasuharu，Tagashira Hideaki，Fukunaga Kohji Dept. of Pharmacol., Grad Sch of Pharm Sciences, Tohoku Univ. The dominant missense mutation（p.E102Q）by SIGMAR1 gene mutation was discovered in the patients of juvenile amyotrophic lateral sclerosis（ALS）. The sigma-1 receptor（Sig-1R）is a chaperone protein localizing in the mitochondrial-associated endoplasmic reticulum（ER）membrane in where the receptor regulates Ca2+ transport from ER to the mitochondria through IP3 receptor（IP3R）. When Sig-1R mutant（Sig-1RE102Q）overexpression in neuroblastoma neuro2A cells, Sig-1RE102Q dissociated from the IP3R and formed aggregations in the cytosol（Biochem Biophys Acta 2014；1840：3320）. Mitochondrial Ca2+ transport induced by IP3R stimulation was also disturbed by Sig-1RE102Q expression, thereby reducing mitochondrial ATP production. The Sig-1RE102Q mutant also reduced the mitochondrial membrane potential and promoted mitophagy. Moreover, the ATP reduction caused the decreased proteasome activity and in turn TAR DNA binding protein（TDP-43）accumulation in the cytosol. These events were recapitulated by pharmacological inhibition of either proteasome or mitochondrial Ca2+ transport. We tried to rescue the ATP reduction by supplementation of mitochondrial TCA cycle substrate, methyl pyruvate. The methyl pyruvate treatment rescued the Sig-1RE102Q-induced ATP reduction, thereby restoring the proteasome activity with concomitant inhibition of cytoplasmic accumulation of TDP-43. Taken together, ATP supplementation with methyl pyruvate can rescue the mitochondrial injury associated with ALS caused by Sig-1RE102Q mutation.		1G3-02 Determination of the key domains of CHRNA7 in the interacting actions of Arctic mutant Aβ JU YE¹，Asahi Toru^1,2，Sawamura Naoya^1,2 ¹Sci. ＆ Engi., Waseda，²Research organization for nano-life innovation Amyloid β protein（Aβ）plays a central role in the pathogenesis of Alzheimer’s disease（AD）. Point mutations within the Aβ sequence that are associated with familial AD（FAD）are clustered around the central hydrophobic core of Aβ. Several types of mutations within the Aβ sequence have been identified, and the‘Arctic’mutation（E22G）has a purely cognitive phenotype typical for AD（Nilsberth, C. et al., Nat. Neurosci. 4（2001））. Previous studies showed an increased formation of Aβ protofibrils as a primary result of the‘Arctic’mutation. However, the molecular mechanism underlying this effect remains un-clarified. Previous reports suggested that Aβ42 binds to one of the neuronal nicotinic acetylcholine receptor’s subunits, neuronal acetylcholine receptor subunit alpha-7（CHRNA7）, with high affinity and thus, may be considered to relate to AD（Wang, H. Y. et al., J. Biol. Chem 275（2000）；Wang, H. Y. et al., J. Biol. Chem 75（2000））. Our previous study indicated that Arctic β binds to CHRNA7 with high affinity, enhances its aggregation further when co-incubated with CHRNA7 and destabilizes the function of CHRNA7 via the inhibition of the Ca²⁺ response and activation of ERK1/2（Ju, Y. et al., J Neurochem. 131（2014））. This study aims at addressing the key domains of CHRNA7 potentially interacting with Arctic Aβ. Site-directed mutagenesis was carried out to study the key domains. We performed an in vitro binding assay using purified mutant CHRNA7 and synthetic Arctic Aβ to search the critical domains for Arctic Aβ-CHRNA7 binding. Furthermore, we are currently over-expressing mutant CHRNA7 in neuronal cells in order to investigate the key domains in CHRNA7 potentially interact with Arctic Aβ to regulate the functions of this receptor.

1G3-03 Function of Cathepsin C and Cystatin F during demyelination LI JIAYI^1,2，Wisessmith Wilaiwan¹，Shimizu Takahiro¹，Tanaka Kenji F^1,4，Kimori Yoshitaka³，Ikenaka Kazuhiro^1,2 ¹National Institutes for Nature Sciences National Institute for Physiological Sciences，²Graduate University for Advanced Studies,School of Life Science，³National Institutes of Natural Sciences, Center for Novel Science Initiatives, Imaging Science Division，⁴Keio University, School of Medicine, Department of Neuropsychiatry Cystatin F, a papain-like lysosomal cysteine proteinase inhibitor, and its main target, cathepsin C, have been demonstrated to be crucial factors in demyelinating diseases. It is found that the expression of cathepsin C and cystatin F are profoundly elevated and matched with ongoing demyelination/remyelination. However, their accurate functional role in demyelinating diseases is still unclear. To clarify their function in the pathological process of demyelination, we used a spontaneous chronic demyelination mouse model, named heterozygous PLP transgenic 4e（PLP4e/-）mouse. Meanwhile, Flexible Accelerated STOP-Tetracycline Operator Knock in（FAST）system is applied to up or down regulate cathepsin C or cystatin F gene expression. Cystatin F as the inhibitor of cathepsin C is predicted as a protective factor. But higher severity of demyelination was observed in cystatin F overexpressing PLP4e/- mice. Additionally, microglia showed highly activated morphology in cystatin F overexpressing mice. Together with the in situ hybridization results that in PLP4e/- mice conditional knock down of cystatin F gene in microglia lead to the down regulation of cathepsin C mRNA level, we predicted that cystatin F induce cathepsin C gene expression in addition to their protein level interaction during demyelination. In order to prove this hypothesis, we plan to check cathepsin C and cystatin F gene and protein expression by using real time PCR and western blot.		1G3-04 Time-lapse imaging of migrating new neurons in the injured adult cerebral cortex Matsumoto Mami，Sawada Masato，Sawamoto Kazunobu Department of Developmental and Regenerative Biology, Nagoya City University Graduate School of Medical Sciences In the adult rodent brain, new neurons are constantly generated from neural stem cells in the ventricular-subventricular zone（V-SVZ）. Under physiological conditions, these new neurons form chains and migrate along the blood vessels in the rostral migratory stream（RMS）toward the olfactory bulbs, where they are integrated into pre-existing neural network. After brain injuries, V-SVZ-derived new neurons also migrate in chains along blood vessels toward the injured sites and differentiate into mature neurons, suggesting the potential of endogenous neural stem cells for neuronal regeneration. Here we show the patterns and dynamics of neuronal migration in the injured adult mouse brain. In the photothrombotic stroke model, we observed new neurons migrating in chains or individually frequently associated with blood vessels in the injured corpus callosum and cerebral cortex, suggesting that new neurons utilize blood vessels as a migratory scaffold. To analyze dynamics of neuronal migration toward the injured sites, we performed time-lapse imaging of migrating new neurons in the cultured adult brain slices. Chain-forming new neurons migrated along the blood vessels from the V-SVZ toward the injured sites. Similar to the neuronal migration in the RMS, new neurons in the injured brain showed saltatory movement, executed by repeated extension of the leading process followed by the advancement of the soma. However, migration speed was significantly slower in the injured brain. These observations provide insights into similarity and difference in neuronal migration between physiological and pathological conditions.

1G3-05 Histamine N-methyltransferase deficiency induced the abnormal sleep-awake cycles and aggressive behavior in mice Naganuma Fumito¹，Yoshikawa Takeo¹，Yanai Atsushi^1,2，Horigome Ai¹，Miura Yamato¹，Nakamura Tadaho¹，Mochizuki Takatoshi²，Yanai Kazuhiko¹ ¹Department pf Pharmacology, Tohoku University Graduate School of Medicine，²Department of Neurology, Harvard Medical School Histamine plays as a neurotransmitter in various physiological events such as sleep-awake cycle and appetite regulation. In order to maintain the homeostasis of histaminergic neuronal activities, the excessive histamine should be inactivated. The previous studies suggested that histamine N-methyltransferase（HNMT）was important for histamine inactivation. However, the role of HNMT in vivo remains almost unclear. In the present study, we generated and investigated the Hnmt knockout mice（KO）to elucidate the importance of HNMT. First, we generated KO by inserting LacZ gene into HNMT gene. LacZ reporter assay revealed that Hnmt were highly expressed in cortex, amygdala, locus coeruleus and Raphe nucleus. Histamine content in the brain lysate of KO was 6 times as abundant as that in wild type mice（WT）. The extracellular histamine in the hypothalamic area was also increased in KO. These results cleared that HNMT was essential for brain histamine clearance. Most of KO was wounded by fighting in home-cage, suggesting the increase of aggressive behavior in KO. We confirmed the aggressive behavior of KO in the resident-intruder test and aggressive biting behavior test. The sleep analysis revealed that the sleep duration of KO in dark period was longer than that of WT, and the KO showed an increase in slow-wave EEG in wakefulness, suggested that Hnmt deficiency caused the dysfunction of sleep-awake cycles. These results indicated that Hnmt was involved in sleep-awake cycles and aggressive behavior through the regulation of histamine concentration and histamine neuronal activities.		1G3-06 The acute immediate effect of X-irradiation and Carbon ion-irradiation on synaptic function and fear memory formation. Anggraeini Puspitasari，Koganezawa Noriko，Kajita Yuki，Shirao Tomoaki Department of Neurobiology and Behavior, Gunma University Graduate School of Medicine Carbon-ion therapy becomes widely known and is used as the advanced therapy for cancer. Carbon-ion irradiation is thought to show a higher biological effectiveness compared to X-irradiation, but still the acute immediate effect on the brain function is poorly understood. In this study, we compared the acute effects of X-ray and carbon-ion irradiation on fear memory formation and accumulation of a synaptic protein. We used 10-12-week old male mice, and administered a single dose of 10 Gy of either X-ray or carbon-ion beam to whole brains. Then fear conditioning was conducted 7 hrs and 24 hrs after the irradiation. We found that the mice irradiated by either X-rays or carbon-ion beam 7 hrs before training did not retrieve the contextual and auditory memories, whereas those irradiated 24 hrs before training did retrieve the both memories. We analyzed drebrin, a marker for synaptic function, immunohistochemically in neuropil of the dentate gyrus of hippocampus. We found there were significant decreases of drebrin intensities 2 hrs and 8 hrs after the irradiation and it returned to the former level 24 hrs after the irradiation. Interestingly, the number of drebrin clusters also decreased with a similar time course in in vitro study. When we analyzed the number of drebrin clusters after we irradiated mature primary hippocampal neurons, it decreased significantly 2 hrs and 8 hrs after X-irradiation and returned to the former level after 24 hrs. Similarly, the number of drebrin clusters significantly decreased 2 hrs after carbon-ion irradiation and tended to return 24 hrs after the carbon-ion irradiation. These results suggest that there are transient effects on the synaptic function of both X-irradiation and carbon-ion irradiation and these may cause fear memory deficits.