Pe-28- 1
withdrawnPe-26-6
Alteredfunctionalconnectivityinrespiratoryimpairment
○AkikoYorita1,TomotakaKawayama1,MasayukiInoue2, TakashiKinoshita1,HanakoOda1,YoshihisaTokunaga1,
YoshihisaShoji2,NaohisaUchimura2,ToshiAbe3,TomoakiHoshino1, TakayukiTaniwaki1
1 Division of Respirology,Neurology,and Rheumatology,Department of Internal Medicine,Kurume University School of Medicine, Japan, 2 Cognitive and Molecular Research Institute of Brain Diseases,Kurume University,
3 Department of Radiology,Kurume University
Objective: Breathing and the perception of breathing impairment are critical function for some neurological disorders. Previous neuroimaging studies have identified brain regions related to respiratory control and perception. However, little is known about the functional connectivity（FC）in respiratory impairment. In this study, we explored FC change at the brain regions involving respiratory impairment. Methods; Sixteenth healthy volunteers underwent rs-fMRI during normal respiration or respiratory impairment induced by breathing effort. Imaging data were preprocessed using SPM 12 and the CONN toolbox. Then seed to voxel analysis was performed using respiratory associated brain regions as a seed. The correlations of significant resting state FC with clinical data were analyzed. Results: Compared with normal respiration, breathing effort showed significantly increased FC between the left supplementary motor area（SMA）and right post central gyrus（Post CG）,between the anterior cingulated cortex（ACC）and right Post CG, and the left anterior insular cortex and right occipital pole. Breathing effort also revealed decreased FC between the brain-stem and right lingual gyrus. That connectivity was significantly correlated with the clinical data（AUC of the Borg scale）.Conclusion: To our knowledge, this study is the first to investigate functional connectivity in the brain during respiratory impairment.
The current results suggest sensory-motor and affective processing in breathing effort.
Pe-26-8
withdrawnPe-27-2
Directreprogrammingofsomaticurine-derived cellstogeneratemultipleneuronalcelllineages○MitsutoSato1,HotakeTakizawa1,AkinoriNakamura2, YoshitsuguAoki1
1 Department of Molecular Therapy, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Japan, 2 Department of Clinical Research, National Hospital Organization Matsumoto Medical Center [Objective] In recent years, cells with stem-like characteristics have been identified in urine samples and have been recognized as useful materials in disease modeling. Very recently, we have reported MyoD1 converted urine-derived cells
（UDCs） as a new primary myoblast model of human muscular dystrophy. Here, we aimed to perform direct reprogramming of UDCs into neuronal cells to model neurological diseases as the next step. [Methods] UDCs were collected from healthy human donors （n=5）, and were cultured in laminin-coated dishes with neuronal differentiation medium, containing B27, retinoic acid, and PDGF-BB, etc.
After culture for 14 days, we evaluated morphological changes, the expression of neuronal differentiation markers by qRT-PCR and immunocytochemistry. [Results]
After neuronal induction of UDCs, morphological changes such as cone-shaped with protrusions formation were observed. The expression of beta III Tubulin and microtubule-associated protein 2 genes were significantly upregulated in differentiated UDCs compared with undifferentiated UDCs evaluated by qRT-PCR and immunocytochemistry. Moreover, the expression of vGLUT1, a glutamatergic neuron marker, and GAD65, a GABAergic neuron marker, were confirmed.
[Conclusions] We show that UDCs have the potential to be differentiated into neuronal cells with a mixed population of glutaminergic and GABAergic neurons.
In vitro direct reprogramming to generate multiple neuronal cell lineages using somatic UDCs may play an essential role to study pathological mechanisms and screen drugs for neuromuscular diseases.
Pe-27-3
Analysisofredbloodcellvelocityinintraparenchymal capillariesandarterialdiameterinmice○YutakaTomita1,2,MiyukiUnekawa1,YoshikaneIzawa1, KazutoMasamoto3,4,KannoIwao4,JinNakahara1
1 Department of Neurology, Keio University School of Medicine, Japan,
2 Tomita Hospital, Japan, 3 Brain Science Inspired Life Support Research Center, University of Electro-Communications, 4 Department of Functional Brain Imaging Research, National Institute of Radiological Sciences BACKGROUND: Control of red blood cell （RBC） velocity in capillaries is essential to meet local neuronal metabolic requirements, while changes of capillary diameter are limited. OBJECTIVE: To further understand the microcirculatory response to neural depolarization, we analyzed the spatiotemporal changes of RBC velocity in intraparenchymal capillaries during cortical spreading depolarization （CSD）.
METHODS: In urethane-anesthetized Tie2-GFP mice （N=21）, sequential images for 60 sec were obtained with high-speed camera （125 fps） laser-scanning confocal fluorescence microscope around CSD induction, simultaneously recording DC potential and regional cerebral blood flow （rCBF） with laser Doppler flowmetry.
The velocity of fluorescence-labeled RBCs in layer I was analyzed with the KEIO-IS2 using Matlab and arterial diameter was analyzed with ImagePro. RESULTS: In total, 265 capillaries were successfully detected in 18 mice （5-19 capillaries/mouse）. The basal velocity and arterial diameter were 0.91±0.36 mm/s and 24±5 µm, respectively.
CSD repeatedly increased the RBC velocity prior to arterial constriction/dilation.
During the first CSD, RBCs remarkably deaccelerated almost together in parallel with marked arterial constriction. The velocity returned to around the basal level, while post-CSD oligemia with slight vasoconstriction remained. CONCLUSION: Taken together with our previous findings, this study supports the idea that RBC flow may be altered independently, at least in part, from arterial regulation, that neuro-capillary coupling plays a role in meeting neural demand.
transmissionintheneocortex
○Yong-chunYu,WuqiangGuan,YinghuiFu Fudan University, China
Objective: During development of the neocortex, sensory experience critically influences neuronal connectivity and synaptic transmission. It has long been postulated that eye opening is crucial for cortical circuit maturation. However, whether eye opening shapes inhibitory synaptic transmission in the neocortex during development has never been reported. Methods: In this study, we performed multiple-electrode whole-cell recordings to record the synaptic transmission between somatostatin-expressing interneurons and pyramidal cells and between parvalbumin-expressing interneurons and pyramidal cells in in the developing visual cortex. Results: We observed that eye opening rapidly weakens the inhibitory synaptic transmission from somatostatin-expressing interneurons to pyramidal cells in the developing visual cortex.
However, the synaptic transmission from parvalbumin-expressing interneurons to pyramidal cells was not significantly changed during eye opening. More importantly, our study provides a postsynaptic mechanism that underlies the reduction of synaptic transmission from somatostatin-expressing interneurons onto pyramidal cells within the timing of eye opening. Conclusion: Our study reveals a selective developmental regulation in inhibitory circuits in the cortex driven by eye opening likely crucial for cortical maturation and function.
Pe-27-1
EfficientgenesuppressionbyDNA/DNA double-strandedoligonucleotideinvivo○YutaroAsami,TetsuyaNagata,KotaroYoshioka,TaikiKunieda, KieYoshida-tanaka,TakanoriYokota
Department of Neurology and Neurological Science, Graduate School of Medical and Dental Sciences, and Center for Brain Integration Research, Tokyo Medical and Dental University, Japan
[Objective] Antisense oligonucleotides （ASOs） are single-stranded nucleic acids that bind to their target RNA in cells and regulate its functions. In recent years, multiple ASOs have been approved for clinical application targeting neurological diseases. However, improving their in vivo potency is highly desirable. As one of the solutions, we recently developed DNA/RNA heteroduplex oligonucleotide
（DNA/RNA HDO） which comprises the ASO and its complementary RNA-strand. This DNA/RNA HDO significantly enhanced the potency of the parent ASO. We here developed a new type of HDO in which the center portion of complementary strand was replaced from RNA to chemically more stable DNA, resulting in a DNA/DNA double-stranded structure （DNA/DNA HDO）.
This study revealed in vivo potency of this DNA/DNA HDO. [Methods] We administered intravenously alpha-tocopherol conjugated DNA/DNA HDOs or DNA/RNA HDOs or original ASOs targeting several sequences of RNA into mice （n = 3 or 4）. We collected liver samples and estimated the gene silencing effect by quantitative real-time （qRT）-PCR assay. We also evaluated the delivery-effect to liver by qRT-PCR. [Results] DNA/DNA HDOs and DNA/RNA HDOs significantly enhanced the inhibitory potency of the parent ASO in sequence specific and dose-dependent manner in mice liver. The efficiency of delivery to liver by DNA/DNA HDO was about two to four-fold higher than the parent ASO.
[Conclusion] Our novel concept of DNA/DNA HDO is useful for the development of therapies for RNA mediated diseases including neurological diseases.
一 般 演 題
ポ ス タ ー （ 英 語 ）
Pe-26-7
EyeopeningshapesinhibitorysynapticPe-28-3
Microglia-astrocytecrosstalkintheperiinfarct areaafterstrokeinrats○ChikageKijima1,YujiUeno1,KenichiroHira1,ToshikiInaba2, ShoNakajima1,NobukazuMiyamoto1,KazuoYamashiro1, TakaoUrabe2,NobutakaHattori1
1 Department of Neurology, Juntendo University School of Medicine, Japan,
2 Department of Neurology, Juntendo University Urayasu Hospital
Purpose: The interaction between microglia and astrocyte is not fully elucidated.
We examined the alteration of the crosstalk between microglia and astrocyte after ischemic stroke in rats. Methods: We subjected adult male Wistar rats to permanent right middle cerebral artery occlusion （MCAO） and sacrificed at 3, 7, 14, 28 and 56 days after MCAO. We compared expressions of GFAP and iba-1 in the peri-infarct area. Cultured microglia and astrocyte were obtained from cerebral cortex of neonatal SD rats. Microglia were exposed to Oxygen-glucose deprivation （OGD）. We examined the expression of iba-1, arginase-1, and iNOs, and morphological alterations of Iba-1+ microglia after OGD. Cultured astrocyte was subjected to OGD, treated with microglia-conditioned medium （MCM）, and then examined in the expression of GFAP, CSPG, C3d . Results: In the peri-infarct area, Iba-1+ microglia increased until 7 days, and then decreased after MCAO, while GFAP+ astrocyte increased from day 7 to 56 days after MCAO. In vitro, after OGD, Iba-1+microglia with resting morphology decreased and bipolar and amoeboid shaped microglia increased, together with an increase in M1 microglia at 24 hours and a decrease in M2 microglia at 96 hours after OGD. MCM reduced the expression of CSPG in cultured astrocyte after OGD. Conclusion: In the peri-infarct area, microglia increased in the acute phase and decreased in the chronic phase, while astrocyte increase until the chronic phase. The expression of CSPG, the main component of astroglial scars, could be potentially regulated by microglia.
Pe-28-5
CRTC2playedanimportantroleunderischemic conditionsatendothelialcellsviap190RhogapA○HideakiKanki1,TsutomuSasaki1,ShigenobuMatsumura2, HiroshiTakemori3,HidekiMochizuki1
1 Department of Neurology, Graduate School of Medicine, Osaka University, Japan, 2 Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, 3 Faculty of Engineering, Department of Chemistry and Biomolecular Science, Gifu University
[Objective] Angiogenesis play crucial roles and cAMP-PKA pathway regulates angiogenisis. CREB pathway is related to cAMP-PKA pathway and very important for cell survival at ischemic conditions. CRTC （CREB regulated transcription coactivator） family are reported to be interacted with CREB and CRTC2 are reported to be predominantly expressed at liver, spleen and lymph node, and regulated glucose metabolism and B cell differentiation, but the role and mechanisms has not yet been elucidated at endothelial cells. Then, we examined the effect of CRTC2 on endothelial function using endothelial cells and knockout mice. [Methods] For evaluating endothelial function, various assays were performed in vitro and luciferase assay were also performed. We used endothelial cell-specific CRTC2-knockout mice for evaluating angiogenesis by aorta ring assay ex vivo and for evaluating the effect of ischemic stroke by photothrombotic stroke in vivo. [Results] CRTC2 was expressed at endothelial cells and only CRTC2, not CRTC1, was activated under ischemic conditions. CRTC2 enhanced endothelial function at various assays in vitro.
Luciferase assay showed CRTC2 was related to p190RhoGapA. Endothelial cell-specific CRTC2-knockout mice showed reduced angiogenesis, and had more severe ischemic stroke and impaired neurological recovery accompanied with increased BBB permeability and reduced blood vessels. [Conclusions] CRTC2 plays a crucial role in endothelial function, especially under ischemic conditions,via p190RhogapA.
CRTC2 is an attractive target for the new treatment of ischemic stroke.
Pe-28-6
TheEffectofNicotineontheChronicInflammation viacAMP-CRTC2signalinginEndothelialCells○TsutomuSasaki1,HideakiKanki1,TomohiroKawano1,HaominYen1, ShintaroSugiyama1,KenichiTodo1,ShigenobuMatsumura2, HiroshiTakemori3,HidekiMochizuki1
1 Department of Neurology, Graduate School of Medicine, Osaka University, Japan, 2 Laboratory of Nutrition Chemistry, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University,
3 Department of Chemistry and Biomolecular Science, Gifu University [Objective]The effect of nicotine or signal transduction via nAchR on the chronic inflammation in cerebral endothelial cells are not well understood. CRTC2 has been found to be a master regulator of glucose and fat metabolism. However, whether CRTC2 plays a role in physiological functions in endothelial cells remains unknown. This study investigated whether nicotine and consequent nAchR signaling have an impact on autophagy dysfunction or cross-talk with chronic inflammation.[Methods] Bovine brain microvasculature endothelial cells （BBMCs） or HUVEC were harvested. OGD was performed described previously. Western blot analyses were performed to examine both CREB-CRTC （CREB-regulated transcription coactivators） axis and autophagy process.
Mice were provided a high fat diet （HFD） from weeks 7 to 11. Mice fed an HFD or a normal diet were used at 11 weeks. Right MCAO occlusion （60 min） was performed using a suture and then reperfused （N=5）. Moreover, we also examined the crosstalk between cAMP-CRTC2 signaling and autophagy using CRTC2 KO mice. [Results] HFD-fed mice showed more severe ischemic injury, BBB damage, and exacerbation of inflammation.
HFD-fed mice showed lower autophagy process in penumbra after stroke. CRTC2 knockout mice exacerbated BBB damage （N=5）. The nAchR signaling contribute to the both chronic inflammation and autophagy pathway. [Conclusions] The chronic inflammation in cerebral endothelial cells contributes to the pathology via autophagy dysfunction in neurovascular unit, at least in part, cAMP-CRTC2 signaling and nAchR signaling.
Pe-29-1
Lightexerciseinducesischemictolerancethrough modulationofmicroRNAsinthegerbilhippocampus○TadayukiTakata1,2,WakakoNonaka2,HisakazuIwama3, HidekiKobara4,KazushiDeguchi2,HisashiMasugata1, TetsuoTouge5,OsamuMiyamoto6,TakehiroNakamura7, ToshifumiItano2,TsutomuMasaki4
1 Department of General Medicine, Kagawa University Faculty of Medicine, Japan, 2 Department of Neurology, Kagawa University Faculty of Medicine, Japan, 3 Department of Life Science Research Center, Kagawa University,
4 Department of Gastroenterology, Kagawa University Faculty of Medicine,
5 Department of Health Sciences, Kagawa University Faculty of Medicine,
6 Department of Physiology 2, Kawasaki Medical School, 7 Department of Medical Technology, Kagawa Prefectural University of Health Sciences [Objective]To investigate the neuroprotective effects of light exercise, without lactate elevation, on ischemia/
reperfusion injury of a gerbil model. [Methods]Transient whole-brain ischemia was induced by occlusion of the bilateral common carotid arteries for 5 min. A group of animals was subjected to treadmill exercise before ischemia induction. Hippocampal neuronal damage and microRNA （miRNA） expression were evaluated, as well as behavioral deficits and plasma lactate levels. [Results]The majority of pyramidal neurons within the Cornet d' Ammon 1 （CA1） area of the gerbils in the ischemia group were loss, when compared to those of the sham group. In contrast, neurons within the same area in gerbils of the exercise plus ischemia group were preserved and appeared normal. short-term memory in the exercise plus ischemia group was preserved compared with that of the ischemia group and was equivalent to that of the sham group. The lactate level and weight of gerbils in each group at sacrifice were not significantly different. 14 miRNAs were upregulated, and 6 miRNAs were downregulated due to ischemia. However, the expression of these miRNAs remained unchanged when animals performed light exercise before the ischemic event. [Conclusions]Histologically, neurons were preserved in CA1, and short-term memory was retained. Although several mechanisms may lead to this effect, it was shown that ischemic tolerance induced by light exercise is associated with miRNA expression level in the hippocampus.
Light exercise with non-elevated lactate levels was shown to induce ischemic tolerance.
○NaokiIwasa1,TakeshiKMatsui1,NaohikoIguchi1,TomoShiota1, NobuyukiEura1,KozueSaito1,EiichiroMori2,KazumaSugie1
1 Department of Neurology, Nara Medical University, Japan, 2 Department of Future Basic Medicine, Nara Medical University
[Objective] Ischemic stroke is one of the most common neurological disease, but the detailed cellular reaction are not evaluated. We used 3D human cerebral organoids derived from human induced pluripotent stem cells（hiPSCs）.
Oxygen-glucose deprivation/reperfusion （OGD/R） is known as an ischemia and reperfusion model. We analyzed human cerebral organoids under OGD/
R condition to detect the true action of neuronal cells and glial cells. [Methods]
Cerebral organoids were generated from hiPSCs. Subsequently, RNA-sequencing and quantitative PCR were performed. Gene expression profile was confirmed, bioinfomatics analysis was done. [Results] Comparing cerebral organoids after OGD/R and non-treated organoids, a total of 52 differentially expressed genes （DEGs） were determined and most of DEGs were downregulated. When False discovery rate （FDR） adjusted p-value ≦ 0.05 was used as a threshold, 15 genes were detected and all genes were downregulated.
The top 5 significant downregulated genes were AFP, TTR, APOA2, ALB and APOA1. RNA sequence analysis and pathway analysis showed the relationship of vitamin digestion and absorption, fat digestion and absorption, PPAR signaling pathway, and complement and coagulation cascades. [Conclusion]
Many genes associated with lipid metabolism are downregulated under OGD/R condition. These findings might indicate the mechanisms underlying ischemic injury, using human cerebral organoids treated by OGD/R.
Pe-28-7
withdrawn一 般 演 題
ポ ス タ ー （ 英 語 ）
Pe-28-4
Oxygen-glucosedeprivationandreperfusioncompromises lipidmetabolisminhumancerebralorganoidsPe-29-2
Impairedmyelinationreducessynchronous neuralactivityrequiredforlearning○DaisukeKato1,NoriyukiMatsukawa2,JunichiNabekura3, MasanoriMatsuzaki4,HiroakiWake1
1 Department of Anatomy and Molecular Cell Biology, Nagoya Univers ity Graduate School of Medicine, Japan, 2 Department of Neurology, Graduate School of Medicine, Nagoya City University, 3 Division of Homeostatic Development, National Institute for Physiological Sciences, 4 Department of Physiology, Graduate School of Medicine, The University of Tokyo Background: Myelination increases the conduction velocity in long-range axons and is pre-requisite for many brain functions. Myelin dysregulation is frequently associated with deficits in learning and cognition, ultimately causing neuropsychiatric disorders. However, it has not been revealed what perturbation of neural activity induced by disruption of myelin regulation impairs learning. Materials and Methods:
WT and PLP-tg mice of 1.5 months age were used. Two photon Ca2+ imaging and in vivo electrophysiology were performed to assess neural activities. Results:
Here, we measured neural activity in the motor cortex during motor learning in transgenic mice with a subtle disruption to their myelin regulation. This disruption impaired motor learning, and was accompanied by a decrease in the amplitude of movement-related activity and an increase in the frequency of spontaneous activity.
Thalamocortical axons showed variability in conduction velocity with a large spread in the timing of postsynaptic cortical responses. Repetitive pairing of forelimb movements with optogenetic stimulation of thalamocortical axon terminals restored learning. Conclusion: Thus, myelin regulation helps to maintain the synchrony of cortical spike-time arrivals through long-range axons. Our results revealed the pathological neuronal circuit activity with impaired myelin and suggest the possibility that pairing of non-invasive brain stimulation with relevant behaviors may ameliorate cognitive and behavioral abnormalities in diseases with impaired myelination.
Pe-29-3
withdrawnPe-29-4
SV2BratherthanSV2Apreferablyinteractswith BACE1asanegativeregulatorofAPPprocessing○MasakazuMiyamoto1,2,AkiraKuzuya2,YasuhaNoda1, KengoUemura2,MegumiAsada-Utsugi1,2,ShinjiIto3, YoshiyasuFukusumi4,HiroshiKawachi4,RyosukeTakahashi2, AyaeKinoshita1
1 Dept. Human Health Sci. Grad. Sch. Med. Kyoto Univ. Kyoto, Japan, 2 Dept.
Neuro. Kyoto Univ. Grad. Sch. Med, Japan, 3 Medical Research Support Center. Grad. Sch. Med. Kyoto, 4 Dept.Cell Biol.Inst of Nephrol.Grad.Sch.Med and Dental Sci.Niigata Univ
<Background> We previously identified synaptic vesicle protein 2B （SV2B）
as a novel binding partner of BACE1 to inhibit amyloidogenic APP processing.
Interestingly, the anti-epileptic drug levetiracetam targeting SV2A, another homologous isoform of SV can restore impaired synaptic transmission and cognitive function in AD model mice and patients. In the present study, thus, we aimed to compare the binding affinity of BACE1 and effects on BACE1-mediated APP processing between SV2A and SV2B. <Methods> Co-transfection of wt BACE1 with either SV2A, SV2B or empty vector was performed in HEK293 cells. The conditioned media were subjected to ELISA assay to detect secreted levels of Aβ40, Aβ42 and sAPPβ, respectively.
The lysates were used for co-immunoprecipitation assay followed by Western blot. <Results> SV2B overexpression led to significant reduction in the levels of Aβ40 and Aβ42 as well as sAPPβ in the conditioned media, compared with SV2A or control overexpression. Further, BACE1 was preferably co-immunoprecipitated with SV2B as compared to SV2A. <Conclusion> SV2B rather than SV2A preferably interacts with BACE1 and exerts significant inhibitory effect on BACE1-mediated APP processing.
Pe-29-5
EfficacyofedaravoneforA-betapathologyinAD modelmicewithchroniccerebralhypoperfusion○YosukeOsakada,JingweiShang,ToruYamashita,TianFeng, XianghongLi,XiaLiu,XiaowenShi,YumikoNakano,
KeiichiroTsunoda,EmiNomura,RyoSasaki,KohTadokoro,KotaSato, MamiTakemoto,NozomiHishikawa,YasuyukiOhta,KojiAbe Department of Neurology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Japan
[Purpose] To investigate the protective effect of edaravone （Eda） for abnormal accumulation of amyloid-beta （Aβ） peptide in the novel AD model mice with chronic cerebral hypoperfusion （CCH）. [Methods] We investigated the expression changes of two main Aβ transport receptors low-density lipoprotein receptor related protein-1（LRP1）
and receptor for advanced glycation end products （RAGE） in a novel AD mice （APP23）
with CCH model, treated by a free radical scavenger Eda. [Results] In contrast to wild type （WT） and APP23 mice, CCH strongly accelerated abnormal Aβ40 depositions and cerebral amyloid angiopathy（CCA） pathology, increased both LRP1 and RAGE expressions in brain parenchyma, while a decrease of LRP1 and an increase of RAGE were observed in vascular endothelial cells at age 12months （M） of AD mice. Furthermore, CCH strongly increased expression of two hypoxia-related preteins hypoxia inducible factor-1α （HIF-1α） and heme oxygenase-1 （HO-1）, two oxidativerelated proteins 4-hydroxy-2-nonenal （4-HNE） and 8-hydroxy-2´-deoxyguanosine （8-OHdG）, and decreased both two vital nutrient transporter proteins major facilitator super family domain containing 2a （Mfsd2a） and glucose transporter1 （Glut1） expressions. Such the above abnormal pathological changes were significantly ameliorated by edaravone treatment.
[Conclusinons] The present study demonstrated that CCH strongly enhanced primary AD pathology causing double imbalances of Aβ efflux and influx transport related proteins in the cortical blood vessels in AD mice, which was greatly ameliorated by Eda.
Pe-29-6
Lithiumchloridereducesphosphorylatedtau protein○ReiAsano1,AyumuKatsuki1,HirohitoSasaki1,
TomohisaYamaguchi1,SoichiEnomoto1,NorimichiShirafuji1, AsakoUeno1,MasamichiIkawa1,OsamuYamamura1,Shi-huiYen2, TadanoriHamano1
1 Department of Neurology, University of Fukui Hospital, Japan, 2 Department of Neuroscience, Mayo Clinic Jacksonville
[Objective] One of the pathological hallmarks of Alzheimer's disease （AD） is neurofibrillary tangles （NFTs）, which are formed by highly phosphorylated tau. Autophagy lysosome system （Eur J Neurosci 27; 1119, 2008） and proteasome are known to play important roles as tau degradation pathways.
Lithium chloride （LiCl） has been shown to inhibit phosphorylated tau by inhibiting glycogen synthase kinase3β （GSK3β）, one of the major tau kinases.
We investigated the decrease of phosphorylated tau by LiCl by using cell culture model if tauopathy expressing wild-type tau via TetOff induction system. [Methods] Ten to 50 mM of LiCl treatment （24 hours） was performed to neuroblastoma cells （M1C） expressing wild-type tau protein （4R0N） via TetOff induction. Total and phosphorylated tau （PHF-1） levels and autophagy activation were examined by Western blot （WB） analysis. [Results] Reduction of total tau （Tau5） and phosphorylated tau （PHF-1, and CP13） with LiCl treatment was detected by WB analysis. C-terminal truncated tau species detected by TauC3 was also reduced by LiCl treatment. In addition, increase of LC3II, and decrease of P62, suggesting autophagy activation was observed.
Major Tau kinase, GSK3β was inactivated by LiCl. [Conclusions] These results imply that LiCl may decrease the phosphorylated tau through suppression of tau phosphokinase and activation of autophagy.
Pe-29-7
TransplantationofMesenchymalStemCellsImproves A-betaPathologybyModifyingMicroglialFunction○KazukiYokokawa,NaotoshiIwahara,ShinHisahara,TaroSaito, HiromiSuzuki,TakashiMatsushita,AkihiroMatsumura, SyuuichirouSuzuki,ShunShimohama
Department of Neurology, School of Medicine, Sapporo Medical University, Japan
[Objective] MSC are increasingly attracting attention as a source of cell therapy for Alzheimer's Disease （AD）. However, the mechanisms of the MSC effects are not fully understood. In this study, we examined the effects of MSC transplantation on Amyloid-β （Aβ） pathology, and on microglial function of AD model mice brain. [Methods] Rat bone marrow derived MSC were transplanted into 7.5-month-old APP/PS1 transgenic （APdE9） mice via the tail vein. At 1.5 months after transplantation, mouse brain samples were removed for various immnohistchemical and biochemical studies. In order to evaluate the microglial Aβ phagocytosis in vitro, mouse microglial cell line MG6 cells and rat bone marrow derived MSC were co-cultured. Changes in microglial Aβ uptake ability with and without treatment were examined by ELISA and flow cytometry. [Results] MSC transplantation reduced Aβ plaques deposition in the APdE9 mouse brain, and reduced Aβ1-42 levels of soluble fraction of brain homogenates. Also, MSC transplantation accelerated accumulation of microglia around Aβ deposits and prompted microglial Aβ uptake as shown by higher frequency of Aβ-containing microglia. MSC transplantation also increased CD14-positive microglia in vivo, which play a critical role in Aβ uptake. In the study using MG6 cells in vitro, co-culture with MSC enhanced Aβ uptake by MG6 cells accompanied by upregulation of CD14 expression. [Conclusions]
These data indicate that MSC transplantation has the potential to modify microglial functions, thereby improving Aβ pathology in AD model mice.
一 般 演 題
ポ ス タ ー （ 英 語 ）
Pe-28-3
Microglia-astrocytecrosstalkintheperiinfarct areaafterstrokeinrats○ChikageKijima1,YujiUeno1,KenichiroHira1,ToshikiInaba2, ShoNakajima1,NobukazuMiyamoto1,KazuoYamashiro1, TakaoUrabe2,NobutakaHattori1
1 Department of Neurology, Juntendo University School of Medicine, Japan,
2 Department of Neurology, Juntendo University Urayasu Hospital
Purpose: The interaction between microglia and astrocyte is not fully elucidated.
We examined the alteration of the crosstalk between microglia and astrocyte after ischemic stroke in rats. Methods: We subjected adult male Wistar rats to permanent right middle cerebral artery occlusion （MCAO） and sacrificed at 3, 7, 14, 28 and 56 days after MCAO. We compared expressions of GFAP and iba-1 in the peri-infarct area. Cultured microglia and astrocyte were obtained from cerebral cortex of neonatal SD rats. Microglia were exposed to Oxygen-glucose deprivation （OGD）. We examined the expression of iba-1, arginase-1, and iNOs, and morphological alterations of Iba-1+ microglia after OGD. Cultured astrocyte was subjected to OGD, treated with microglia-conditioned medium （MCM）, and then examined in the expression of GFAP, CSPG, C3d . Results: In the peri-infarct area, Iba-1+ microglia increased until 7 days, and then decreased after MCAO, while GFAP+ astrocyte increased from day 7 to 56 days after MCAO. In vitro, after OGD, Iba-1+microglia with resting morphology decreased and bipolar and amoeboid shaped microglia increased, together with an increase in M1 microglia at 24 hours and a decrease in M2 microglia at 96 hours after OGD. MCM reduced the expression of CSPG in cultured astrocyte after OGD. Conclusion: In the peri-infarct area, microglia increased in the acute phase and decreased in the chronic phase, while astrocyte increase until the chronic phase. The expression of CSPG, the main component of astroglial scars, could be potentially regulated by microglia.
Pe-28-5
CRTC2playedanimportantroleunderischemic conditionsatendothelialcellsviap190RhogapA○HideakiKanki1,TsutomuSasaki1,ShigenobuMatsumura2, HiroshiTakemori3,HidekiMochizuki1
1 Department of Neurology, Graduate School of Medicine, Osaka University, Japan, 2 Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, 3 Faculty of Engineering, Department of Chemistry and Biomolecular Science, Gifu University
[Objective] Angiogenesis play crucial roles and cAMP-PKA pathway regulates angiogenisis. CREB pathway is related to cAMP-PKA pathway and very important for cell survival at ischemic conditions. CRTC （CREB regulated transcription coactivator） family are reported to be interacted with CREB and CRTC2 are reported to be predominantly expressed at liver, spleen and lymph node, and regulated glucose metabolism and B cell differentiation, but the role and mechanisms has not yet been elucidated at endothelial cells. Then, we examined the effect of CRTC2 on endothelial function using endothelial cells and knockout mice. [Methods] For evaluating endothelial function, various assays were performed in vitro and luciferase assay were also performed. We used endothelial cell-specific CRTC2-knockout mice for evaluating angiogenesis by aorta ring assay ex vivo and for evaluating the effect of ischemic stroke by photothrombotic stroke in vivo. [Results] CRTC2 was expressed at endothelial cells and only CRTC2, not CRTC1, was activated under ischemic conditions. CRTC2 enhanced endothelial function at various assays in vitro.
Luciferase assay showed CRTC2 was related to p190RhoGapA. Endothelial cell-specific CRTC2-knockout mice showed reduced angiogenesis, and had more severe ischemic stroke and impaired neurological recovery accompanied with increased BBB permeability and reduced blood vessels. [Conclusions] CRTC2 plays a crucial role in endothelial function, especially under ischemic conditions,via p190RhogapA.
CRTC2 is an attractive target for the new treatment of ischemic stroke.
Pe-28-6
TheEffectofNicotineontheChronicInflammation viacAMP-CRTC2signalinginEndothelialCells○TsutomuSasaki1,HideakiKanki1,TomohiroKawano1,HaominYen1, ShintaroSugiyama1,KenichiTodo1,ShigenobuMatsumura2, HiroshiTakemori3,HidekiMochizuki1
1 Department of Neurology, Graduate School of Medicine, Osaka University, Japan, 2 Laboratory of Nutrition Chemistry, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University,
3 Department of Chemistry and Biomolecular Science, Gifu University [Objective]The effect of nicotine or signal transduction via nAchR on the chronic inflammation in cerebral endothelial cells are not well understood. CRTC2 has been found to be a master regulator of glucose and fat metabolism. However, whether CRTC2 plays a role in physiological functions in endothelial cells remains unknown. This study investigated whether nicotine and consequent nAchR signaling have an impact on autophagy dysfunction or cross-talk with chronic inflammation.[Methods] Bovine brain microvasculature endothelial cells （BBMCs） or HUVEC were harvested. OGD was performed described previously. Western blot analyses were performed to examine both CREB-CRTC （CREB-regulated transcription coactivators） axis and autophagy process.
Mice were provided a high fat diet （HFD） from weeks 7 to 11. Mice fed an HFD or a normal diet were used at 11 weeks. Right MCAO occlusion （60 min） was performed using a suture and then reperfused （N=5）. Moreover, we also examined the crosstalk between cAMP-CRTC2 signaling and autophagy using CRTC2 KO mice. [Results] HFD-fed mice showed more severe ischemic injury, BBB damage, and exacerbation of inflammation.
HFD-fed mice showed lower autophagy process in penumbra after stroke. CRTC2 knockout mice exacerbated BBB damage （N=5）. The nAchR signaling contribute to the both chronic inflammation and autophagy pathway. [Conclusions] The chronic inflammation in cerebral endothelial cells contributes to the pathology via autophagy dysfunction in neurovascular unit, at least in part, cAMP-CRTC2 signaling and nAchR signaling.
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Lightexerciseinducesischemictolerancethrough modulationofmicroRNAsinthegerbilhippocampus○TadayukiTakata1,2,WakakoNonaka2,HisakazuIwama3, HidekiKobara4,KazushiDeguchi2,HisashiMasugata1, TetsuoTouge5,OsamuMiyamoto6,TakehiroNakamura7, ToshifumiItano2,TsutomuMasaki4
1 Department of General Medicine, Kagawa University Faculty of Medicine, Japan, 2 Department of Neurology, Kagawa University Faculty of Medicine, Japan, 3 Department of Life Science Research Center, Kagawa University,
4 Department of Gastroenterology, Kagawa University Faculty of Medicine,
5 Department of Health Sciences, Kagawa University Faculty of Medicine,
6 Department of Physiology 2, Kawasaki Medical School, 7 Department of Medical Technology, Kagawa Prefectural University of Health Sciences [Objective]To investigate the neuroprotective effects of light exercise, without lactate elevation, on ischemia/
reperfusion injury of a gerbil model. [Methods]Transient whole-brain ischemia was induced by occlusion of the bilateral common carotid arteries for 5 min. A group of animals was subjected to treadmill exercise before ischemia induction. Hippocampal neuronal damage and microRNA （miRNA） expression were evaluated, as well as behavioral deficits and plasma lactate levels. [Results]The majority of pyramidal neurons within the Cornet d' Ammon 1 （CA1） area of the gerbils in the ischemia group were loss, when compared to those of the sham group. In contrast, neurons within the same area in gerbils of the exercise plus ischemia group were preserved and appeared normal. short-term memory in the exercise plus ischemia group was preserved compared with that of the ischemia group and was equivalent to that of the sham group. The lactate level and weight of gerbils in each group at sacrifice were not significantly different. 14 miRNAs were upregulated, and 6 miRNAs were downregulated due to ischemia. However, the expression of these miRNAs remained unchanged when animals performed light exercise before the ischemic event. [Conclusions]Histologically, neurons were preserved in CA1, and short-term memory was retained. Although several mechanisms may lead to this effect, it was shown that ischemic tolerance induced by light exercise is associated with miRNA expression level in the hippocampus.
Light exercise with non-elevated lactate levels was shown to induce ischemic tolerance.
○NaokiIwasa1,TakeshiKMatsui1,NaohikoIguchi1,TomoShiota1, NobuyukiEura1,KozueSaito1,EiichiroMori2,KazumaSugie1
1 Department of Neurology, Nara Medical University, Japan, 2 Department of Future Basic Medicine, Nara Medical University
[Objective] Ischemic stroke is one of the most common neurological disease, but the detailed cellular reaction are not evaluated. We used 3D human cerebral organoids derived from human induced pluripotent stem cells（hiPSCs）.
Oxygen-glucose deprivation/reperfusion （OGD/R） is known as an ischemia and reperfusion model. We analyzed human cerebral organoids under OGD/
R condition to detect the true action of neuronal cells and glial cells. [Methods]
Cerebral organoids were generated from hiPSCs. Subsequently, RNA-sequencing and quantitative PCR were performed. Gene expression profile was confirmed, bioinfomatics analysis was done. [Results] Comparing cerebral organoids after OGD/R and non-treated organoids, a total of 52 differentially expressed genes （DEGs） were determined and most of DEGs were downregulated. When False discovery rate （FDR） adjusted p-value ≦ 0.05 was used as a threshold, 15 genes were detected and all genes were downregulated.
The top 5 significant downregulated genes were AFP, TTR, APOA2, ALB and APOA1. RNA sequence analysis and pathway analysis showed the relationship of vitamin digestion and absorption, fat digestion and absorption, PPAR signaling pathway, and complement and coagulation cascades. [Conclusion]
Many genes associated with lipid metabolism are downregulated under OGD/R condition. These findings might indicate the mechanisms underlying ischemic injury, using human cerebral organoids treated by OGD/R.