e ポスター

e ポスター 13. 神経疾患の分子基盤 e Poster 13. Molecular basis of neurological disease

2020/9/12　13:40～14:40　オンデマンドB-1 P3-15 D2受容体発現細胞におけるNMDA受容体のマグネシウムブロックの減少により引き起こされる運動制御と学習および記憶の変化の分析 Analysis of alteration in motor control and learning and memory caused by reduced magnesium block of NMDA receptors in the D2 receptor expressing cells of the indirect pathway 笹岡俊邦¹、齊藤奈英¹、知見聡美²、阿部学¹、川村名子¹、鍋島曜子³、鍋島陽一³、田井中一貴¹、崎村建司¹、南部篤² 1. 新潟大学脳研究所、2. 生理学研究所、3. 先端医療研究センター Toshikuni Sasaoka¹, Nae Saito¹, Satomi Chiken², Manabu Abe¹, Meiko Kawamura¹, Yoko Nabeshima³, Yo-ichi Nabeshima³, Kazuki Tainaka¹, Kenji Sakimura¹, Atsushi Nambu² 1. Brain Research Institute, Niigata University, 2. National Institute for Physiological Sciences, 3. Foundation for Biomedical Research and Innovation at Kobe Parkinson's disease (PD) is a neurological disorder that exhibits motor and non-motor symptoms. Midbrain dopaminergic neurons mainly project to the striatum, a main input station of the basal ganglia. In the striatum, there are two types of projection neurons: "direct pathway" neurons that express dopamine D1 receptors (D1R) and "indirect pathway" neurons that express dopamine D2 receptors (D2R). Imbalances between neural activity in these two pathways have been proposed to underlie the profound motor and non-motor symptoms in PD. However, differences in cellular and synaptic properties in these circuits remain unclear. To understand neural mechanisms of motor and non-motor symptoms of PD and the roles of NMDA receptor activation in information processing through the basal ganglia, we developed genetically modified mice expressing activated NMDA receptors specifically in striatal indirect pathway neurons. A single asparagine residue (N595) in the second membrane-associated segment of GluN2 subunits is critical for voltage-dependent Mg²⁺ blockade. We have generated the GluN2 mutant mice by a novel method for introducing a desired mutation into the gene of interest in a spatially restricted manner (Hayashi et al. 2014). Before Cre-loxP recombination, the wild-type exon 10 (N595) of GluN2 subunit was expressed in the GluN2 mutant mice. Only when the wild-type exon 10 between two loxP sequences was deleted by the action of Cre recombinase, the mutant exon 10 would be spliced into mRNA. This mutation reduced the Mg²⁺block property of the NMDA receptors. We generated the mutant mice in which the mutation (N595Q) of GluN2 was selectively introduced in D2R-expressing indirect pathway neurons using the Cre-expressing mice under the control of D2R gene. Then we analyzed the distribution of Cre-expressing cells by using three-dimensional imaging after whole brain transparency technique and performed several behavior tests of the mice to examine spontaneous motor activity and learning and memory functions.		2020/9/12　13:40～14:40　オンデマンドB-1 P3-16 Loss of collapsin response mediator protein 4 attenuates 6-hydroxydopamine-induced impairments in a mouse model of Parkinson's disease LI WENTING¹、Goshima Yoshio²、Ohshima Toshio¹ 1. Waseda University、2. Yokohama City University WENTING LI¹, Yoshio Goshima², Toshio Ohshima¹ 1. Waseda University, 2. Yokohama City University Parkinson's disease (PD) is a chronic neurodegenerative disorder characterized by impaired motor symptoms induced by the degeneration of dopaminergic neurons of the substantia nigra pars compacta (SNc). Many factors are speculated to operate in the mechanism of PD, including oxidative stress, mitochondrial dysfunction, abnormal protein handling, and PD induced apoptosis. Besides, researchers have recently shown that inflammatory secretions may engage neighboring cells such as astrocytes, which then induce autocrine and paracrine responses that amplify the inflammation, leading to neurodegeneration. In the present study, we analyzed the neuroprotective and anti-inflammatory effects of collapsin response mediator protein 4 (CRMP4) deletion in 6-hydroxydopamine (6-OHDA)-injected male mice, as well as its effects on motor impairments. Our findings indicated that the deletion of CRMP4 could maintain the TH-positive fibers in the striatum and the TH-positive cells in SNc, attenuate the inflammatory responses, and improve motor coordination and rotational behavior. Furthermore, based on our findings at the early time points, we hypothesized that primary differences between the Crmp4+/+ and Crmp4-/- mice may occur in microglia instead of neurons. Although further work should be carried out to clarify the specific role of CRMP4 in the pathogenesis of PD, our findings suggest that it could be a possible target for the treatment of PD.

2020/9/12　13:40～14:40　オンデマンドB-1 P3-17* システイン残基へのDA酸化物の修飾がTHの活性を減少させる Dopamine oxides inactivate tyrosine hydroxylase by modification of the cysteine residue 犬飼駿弥¹、原怜¹、一瀬宏¹ 1. 東京工業大学・生命理工学院 Shunya Inukai¹, Satoshi Hara¹, Hiroshi Ichinose¹ 1. Tokyo Institute of Technology, School of Life Science and Technology Parkinson's disease (PD) is a progressive neurodegenerative disease, characterized by the progressive loss of dopaminergic neurons in the nigrostriatal pathway. Although the detailed mechanism of the neuronal degeneration has not yet been identified, mitochondrial dysfunction, accumulation of aggregated proteins, oxidative stress, and dysfunction of proteolytic system are implicated in the pathogenesis of PD. Dopamine (DA) is synthesized in dopaminergic neurons, and spontaneously converted into DA oxides in the cells because of its susceptibility to oxidation. DA oxides are thought to be one of the causes of PD because it has neurotoxicity through covalent modification to cysteine residues of proteins. Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the DA-biosynthesis. TH activity is regulated by feedback inhibition induced by binding of catecholamines such as DA and norepinephrine, and the inhibition is suppressed by the phosphorylation at the N-terminal region. The N-terminal region of TH (particularly 1-65 a.a.) is thought to be an intrinsically disordered region, which may cause protein aggregation. In this study, we examined the effect of DA oxides to TH. We prepared DA oxides by treating DA with tyrosinase, and determined major DA oxides as aminochrome (AC) by LC-MS. We found that recombinant TH activity was reduced by incubation with DA oxides. The S40E mutant of TH can mimic the activated and phosphorylated state of TH, and DA-induced feedback inhibition was not observed in S40E mutant. We found that the incubation of TH with DA oxides strongly inhibited the activity of S40E mutant. Using MALDI-TOF-MS to determine the DA oxides modification of TH, we observed increasing mass of 147 m/z corresponding to AC modification. The increase was not detected in C176A mutant of TH, suggesting Cys176 was modified with AC. These results suggest that the modification of AC to the cysteine residue of TH can regulate its activity regardless of the phosphorylation states of TH.		2020/9/12　13:40～14:40　オンデマンドB-1 P3-18 プロスタグランジンE合成酵素-1の誘導はマウスコラゲナーゼ誘発脳出血モデルにおける神経炎症と神経学的運動障害に寄与する Microsomal prostaglandin E synthase-1 contributes to neuroinflammation and neurological dysfunctions in a collagenase-induced mouse intracerebral hemorrhage model 松尾由理¹、宮原伸卓¹、柚木紀香¹、與澤智佳²、植松智³、審良静男⁴、高橋達雄¹、田辺光男² 1. 北陸大学、2. 北里大学、3. 大阪市立大学、4. 大阪大学 Yuri Ikeda-Matsuo¹, Nobutaka Miyahara¹, Norika Yunoki¹, Chika Yozawa², Satoshi Uematsu³, Shizuo Akira⁴, Tatsuo Takahashi¹, Mitsuo Tanabe² 1. Hokuriku Univ, 2. Kitasato Univ, 3. Osaka City Univ, 4. Osaka Univ We have demonstrated that microsomal prostaglandin E synthase-1 (mPGES-1), an inducible terminal enzyme for PGE₂ synthesis, is a critical factor of stroke-reperfusion injury and neurodegeneration in Parkinson's disease. In this study, we investigated the role of mPGES-1 in neuroinflammation and neurological dysfunctions observed after intracerebral hemorrhage (ICH). Collagenase was injected into the left striatum of adult mPGES-1 knockout (KO) and wild-type (WT) mice. In WT mice, mRNA and protein of mPGES-1 were significantly up-regulated in striatum and cerebral cortex after ICH. In mPGES-1 KO mice, although the hemorrhage and edema size were almost the same as WT mice, survival rate was significantly higher than WT mice. The PGE₂ production, TNF-α induction and glial activation after ICH in mPGES-1 KO brain were significantly less than those in WT brain. DAPI and TUNEL staining showed ICH-induced nuclear condensation and DNA fragmentation in mPGES-1 KO striatum were less than those in WT striatum. Furthermore, mPGES-1 KO mice showed better performance in stepping error test, rotarod test and neurological dysfunction scoring compared with the WT mice. Because significant microglial activation was observed after ICH, the role of microglial mPGES-1 was investigated using mice primary microglial culture. Hemoglobin significantly increased PGE₂ production in WT microglia, but not in mPGES-1 KO microglia. The hemoglobin-induced expressions of TNF-α and IL-1β in mPGES-1 KO microglia were significantly less than those in WT microglia. These results suggest that microglial mPGES-1 contributes to ICH-induced neuroinflammation, neuronal apoptosis, neurological dysfunctions and mortality through PGE₂ production. Thus, mPGES-1 may be a new therapeutic target for ICH.

2020/9/12　13:40～14:40　オンデマンドB-1 P3-19 大脳皮質発達過程におけるオートファジー関連分子WDR45の役割 Role of WDR45, the autophagy-related gene, in the development of mouse cerebral cortex 野田万理子¹、岩本郁子¹、田畑秀典¹、伊東秀記¹、永田浩一¹ 1. 愛知県医療療育総合センター発達障害研究所 Mariko Noda¹, Ikuko Iwamoto¹, Hidenori Tabata¹, Hidenori Ito¹, Kouichi Nagata¹ 1. Institute for Developmental Research, Aichi Developmental Disability Center (ADDC) WDR45 is highly conserved during evolution and essential in autophagy. WDR45 gene de novo mutations are reported to cause static encephalopathy of childhood with neurodegeneration in adulthood (SENDA), which is a subtype of neurodegeneration with brain iron accumulation (NBIA). These mutations lead lower expression of WDR45 protein. Recently, the whole-exome analysis of individuals with severe and undiagnosed developmental disorders revealed that their genomes are enriched in de novomutations in developmentally important genes, including WDR45. Although these reports suggested that the WDR45 mutations play a causative role in developmental disorders, its molecular pathophysiological mechanism in embryonic stage remains largely unknown. We here examined the role of WDR45 in mouse brain development. We first performed immunohistochemistry and western blotting to determine the WDR45 protein expression profile in the developing mouse brain. WDR45 was expressed throughout the developmental process. Acute silencing of WDR45 by in utero electroporation was then carried out to determine the knock down effects on cortical neurogenesis. It had little effects on excitatory neuron positioning during corticogenesis, but dendritic arbor formation and synaptogenesis was severely impaired. Based on these results, we propose that WDR45 is critical for the maturation of cortical excitatory neurons and it is likely to be related to pathophysiology of SENDA.		2020/9/12　13:40～14:40　オンデマンドB-1 P3-20 【誌上発表】脳出血モデルラットにおける超早期リハビリテーションは炎症促進因子の発現および神経細胞死を促進させる Very early rehabilitation promotes pro-inflammatory factors and neuronal cell death in cerebral hemorrhage rats 玉越敬悟¹、前田まどか²、中村慎之介³、室橋七衣¹ 1. 新潟医療福祉大学、2. 桑名病院、3. 岩室リハビリテーション病院 Keigo Tamakoshi¹, Madoka Maeda², Shinnosuke Nakamura³, Nae Murohashi¹ 1. Niigata University of Health and Welfare, 2. Kuwana Hospital, 3. Iwamuro Rehabilitation Hospital The present study investigated the effect of exercise on recovery of motor function following intracerebral hemorrhage (ICH) in rats. Under deep anesthesia, rats were placed inside a stereotaxic apparatus, and ICH was induced by injecting a collagenase solution in 0.9% saline into the left striatum. Rats were randomly assigned to three groups: no training-ICH (ICH), no training-placebo-surgery (SHAM), and very early exercise (ICH+VET). ICH+VET rats ran on a treadmill for 60 min at 6 h and 24 h after surgery. Horizontal ladder and rotarod tests evaluated motor function. Brain tissue was collected after exercise at 27 h after surgery after surgery from all groups. Hematoma volume and cerebral edema were measured in brain tissue as well as expression of IL-1b, TGF-b1, and IGF-1 mRNA in the sensorimotor cortex and striatum using real-time PCR. The protein expression levels of NeuN and PSD95 were analyzed using Western blotting method. Horizontal ladder testing showed a significant increase in error rate for ICH+VET rats compared to the ICH group. IL-1b mRNA levels in ICH+VET rats were significantly higher than those in the ICH group in the striatum. TGF-b1 mRNA expression in ICH+VET group was significantly lower than that in the ICH group in the sensorimotor cortex. The protein expression levels of NeuN and PSD95 in the ICH + VET group were significantly lower than those in the SHAM group in the sensorimotor cortex and striatum. This study revealed that starting exercise within 24 h after cerebral hemorrhage exacerbates motor dysfunction. While this was not due to an increase in hematoma volume or cerebral edema, it may be related to suppression of anti-inflammatory and nerve growth factor upregulation and promotion of pro-inflammatory factor elevation in sensorimotor areas. Furthermore, alterations in these factors may be involved in neuronal cell death and synaptic regression.

2020/9/12　13:40～14:40　オンデマンドB-1 P3-21* CRMP2恒常的活性化における脊髄損傷脳病態とトランスクリプトーム解析 The brain pathology and transcriptome analysis after spinal cord injury in constitutively activated CRMP2 mice 菅野彩佳^1,2、朴文惠¹、山崎美輝^1,2、高橋清文³、松永浩子³、細川正人^3,4、富永大介^2,5、五嶋良郎⁶、竹山春子^1,2,3,4、大島登志男^1,4 1. 早大生命医科、2. 早大・産総研 CBBD-OIL、3. 早大・ナノライフ創新研究機構、4. 早稲田大学先進生命動態研究所、5. 産総研細胞分子バイオテクノロジー、6. 横浜市・医 Ayaka SUGENO Sugeno^1,2, Wenhui Piao¹, Miki Yamazaki^1,2, Kiyofumi Takahashi³, Hiroko Matsunaga³, Masahito Hosokawa^3,4, Daisuke Tominaga^2,5, Yoshio Goshima⁶, Haruko Takeyama^1,2,3,4, Toshio Ohshima^1,4 1. Life Science and Medical Bioscience Dept.,Waseda Univ. , 2. CBBD-OIL, Waseda-AIST, 3. Research Organization for Nano and Life Innovation, Waseda, 4. Institute for Advanced Research of Biosystem Dynamics, Waseda, 5. Cellular and Molecular Biotechnology Research Inst., AIST, 6. Molecular Pharmacology and Neurobiology Dept., Yokohama City Univ. Spinal cord injury (SCI) has been researched for a long time as representative of CNS axon injury in vivo. Recent study showed that the mutation by inhibition of CRMP2 phosphorylation at Ser522 (CRMP2KI) induces axon elongation and partial recovery of the lost sensorimotor function after SCI. Here we attempt to reveal the intracellular mechanism of SCI pathophysiology in CRMP2KI by analyzing structural pathology observed in sensorimotor cortex and by performing transcriptome analysis in the same micro-dissected region. We identified significantly large upregulation of 4 Hb genes in WT with upregulation of oxidative phosphorylation and ribosomal pathways after SCI. On the other hand, we revealed upregulation in channel activity genes and downregulation of genes regulating vesicles, synaptic function, glial cell differentiation and myelin sheath in CRMP2KI mice. In addition, spine reduction and neural cell body shrinkage were suppressed in CRMP2KI after SCI. We also revealed transcriptome profile of CRMP2KI mice for the first time and found that energy metabolism and neuronal function pathways were differentially expressed broadly. Our results suggest that CRMP2KI seems to improve SCI pathophysiology not only via microtubule stabilization in neurons but via the whole metabolic system in CNS and maybe response changes in glial cells and synapses. To summarize, we revealed new insights on SCI pathophysiology and regenerative mechanism after SCI by the inhibition of CRMP2 phosphorylation.