神経保護、神経毒性と神経炎症 Neuroprotection, Neurotoxicity and Neuroinflammation
P1-1-223 ALSマウスの疾患進行におけるTGF-β1の関与 The roles of TGF-β1 in the disease progression of ALS mice ○遠藤史人1, 小峯起1, 山中宏二1 ○Fumito Endo1, Okiru Komine1, Koji Yamanaka1 理研・BSI・運動ニューロン変性1 Lab for MND, RIKEN BSI, Saitama1 Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease characterized by the selective loss of motor neurons. Transforming growth factor-βs (TGF-βs) are pleiotropic cytokines that have key roles in immune-regulation, inflammation, T lymphocyte differentiation and tissue repair after injury. Although TGF-β1 was increased in the plasma and CSF of ALS patients, its exact role in ALS pathogenesis remains unknown. In end-stage SOD1G93A mice, TGF-β1 mRNA was highly up-regulated and TGF-β1 was expressed in astrocytes of the lumbar spinal cord. To explore role of TGF-β1 in non-cell autonomous cell death mediated by glial cells in ALS, we genetically enhanced the expression of TGF-β1 in astrocytes of SOD1G93A mice by using the mice expressing porcine TGF-β1 under GFAP promoter. Double transgenic mice (SOD1G93A/TGF-β1 mice) showed accelerated disease progression. The expression levels of IFN-γ, IL-4, IGF-I, CD68 and CCL5 mRNA were reduced in the lumbar spinal cord of end-stage SOD1G93A/TGF-β1 mice. Immunohistochemical study revealed that the expression levels of Mac-2, CD68, CD11c and IGF-I in microglia were reduced in the anterior horn of end-stage SOD1G93A/TGFβ1 mice. Although the number of infiltrating lymphocytes was reduced in the spinal cord of end-stage SOD1G93A/TGFβ1 mice, the ratio of IFN-γ-producing and IL-4-producing lymphocytes was significantly increased. In end-stage SOD1G93A mice, the expression level of TGF-β1 mRNA negatively correlated not only with the ratio of IFN-γ and IL-4 but with the survival time of SOD1G93A mice. Furthermore, IFN-γ and IL-4 antagonistically regulated IGF-I mRNA level in SOD1G93A primary microglia. These findings suggest that astrocytes producing TGF-β1 accelerate the disease progression of possibly through controlling IFN-γ/IL-4 balance of lymphocytes and activation of microglia.	P1-1-224 プログラニュリン欠損は脳傷害後の活性化ミクログリアにおけるリソソーム生合成と神経細胞損傷を増大する Progranulin deficiency leads to increased lysosomal biogenesis in activated microglia and exacerbated neuronal damage after traumatic brain injury in mice ○田中良法1, 松脇貴志1, 山内啓太郎1, 西原真杉1 ○Yoshinori Tanaka1, Takashi Matsuwaki1, Keitaro Yamanouchi1, Masugi Nishihara1 東京大学大学院　獣医生理学教室1 Dept of Veterinary Physiology, Univ of Tokyo, Tokyo1 Progranulin (PGRN) is a multifunctional growth factor. We have identified PGRN as one of the factors involved in sex differentiation of the brain and adult neurogenesis in rodents. We have also shown that PGRN expression increases after traumatic brain injury (TBI), and the major source of PGRN is CD68-positive activated microglia. Further, PGRN deficiency induces exacerbated inflammatory responses associated with increased CD68-positive activated microglia. CD68 is a member of the lysosome associated membrane protein (Lamp) family. There is increasing evidence that PGRN is involved in lysosomal function, and it is also recently reported that the patients with the homozygous mutation present with adult onset neuronal ceroid lipofuscinosis, which is characterized by storage of abnormal lipopigment in lysosomes. Therefore, we investigated the role of PGRN in lysosomal biogenesis of activated microglia in the cerebral cortex after TBI. PGRN has two CLEAR sequences where transcription factor EB (TFEB), the master regulator of lysosomal proteins, binds in the promoter region. PGRN was colocalized with the lysosomal marker Lamp1, and PGRN deficiency resulted in increased Lamp1-positive area after TBI. PGRN deficiency also increased expression of lysosome-related genes after TBI. Further, PGRN deficiency increased the numbers of microglia with TFEB localized to nucleus, and decreased ribosomal S6 kinase 1 phosphorylation that reflects mammalian target of rapamycin complex 1 (mTORC1) activity after TBI. Additionally, PGRN deficiency decreased nissl-positive living neurons and increased fluoro-jade B-positive degenerating neurons. These findings suggest that PGRN production is increased in activated microglia after TBI, and then PGRN is transported to lysosomes and involved in the activation of mTORC1 with a resultant decrease in TFEB translocation to the nucleus, which suppresses excessive expression of lysosomal genes.
P1-1-225 ミクログリアの炎症反応におけるモノアシルグリセロールリパーゼの生理機能 Physiological function of monoacylglycerol lipase in microglial response to inflammation ○河内全1, 細川昌則1, 榎戸靖1 ○Zen Kouchi1, Masanori Hosokawa1, Yasushi Enokido1 愛知県心身障害者コロニー発達障害研究所 病理学部1 Dept Pathology, Inst Dev Res, Aichi Human Service Ctr, Aichi, Japan1 Monoacylglycerol lipase (MAGL) hydrolyzes 1 or 2-monoacylglycerol, and endocannabinoid 2-arachidonylglycerol (2-AG), which is one of the substrates and an arachidonate precursor, is important for the regulation of axonal pathfinding and neocortical pyramidal cell specification in developmental brain. Recently, it was reported that MAGL knockout mice show impaired neuroinflammatory responses including eicosanoid biosynthesis and production of inflammatory cytokines (Science (2011) 334, 809-813); however, its role in microglial response to pathological environment is still largely unknown. LPS treatment or hypoxic condition modulates microglial functions, such as production of inflammatory cytokines and phagocytosis. In order to elucidate physiological role of MAGL in microglia in pathological conditions, we carried out expression and knockdown analysis by using microglia treated with or without 1 μg/ml LPS in normoxic or hypoxic condition (1%O2 or CoCl2 treatment). Both hypoxia and LPS treatment negatively regulated expression of monoacylglycerol lipase at transcriptional levels in microglia. On the other hand, LPS-dependent up-regulation of inflammatory cytokines and Fcγ receptor was observed in normoxic or hypoxic condition. Knockdown of MAGL in microglia by lentivirus-mediated shRNAs inhibited Fcγ receptor-dependent phagocytosis. However, loss of MAGL activity did not affect LPS-induced upregulation of inflammatory cytokines, such as TNFα and IL-6 in either normoxia or hypoxia. Introduction of FLAG-tagged MAGL into BV-2 microglial cell line, which lacks endogenous MAGL expression, promoted Fcγ receptor-mediated phagocytosis of IgG beads regardless of LPS or CoCl2 treatment. We will discuss the specific role of MAGL in microglial response to inflammatory stimuli and its effect on neuroinflammation.	P1-1-226 Cdk5コンディショナルノックアウトマウスを用いた神経変性の治療 PPARγ agonist treatment of neurodegeneration in Cyclin-dependent kinase 5conditional KO mice ○浜田隆介1, 高橋悟2,3大島登志男1 ○Ryusuke Hamada1, Satoru Takahashi2,3, Ashok Kulkarni2, Toshio Ohshima1 早大院・先理・生医1, NIDCR・細胞生物学/発生生物学研究室2, 旭川医科大学小児科3 Dept. Life Sci. Med. Biosci., Grad. Sch. Adv. Eng. Sci., Waseda Univ., Tokyo1, Lababoratory for Cell and Developmental Biology, NIDCR, NIH, USA2, Department of Pediatrics, Asahikawa Medical University, Hokkaido3 There is no way to treat perfectly the neurodegenerative disorders such as Alzheimer's disease, Huntington's disease and Parkinson's disease. Cyclin-dependent kinase 5 (Cdk5) is related to these disorders and involved in the formation of brain, particularly in the neuronal migration. Previous study identified that CaMKII-cre Cdk5 conditional KO (Cdk5 cKO) mice have following phenotypes; 1) defect of cortical development, 2) age-dependent neuronal loss in the forebrain, 3) age-dependent astrocytic and microglial activation in the forebrain, 4) seizure and 5) increase of mortality after weaning and after 2 months. We hypothesized that the reason of this age-dependent neurodegeneration is related to inflammatory reactions including microglial activation. Thus, we used pioglitazone (PPARγ agonist) to inhibit the expression of proinflammatory genes and examined the effect of this drug on the progression of neurodegeneration. We compared 2- and 3-month-old control mice, Cdk5 cKO mice and treated Cdk5 cKO mice in several aspects. Comparison of survival curves between Cdk5 cKO and treated Cdk5 cKO mice showed the improvement of viability in treated ones. Histological analysis also showed the improvement of neuronal loss, astrocytic activation and microglial activation. These results suggest that inflammation deteriorates neurodegeneration in Cdk5 cKO mice and PPARγ agonist can be possible therapeutic drug for inflammation-induced neurodegeneration.
P1-1-227 15-デオキシ-デルタ12,14-プロスタグランジンJ2による神経細胞死はその膜標的タンパク質に対する抗体によって抑制される The 15-deoxy-Δ12,14-prostaglandin J2-induced neuronal cell death was suppressed by an antibody against its membrane target ○矢上達郎1, 山本泰博1, 高馬宏美1 ○Tatsurou Yagami1, Yasuhiro Yamamoto1, Hiromi Kohma1 姫路獨協大・薬・生理1 Dept Physiol, Facl Pharmaceutic Sci, Himeji Dokkyo Univ, Himeji, Hyogo, JPN1 We have identified plasmalemmal glyceraldehyde-3-phosphate dehydrogenase (pl-GAPDH) was one of membrane targets for 15-deoxy-Δ12,14-prostaglandin J2(15d-PGJ2). pl-GAPDH was expressed on the cell surface of cortical neurons, and colocalized with membrane proteins labeled by biotinylated 15d-PGJ2. 15d-PGJ2 induced neuronal cell death in a concentration-dependent manner. No neurotoxicity of anti-GAPDH antibody was detected. The antibody suppressed the neurotoxicity of 15d-PGJ2. 15d-PGJ2 shortened neurites and shrunk cell bodies. The anti-GAPDH antibody ameliorated the 15d-PGJ2-altered neuronal morphology. Thus, the anti-GAPDH antibody prevented neurons from undergoing the 15d-PGJ2-induced cell death.	P1-1-228 セレコキシブはLPSによる脳炎症および神経機能障害を軽減する Celecoxib attenuates systemic lipopolysaccharide-induced brain inflammation and injury, and improves behavioral performance in neonatal rats ○貝崎明日香1,2, 田中佐知子1, 沼澤聡1 ○Asuka Kaizaki1,2, Lir-Wan Fan2, Lu-Tai Tien3, Sachiko Tanaka1, Satoshi Numazawa1, Zhengwei Cai2 昭和大・薬・毒物学1, ミシシッピ医療センター大・新生児医学2, 輔仁カトリック大・医3 Dept Pharmacol, Toxicol & Therapeut, Divi Toxicol, Sch Pharm, Showa Univ1, Dept Pediatrics, Divi Newborn Med, Univ of Mississippi Med Cent, Jackson, USA2, Sch Med, Fu Jen Catholic Univ, New Taipei City, Taiwan3 Our previous study showed that intraperitoneal injection of lipopolysaccharide (LPS) resulted in brain inflammation and injury in neonatal rats. Cyclooxygenase-2 (COX-2) is induced in inflammatory cells in response to cytokines and pro-inflammatory molecules, suggesting that COX-2 has a role in the inflammatory processes. The objective of the current study was to examine whether celecoxib, a selective COX-2 inhibitor, can ameliorate LPS-induced brain inflammation, brain damage, and neurological dysfunction. Intraperitoneal (i.p.) injection of LPS (2 mg/kg) was performed in P5 rat pups and celecoxib (20 mg/kg) or vehicle was administered (i.p.) 5 min after LPS injection. The control rats were injected (i.p.) with sterile saline. Neurobehavioral tests were carried out before LPS exposure (P5) and 24 hr after the LPS exposure (P6), and brain injury was examined after these tests. Our results showed that celecoxib provided protection against LPS-induced impairments of neurobehavioral performance, such as impaired righting reflex, negative geotaxis, and wire hanging maneuver. Celecoxib treatment also provided protection against LPS-induced severe brain damage including loss of oligodendrocytes (O4+) and tyrosine hydroxylase immunoreactive cells, as well as decreases in mitochondria activity. Celecoxib administration significantly attenuated LPS-induced increments in the concentration of IL-1beta, protein levels of p38 MAPK and phosphorylated-p38 MAPK, and the number of activated microglia in the neonatal rat brain. These results suggest that celecoxib may provide protection against systemic LPS exposure-induced brain injury and neurobehavioral disturbance, and the protective effects are associated with its anti-inflammatory properties.(Supported by NIH grant NS 54278 and Newborn Medicine Funds from the Department of Pediatrics, UMMC)
P1-1-229 炎症初期において P2X4 受容体を介したミクログリア活性化の亢進がミクログリアからのグルタミン酸放出に重要である P2X4 receptor-mediated acceleration of microglial activation is important for the L-glutamate release from activated microglia in the early stage of inflammation ○佐藤薫1, 藤森康希1,2, 高木淳平1,2, 鈴木岳志2, 関野祐子1 ○Kaoru Sato1, Koki Fujimori1,2, Junpei Takaki1,2, Takeshi Suzuki2, Yuko Sekino1 国立医薬品食品衛生研究所薬理部1, 慶應大学薬学部基礎生物2 Div Parmacol, NIHS, Tokyo1, Div Basic Biol Sci, Fac Pharm, Keio Univ, Tokyo2 Recently we clarified the mechanisms underlying the decrease in astrocytic L-glutamate (L-Glu) transporter activity in the early stage of inflammation using in vitro inflammation model without cell death: activated microglia releases L-Glu through connexin hemichannel and the resulting high concentration of extracellular L-Glu down-regulated L-Glu transporter expression in astrocytes. In inflammation, up-regulation of microglial P2X4 receptor expression has been reported. Thus, in this study, we examined the involvement of microglial P2X4 receptor activation in the microglial activation and L-Glu release. We confirmed that LPS treatment for in vitro inflammation model without cell death (10 ng/ml, 72 hr) caused increased expression of connexin-32 (Cx32) but not Cx43. LPS-induced microglial activation, increase in Cx32 expression, and L-Glu release were inhibited by 5-BDBD, P2X4 receptor inhibitor. P2X4 receptor allosteric modulator, ivermectin, induced microglial activation, increase in Cx32 expression, and L-Glu release. Further, among SSRI, only paroxetin which have the interaction with P2X4 receptor suppressed these phenomena. Our study indicates that P2X4-mediated acceleration of microglial activation is important for the L-Glu release from activated microglia in the early stage of inflammation, suggesting that the microglial P2X4 receptor activation was one of the key signal pathways of the onset of neuroinflammation.

上部に戻る

前に戻る