| 細胞死/神経保護 |
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| P3-20
リポポリサッカライドはBV-2細胞のUVによるアポトーシスを抑制する
金子 葉子1，中島 昭1，長崎 弘1，小谷 侑1，永津 俊治2，太田 明1
藤田保健衛生大・医・生理学I1，藤田保健衛生大・医・薬理学2
We previously reported that an optimal dose of lipopolysaccharide（LPS）markedly extends the lifespan of murine primary-cultured microglia by suppressing apoptotic and autophagic cell death pathways. In the present study, we investigated the effects of LPS pretreatment on ultraviolet（UV）-induced apoptosis of cells from the microglial cell line BV-2. BV-2 cells underwent apoptosis within 1 h of UV irradiation. At 3 h, more than half of cells were apoptotic, and procaspase-3 was cleaved into its active form. In contrast, in BV-2 cells treated with LPS for 24 h, UV irradiation caused neither apoptosis nor procaspase-3 cleavage. LPS treatment arrested the cell cycle in G1 phase and in effect protected the BV-2 cells from UV-induced apoptosis. LPS treatment upregulated cyclin-dependent kinase inhibitor p21Waf1/Cip1 and growth arrest and DNA damage-inducible（GADD）45α in BV-2 cells. When p21Waf1/Cip1 and GADD45α were knocked down by small interfering RNA, procaspase-3 was cleaved into its active form to induce apoptosis. Collectively, our findings suggest that LPS inhibits UV-induced apoptosis in BV-2 cells, at least partly through arrest of the cell cycle in G1 phase by upregulation of p21Waf1/Cip1 and GADD45α. Excessive activation of microglia may play a critical role in the exacerbation of neurodegeneration, therefore normalizing the precise regulation of apoptosis in these cells may be a new strategy to prevent the deterioration caused by neurodegenerative disorders. |
| P3-21
アストロサイトにおけるヌクレオシドトランスポーターの機能と過酸化水素による細胞死
田中 康一1,2,3，北中 順惠2，北中 純一2，山際 友花1，糸井 正二郎1，塚原 飛央3，佐藤 友昭3，竹村 基彦2，馬場 明道1，西山 信好1
兵庫医療大学薬学部医療薬学科薬理学分野1，兵庫医科大学薬理学分野2，鹿児島大学大学院医歯学総合研究科生体機能制御学講座歯科応用薬理学分野3
We have found that cultured astrocytes pretreated with N6, 2'-O-dibutyryladenosine 3',5'-cyclic monophosphate（DBcAMP）, a permeable analogue of cAMP, but not astrocytes pretreated without DBcAMP and neurons, have the ability to incorporate thymidine into acid insoluble fraction via equilibrative nucleoside transporter 2（ENT2）and concentrative nucleoside transporter（CNT3）at an early time（15-30 min）for repair on hydrogen peroxide（H2O2）-induced DNA injury.
This time we studied the relation between the system of nucleoside transport-DNA repair and cell death induced by H2O2 in cultured astrocytes. Cell death was assessed by staining of propidium iodide and MTT assay. The treatment of H2O2 for 60 and 30 min induced acute and delayed cell death in the different concentration dependent manners, respectively. The inhibiton of CNT（removal of extracellular Na+）or ENT（addition of nitrobenzylthioinosine, dilazep, dipyridamole）promoted delayed cell death.
These finding indicate that the inhibition of the system of nucleoside transport-DNA repair might induce delayed cell death in cultured differentiated astrocytes on H2O2-induced DNA injury. |
| P3-22
アポE含有リポタンパク質とalpha2-マクログロブリンの神経保護効果における役割
林 秀樹，袁 博，高木 教夫
東京薬科大学薬学部応用生化学
Lipoproteins secreted from glia are important for maintainance of lipid homeostasis in the central nervous system（CNS）and contain apolipoprotein E（apo E）in high density lipoprotein-like particles. We have previously demonstrated that apo E-containing lipoproteins protect primary cultured retinal ganglion cells as the CNS neurons from apoptosis induced by glutamate neurotoxicity, and intravitreal injection of the liporpoteins decreases optic nerve degeneration in glutamate-aspartate transporter（GLAST）-deficient mice, an animal model of glaucoma. Neuronal apoptosis was prevented when the lipoproteins bound to the low density lipoprotein receptor-related protein 1（LRP1）, a multi-functional receptor of the low density lipoprotein receptor family. It has been reported that alpha2-macroglobulin（a2M）is increased in aqueous humor of glaucoma patients. We also determined that the increase of a2M in vitreous humor of GLAST-deficient mice. Here, we demonstrate that a2M blocked the neuroprotective effect of apo E-containing lipoproteins against glutamate neurotoxicity, and on the other hand, the lipoproteins prevented a secretion of a2M from glia. Since both apo E and a2M are ligands for LRP1, our findings indicate that a balance between apo E-containing lipoproteins and a2M might be important for neuronal survival in retinal ganglion cells against glutamate neurotoxicity. |
| P3-23
小脳顆粒神経細胞に発現するGPR3は細胞生存に関与する
田中 茂，宮城 達博，秀 和泉，白藤 俊彦，酒井 規雄
広島大学大学院 医歯薬保健学研究科 神経薬理学
G-protein coupled receptor（GPR）3 belongs to a member of constitutively active Gs-coupled receptors that increase the level of 3'-5'-cyclic adenosine monophosphate（cAMP）and is highly expressed in the brain. We have previously reported that developmental expression of GPR3 in rodent cerebellar granule neurons is associated with neurite outgrowth and cell survival；however, the physiological functions of GPR3 remain to be fully elucidated. In the present study, we investigated the survival and antiapoptotic functions of GPR3 under normal and apoptosis-inducing culture conditions. Under normal culture conditions, CGNs from GPR3-knockout mice indiacted lower survival than did CGNs from wild-type mice. The antiapoptotic effect of GPR3 was also observed under hypoxic（1% O2/5% CO2）and reactive oxygen species（ROS）-induced apoptotic conditions. Next, we investigated the signaling pathways involved in the GPR3-mediated antiapoptotic effect. The addition of the PKA inhibitor KT5720, the MAP kinase inhibitor U0126, and the PI3 kinase inhibitor LY294002 abrogated the GPR3-mediated antiapoptotic effect in a potassium-deprivation model of apoptosis. Furthermore, downregulation of endogenous GPR3 expression in CGNs resulted in a marked reduction in the basal levels of ERK and Akt phosphorylation under normal culture conditions. Finally, we used a transient middle cerebral artery occlusion（tMCAO）model in wild-type and GPR3-knockout mice. After tMCAO, GPR3-knockout mice indicated a significantly larger infarct area than did wild-type mice. These results suggest that the developmental expression of constitutively active Gs-coupled GPR3 activates the ERK and Akt signaling pathways at the basal level, thereby protecting neurons from apoptosis-inducing stimuli. | |
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