Cloning and functional analysis of Cl-ATPase 51kDa subunit

Cl-ATP酶51kDa亚基的克隆及功能分析

• 批准号: 14580753
• 负责人: HATTORI Naoki
• 金额: $ 2.3万
• 依托单位: Kansai Medical University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14580753/
• 关键词: chloridw; ATPase; amyloid β; Alzheimer's disease; phosphatidylinositol-4-phosphate; Cl^--ATPase; イノシトール; グルタミン酸; PI; PIP4

Cl-ATPase enables the inhibitory neurotransmission by GABA by keeping the intracellular chloride concentration l* we previously reported that Cl-ATPase activity decreased in Alzheimer's disease brains and that low dose amyloid β(Aβ) inhibited Cl-ATPase activity leading to the elevation of intracellular chloride levels. In. the pres* study, (1)we performed the cloning of Cl-ATPase 51kDa subunit and (2)we examined the effects phosphatidylinositol 4 phosphate on Ap-induced inhibition of Cl-ATPase.(1)We introduced reactive red column for the purification of Cl-ATPase to increase the recovery of the protein and set up sensitive assay for Cl-ATPase activity. We have identified several candidate proteins by mass spectrometric analysis on prot* spots separated two-dimensional electrophoresis.(2)Low dose(10nM) of Aβ treatment for 2 days decreased Cl-ATPase activity by 47%, while did not affect Na/K-ATPase * anion insentive ATPase activities. Phosphatidylinositol(PI 50-750nM) and phosphatidylinositol-4-phosphate do* dependency recovered the Cl-ATPase activity that was suppressed by Aβ. Intracellular Cl concentration that was increa* three times by Aβ returned to normal by adding 75nM PI or PI4P. Low dose of Aβ(10nM) plus 10μM glutam* administration to neurons induced DNA fragmentation and reduced cell viability as shown by increased LDL release and WST assay. Seventy-five nM PI or PI4P recovered these cell damages. It is widely accepted that glutamate neurotoxicity involved in the pathogenesis of Alzheimer's disease. We demonstrated that elevation of intracellular chloride concentration * to the reduction of Cl-ATPase was possibly involved in the pathogenesis of neurodegeneration in Alzheimer's disease, * addition, we suggested that inositol might be used for the treatment or prevention of the progression of neurodegeneraton Alzheimer's disease.

CL-ATPase通过保持细胞内氯化物浓度L*来实现GABA的抑制性神经传递，我们先前报道说，阿尔茨海默氏病大脑中的Cl-ATPase活性降低，而低剂量的淀粉样蛋白β（Aβ）抑制CL-ATPase的活性导致了内细胞内氯化物含量的升高。在。 the pres* study, (1)we performed the cloning of Cl-ATPase 51kDa subunit and (2)we examined the effects phosphatidylinositol 4 phosphate on Ap-induced inhibition of Cl-ATPase.(1)We introduced reactive red column for the purification of Cl-ATPase to increase the recovery of the protein and set up sensitive assay for Cl-ATPase activity.We have identified several candidate通过质谱分析对蛋白质的蛋白质分离二维电泳。（2）低剂量（10nm）Aβ处理2天，将Cl-ATPase活性降低了47％，而不会影响Na/k-ATPase * anion * anion intentive intentive ATPase ATPase活性。磷脂酰肌醇（PI 50-750nm）和磷脂酰肌醇4-磷酸*依赖性恢复了被Aβ抑制的Cl-ATPase活性。通过添加75nm Pi或PI4P，Aβ恢复了3倍的细胞内CL浓度。低剂量的Aβ（10nm）加10μM谷氨酸*神经元的给药诱导DNA片段化并降低细胞活力，如LDL释放和WST分析增加所示。 75个NM PI或PI4P恢复了这些细胞损伤。人们普遍认为，谷氨酸神经毒性涉及阿尔茨海默氏病的发病机理。我们证明，在阿尔茨海默氏病中，细胞内氯化物浓度 *升高与降低Cl-ATPase的降低是可能参与神经退行性的发病机理， *另外，我们建议肌醇可用于治疗或预防阿尔茨海默氏病神经变性的阿尔茨海默氏病的进展。

项目成果

Liu B, Hattori N, Jiang B, Nakayama Y, Zhang NY, Wu B, Kitagawa K, Taketo M, Matsuda H, Inagaki C.: "Single cell RT-PCR demonstrates differential expression of GABAc receptor p subunits in rat hippoeampal pyramidal and granule cells."Mol Brain Res. 123. 1

Liu B、Hattori N、Jiang B、Nakayama Y、Zhang NY、Wu B、Kitakawa K、Taketo M、Matsuda H、Inagaki C.：“单细胞 RT-PCR 证明大鼠海马锥体和大鼠海马锥体和 GABAc 受体 p 亚基的差异表达

Liu B et al.: "Single cell RT-PCR demonstrates differential expression of GABAc receptor rho subunits in rat hippocampal pyramidal and granule cells"Mol Brain Res. (In press). (2004)

Liu B 等人：“单细胞 RT-PCR 证明大鼠海马锥体细胞和颗粒细胞中 GABAc 受体 rho 亚基的差异表达”Mol Brain Res。

Jiang B et al.: "Expression and roles of Cl channel CIC5 in cell cycles of myeloid cells."Biochem.Biophys.Res.Commun.. 317. 192-197 (2004)

Jiang B 等：“Cl 通道 CIC5 在骨髓细胞细胞周期中的表达和作用”。Biochem.Biophys.Res.Commun.. 317. 192-197 (2004)

Jiang B et al.: "Expression and roles of Cl^-channel ClC-5 in cell cycles of myeloid cells"Biochem Biophys Res Commun. 317. 192-197 (2004)

Jiang B等：“Cl^-通道ClC-5在骨髓细胞细胞周期中的表达和作用”Biochem Biophys Res Commun。

Wu B, Kitagawa K, Yagyu K, Zhang NY, Hattori N, Inagaki C.: "Phosphatidylinositol and PI-4-monophosphate recover amyloid β protein-induced inhibition of Cl-ATPase activity."Life Sci. 72. 455-463 (2002)

Wu B、Kitakawa K、Yagyu K、Zhang NY、Hattori N、Inagaki C.：“磷脂酰肌醇和 PI-4-单磷酸恢复淀粉样蛋白 β 蛋白诱导的 Cl-ATP 酶活性抑制。” 72. 455-463（生命科学） 2002）