The molecularbiological effect of AD disease genes on APP processing in cultured cells

AD疾病基因对培养细胞APP加工的分子生物学影响

• 批准号: 10470204
• 负责人: FUKATSU Ryo
• 金额: $ 7.87万
• 依托单位: Saitama Medical School
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1998
• 资助国家: 日本
• 起止时间: 1998 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10470204/
• 关键词: Amyloid β protein; Amyloidprecursor protein; Presenilin-1; Chloroquine (CQN); Endosomal; lysosomal pathway; CQN myocytes system; Experimental model; Disease gene; アルツハイマー病; 細胞内局在; chloroquine,; monennsin; concanamycin

The cellular mechanisms underlying production of amyloid β protein (A β), remain key but open issue. An endosomal/lysosomal pathway has been suggested as a site, whereby A β is cleaved from amyloid precursor protein (APP). Previous studies from our laboratory demonstrated that APP, Aβ , and amyloid-associated proteins are involved in the pathogenesis of experimental myopathy induced by chloroquine (CQN) in rats, which is potent agents affecting acidic compartments, and endosomal/lysosomal pathway. Recent immunocytochemical studies showed that presenilin-1 (PS-1) is involved in protein trafficking, including APP. This evidence prompted us to study APP and PS-I processing in cultured myocytes under CQN.Microvacuoles were recognized in the perinuclear region of cultured myocytes after CQN treatment. These vacuoles were stained with anti APP and PS-I. Double labeling with anti APP and PS-1showed that APP, and PS-I were co-localized in the vacuoles. Northern ELISA demonstrated upregulated m … More RNA level of APP, but mRNA of PS-I were unchanged during CQN treatment. About 12 kDa amyloidogenic band was detactable by Western blot analyses using various anti-APP and anti-Aβ antibodies in mitochondrial and microsomal fraction of CQN-treated cells. About 4 kDa band, presumably Aβwas detectable in mitochondrial fraction of both control and CQN-treated cells. 27 and 47 kDa PS-I fragments were detectable in lysate, mitochondrial and microsomal fractions obtained from CQN untreated myocytes with anti-PS-I, N-terminal, and full-length PS band, respectively.Under Brefeldin, Monensin, and Concanamycin treatment, immunopathological, biochemical, molecular biological studies were performed on cultured myocytes. Our data indicate that the endosomal/lysosomal pathway play an important role in generating amyloidogenic fragments and Aβ from full-length APP and that β-coatmer protein, non-clathrin coated vesicles pathway might 'be involved in APP trafficking to CQN sensitive endosomes.In conclusion, the experimental cultured myocyte systems with or without CQN treatment provide an excellent model to shed light on the cellular mechanism by which Aβ is produced from APP in terms of the pathogenesis of AD. Less

淀粉样β蛋白（A β）产生的细胞机制仍然是关键但尚未解决的问题。内体/溶酶体途径被认为是A β从淀粉样前体蛋白（APP）裂解的位点。本实验室的前期研究表明，APP、Aβ和淀粉样蛋白相关蛋白参与了氯喹（CQN）诱导的大鼠实验性肌病的发病机制，CQN是影响酸性区室和内体/溶酶体途径的强效药物。最近的免疫细胞化学研究表明，早老素-1（PS-1）参与了包括APP在内的蛋白质运输，这一证据促使我们研究CQN处理下培养的心肌细胞中APP和PS-1的加工。这些空泡用抗APP和PS-I染色。抗APP和PS-1双标记显示APP和PS-1共定位于空泡内。北方ELISA显示， ...更多信息 在CQN处理期间，APP的RNA水平，而PS-I的mRNA水平没有变化。在CQN处理的细胞的线粒体和微粒体部分中，使用各种抗APP和抗A β抗体通过Western印迹分析可检测到约12 kDa的淀粉样蛋白生成带。在对照细胞和CQN处理细胞的线粒体部分中均可检测到约4 kDa条带，推测为Aβ。在CQN未处理的心肌细胞的裂解物、线粒体和微粒体组分中分别检测到27和47 kDa的PS-Ⅰ片段，其具有抗PS-Ⅰ、N-末端和全长PS带。我们的数据表明，内体/溶酶体途径在从全长APP产生淀粉样蛋白片段和Aβ中起重要作用，β-包被蛋白，非网格蛋白包被的囊泡途径可能参与APP向CQN敏感内体的运输。有或没有CQN处理的实验培养的肌细胞系统提供了一个很好的模型，以阐明Aβ产生的细胞机制。APP在AD发病机制中的作用。少

项目成果

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