MOLECULAR BIOLOGICAL STUDIES OF APP PROCESSING

APP 处理的分子生物学研究

• 批准号: 03670568
• 负责人: TAKAHATA Naohiko
• 金额: $ 1.02万
• 依托单位: SAPPORO MEDICAL UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1991
• 资助国家: 日本
• 起止时间: 1991 至 1992
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-03670568/
• 关键词: Alzheimer's disease; monoclonal antibody; amyloid precursor protein; processing; amyloid beta protein; cell culture; leupeptin; endosomal; lysosomal pathway; アルツハイマ-病; soluble APP; membrane bound APP; β蛋白

The principal neuropathological feature of Alzheimer's disease is extracellular deposition of about 4 kDa proteinous fragment, designated as amyloid beta peptides (Abeta) derived by proteolytic cleavage from amyloid precursor protein (APP), large cell surface receptor like protein. There have been evidences that APP is proteolytically degraded in the secretory pathway, and endosomal/lysosomal pathway. The pathway is not still identified, in which APP is cleaved to generate Abeta. To clarify the intracellular processing of APP into the generation of Abeta, we detected and characterized potentially amyloidogenic or non-amyloidogenic fragments using newly established monoclonal and polyclonal antibodies, which were raised against eight different synthetic peptides deduced from the sequence of APP770 cDNA,with subcellular fractions obtained from the cultured cells with or without leupeptin, potent lysosomal protease inhibitor of lysosome. APP fragments of 50 and 20 kDa molecular mass containing full-length Abeta were identified in the cultured cells. Immunoblot analysis, biochemical study for specific marker enzyme activity of the fractions obtained from subcellular fractionation, sucrose density gradient centrifugation, together with immunohistochemical observations indicated that the 50 kDa APP fragment was produced in the compartment closely related to endosomal/lysosomal system. Our data suggest that the endosomal/lysosomal pathway is involved in the processing and generation of Abeta.

阿尔茨海默病的主要神经病理学特征是约4kDa蛋白质片段的细胞外沉积，该蛋白质片段被称为淀粉样β肽（Abeta），该淀粉样β肽通过蛋白水解裂解来自淀粉样前体蛋白（APP），大细胞表面受体样蛋白。有证据表明APP在分泌途径和内体/溶酶体途径中被蛋白水解降解。该途径尚未确定，其中APP被切割以产生Abeta。为了阐明APP的细胞内加工成Abeta的产生，我们检测和表征潜在的淀粉样蛋白或非淀粉样蛋白的片段，使用新建立的单克隆和多克隆抗体，这是提出了对八个不同的合成肽推导出的序列的APP 770 cDNA，亚细胞级分从培养的细胞与或不与亮抑酶肽，有效的溶酶体蛋白酶抑制剂的溶酶体。在培养的细胞中鉴定了含有全长Abeta的50和20 kDa分子量的APP片段。免疫印迹分析、蔗糖密度梯度离心和亚细胞分级分离后的特异性标志酶活性的生化研究以及免疫组织化学观察表明，50 kDa APP片段产生于与内体/溶酶体系统密切相关的隔室中。我们的数据表明，内体/溶酶体途径参与了Abeta的加工和产生。

项目成果

Takamaru Y et al: "Apolipoprotein E in Alzheimer's disease affected brains" Brain Research. (submitted). (1995)

Takamaru Y 等人：“阿尔茨海默病影响大脑中的载脂蛋白 E”大脑研究。

Takamaru Y et al: "Alzheimer's and Parkinson's Disease: Recent Developments" Plenum Press, 7 (1995)

Takamaru Y 等人：“阿尔茨海默病和帕金森病：最新进展”Plenum Press，7 (1995)

Takamaru Y,Obara T,Fukatsu R,Tsuzuki K,Aizawa Y,Fujii M,Kobayashi M,Gotoda T,Yanagihara R,Garruto R,Oguma K and Takahata N: "Monoclonal antibodies against paired helical filament in Alzheimer's disease and parkinsonism-dementia complex of guam" In Nagatsu

Takamaru Y、Obara T、Fukatsu R、Tsuzuki K、Aizawa Y、Fujii M、Kobayashi M、Gotoda T、Yanagihara R、Garruto R、Oguma K 和 Takahata N：“针对阿尔茨海默病和帕金森痴呆症中配对螺旋丝的单克隆抗体

Aizawa Y,Fukatsu R,Takamaru Y,Tsuzuki K,Obara T,Fujii M,Kobayashi M,Takahata N,Gotoda T,Nagasawa S,Oguma K,Kunishita T and Tabira T: "Monoclonal antibodies against senile plaque amyloid in Alzheimer's disease" In Nagatsu T,Fisher A,Yoshida M (eds). Plenum

Aizawa Y、Fukatsu R、Takamaru Y、Tsuzuki K、Obara T、Fujii M、Kobayashi M、Takahata N、Gotoda T、Nagasawa S、Oguma K、Kunishita T 和 Tabira T：“针对阿尔茨海默病中老年斑淀粉样蛋白的单克隆抗体”

Yuji Aizawa: "Monoclonal antibodies raised against native amyloid in Alzheimer's disease ーestablishment and characterizationー"

Yuji Aizawa：“针对阿尔茨海默病中的天然淀粉样蛋白产生的单克隆抗体 - 建立和表征 -”