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

淀粉样蛋白β蛋白(Aβ)产生的细胞机制仍然是关键但仍是悬而未决的问题。内切/溶酶体途径被认为是从淀粉样前体蛋白(APP)中裂解Aβ的位点。本实验室以往的研究表明，APP、Aβ和淀粉样蛋白相关蛋白参与了氯喹诱导的大鼠实验性肌病的发病机制，氯喹是影响酸性脑室的有效药物，也参与了内质体/溶酶体途径。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在内的蛋白质转运。这一证据促使我们对CQN作用下培养的心肌细胞进行APP和PS-I处理的研究。这些空泡用抗APP和PS-I染色。抗APP和PS-1双标结果显示APP和PS-I共定位于空泡中。Northern EL ISA证实m…表达上调在CQN处理过程中，APP的RNA水平增加，而PS-I的mRNA水平没有变化。用不同的抗APP和抗Aβ抗体进行Western印迹分析，在CQN处理的细胞线粒体和微粒体部分可分离出约12 kDa的淀粉样变性条带。在对照组和氯喹酮处理的细胞线粒体片段中均可检测到约4 kDa的Aβ条带。在CQN处理的心肌细胞的裂解液、线粒体和微粒体中分别检测到27和47 kDa的PS-I片段。在Brefeldin、莫能菌素和刀豆素处理下，对培养的心肌细胞进行免疫病理、生化和分子生物学研究。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的过程中起着重要作用，而β-Coatmer蛋白、非笼络蛋白包裹的囊泡途径可能参与了APP运输到CqN敏感内体的过程。总之，无论有没有CqN处理的实验培养的心肌细胞系统都提供了一个很好的模型，从AD的发病机制来阐明APP产生Aβ的细胞机制。

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