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

β淀粉样蛋白（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从淀粉样蛋白前体蛋白（APP）中裂解的一个位点。我们实验室之前的研究表明，APP、Aβ和淀粉样蛋白相关蛋白参与了氯喹（CQN）引起的大鼠实验性肌病的发病机制，氯喹是影响酸性区室和内体/溶酶体途径的有效药物。最近的免疫细胞化学研究表明，早老素-1 （PS-1）参与蛋白质运输，包括APP。这一证据促使我们研究CQN下培养的肌细胞中APP和PS-1的加工。经CQN处理后，在培养的肌细胞核周区发现了微空泡。对这些空泡进行抗APP和PS-I染色。抗APP和ps -1的双重标记表明APP和ps -1在液泡中共定位。Northern ELISA结果显示，CQN处理后，APP mRNA水平上调，而PS-I mRNA水平不变。在cqn处理细胞的线粒体和微粒体部分中，使用各种抗app和抗a β抗体进行Western blot分析，可分离出约12 kDa的淀粉样蛋白条带。在对照组和cqn处理的细胞的线粒体部分中都检测到大约4 kDa波段的a β。在CQN未处理的肌细胞的裂解液、线粒体和微粒体中分别检测到27和47 kDa的PS- i片段，分别具有抗PS- i、n端和全长PS带。在布雷菲尔丁、莫能菌素和康那霉素的作用下，对培养的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/溶酶体途径在全长APP生成淀粉样蛋白片段和Aβ中起重要作用，β包覆蛋白、非网格蛋白包覆的囊泡途径可能参与了APP向CQN敏感内体的运输。综上所述，CQN处理或不处理的实验培养肌细胞系统提供了一个很好的模型，可以揭示APP产生Aβ的细胞机制在AD发病机制中的作用。少

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