AMYLOID PRECURSOR PROTEIN IN BIOLOGY & PATHOGENESIS OF A

淀粉样β肽（β/A4）在家族性糖尿病中的蓄积机制 阿尔茨海默氏病（FAD）和唐氏综合征是未知的，可能 异质的 β PP 770基因第17外显子点突变的发现 在几个AD家庭的受影响成员中， 异常的β淀粉样前体蛋白（betaAPP）可以导致这种疾病 在某些情况下，这是一个很大的挑战。 没有遗传缺陷的报告，在大加拿大 和已知与21号染色体连锁的意大利FAD家系。 基因复制与唐氏综合症有关。 我们的目标是阐明 在这些条件中的每一个中β/A4积累的原因将是 通过以下具体实验解决：1）检查 在受影响的人与受影响的人之间， 以及加拿大和意大利家庭中未受影响的成员。 我们要求 FAD中β APP mRNA或蛋白水平是否存在差异 如果是，这是否反映了启动子功能的改变 区域或mRNA稳定性的变化。 分裂期总内源性mRNA 来自家族成员的应激成纤维细胞系将与 通过免疫印迹检测β APP蛋白水平。 同样的细胞系也将 用全长β App启动子-报告基因瞬时转染 基因构建体 2)为了研究β APP水平如何影响 作为基因剂量的函数进行调节，我们将解决未满足的需求 为了严格分析细胞中betaAPP转录物的水平， 存活的21三体患者和对照组。 总β APP mRNA和 淋巴细胞裂解物中的蛋白质水平将被定量， 21号染色体上或染色体外的其他基因产物。 30、判断是否 β APP外显子17突变a）有利于选择性细胞内切割 产生含有完整的羧基末端片段的事件 β A4或B）仅在特定条件下才导致此类片段的出现。 细胞条件（例如应激）。 脉冲追踪实验和 用转染细胞的各种β APP抗体进行免疫沉淀 将用突变的β APP cDNA进行。 40、检查 假设a）完整膜的细胞外部分 糖蛋白，β APP，作为底物粘附分子发挥作用 受体，类似于整合素;和B）细胞内部分 与细胞骨架结合。 细胞配体，其结合β-APP， 正常条件将被鉴定为SDS上的共沉淀蛋白质。 用35 S甲硫氨酸代谢标记培养物中的细胞后的PAGE。 外显子17突变对转染细胞粘附于细胞膜的影响 还将研究基层。 betaAPP基因的研究进展 转录、mRNA稳定性、蛋白质降解及其正常 功能对于预防β/A4积累至关重要。

The mechanism of amyloid beta peptide (beta/A4) accumulation in familial Alzheimers disease (FAD) and Down's syndrome is unknown and probably heterogeneous. The discovery of point mutations in exon 17 of betaPP770 in affected members of several families with AD is evidence that an abnormal beta amyloid precursor protein (betaAPP) can cause the disease in some cases. No genetic defect has been reported in the large Canadian and Italian FAD pedigrees which have known linkage to chromosome 21. Gene duplication is implicated in Down's syndrome. Our goal to elucidate the cause of beta/A4 accumulation in each of these conditions -will be addressed by the following specific experiments: 1) To examine the possibility of altered transcription of the betaAPP gene between affected and unaffected members of the Canadian and Italian families. We ask whether differences in the levels betaAPP MRNA or protein in FAD occur and if so, whether this reflects altered functioning of the promotor region or changes in MRNA stability. Total endogenous MRNA in dividing and stressed fibroblast cell lines from family members will be correlated with betaAPP protein levels by immunoblot. The same cell lines will also be transiently transfected with a full length betaApp promotor-reporter gene construct. 2) To examine the question, of how betaAPP levels are regulated as a function of gene dosage, we will address the unmet need to analyze rigorously the level of betaAPP transcripts in cells from living patients with trisomy 21 and controls. Total betaAPP MRNA and protein levels in lymphocyte lysates will be quantitated and compound to other gene products on or off chromosome 21. 30 To determine whether betaAPP exon 17 mutations a) favor an alternative intracellular cleavage event that generates carboxyl terminal fragment(s) containing the intact betaA4 or b) cause the appearance of such fragments only under special cellular conditions (e.g. stress). Pulse chase experiments and immunoprecipitations with various betaAPP antibodies of cells transfected with the mutant betaAPP CDNA'S will be conducted. 40 To examine the hypothesis that a) the extracellular portion of the integral membrane glycoprotein, betaAPP, functions as a substrate adhesion molecule receptor, similar to the Integrins; and b) the intracellular portion binds to the cytoskeleton. Cellular ligands that bind to betaAPP under normal conditions will be identified as co-precipitating proteins on SDS- PAGE after cells in culture are metabolic labeled with 35 S methionine. The effect of the exon 17 mutations on transfected cell adhesion to the substratum will also be studied . An understanding of betaAPP gene transcription, MRNA stability, protein degradation and its normal function is critical to the prevention of beta/A4 accumulation.