FACTORS GOVERNING ALZHEIMERS ABETA PROTEIN

4kD(39-43个残基)的淀粉样β蛋白(AB) 在阿尔茨海默病(AD)中被称为淀粉样蛋白，编码为一种内源性多肽 从一组695-770的羧基末端开始99个残基 残基糖蛋白被称为淀粉样β蛋白前体 (BAPP)。我们实验室和其他实验室的最新数据已经证实 BAPP的正常处理包括：(I)构成分泌 BAPP在AB内被切割以产生一个大的， 分泌的NH2-末端衍生物和8.7kD的COOH-末端片段， 它们都不能产生淀粉样蛋白，因为它们不包含 整个AB，(Ii)产生复合体的内切/溶酶体加工 一组COOH末端的衍生物，包括潜在的淀粉样变性 具有整个AB a或靠近其NH2末端的形式，以及(Iii) 4kD AB的生产(与沉积的AB基本相同) AD中的淀粉样蛋白)是从培养的细胞中释放出来的，很容易检测到 在脑脊液中。强有力的证据表明淀粉样蛋白沉积起着关键作用 AD的发展源于对家族性AD的识别 (FAD)AD表型与基因突变协同聚集的家系 BAPP基因。这些突变中的三个改变了Valine突变(NL) 将直接位于氨基位置的赖氨酸-蛋氨酸改变为AB1 (在BAPP中lys670=met671)到异亮氨酸、苯丙氨酸或甘氨酸。一个 第四双突变(NL)改变赖氨酸-蛋氨酸定位 立即将A1(BAPP770中的lys670-met671)氨基转变为天冬酰胺-亮氨酸。 这些突变的位置紧邻AB 提示它们可能通过改变BAPP的处理方式而导致AD 是淀粉样变性。为了评估这种可能性，我们最近比较了 表达正常BAPP695或FAD连锁的人神经母细胞瘤(M17)细胞 突变的BAPP 695。表达BAPP NL突变体的细胞显示出5倍的 11.4kD含AB羧基的相对含量增加- 末端BAPP衍生物，他们将6倍以上的5kD AB释放到 5~6成熟。这些观察结果提供了强有力的证据：(I)这个突变体 BPP导致AD是因为它经历了改变的处理，从而释放 AB的量增加，以及(Ii)在 培养的细胞与阿尔茨海默病高度相关。在此应用程序中，我们建议 通过试验扩大对AB生产的分析 设计用来(1)检验4kD AB产生和 由正常细胞处理释放的几种多肽 可变COOH末端(AB39-43)，类似于已被 在AD淀粉样蛋白中鉴定，(2)检验BAPP717的假设 突变体倾向于生产较长的AB1-42或43型，这 选择性沉积为斑块核心淀粉样蛋白，而不增加 根据我们小组和其他人的观察，AB的生产符合预期 佛波醇酯大大增加了大型 秘密的BAPP衍生品。此外，我们还建议(4)比较BAPP 加工与转基因小鼠或人细胞释放AB 表达野生型BAPP或FAD连锁突变体BAPPS以(I) 确定迄今为止在转基因领域取得的令人失望的结果 阿尔茨海默病小鼠模型与小鼠AB产生率低有关 表达人BAPP的细胞，以及(Ii)识别可能 引入CNS AB后显著提高产量 转基因小鼠。最后，我们计划(5)开展系统的 阿尔茨海默病不同部位可溶性AB含量的比较 控制大脑，努力确定可溶性AB的量是否 与AD和/或淀粉样蛋白沉积相关。

The 4 kD (39-43 residue) amyloid beta protein (AB), which is deposited as amyloid in Alzheimer's disease (AD), is encoded as an internal peptide that begins 99 residues from the carboxyl terminus of a set of 695-770 residue glycoproteins referred to as the amyloid Beta protein precursor (BAPP). Recent data from our laboratory and others has established that normal processing of the BAPP involves (i) a constitutive secretory pathway in which the BAPP is cleaved within AB to produce a large, secreted, NH2-terminal derivative and an 8.7 kD COOH-terminal fragment, neither of which can produce amyloid because they do not contain the entire AB, (ii) endosomal/lysosomal processing which produces a complex set of COOH-terminal derivatives that includes potentially amyloidogenic forms with the entire AB a or near their NH2 terminus, and (iii) the production of 4 kD AB(essentially identical to the AB deposited as amyloid in AD) that is released from cultured cells and readily detected in CSF. Strong evidence that amyloid deposition plays a critical role in the development of AD has come from the identification of familial AD (FAD) kindreds in which the AD phenotype cosegregates with mutations in the BAPP gene. Three of these mutations alter the valine mutation (NL) alters the lysine-methionine located immediately amino to AB1 (lys670=met671 in BAPP) to isoleucine, phenylalanine, or glycine. A fourth double mutation (NL) alters the lysine-methionine located immediately amino to A1 (lys670-met671 in BAPP770) to asparagine-leucine. The location of these mutations in close proximity to AB immediately suggests that they may cause AD by altering BAPP processing in a way that is amyloidogenic. To evaluate this possibility, we have recently compare human neuroblastoma (M17) cells expressing normal BAPP695 or FAD-linked mutant BAPP 695. Cells expressing the BAPP NL mutant showed a 5-fold increase in the relative amount of an 11.4 kD AB-bearing carboxyl- terminal BAPP derivative, and they released 6-fold more 5 kD AB into the medium. These observations provide strong evidence (i) that this mutant BPP causes AD because it undergoes altered processing that releases increased amounts of AB, and (ii) that the pathway producing AB in cultured cells is highly relevant to AD. In this application, we propose to expand our analysis of AB production by undertaking experimentation designed t o (1) test the hypothesis that the 4kD AB produced and released by normal cellular processing consists of several peptides with variable COOH-termini (AB39-43) similar to the AB peptides that have been identified in AD amyloid, (2) test the hypothesis that the BAPP717 mutants favor production of the longer AB1-42 or 43 forms, which selectivity deposit as plaque core amyloid, without increasing the production of AB as expected from the observation by our group and others that phorbol esters substantially increase the production of the large secreted BAPP derivative. In addition, we propose (4) to compare BAPP processing and the AB released by transfected mouse or human cells expressing wild type BAPP or the FAD-linked mutant BAPPs in order to (i) determine if the disappointing results obtained to date in transgenic mouse model of AD are related to low rates of AB production in mouse cells expressing human BAPPs, and (ii) identify constructs likely to significantly increase CNS AB production when they are introduced into transgenic mice. Finally, we plan (5) to carry out a systematic comparison of the amount of soluble AB in various regions of AD and control brains in an effort to determine if the amount of soluble AB is correlated with AD and/or amyloid deposition.