ACHE, CHAT AND CHOLINERGIC NEURONS IN AGING AND AD

我们实验室和其他机构最近的数据表明， 淀粉样β蛋白前体（BetaAPP）的加工产生并 释放4 kD淀粉样β蛋白（ABeta）， A型淀粉样蛋白沉积到阿尔茨海默病（AD）。强 有证据表明淀粉样蛋白沉积在 AD的诊断来自于家族性AD（FAD）激酶的鉴定， AD表型与β APP基因突变共分离。 这些突变中有三个改变了位于三个羧基残基的缬氨酸 至A β 43（β APP中的val 717）至异亮氨酸（delta I）、苯丙氨酸 （Δ F）或甘氨酸（Δ G）。第四个双突变（delta NL） 将位于紧邻氨基的赖氨酸-蛋氨酸改变为A β 1， 天冬酰胺-亮氨酸。这些突变的位置非常接近 直接表明他们可能通过改变β APP而导致AD 以淀粉样蛋白生成的方式加工。为了评估这种可能性， 我们比较了表达正常或FAD的人神经母细胞瘤（M17）细胞， 连锁突变体betaAPP 695。表达betaAPPdeltaNL突变体的细胞 显示出大约11.4的相对量增加了5倍 携带kD A-β羧基末端β APP衍生物，并且它们释放 6-将4kD Abeta增加一倍。这些观察提供了强有力的 证据表明betaAPPdeltaNL导致AD，因为它经历了改变 处理过程中释放出更多的ABeta。值得注意的是， 表达β APPdeltaI的转染细胞在11.4 kD携带A β COOH-末端β APP衍生物， 分泌4kD Abeta。为了进一步检查FAD相关（betaAPP 717）， 突变体（delta-I，delta-F），我们通过（i）分析转染的M17细胞， 从条件培养基中分离代谢标记的4kD A β， 用CNBr消化，并分析释放的COOH末端肽 或（ii）使用夹心ELISA评估条件培养基中的A-β 区分A β 1 -40和较长的A β 1 -42的基因。两种方法 证明β APP 717突变导致1.5至1.9倍的 增加长Abeta 1 -42的百分比。公 确定长的A β（例如A β 1 -42）形成不溶性淀粉样蛋白 比Abeta 1 -40更快地形成原纤维。因此，β APP 717突变体，如 deltaNL突变体，经历改变的加工，增强了 淀粉样蛋白沉积的可能性。综合这些观察， 提供了强有力的证据（i）淀粉样蛋白沉积在AD中至关重要， 和（ii）在培养细胞中产生A-的途径是高度 与AD中的淀粉样蛋白沉积有关。现在很明显， 淀粉样蛋白沉积将取决于（i）BetaAPP沉积的速率， （ii）分泌的Abeta被加工成分泌的Abeta的速率， 去除，和（iii）不溶性淀粉样蛋白原纤维形成的速率 在任何可溶性细胞外A β的普遍浓度下。在这 建议我们关注的因素，支配A-浓度，因为我们的 对AD遗传形式的研究表明， 在确定是否有足够的淀粉样蛋白沉积 导致疾病因此，本建议的第一个具体目标是 鉴定确定所述蛋白酶的所述组， 各种Abeta肽被释放。第二个具体目标是 确定负责清除分泌的Abeta的机制， 目前完全未知的机制。我们最后的目标 是分析血浆中的Abeta，以确定 总A β或以A β 42结尾的A β与AD相关。

Recent data from our laboratory and others has established that normal processing of the amyloid beta protein precursor (BetaAPP) produces and releases 4 kD amyloid Beta protein (ABeta) that is essentially identical to the ABeta deposited as amyloid iA Alzheimer's disease (AD). 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 betaAPP gene. Three of these mutations alter the valine located three residues carboxyl to Abeta43 (val717 in betaAPP) to isoleucine (delta I), phenylalanine (delta F), or glycine (delta G). A fourth double mutation (delta NL) alters the lysine-methionine located immediately amino to Abeta1 to asparagine-leucine. The location of these mutations in close proximity to A-beta immediately suggests that they may cause AD by altering betaAPP processing in a way that is amyloidogenic. To evaluate this possibility, we compared human neuroblastoma (M17) cells expressing normal or FAD- linked mutant betaAPP695. Cells expressing the betaAPPdeltaNL mutant showed a 5-fold increase in the relative amount of an approximate 11.4 kD A-beta-bearing carboxyl-terminal betaAPP derivative, and they released 6-fold more 4 kD Abeta into the medium. These observations provide strong evidence that betaAPPdeltaNL causes AD because it undergoes altered processing that releases increased amounts of ABeta. Significantly, transfected cells expressing betaAPPdeltaI showed no increase in the 11.4 kD Abeta-bearing COOH-terminal betaAPP derivative and no increase in secretion of 4 kD Abeta. To further examine the FAD-linked (betaAPP717 mutants (delta-I, delta-F), we analyzed transfected M17 cells by (i) isolating metabolically labeled 4 kD Abeta from conditioned medium, digesting with CNBr, and analyzing the COOH-terminal peptides released or (ii) assessing the A-beta in conditioned medium using sandwich ELISAs that discriminate Abeta1-40 from the longer Abeta1-42. Both methods demonstrated that the betaAPP717 mutations cause a 1.5 to 1.9-fold increase in the percentage of long Abeta1-42 generated. It is well established that long Abeta (e.g. Abeta1-42) forms insoluble amyloid fibrils more rapidly than Abeta1-40. Thus the betaAPP717 mutants, like the deltaNL mutant, undergo altered processing that enhances the likelihood of amyloid deposition. Taken together these observations provide strong evidence (i) that amyloid deposition is critical in AD, and (ii) that the pathway producing A- in cultured cells is highly relevant to amyloid deposition in AD. It is now evident that the rate of amyloid deposition will depend on (i) the rate at which BetaAPP is processed into secreted Abeta, (ii) the rate at which secreted Abeta is removed, and (iii) the rate at which insoluble amyloid fibrils are formed at any prevailing concentration of soluble, extracellular Abeta. In this proposal we focus on the factors that govern A- concentration because our studies of the genetic forms of AD indicate that Abeta concentration is critically important in determining whether enough amyloid is deposited to cause disease. Thus the first specific aim of this proposal is to identify the set of proteases that determine the rate at which the various Abeta peptides are released. Our second specific aim is to identify the mechanism(s) responsible for clearing secreted Abeta, mechanism(s) that currently are completely unknown. Our last specific aim is to analyze Abeta in plasma to determine whether the concentrations of total Abeta or Abeta ending at Abeta42 are correlated with AD.