ACHE, CHAT AND CHOLINERGIC NEURONS IN AGING AND AD

衰老和 AD 中的疼痛、聊天和胆碱能神经元

• 批准号: 2049586
• 负责人: STEVEN G YOUNKIN
• 金额: $ 24.61万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-12-01 至 1998-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2049586
• 关键词: Alzheimer's disease; acetylcholine; acetylcholinesterase; amyloid proteins; blood chemistry; choline acetyltransferase; disease /disorder proneness /risk; endopeptidases; enzyme linked immunosorbent assay; gene expression; gene mutation; guinea pigs; human subject; immunocytochemistry; neuritic plaques; protein metabolism; protein sequence; protein structure function; tissue /cell culture; transfection

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.

我们实验室和其他实验室的最新数据表明，正常情况下 淀粉样β蛋白前体（BetaAPP）的加工产生并 释放本质上相同的 4 kD 淀粉样β蛋白 (ABeta) Aβ 沉积为淀粉样蛋白 iA 阿尔茨海默病 (AD)。强的 有证据表明淀粉样蛋白沉积在发育中起关键作用 AD 的诊断来自于对家族性 AD (FAD) 亲属的识别 AD 表型与 betaAPP 基因突变共分离。 其中三个突变改变了位于三个羧基残基的缬氨酸 至 Abeta43（betaAPP 中的 val717）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） 改变位于 Abeta1 紧邻氨基的赖氨酸-蛋氨酸 天冬酰胺-亮氨酸。这些突变的位置非常接近 A-beta 立即表明它们可能通过改变 betaAPP 引起 AD 以淀粉样蛋白生成的方式进行加工。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 betaAPP695。表达 betaAPPdeltaNL 突变体的细胞 显示相对量增加了 5 倍，约为 11.4 kD A-β 带有羧基末端 betaAPP 衍生物，他们释放了 将 6 倍以上的 4 kD Abeta 添加到培养基中。这些观察提供了强有力的 有证据表明 betaAPPdeltaNL 会导致 AD，因为它发生了改变 释放更多 Aβ 的处理。显著地， 11.4 表达 betaAPPdeltaI 的转染细胞未显示出增加 kD 带有 Abeta 的 COOH 末端 betaAPP 衍生物，并且没有增加 4 kD Abeta 的分泌。为了进一步检查 FAD 链接 (betaAPP717 突变体（delta-I、delta-F），我们通过以下方式分析转染的 M17 细胞： 从条件培养基中分离代谢标记的 4 kD Abeta， 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 Abeta1-40 和较长的 Abeta1-42。两种方法 证明 betaAPP717 突变导致 1.5 至 1.9 倍 长 Abeta1-42 生成的百分比增加。很好 确定长 Abeta（例如 Abeta1-42）形成不溶性淀粉样蛋白 原纤维形成速度比 Abeta1-40 更快。因此 betaAPP717 突变体，例如 deltaNL突变体，经过改变的处理，增强了 淀粉样蛋白沉积的可能性。综合这些观察结果 提供强有力的证据 (i) 淀粉样蛋白沉积在 AD 中至关重要， (ii) 在培养细胞中产生 A- 的途径高度 与 AD 中淀粉样蛋白沉积有关。现在可以看出，比率 淀粉样蛋白沉积取决于 (i) BetaAPP 的速率 加工成分泌型 Abeta，(ii) 分泌型 Abeta 被加工成的速率 去除，以及（iii）不溶性淀粉样原纤维形成的速率 任何可溶性细胞外 Abeta 的主要浓度。在这个 建议我们重点关注控制 A 浓度的因素，因为我们的 AD 遗传形式的研究表明 Abeta 浓度为 对于确定是否沉积了足够的淀粉样蛋白至关重要 导致疾病。因此，该提案的第一个具体目标是 确定一组蛋白酶，这些蛋白酶决定了 释放各种 Abeta 肽。我们的第二个具体目标是 确定负责清除分泌的 Abeta 的机制， 目前完全未知的机制。我们最后的具体目标 是分析血浆中的 Abeta 以确定是否 总 Abeta 或以 Abeta42 结尾的 Abeta 与 AD 相关。