GENETIC AND BIOCHEMICAL EVENTS IN AB-INDUCED APOPTOSIS

描述：小肽β-淀粉样蛋白，AB，被认为与 阿尔茨海默病的神经退变过程。申请人有 在确定AB毒性方面发挥了重要作用，现在已经获得了数据 表明它会导致培养的神经元通过凋亡途径死亡 表现为膜爆裂、染色质凝集和DNA 碎片化。经典的定义是凋亡性或程序性细胞死亡 从形态上看，最初的表面磨擦，染色质浓缩和 核收缩，随后核碎裂成核体 和多核糖体分散，而内质网残留和线粒体有限 保持正常。只有在继发性坏死阶段的后期， 质膜解体。相比之下，坏死性细胞死亡 以内质网、线粒体和膜早期扩张为特征 颠覆。该建议是定义细胞和 AB反应引发的分子事件主要集中在 即刻早期基因的诱导，IEGs，(c-fos和c-jun以及后来的c-fos MYC和JunB)以及钙和其他第二信使在 诱导FOS和JUN及DNA片段化。申请人有 初步数据表明AB诱导了IEGs、c-fos和c-jun。在其他 细胞系统中，反义抑制fos和jun已抑制 细胞凋亡。目标1建议定义c-jun的详细时间进程 AB诱导c-fos表达。目标2建议使用反义 以阻断这种诱导，并确定细胞死亡是否 被阻止了。在其他系统中，细胞凋亡已被证明会导致 Fos和Jun诱导的时间进程与DNA的时间进程平行 碎片化。目标4是确定DNA的时间进程 分段并查看c-fos和c-jun的时间进程是否相同 归纳法。还建议确定反义基因是否 目标2中的处理可防止DNA片段化。C-myc是 另一种IEG被证明通过反义和 表达研究，还有其他一些研究，包括bcl2、bcl2、bcl1、bc1、bc1-2、b X、CED-3和CED-9。目标3旨在确定其他方面的变化 重点关注c-myc和JunB对AB的反应。基于中的工作 其他凋亡系统和其他实验室也在研究AB毒性 作为他们自己的数据，申请者假设AB细胞凋亡是 通过细胞内钙的短暂升高，随后是一种 DNA片段化之前的延迟上升。在目标5中，申请人 寻求更好地定义钙作为AB的介体的作用 使用钙成像、DNA组织化学评估的反应 3‘-羟基自由基团原位末端标记的碎裂 碎片和光学显微镜。

DESCRIPTION: The small peptide beta-amyloid, AB, has been implicated in the neurodegenerative process in Alzheimer's disease. The applicant has played a major role in defining AB toxicity and has now obtained data showing that it causes cultured neurons to die by an apoptotic pathway exhibiting membrane blabbing, chromatin condensation and DNA fragmentation. Classically apoptotic or programmed cell death was defined morphologically by initial surface blabbing, chromatin condensation and nuclear shrinkage, followed by nuclear fragmentation into nuclear bodies and polyribosome dispersal while limited ER remains and mitochondria remain normal. Only late in the secondary necrosis stage does the plasma membrane disintegrate. In contrast, necrotic cell death is characterized by early dilation of the ER and mitochondria and membrane disruption. The proposal is to define the sequence of cellular and molecular events initiated by the response to AB focussing on the induction of immediate early genes, IEGs, (c-fos and c-jun and later c- myc and junB) and the role of calcium and other second messengers in inducing fos and jun and DNA fragmentation. The applicant has preliminary data that AB induces the IEGs, c-fos and c-jun. In other cell systems, antisense inhibition of fos and jun has inhibited apoptosis. Aim 1 proposes to define the detailed time course of c-jun and c-fos induction in response to AB. Aim 2 proposes to use antisense oligos to block this induction and determine whether cell death is prevented. In other systems where apoptosis has been shown to result in fos and jun induction, the time course parallels that of DNA fragmentation. Aim 4 is to determine the time course for DNA fragmentation and see if the time course is the same for c-fos and c-jun induction. It is also proposed to determine whether anti-sense treatment as in aim 2 protects against DNA fragmentation. C-myc is another IEG shown to be involved in apoptosis by antisense and expression studies and there are a number of others including bcl-2, bcl- x, ced-3 and ced-9. Aim 3 is directed at determining changes in other IEGs, focussing on c-myc and junB in response to AB. Based on work in other apoptotic systems and other labs working on AB toxicity as well as their own data, the applicants hypothesize that AB apoptosis is mediated by a transient rise in intracellular calcium, followed by a delayed rise which precedes DNA fragmentation. In aim 5, the applicant seeks to better define the role of calcium as a mediator of the AB response using calcium imaging, histochemical assessment of DNA fragmentation using in situ end labelling of free 3'-OH groups on fragments and light microscopy.