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GENETIC AND BIOCHEMICAL EVENTS IN AB-INDUCED APOPTOSIS

AB 诱导的细胞凋亡中的遗传和生化事件

基本信息

批准号：
2001588
负责人：
Carl Wayne Cotman
金额：
$ 15.72万
依托单位：
UNIVERSITY OF CALIFORNIA-IRVINE
依托单位国家：
美国
项目类别：
财政年份：
1995
资助国家：
美国
起止时间：
1995-01-15 至 1997-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/2001588
关键词：
DNA damage amyloid proteins antisense nucleic acid apoptosis calcium flux cellular pathology cytotoxicity gene induction /repression histochemistry /cytochemistry regulatory gene synthetic peptide tissue /cell culture

项目摘要

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.

描述：小肽β-淀粉样蛋白，AB，已被牵连，阿尔茨海默病的神经退化过程申请人已经在定义AB毒性方面发挥了重要作用，现在已经获得了数据表明它通过凋亡途径导致培养的神经元死亡表现出膜泄漏，染色质浓缩和DNA 碎片化经典的凋亡或程序性细胞死亡被定义为形态学上通过最初的表面blabbing，染色质凝聚和核收缩，随后核碎裂成核体和多核糖体分散，而有限的ER仍然和线粒体保持正常。只有在继发性坏死的晚期，质膜解体。相反，坏死性细胞死亡是以ER、线粒体和膜的早期扩张为特征破坏该建议是定义细胞的序列，由对AB的反应引发的分子事件，诱导立即早期基因，IEGs，（c-fos和c-jun以及随后的c-fos和c-jun）， myc和junB）以及钙和其他第二信使在诱导fos和jun和DNA断裂。申请人已经初步数据表明，AB诱导IEG，c-fos和c-jun。细胞系统中，反义抑制fos和jun可抑制凋亡目标1提出定义c-jun的详细时间过程和c-fos诱导。目标2建议使用反义寡核苷酸阻断这种诱导，并确定细胞死亡是否是防止。在其他系统中，细胞凋亡已被证明会导致 fos和jun诱导，时间过程与DNA的时间过程平行碎片化目的4是确定DNA的时间过程碎片化，看看c-fos和c-jun的时间过程是否相同诱导还建议确定反义是否如目的2中的处理防止DNA片段化。 C-myc是另一种IEG通过反义和反义寡核苷酸参与细胞凋亡，表达的研究，还有一些其他的，包括bcl-2，bcl-2， x、ced-3和ced-9。目标3旨在确定其他方面的变化 IEGs，重点关注对AB的c-myc和junB反应。根据工作，其他凋亡系统和其他研究AB毒性的实验室作为他们自己的数据，申请人假设AB细胞凋亡是介导的细胞内钙离子的瞬时上升，随后是在DNA断裂之前的延迟上升。在目标5中，申请人旨在更好地定义钙作为AB介导剂的作用，使用钙成像的反应，DNA的组织化学评估使用游离3 '-OH基团的原位末端标记的片段化，碎片和光学显微镜。