ADAPT 78 IN OXIDANT STRESS, AGING AND NEURODEGENERATION

ADAPT 78 对抗氧化应激、衰老和神经退行性变

• 批准号: 6497189
• 负责人: Kelvin J. A. Davies
• 金额: $ 32.51万
• 依托单位: UNIVERSITY OF SOUTHERN CALIFORNIA
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-02-01 至 2005-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6497189
• 关键词: Alzheimer's disease; Downs syndrome; PC12 cells; Parkinson's disease; aging; antisense nucleic acid; glutathione; human tissue; hydrogen peroxide; in situ hybridization; neural degeneration; neurogenetics; neuroprotectants; oxidative stress; polymerase chain reaction; postmortem; protein isoforms; stress proteins; western blottings

We are interested in a possible link between oxidative stress, aging and neurodegenerative diseases. In recent studies of adaptation to oxidative stress we have identified several previously unknown genes (in addition to confirming the overexpression of several known genes) that appear to provide stress protection in isolated hamster cells in culture. These "adapt" genes include: adapt15, adapt33, adapt66, adapt73, adapt78, and adapt 116. Although our studies indicate that full adaptation depends upon both transcription and translation, it is not clear which genes are actually required. Although each of these newly discovered genes is worthy of detailed study, adapt78 whose mRNA levels increase more than 50 fold in adaptation, in particular stands out. In screening studies employing autopsy samples from human brains, we have now found that the human homologue of adapt78 exhibits extremely high levels of expression in brain autopsy samples from Alzheimer's disease patients, and low levels of expression in brain samples encompassing the substantia nigra from patients who died with Parkinson's disease. Recently, it has become clear that our adapt78 is identical to (or at least highly homologous with) the simultaneously independently discovered Down syndrome critical region 1 (DSCR1) gene of chromosome 21. Furthermore, two different isoforms of both adapt78 and DSCR1 are differentially expressed; corresponding to differentially spliced forms of exons 1-5, 6, 7 and exons 4-5, 6, 7. We propose to now carefully study expression of both isoforms of the human adapt78 gene in different brain regions, using the more sensitive techniques of RT-PCR and in situ hybridization. We plan to study adapt78 expression as a function of age, in brain autopsy samples from otherwise healthy individuals, since adapt78 expression may well vary with age. We will perform detailed studies of brain samples from Alzheimer's disease patients, Parkinson's disease patients, and Down syndrome patients in order to carefully determine both qualitative and quantitative differences in expression of both isoforms of adapt78 mRNA. Localization of adapt78 mRNA expression by cell type will also be studied. We also will synthesize and characterize the (1-5, 6, 7 and 4-5, 6, 7) Adapt78 proteins and generate antibodies to them in order to study expression of the actual proteins in all cell and brain samples. In cell culture studies, with PC-12 cells, we will test the hypothesis that inducible overexpression of adapt78 may confer an oxidative stress resistance phenotype. We will also test the ability of inducible adapt78 overexpressing cells to overcome the lethal oxidizing effects of glutathione deficiency, caused by expression of antisense message to gamma glutamylcysteine synthetase. These studies will allow us to begin to investigate our hypothesis that aging, and perhaps certain neurodegenerative diseases, involving defects in the expression of adapt78 and other adaptive genes required to cope with the deleterious effects of oxidative stress.

我们对氧化应激，衰老和神经退行性疾病之间的可能联系感兴趣。 在最新的对氧化应激的适应研究中，我们已经确定了几个以前未知的基因（除了确认了几种已知基因的过表达），这些基因似乎在培养的孤立仓鼠细胞中提供了应力保护。 这些“适应”基因包括：ADAPT15，ADAPT33，ADAPT66，ADAPT73，ADAPT78和ADAPT116。尽管我们的研究表明完全适应取决于转录和翻译，但实际上需要哪些基因。 尽管这些新发现的基因中的每一个都值得详细研究，但Adapt78的mRNA水平在适应性上增加了50倍以上，特别是脱颖而出。 在筛查研究的研究中，我们现在发现，ADAPT78的人类同源物在阿尔茨海默氏病患者的脑尸检样品中表现出极高的表达水平，而在脑样本中，脑样本的表达较低，涵盖死于帕金森氏病的患者的脑质体。最近，很明显，我们的Adapt78与（或至少与高度同源）同时独立发现的唐氏综合征临界区域1（DSCR1）基因21染色体。此外，另外两种不同的ADAPT78和DSCR1的同工型也差异地表达了；对应于外显子1-5、6、7和外显子4-5、6、7的差异剪接形式。我们建议使用RT-PCR和原位杂交的更敏感的技术技术，仔细研究了不同大脑区域中人类Adapt78基因的两种同工型的表达。 我们计划在其他健康个体的脑尸检样本中研究Adapt78的表达，因为Adapt78的表达可能随着年龄的增长而变化。 我们将对来自阿尔茨海默氏病患者，帕金森氏病患者和唐氏综合症患者的大脑样本进行详细研究，以便仔细确定ADAPT78 mRNA的两种同工型表达的定性和定量差异。还将研究Adapt78通过细胞类型的MRNA表达的定位。 我们还将合成并表征（1-5、6、7和4-5、6、7）Adapt78蛋白质并生成抗体，以研究所有细胞和脑样品中实际蛋白的表达。 在细胞培养研究中，使用PC-12细胞，我们将检验以下假设：Adapt78的可诱导过表达可能赋予氧化应激抗性表型。 我们还将测试可诱导的Adapt78过表达细胞克服谷胱甘肽缺乏症的致命氧化作用的能力，这是由于反义信息与γ-谷氨酰胺顺氨酸合成酶的表达引起的。这些研究将使我们能够开始研究我们的假设，即衰老或某些神经退行性疾病，涉及Adapt78表达中的缺陷以及应对氧化应激的有害作用所需的其他适应性基因。