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: 6703655
• 负责人: Kelvin J. A. Davies
• 金额: $ 34.04万
• 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 2007-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6703655
• 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.

我们感兴趣的是氧化应激、衰老和神经退行性疾病之间可能存在的联系。在最近关于氧化应激适应的研究中，我们已经确定了几个以前未知的基因(除了证实几个已知基因的过度表达外)，它们似乎在培养的分离的仓鼠细胞中提供了应激保护。这些“适应”基因包括：适应15、适应33、适应66、适应73、适应78和适应116。尽管我们的研究表明，完全适应依赖于转录和翻译，但实际上需要哪些基因尚不清楚。尽管这些新发现的基因中的每一个都值得详细研究，但其中尤其突出的是Adapt78，它的mRNA水平在适应过程中增加了50倍以上。在使用人脑尸检样本的筛选研究中，我们现在发现，在阿尔茨海默病患者的大脑尸检样本中，Adapt78的人类同源物表现出极高的表达水平，而在帕金森氏病死亡患者的大脑样本中，包括黑质在内的大脑样本的表达水平很低。最近，我们的Adapt78与同时独立发现的21号染色体的唐氏综合症临界区1(DSCR1)基因完全相同(或至少高度同源)，这一点已经变得明显。此外，Adapt78和DSCR1的两个不同的亚型都有不同的表达；对应于不同剪接形式的外显子1-5，6，7和外显子4-5，6，7。我们建议现在使用更敏感的RT-PCR和原位杂交技术，仔细研究人类Adapt78基因的两个亚型在不同脑区的表达。我们计划在其他健康个体的脑部尸检样本中研究Adapt78的表达与年龄的关系，因为Adapt78的表达可能会随着年龄的变化而变化。我们将对阿尔茨海默病患者、帕金森病患者和唐氏综合症患者的大脑样本进行详细研究，以仔细确定Adapt78mRNA两种异构体表达的质和量差异。还将研究Adapt78mRNA按细胞类型的定位表达。我们还将合成和表征(1-5，6，7和4-5，6，7)Adapt78蛋白，并产生抗体，以研究实际蛋白在所有细胞和大脑样本中的表达。在细胞培养研究中，以PC-12细胞为对象，我们将检验一种假设，即可诱导的Adapt78过表达可能赋予氧化应激抵抗表型。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死氧化效应的能力，谷胱甘肽缺乏症是由表达γ谷氨酰半胱氨酸合成酶的反义信息引起的。这些研究将使我们能够开始调查我们的假设，即衰老，也许还有某些神经退行性疾病，涉及应对氧化应激有害影响所需的Adapt78和其他适应基因表达的缺陷。