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: 6071182
• 负责人: Kelvin J. A. Davies
• 金额: $ 31.26万
• 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/6071182
• 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。 在最近的适应氧化应激的研究中，我们已经确定了几个以前未知的基因（除了确认几个已知基因的过表达），似乎在培养的仓鼠细胞中提供应激保护。 这些“adapt”基因包括：adapt 15、adapt 33、adapt 66、adapt 73、adapt 78和adapt 116。 虽然我们的研究表明，完全适应取决于转录和翻译，但尚不清楚实际上需要哪些基因。 虽然这些新发现的基因中的每一个都值得详细研究，但adapt 78的mRNA水平在适应中增加了50倍以上，特别突出。 在使用人类大脑尸检样本的筛选研究中，我们现在发现adapt 78的人类同源物在阿尔茨海默病患者的大脑尸检样本中表现出极高的表达水平，而在帕金森病患者死亡的黑质大脑样本中表现出低水平的表达。最近，已经清楚我们的adapt 78与同时独立发现的21号染色体的唐氏综合征关键区1（DSCR 1）基因相同（或至少高度同源）。 此外，adapt 78和DSCR 1两者的两种不同同种型差异表达;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。我们建议现在仔细研究人类adapt 78基因的两种亚型在不同大脑区域的表达，使用更灵敏的RT-PCR和原位杂交技术。 我们计划研究adapt 78的表达作为年龄的函数，在脑尸检样本，否则健康的人，因为adapt 78的表达可能会随着年龄的变化。 我们将对阿尔茨海默病患者、帕金森病患者和唐氏综合征患者的脑样本进行详细研究，以仔细确定adapt 78 mRNA两种亚型表达的定性和定量差异。还将通过细胞类型研究adapt 78 mRNA表达的定位。 我们还将合成和表征（1-5，6，7和4-5，6，7）Adapt 78蛋白并产生针对它们的抗体，以研究所有细胞和大脑样品中实际蛋白的表达。 在细胞培养研究中，用PC-12细胞，我们将测试adapt 78的诱导性过表达可能赋予氧化应激抗性表型的假设。 我们还将测试诱导型adapt 78过表达细胞克服谷胱甘肽缺乏的致死氧化作用的能力，谷胱甘肽缺乏是由γ谷氨酰半胱氨酸合成酶的反义信息表达引起的。这些研究将使我们开始调查我们的假设，即衰老，也许某些神经退行性疾病，涉及缺陷的adapt 78和其他适应性基因的表达需要科普氧化应激的有害影响。