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可能存在的联系感兴趣。 在最近的氧化应激适应研究中，我们发现了几个以前未知的基因（除了确认了几个已知基因的过度表达之外），它们似乎可以为培养的分离仓鼠细胞提供应激保护。 这些“适应”基因包括：adapt15、adapt33、adapt66、adapt73、adapt78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。 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水平在适应过程中增加了50倍以上，尤其引人注目。 在使用人类大脑尸检样本的筛选研究中，我们现在发现adapt78的人类同源物在阿尔茨海默病患者的脑部尸检样本中表现出极高水平的表达，而在死于帕金森病的患者的黑质脑样本中表现出低水平的表达。最近，我们已经清楚我们的adapt78与同时独立发现的21号染色体的唐氏综合症关键区1（DSCR1）基因相同（或至少高度同源）。此外，adapt78和DSCR1的两种不同亚型存在差异表达；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。我们建议现在使用更灵敏的RT-PCR和原位杂交技术仔细研究人类adapt78基因的两种亚型在不同大脑区域的表达。 我们计划在其他健康个体的脑尸检样本中研究作为年龄函数的适应性78表达，因为适应性78表达可能会随着年龄而变化。 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，以便仔细确定 Adapt78 mRNA 两种亚型表达的定性和定量差异。还将研究适应78 mRNA 表达按细胞类型的定位。 我们还将合成和表征 (1-5, 6, 7 和 4-5, 6, 7) Adapt78 蛋白，并生成针对它们的抗体，以便研究所有细胞和大脑样本中实际蛋白的表达。 在细胞培养研究中，我们将使用 PC-12 细胞来检验这样的假设：adapt78 的诱导性过度表达可能会赋予氧化应激抵抗表型。 我们还将测试诱导型 Adapt78 过表达细胞克服谷胱甘肽缺乏的致命氧化作用的能力，谷胱甘肽缺乏是由表达对 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研究我们的假设，即衰老，也许还有某些神经退行性疾病，都涉及适应78和其他应对氧化应激有害影响所需的适应性基因的表达缺陷。