MT COBRE: GLUTATHIONE: LINKING DNA REPAIR REGULATION AND NEURONAL VULNERABILITY

MT COBRE：谷胱甘肽：将 DNA 修复调节与神经元脆弱性联系起来

• 批准号: 7720406
• 负责人: FERNANDO CARDOZO-PELAEZ
• 金额: $ 15.18万
• 依托单位: UNIVERSITY OF MONTANA
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-05-01 至 2009-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7720406
• 关键词: 8-hydroxy-2' -deoxyguanosine; 8-oxoguanine; Acetylcysteine; Alzheimer' s Disease; Amyotrophic Lateral Sclerosis; Antimycin A; Antioxidants; Base Pairing; Buthionine Sulfoximine; Cell Line; Cells; Computer Retrieval of Information on Scientific Projects Database; Condition; DNA; DNA Repair; Data; Deletion Mutagenesis; Dose; Excision; Fluorescence; Funding; Glutathione; Grant; Green Fluorescent Proteins; Hydrogen Peroxide; Indium; Institution; Link; Mediating; Mitochondria; Molecular; Neurons; Onset of illness; Oxidation-Reduction; Parkinson Disease; Play; Polymerase Chain Reaction; Population; Production; Promoter Regions; Proteins; Reduced Glutathione; Regulation; Reporter Genes; Reporting; Research; Research Personnel; Resources; Respiration; Role; S-ethyl glutathione; Site; Site-Directed Mutagenesis; Source; Stimulus; Stream; Superoxides; Testing; Time; Tissues; Translations; United States National Institutes of Health; base; buthionine; diethyl maleate; fluorescence activated cell sorter device; inhibitor/antagonist; neurological pathology; normal aging; promoter; repaired; response; transcription factor; vector

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 8-Oxoguanine glycosylase 1 (Ogg1) is a constitutive glycosylase responsible for the removal of the promutagenic modified base 8-hydroxy-2'-deoxyguanosine (oxo8dG) from DNA. Ogg1 is expressed in all tissues and it is suggested that its expression is modulated by the redox status of the cell, as evidenced by the presence of antioxidant responsible elements (ARE) in the promoter region. Additionally, reports of altered Ogg1 activity and expression under conditions that shift the redox status support this idea. However, little is known of the specific triggers in the redox milieu responsible for the regulation of its expression or activity. Accumulation of oxo8dG has been linked to neuronal cell loss in major neurological pathologies such as Parkinson's and Alzheimer disease, as well as in amyotrophic lateral sclerosis, and during normal aging. It has been shown that some of these high oxo8dG levels are a consequence of a decreased Ogg1 activity. Although oxo8dG can form and accumulate in DNA due to diverse noxious stimuli, its removal from DNA is solely dependent in the activity of Ogg1. Thus, understanding the cellular mechanisms responsible for the regulation and expression of Ogg1 in neurons is needed to identify pathological and/or toxicological conditions responsible for the accumulation of oxo8dG and the onset of disease. Our data, not only supports the notion that the redox status of the cell plays a major role in Ogg1 regulation, but suggests that the level of reduced glutathione (GSH) is the major component in such regulation. We hypothesize that GSH acts as a molecular switch that regulates the oxo8dG levels in DNA via modulation of Ogg1 expression and/or activity. Additionally, alterations in cell response capacity to repair DNA in distinct cell populations will allow identifying differential neuronal vulnerability. This hypothesis will be tested with the following specific aims. Specific Aim 1. To fully delineate temporal GSH-mediated regulation of Ogg1 expression and its consequences in changes in ox8dG levels in neurons. Neuronal cell lines expressing a pAM/HOGG1promoter-hrGFP vector will be exposed to GSH depleting (L-buthionine-(S,R)-sulfoximine (BSO), Diethylmaleate(DEM)) or GSH inducing agents (N-acetyl cysteine(NAC)), and glutathione ethyl ester (GSHEt); time-course changes in Ogg1 expression will be visualized via changes in GFP expression with a fluorescent cell sorter. Changes in Ogg1 at critical times and/or doses will be further corroborated by Real-Time PCR and Ogg1 activity. Additionally, oxo8dG (HPLC-EC) levels will be assessed in relation to alterations in Ogg1 expression. Specific Aim 2. To determine the degree of neuronal vulnerability associated with Ogg1 expression changes after redox modulation. Neuronal cell lines will be treated with BSO, DEM, NAC GSHEt at doses and times that cause maximum change in Ogg1 expression, as identified in Specific Aim1. Then, cells will be challenged with increasing doses of H2O2, a known inducer of oxo8dG or antimycin A, an inhibitor of mitochondrial respiration and inducer of H2O2 and superoxide production. Specific Aim 3. To determine the necessary and/or sufficient role of recognition sites of transcription factors in the promoter region that allow for basal and inducible expression of Ogg1. Site directed mutagenesis deletion of recognition sites of transcription factors (AP4, Nrf2, and two Sp1) located in the 350 base pair up stream to the translation origin for Ogg1 will be done and expression of green fluorescence protein (reporter gene) will be determined via fluorescence activated cell sorter. The relevance for putative recognition sites in Ogg1 promoter will be established for basal and GSH-modulated expression of Ogg1.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 8-氧气甘氨酸糖基化酶1（OGG1）是一种负责去除前毒素改性碱基8-羟基-2'-脱氧鸟苷（OXO8DG）的组成型糖基酶。 OGG1在所有组织中均表达，并提出其表达是由细胞的氧化还原状态调节的，如启动子区域中存在抗氧化剂负责任元件（IS）所证明的那样。此外，在改变氧化还原状态的条件下，OGG1活动和表达改变的报告支持了这一想法。但是，氧化还原环境中负责调节其表达或活性的特定触发因素知之甚少。 OXO8DG的积累与主要神经病理学的神经元细胞损失有关，例如帕金森氏病和阿尔茨海默氏病，以及肌萎缩性侧向硬化症以及正常衰老期间。已经表明，其中一些高OXO8DG水平是OGG1活性降低的结果。尽管由于多种有害刺激，OXO8DG可以在DNA中形成并积聚，但它从DNA中的去除仅取决于OGG1的活性。因此，需要了解对神经元中OGG1调控和表达的细胞机制，以识别导致OXO8DG积累和疾病发作的病理和/或毒理学条件。我们的数据不仅支持这样的观念：细胞的氧化还原状态在OGG1调节中起主要作用，而且表明降低的谷胱甘肽（GSH）的水平是该调节中的主要组成部分。我们假设GSH充当分子开关，该开关通过调节OGG1表达和/或活性来调节DNA中的OXO8DG水平。另外，在不同细胞群体中修复DNA的细胞反应能力的改变将允许识别差异神经元脆弱性。该假设将以以下特定目的进行检验。 具体目的1。完全描绘了临时GSH介导的OGG1表达调节及其在神经元中OX8DG水平变化中的后果。 表达PAM/HOGG1促进剂-HRGFP载体的神经元细胞系将暴露于GSH耗竭（L-buthionine-（S，R） - 硫胺（BSO），二乙基甲基甲甲基（DEM））或GSH诱导剂（N-乙酰基酯（N-acetyl）（NAC）（NAC）（NAC）（nAc）和GLOTHEL（NAC）和GLOTHER（NAC）和GLOTHER（GESER）（GESER）（gnaC）和glite（glute）（glute）（glute）; OGG1表达的时间顺序变化将通过使用荧光细胞分类器的GFP表达的变化来可视化。实时PCR和OGG1活动将进一步证实OGG1在关键时间和/或剂量下的变化。此外，将根据OGG1表达的改变评估OXO8DG（HPLC-EC）水平。 具体目标2。确定氧化还原调制后与OGG1表达变化相关的神经元脆弱性程度。 如在特定AIM1中所确定的那样，将以BSO，DEM，NAC GSHET的剂量和最大变化的时间对神经元细胞系进行处理。然后，将通过增加剂量的H2O2（一种已知的OXO8DG或抗霉素A）（线粒体呼吸的抑制剂，H2O2和超氧化物产生的诱导剂）来挑战细胞。 具体目的3。确定启动子区域中转录因子识别位点的必要和/或足够作用，允许OGG1的基础和诱导表达。 位于位于350碱基对的转录因子识别位点（AP4，NRF2和两个SP1）的识别位点的诱变缺失将完成，并将通过荧光激活的细胞差来确定绿色荧光蛋白（Reporter Gene）的绿色荧光蛋白（Reporter Gene）的表达。将建立与OGG1启动子中假定识别位点的相关性，以用于OGG1的基础和GSH调节表达。