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: 7959449
• 负责人: FERNANDO CARDOZO-PELAEZ
• 金额: $ 14.75万
• 依托单位: UNIVERSITY OF MONTANA
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-05-01 至 2010-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7959449
• 关键词: 8-hydroxy-2' -deoxyguanosine; Acetylcysteine; Alzheimer' s Disease; Amyotrophic Lateral Sclerosis; Antimycin A; Antioxidants; Base Pairing; Cell Line; Cells; Computer Retrieval of Information on Scientific Projects Database; DNA; DNA Repair; Data; Dose; Excision; Fluorescence; Funding; Glutathione; Grant; Hydrogen Peroxide; Indium; Institution; Link; Mediating; Mitochondria; Molecular; Neurons; Neurosciences; Onset of illness; Oxidation-Reduction; Parkinson Disease; Play; 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.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 8-氧代鸟嘌呤糖基化酶1（Ogg 1）是一种组成型糖基化酶，负责从DNA中去除前诱变修饰的碱基8-羟基-2 '-脱氧鸟苷（oxo 8dG）。Ogg 1在所有组织中表达，并建议其表达由细胞的氧化还原状态调节，如在启动子区域中存在抗氧化剂责任元件（ARE）所证明的。此外，在改变氧化还原状态的条件下改变Ogg 1活性和表达的报告支持这一想法。然而，很少有人知道的特定触发器在氧化还原环境中负责其表达或活性的调节。 oxo 8dG的积累与主要神经病理学如帕金森病和阿尔茨海默病以及肌萎缩性侧索硬化症和正常衰老期间的神经元细胞损失有关。已经表明，这些高oxo 8dG水平中的一些是Ogg 1活性降低的结果。虽然oxo 8dG可以形成并积累在DNA中由于不同的有害刺激，它从DNA中的去除是完全依赖于Ogg 1的活性。因此，需要了解负责神经元中Ogg 1的调节和表达的细胞机制，以确定负责oxo 8dG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细胞的氧化还原状态在Ogg 1调节中起主要作用的观点，而且表明还原型谷胱甘肽（GSH）的水平是这种调节的主要组成部分。我们假设GSH作为一个分子开关，通过调节Ogg 1的表达和/或活性来调节DNA中的oxo 8dG水平。此外，在不同的细胞群体中修复DNA的细胞反应能力的改变将允许识别不同的神经元脆弱性。将以下列具体目标检验这一假设。 具体目标1。充分描绘时间GSH介导的Ogg 1表达的调节及其在神经元ox 8dG水平变化中的后果。 将表达pAM/HOGG 1启动子-hrGFP载体的神经元细胞系暴露于GSH消耗剂（L-丁硫氨酸-（S，R）-亚砜亚胺（BSO）、马来酸二乙酯（DEM））或GSH诱导剂（N-乙酰半胱氨酸（NAC））和谷胱甘肽乙酯（GSHEt）;通过荧光细胞分选仪观察Ogg 1表达的时程变化。通过实时PCR和Ogg 1活性进一步证实关键时间和/或剂量时Ogg 1的变化。此外，将评估与Ogg 1表达变化相关的oxo 8dG（HPLC-EC）水平。 具体目标2。确定氧化还原调节后与Ogg 1表达变化相关的神经元易损性程度。 神经元细胞系将用BSO、DEM、NAC GSHEt以引起Ogg 1表达的最大变化的剂量和时间处理，如特异性Aim 1中所确定的。然后，细胞将用增加剂量的H2 O2进行攻击，H2 O2是oxo 8dG或抗霉素A的已知诱导剂，是线粒体呼吸的抑制剂和H2 O2和超氧化物产生的诱导剂。 具体目标3。 确定启动子区域中转录因子识别位点的必要和/或充分作用，以允许Ogg 1的基础和诱导表达。 将对位于Ogg 1翻译起点上游350个碱基对中的转录因子（AP 4、Nrf 2和两个Sp1）的识别位点进行定点突变缺失，并通过荧光激活细胞分选仪测定绿色荧光蛋白（报告基因）的表达。Ogg 1启动子中的推定识别位点的相关性将被建立用于Ogg 1的基础和GSH调节的表达。