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

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 8-氧鸟嘌呤糖基酶1(Ogg1)是一种结构型糖基酶，负责从DNA中去除致突变的修饰碱基8-羟基-2‘-脱氧鸟苷(Oxo8dG)。Ogg1在所有组织中都有表达，它的表达受到细胞氧化还原状态的调节，启动子区存在抗氧化反应元件(ARE)就是明证。此外，有关Ogg1活性和表达在改变氧化还原状态的条件下发生变化的报道支持这一观点。然而，人们对氧化还原环境中调控其表达或活性的具体触发因素知之甚少。Oxo8dG的积累与帕金森氏症和阿尔茨海默病等主要神经系统疾病以及肌萎缩侧索硬化症和正常衰老过程中的神经细胞丢失有关。已经证明，这些高oxo8dG水平中的一些是Ogg1活性下降的结果。虽然oxo8dG可以由于各种有害刺激而在DNA中形成和积累，但它从DNA中的去除完全依赖于Ogg1的活性。因此，需要了解Ogg1在神经元中调控和表达的细胞机制，以确定导致oxo8dG积累和疾病发生的病理和/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Ogg1调控中起主要作用的观点，而且表明还原型谷胱甘肽(GSH)的水平是这种调控的主要组成部分。我们假设GSH作为一个分子开关，通过调节Ogg1的表达和/或活性来调节DNA中的oxo8dG水平。此外，不同细胞群中修复DNA的细胞反应能力的改变将有助于识别不同的神经元脆弱性。这一假设将通过以下具体目标进行检验。 具体目的1.全面描述GSH介导的Ogg1表达的时间调控及其对神经元ox8dG水平变化的影响。 表达Pam/HOGG1启动子-hrGFP载体的神经细胞株将暴露于GSH耗竭(L-丁硫氨酸-(S，R)-亚磺胺(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)；Ogg1表达的时程变化将通过荧光细胞分类器观察GFP表达的变化。Ogg1在关键时间和/或剂量的变化将得到实时聚合酶链式反应和Ogg1活性的进一步证实。此外，还将根据Ogg1表达的变化来评估oxo8dG(高效液相色谱-EC)水平。 具体目的2.确定氧化还原调节后Ogg1表达变化对神经元易损性的影响程度。 BSO、DEM、NAC GSHEt将在特定的Aim1中确定的引起Ogg1表达最大变化的剂量和时间处理神经元细胞系。然后，细胞将受到剂量增加的过氧化氢的挑战，过氧化氢是一种已知的oxo8dG诱导剂或抗霉素A，它是线粒体呼吸的抑制剂，也是过氧化氢和超氧化物产生的诱导剂。 具体目的3.确定转录因子在启动子区域的识别位点的必要和/或充分作用，以允许Ogg1的基础和诱导表达。 将对位于Ogg1翻译起始处上游350个碱基对的转录因子(AP4、NRF2和两个Sp1)的识别位点进行定点突变，并通过荧光激活细胞分选器检测绿色荧光蛋白(报告基因)的表达。Ogg1启动子中假定的识别位点与Ogg1的基础表达和GSH调控的表达之间的相关性将被建立。

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.