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

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。8-氧鸟嘌呤糖基酶1 （Ogg1）是一种组成型糖基酶，负责从DNA中去除促生修饰的碱基8-羟基-2¿-脱氧鸟嘌呤（oxo8dG）。Ogg1在所有组织中均有表达，其表达受细胞氧化还原状态的调节，如在启动子区域存在抗氧化负责元件（ARE）所证明。此外，在改变氧化还原状态的条件下，Ogg1活性和表达改变的报道支持了这一观点。然而，对氧化还原环境中负责调节其表达或活性的特定触发器知之甚少。oxo8dG的积累与帕金森病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元细胞损失有关。研究表明，其中一些高氧8dg水平是Ogg1活性降低的结果。虽然oxo8dG可以由于各种有害刺激而在DNA中形成和积累，但其从DNA中的去除仅依赖于Ogg1的活性。因此，了解神经元中Ogg1的调控和表达的细胞机制是确定oxo8dG积累和疾病发病的病理和/或毒理学条件的必要条件。我们的数据不仅支持细胞的氧化还原状态在Ogg1调节中起主要作用的观点，而且表明还原型谷胱甘肽（GSH）水平是这种调节的主要成分。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，在不同细胞群中修复DNA的细胞反应能力的改变将允许识别不同的神经元易感性。这一假设将通过以下具体目标进行检验。具体目标全面描述gsh介导的Ogg1表达的时间调控及其对神经元中ox8dG水平变化的影响。表达pAM/ hogg1启动子- hrgfp载体的神经细胞系将暴露于GSH消耗(l -丁硫氨酸-（S，R)-亚砜亚胺（BSO），二乙基马来酸（DEM））或GSH诱导剂（n -乙酰半胱氨酸（NAC））和谷胱甘肽乙酯（GSHEt）；利用荧光细胞分选器，通过GFP表达的变化来观察Ogg1表达的时间变化。在关键时刻和/或剂量Ogg1的变化将通过Real-Time PCR和Ogg1活性进一步证实。此外，将评估oxo8dG （HPLC-EC）水平与Ogg1表达变化的关系。具体目标2。测定氧化还原调控后Ogg1表达变化与神经元易损性的关系。神经细胞系将用BSO、DEM、NAC GSHEt处理，剂量和时间可引起Ogg1表达的最大变化，如Specific aim中所示。然后，细胞将被增加剂量的H2O2挑战，H2O2是一种已知的oxo8dG诱导剂或抗霉素a，一种线粒体呼吸抑制剂和H2O2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Ogg1的基础和诱导表达。对Ogg1翻译起点上游350碱基对的转录因子（AP4、Nrf2和两个Sp1）的识别位点进行定点诱变，通过荧光激活细胞分选仪检测绿色荧光蛋白（报告基因）的表达。Ogg1启动子中假定的识别位点的相关性将与基础和gsh调节的Ogg1表达建立联系

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