Alcohol Impairs Neonatal Astrocyte GSH Homeostasis

酒精损害新生儿星形胶质细胞 GSH 稳态

• 批准号: 6620857
• 负责人: GEORGE I HENDERSON
• 金额: $ 14.6万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-03-01 至 2005-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6620857
• 关键词: acid aminoacid ligase; aminoacid transport; astrocytes; brain; cell death; chemical kinetics; cysteine; cystine; cytotoxicity; developmental neurobiology; enzyme activity; ethanol; free radical oxygen; glutamates; glutamyltransferase; glutathione; glycine; high performance liquid chromatography; homeostasis; laboratory rat; mitochondria; neurotoxicology; newborn animals; oxidative stress; protein biosynthesis; protein transport; tissue /cell culture

Prior studies in our laboratory and others have documented ethanol- related oxidative stress in the developing brain and there is evidence suggesting that it may cause at least some of the neurotoxic effects of ethanol (E). The mechanism(s) by which E causes this oxidative stress remain to be determined, but it is likely related to relatively low anti- oxidant status. There is a compelling literature illustrating that astrocytes play a vital neuroprotective role by producing and exporting reduced glutathione (GSH) and we include, as preliminary data, evidence that astrocyte GSH homeostasis may be perturbed by E in the developing brain. Hypothesis. We hypothesize that one mechanism underlying the E- mediated oxidative stress and subsequent damage to the developing brain is an impairment of the ability of astrocytes to generate and/or export GSH. Hypothesis: We hypothesize that one mechanisms underlying the E- mediated oxidative stress and subsequent damage to the developing brain is an impairment of the ability of astrocytes to generate and/or export GSH. Specific Aims. The following specific aims are extensions of our preliminary studies and those of others which document an E-mediated reduction of GSH in cultured neonatal cortical astrocytes. They will determine the means by which E causes this effect. They focus on E effects on well established control points in GSH synthesis and on its efflux. Additionally, experiments will determine the effect of E on viability of astrocytes with respect to mitochondrial functions key to survival and generation of oxygen species which may deplete astrocyte GSH. Specific Aim One will determine the effect(s) of E on astrocyte uptake of precursors required for the synthesis of GSH. We will focus on the effects of E on inward directed transport of cystine and cysteine, which are known determinants of GSH synthesis, but we will also determine effects of E on cellular content of other relevant amino acids e.g. glutamate glycine. These studies will also determine effects of E on GSH uptake and efflux by astrocytes. Specific Aim Two will determine effects of E on the enzymatic synthesis of GSH by astrocytes with a focus on the impact on activity of gamma- glutamylcysteine synthetase, the rate-limiting enzyme in GSH synthesis. Additionally, effects of E on astrocyte-bound gamma-glutamyl transpeptidas4e as a source on CysGly for neuronal GSH synthesis. will be determined. Specific Aim Three will determine the effects of E on astrocyte variability at the mitochondrial . The emphasis on the mitochondrion is due to it being a target for E toxicity, the source of ATP for GSH synthesis, and a primary source of oxidative stress. These experiments will determine E-related depletion of mitochondrial GSH, production of oxidative products which deplete mitochondria of GSH, and induction of mitochondrially-mediated cell death which we document in the appendix.

我们实验室和其他人的先前研究已经记录了发育中大脑中与乙醇相关的氧化应激，并且有证据表明它可能导致乙醇（E）的至少一些神经毒性作用。E引起这种氧化应激的机制仍有待确定，但可能与相对较低的抗氧化状态有关。有一个令人信服的文献表明，星形胶质细胞发挥重要的神经保护作用，通过生产和出口还原型谷胱甘肽（GSH），我们包括，作为初步数据，证据表明，星形胶质细胞GSH稳态可能会受到干扰E在发育中的大脑。假说.我们假设，一个潜在的E-介导的氧化应激和随后的损害发育中的大脑的机制是星形胶质细胞产生和/或输出GSH的能力受损。假设：我们推测，E介导的氧化应激和随后对发育中大脑的损伤的一个机制是星形胶质细胞产生和/或输出GSH的能力受损。具体目标。以下具体的目标是我们的初步研究和其他文件的E-介导的还原型谷胱甘肽在培养的新生儿皮质星形胶质细胞的扩展。它们将决定E引起这种效应的手段。他们专注于E对GSH合成及其外排的既定控制点的影响。此外，实验将确定E对星形胶质细胞存活力的影响，这与线粒体功能有关，线粒体功能对存活和产生可能耗尽星形胶质细胞GSH的氧物种至关重要。具体目标一将确定E对星形胶质细胞摄取GSH合成所需前体的影响。我们将重点关注E对胱氨酸和半胱氨酸（GSH合成的已知决定因素）内向转运的影响，但我们也将确定E对其他相关氨基酸（如谷氨酸甘氨酸）细胞含量的影响。这些研究还将确定E对星形胶质细胞摄取和排出GSH的影响。具体目标二将确定E对星形胶质细胞GSH酶促合成的影响，重点是对GSH合成限速酶γ-谷氨酰半胱氨酸合成酶活性的影响。此外，E对星形胶质细胞结合的γ-谷氨酰转肽酶4 e作为神经元GSH合成的CysGly来源的影响。将被确定。具体目标三将确定E对星形胶质细胞线粒体变异性的影响。对Escherichia coli的强调是因为它是E毒性的靶点，是GSH合成的ATP来源，也是氧化应激的主要来源。这些实验将确定线粒体GSH的E相关消耗、消耗线粒体GSH的氧化产物的产生以及神经介导的细胞死亡的诱导，我们在附录中记录。