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: 6422536
• 负责人: GEORGE I HENDERSON
• 金额: $ 14.58万
• 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/6422536
• 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.

在我们的实验室和其他实验室之前的研究已经证明了乙醇相关的氧化应激在发育中的大脑中，有证据表明它可能至少导致乙醇的一些神经毒性作用(E)。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。有令人信服的文献表明，星形胶质细胞通过产生和输出还原型谷胱甘肽（GSH）发挥重要的神经保护作用，我们包括，作为初步数据的证据，星形胶质细胞GSH稳态可能被发育中的大脑中的E扰乱。假设。我们假设，E介导的氧化应激和随后对发育中的大脑的损伤的一种机制是星形胶质细胞产生和/或输出GSH的能力受损。假设：我们假设，E介导的氧化应激和随后对发育中的大脑的损伤的一种机制是星形胶质细胞产生和/或输出GSH的能力受损。具体的目标。以下具体目标是我们的初步研究的延伸，以及其他记录了e介导的新生皮层星形胶质细胞中谷胱甘肽减少的研究。他们将确定E产生这种效果的方法。他们关注的是E对谷胱甘肽合成和外排的控制点的影响。此外，实验将确定E对星形胶质细胞活力的影响，这与线粒体功能有关，线粒体功能对星形胶质细胞存活和产生可能耗尽星形胶质细胞GSH的氧气至关重要。具体目的一将确定E对星形胶质细胞摄取合成谷胱甘肽所需的前体的影响。我们将重点关注E对胱氨酸和半胱氨酸向内转运的影响，这是已知的GSH合成的决定因素，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谷胱甘肽的影响。特异性目的二将确定E对星形胶质细胞酶促合成谷胱甘肽的影响，重点关注对γ -谷氨酰半胱氨酸合成酶活性的影响，这种合成酶是谷胱甘肽合成的限速酶。此外，E对星形胶质细胞结合的γ -谷氨酰转肽酶4e的影响，作为神经元GSH合成的CysGly来源。将被决定。特异性目的三将确定E对线粒体星形胶质细胞变异性的影响。强调线粒体是由于它是E毒性的靶点，是GSH合成ATP的来源，也是氧化应激的主要来源。这些实验将确定e相关的线粒体GSH消耗，氧化产物的产生，消耗GSH的线粒体，以及线粒体介导的细胞死亡的诱导，我们在附录中记录。