Alcohol Impairs Neonatal Astrocyte GSH Homeostasis

我们实验室和其他实验室之前的研究已经证明，与酒精相关的氧化应激在发育中的大脑中存在，有证据表明，它可能至少引起乙醇(E)的部分神经毒性作用。E引起这种氧化应激的机制(S)尚不清楚，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，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害的一个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害背后的一个机制是星形胶质细胞产生和/或输出GSH的能力受损。明确的目标。以下具体目标是我们和其他人的初步研究的延伸，这些研究记录了E介导的培养的新生儿皮质星形胶质细胞中GSH的减少。他们将确定E导致这种效果的方法。他们关注的是E对谷胱甘肽合成控制点及其外流的影响。此外，实验将确定E对星形胶质细胞活力的影响，涉及线粒体功能，线粒体功能是生存和产生可能耗尽星形胶质细胞GSH的氧物种的关键。具体目的一是确定E对星形胶质细胞摄取合成GSH所需前体的影响(S)。我们将集中于E对半胱氨酸和半胱氨酸内向转运的影响，这是GSH合成的已知决定因素，但我们也将确定E对细胞中其他相关氨基酸含量的影响，如谷氨酸和甘氨酸。这些研究还将确定E对星形胶质细胞摄取和外排GSH的影响。具体目标二将确定E对星形胶质细胞酶促合成GSH的影响，重点是对GSH合成的限速酶--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4e的影响作为CysGly的来源，用于神经元GSH的合成。将会被确定。具体目标三将确定E在线粒体对星形胶质细胞变异性的影响。之所以强调线粒体，是因为它是E毒性的目标，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E相关的线粒体GSH耗竭，氧化产物的产生耗尽线粒体的GSH，以及我们在附录中记录的线粒体介导的细胞死亡的诱导。

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.