Alcohol Impairs Neonatal Astrocyte GSH Homeostasis

酒精损害新生儿星形胶质细胞 GSH 稳态

基本信息

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
项目类别：
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

项目摘要

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记录了发育中的大脑中与乙醇相关的氧化应激，并且有证据表明它可能至少会引起乙醇（E）的至少某些神经毒性作用。 E导致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剂状态有关。有令人信服的文献表明，星形胶质细胞通过产生和出口减少的谷胱甘肽（GSH）发挥了至关重要的神经保护作用，我们包括作为初步数据，证据表明，在发育中E中E可能会受到星形胶质细胞GSH的稳态。假设。我们假设，介导的氧化应激和随后对发育中大脑的损害的一种机制是对星形胶质细胞产生和/或导出GSH的能力的损害。假设：我们假设，介导的氧化应激和随后对发育中大脑的损害的一种机制是星形胶质细胞产生和/或导出GSH的能力的损害。具体目标。以下具体目的是我们的初步研究的扩展，以及其他研究，这些研究记录了培养的新生儿皮质星形胶质细胞中GSH的降低。他们将确定e引起这种效果的手段。他们着重于对GSH合成中良好的控制点及其外排的E效应。此外，实验将确定E相对于线粒体功能的星形胶质细胞生存能力的影响，这是可能耗尽星形胶质细胞GSH的氧气生存和产生的关键。特定的目标将确定E对GSH合成所需的前体的星形胶质细胞吸收的影响。我们将重点关注E对Cystine和半胱氨酸的向内定向转运的影响，这是GSH合成的决定因素，但我们还将确定E对E对其他相关氨基酸的细胞含量的影响，例如谷氨酸甘氨酸。这些研究还将确定E对星形胶质细胞的GSH吸收和外排的影响。具体目标两个将确定E对星形胶质细胞对GSH的酶促合成的影响，重点是对GMH合成中γ- - 谷氨酰胺合成酶的影响，GSH合成中的速率限制酶。另外，E对星形胶质细胞结合的γ-谷氨酰基转肽AS4E的影响是神经元GSH合成的cysgly的来源。将确定。具体目标三将确定E对线粒体上星形胶质细胞变异性的影响。对线粒体的强调是由于它是E毒性的靶标，GSH合成的ATP来源以及氧化应激的主要来源。这些实验将确定线粒体GSH的电子相关耗竭，耗尽GSH的氧化产物的产生以及我们在附录中记录的线粒体介导的细胞死亡的诱导。