Astrocyte Control of Toxin-Mediated Neuron Death: Role of the Gamma-Glutamyl Path

描述(由首席调查员提供)： 背景毒素介导的氧化应激(OS)是最终导致神经元凋亡的触发因素。这一事件可能与细胞谷胱甘肽(GSH)的减少有关，细胞死亡可以通过增加神经元GSH来缓解。星形胶质细胞通过β-谷氨酰循环保护神经元免受毒素相关OS和随后的细胞凋亡的影响，从而维持神经元GSH的动态平衡。了解这一神经保护途径，特别是其调节成分和物种比较，最终可能导致针对破坏性神经退行性疾病的新的临床干预措施，以及建立准确的模型系统。假设：我们假设，在星形胶质细胞-神经元轴中，存在一条由多种成分组成的高效、受调控的途径，该途径可以增强神经元GSH的稳态，以响应乙醇(E)和帕金森病产生的环境毒素，从而将神经元的死亡降至最低。然而，存在暴露于这些环境交叉应激源的模式，这些模式破坏了通路的组成部分，从而削弱了其神经保护能力。具体目标1：目的利用神经元和星形胶质细胞的单独和联合培养，研究鱼藤酮、百草枯和E对星形胶质细胞和星形胶质细胞-谷氨酰循环中神经元成分的直接影响。实验参数将是环境毒素单独或组合对GSH、GSH酶合成的关键前体的向内运输的影响，以及发生神经保护的这种机制的组件。具体目标2：目标2将涉及神经保护途径的三个基本组成部分的调节。有待确定的是对毒素的调节和破坏性反应的基本机制。这将是通过多药耐药蛋白(MRP)和两种质膜外肽酶--谷氨酰转肽酶(GGT)和氨基肽酶N(APN)来调节GSH的外流。具体目标3：AIM 3将使用活体模型将体外研究结果扩展到完整的动物。实验将利用双光子激发显微镜来确定毒素效应，特别是在活的大脑中的皮质星形胶质细胞和神经元。这将是对毒素暴露过程中GGT和APN表达、GSH含量和细胞凋亡相关事件的时间推移测定。他们将利用缺乏MRPL、MRP4或GGT的转基因小鼠。概述：该提案提出了一种新的系统，通过该系统，神经胶质细胞可以保护神经元免受三种环境毒素的毒性影响。这些化合物可以导致神经元因氧化损伤而死亡，而被称为星形胶质细胞的细胞通过维持神经元抗氧化剂来防止这种情况发生。我们将确定毒素是如何破坏它的(以防止这种情况发生)，以及这个系统是如何正常控制的。

DESCRIPTION (provided by principal investigator): Background Toxin-mediated oxidative stress (OS) is a trigger that ultimately commits neurons to apoptotic death. The event can be associated with decreased cell glutathione (GSH) and cell death is mitigated by augmenting neuron GSH. Astrocytes protect neurons from toxin-related OS and subsequent apoptosis via the ?-glutamyl cycle, which maintains neuron GSH homeostasis. An understanding of this neuroprotective pathway, especially its regulatory components and species comparisons, could ultimately lead to novel clinical interventions for devastating neurodegenerative disorders as well as establish accurate model systems. Hypothesis: We hypothesize that within the astrocyte-neuron axis, there is a highly effective, regulated pathway consisting of components which can enhance neuron GSH homeostasis in response to EtOH (E) and Parkinson's Disease-producing environmental toxins, thereby minimizing death of neurons. However, there are exposure patterns to these environmental cross-stressors which damage components of the pathways, thereby impairing its neuroprotective capacity. Specific Aim 1: Aim one will utilize cultures of neurons and astrocytes, alone and in co-culture, to address the direct impact of rotenone, paraquat, and E on astrocyte and neuron components of the ?-glutamyl cycle. Experimental parameters will be effects of the environmental toxins, alone or in combination, on inward transport of key precursors of GSH, GSH enzymatic synthesis, and components of this machinery at which neuroprotection occurs. Specific Aim 2: Aim two will address regulation of three essential components of the neuroprotective pathway. To be determined will be basic mechanisms underlying regulatory and damaging responses to the toxins. These will be regulation of GSH efflux via multidrug resistance protein(s) (Mrp), and two plasma membrane ectopeptidases, ?-glutamyl transpeptidase (GGT) and aminopeptidase N (ApN). Specific Aim 3: Aim 3 will use an in vivo model to extend the in vitro findings to the intact animal. Experiments will utilize two-photon excitation microscopy to determine toxin effects specifically in cortical astrocytes and neurons in the living brain. These will be time-lapse determinations of expressions of GGT and ApN, GSH content and apoptosis-related events during toxin exposure. They will utilize transgenic mice deficient in Mrpl, Mrp4, or GGT. General Description: The proposal addresses a new system by which glial cells protect neurons from the toxic effects of three environmental toxins. These compounds can cause neurons to die as a result of oxidative damage and cells called astrocytes prevent this by maintaining neuron antioxidants. We will determine how the toxins damage it (to prevent this from happening), and how this system is normally controlled.