Impairment of Glial Glutamate Transporter GLT1 in ALS

描述（由申请人提供）：本提案的目的是研究肌萎缩性侧索硬化症（ALS）中导致胶质谷氨酸转运体GLT1损伤和丢失的分子机制。ALS是一种年龄依赖性的神经退行性疾病，发生在脊髓、运动皮层和脑干的运动神经元。越来越多的证据表明，缺乏谷氨酸摄取可能是肌萎缩侧索硬化症运动神经元丧失的一个因素。据报道，在ALS患者中，突触体谷氨酸摄取的最大速度显著降低。在受疾病影响的区域发现了损伤，例如脊髓和运动皮层，并且据报道，60%的散发性ALS患者的运动皮层GLT1免疫反应性特异性降低（30- 90%）。在表达SOD1（G85R）突变的转基因小鼠中也检测到GLT1免疫反应性的显著丧失，这表明散发性和家族性ALS （SOD成分）具有共同的分子机制。导致ALS患者GLT1丧失的确切事件尚不清楚。GLT1 mRNA的水平没有变化，这让研究者怀疑GLT1的减少不是由于mRNA转录的减少，而是由于翻译或翻译后水平的一些其他事件。我们最近报道了表达SOD突变的细胞内H2O2的细胞内传递导致选择性GLT1抑制。此外，我们发现GLT1的胞质c端结构域参与了抑制作用。这些观察结果提供了SOD1突变与ALS患者GLT1损伤之间的第一个联系。为什么GLT1选择性受损而其他谷氨酸转运蛋白不敏感？GLT1具有最多的易氧化氨基酸残基，如半胱氨酸、组氨酸和酪氨酸，因此最容易发生氧化修饰和损伤。我们预计由SOD1突变引起的GLT1损伤将经历持续的内化和/或选择性降解。关于谷氨酸转运体的调节和降解，以及它们的功能抑制或刺激如何影响神经退行性疾病（如ALS）的病理生理事件，人们知之甚少。我们计划通过以下方法来确定导致ALS中GLT1丢失的途径：1)定义由SOD1突变体介导的GLT1失活的分子事件，并确定GLT1 c端区域中失活的目标位点；2)定义正常条件下和由ALS相关SOD1突变引发的条件下GLT1降解的途径3)使用转基因技术，我们将在体内确定GLT1失活在ALS的发生和进展中所起的作用。

DESCRIPTION (provided by applicant): The objective of this proposal is to study the molecular mechanisms leading to impairment and loss of the glial glutamate transporter GLT1 in amyotrophic lateral sclerosis (ALS). ALS is an age-dependent neurodegenerative disorder of motor neurons in the spinal cord, motor cortex and brain stem. There is a growing body of evidence indicating that deficient glutamate uptake may be a contributory factor to motor neuron loss in ALS. In ALS patients, a marked decrease in the maximal velocity of synaptosomal glutamate uptake was reported. The impairment was found in regions affected by the disease, such as the spinal cord and motor cortex and a specific reduction of GLT1 immunoreactivity (30-90 percent) in the motor cortex of 60 percent of sporadic ALS patients was reported. A marked loss of GLT1 immunoreactivity was also detected in transgenic mice expressing the SOD1(G85R) mutation, suggesting that the sporadic and the familial form of ALS (SOD component) share common molecular mechanisms. The precise events leading to GLT1 loss in ALS are not yet understood. The levels of GLT1 mRNA are unchanged, letting investigator suspect that the reduction of GLT1 is not due to decreased transcription of mRNA, but rather to some other events at the translation or post translational level. We have recently reported that intracellular delivery of H2O2 in cells expressing SOD mutations led to selective GLT1 inhibition. Moreover, we showed that the cytoplasmic C-terminal domain of GLT1 is involved in the inhibition. These observations provided the first link between the SOD1 mutations and GLT1 impairment in ALS. Why is GLT1 selectively damaged while other glutamate transporters are insensitive? GLT1 has the highest number of oxidant vulnerable amino acid residues, such as cysteines histidines and tyrosines and therefore is the most prone to oxidative modifications and damage. We expect that a damaged GLT1 caused by the SOD1 mutants would undergo to a sustained internalization and/or selective degradation. Little is known about glutamate transporters regulation and degradation and how their functional inhibition or stimulation affects the pathophysiological events of neurodegenerative diseases such as ALS. We plan to determine the pathways that lead to GLT1 loss in ALS by taking the following approaches: I) defining the molecular events responsible for the inactivation of GLT1 mediated by the SOD1 mutants and identifying the sites in the C-terminal domain of GLT1 that are targets for the inactivation; 2) defining the pathways of GLT1 degradation under normal conditions and under conditions initiated by the ALS-linked SOD1 mutations 3) using transgenic technology we will establish in vivo what role the inactivation of GLT1 is playing in the onset and progression of ALS.