Impairment of Glial Glutamate Transporter GLT1 in ALS

描述(申请人提供)：这项建议的目的是研究在肌萎缩侧索硬化症(ALS)中导致胶质谷氨酸转运体GLT1损伤和丢失的分子机制。肌萎缩侧索硬化症是一种年龄依赖性的脊髓、运动皮质和脑干运动神经元退行性疾病。越来越多的证据表明，谷氨酸摄取不足可能是肌萎缩侧索硬化症运动神经元丢失的一个因素。在ALS患者中，突触小体谷氨酸摄取的最大速度明显减慢。这种损害发生在受疾病影响的区域，如脊髓和运动皮质，据报道，在60%的散发性ALS患者的运动皮质中，GLT1免疫反应特异性降低(30%-90%)。在表达SOD1(G85R)突变的转基因小鼠中也检测到明显的GLT1免疫反应性丧失，这表明散发性和家族性ALS(SOD成分)具有共同的分子机制。导致肌萎缩侧索硬化症患者GLT1缺失的确切事件尚不清楚。GLT1的mRNA水平没有变化，这让研究者怀疑GLT1的降低并不是由于mRNA转录减少，而是由于翻译或翻译后水平上的其他事件。我们最近报道，在表达SOD突变的细胞中，过氧化氢的细胞内传递导致选择性的GLT1抑制。此外，我们还发现GLT1的胞质C-末端结构域参与了抑制作用。这些观察提供了SOD1突变和ALS患者GLT1损伤之间的第一个联系。为什么GLT1选择性受损，而其他谷氨酸转运体不敏感？GLT1含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突变体引起的GLT1损伤将经历持续的内化和/或选择性降解。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。我们计划通过采取以下方法来确定导致ALS中GLT1丢失的途径：i)确定由SOD1突变体介导的GLT1失活的分子事件，并确定GLT1 C-末端结构域中的失活靶点；2)确定在正常条件下和在ALS连锁的SOD1突变引发的条件下GLT1降解的途径3)利用转基因技术，我们将在体内建立GLT1失活在ALS的发生和发展中所起的作用。

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.