EXPRESSION OF EAAT2 IN THE DEVELOPING HUMAN BRAIN

本研究的长期目标是表征谷氨酸转运在人类中枢神经系统中的发育和区域表达，从而更好地了解谷氨酸转运在发育中的大脑疾病中的作用。谷氨酸转运是负责清除兴奋性氨基酸的主要机制，并保护中枢神经元免受兴奋性毒性。矛盾的是，谷氨酸转运体似乎也是缺氧/缺血中积累的细胞外谷氨酸毒性浓度的来源。谷氨酸转运蛋白GLT1（人类中的EAAT2）在前脑中似乎是最重要的。虽然GLT1被认为主要定位于星形胶质细胞，但我们最近发现了一种剪接变体（GLT1b）在神经元中表达。此外，利用胚胎大鼠神经元培养的研究表明，GLT1转运活性和蛋白表达都受神经元活动的调节。在这个项目中，目标是表征EAAT2在人脑中的发育和区域表达，并了解这种转运体是如何被调节的。这一信息将是重要的，因为谷氨酸转运蛋白大量表达的位置很可能是正常情况下谷氨酸能机制运作的位置，并且在缺氧/缺血时，潜在的兴奋毒性机制也在运作。本项目的假设是EAAT2变异形式在人脑中表现出独立的发育和区域变异，其活性和表达受到不同机制的调控。该项目的具体目标是：1。表征人脑中eaat2mrna的发育和区域表达。2. 表征EAAT2蛋白在人脑中的发育和区域表达。3. 表征神经元活动对培养大鼠胚胎神经元GLT1摄取谷氨酸和GLT1a、GLT1b表达的影响。这些研究将对我们了解脑室周围白质软化症、胆红素脑病、缺氧/缺血、癫痫和精神分裂症的发病机制以及用于治疗这些疾病的药物对儿童的影响产生重要影响。

The long-term objective of this research is to characterize the developmental and regional expression of glutamate transport in the human central nervous system, leading to a better understanding of the role of glutamate transport in disorders of the developing brain. Glutamate transport is the major mechanism responsible for clearance of excitatory amino acids, and protects central neurons against excitotoxicity. Paradoxically, glutamate transporters also appear to be the source of toxic concentrations of extracellular glutamate that accumulate in hypoxia/ischemia. The glutamate transporter GLT1 (EAAT2 in the human) appears to be the most important in the forebrain. Although GLT1 has been thought to be primarily localized in astrocytes, we have recently found a splice variant (GLT1b) that is expressed in neurons. In addition, studies using embryonic rat neurons in culture suggest that GLT1 transport activity and protein expression are both regulated by neuronal activity. In this project, the goal is to characterize the developmental and regional expression of EAAT2 in the human brain, and to understand how this transporter is modulated. This information will be important because the locations in which glutamate transporters are heavily expressed are likely to be sites in which glutamatergic mechanisms are operating under normal circumstances, and potentially, excitotoxic mechanisms are operating during hypoxia/ischemia. The hypothesis motivating this project is that EAAT2 variant forms will show independent developmental and regional variation in the human brain and that their activity and expression will be regulated by different mechanisms. The specific aims of this project are to: 1. Characterize the developmental and regional expression of EAAT2 mRNA in the human brain. 2. Characterize the developmental and regional expression of EAAT2 protein in the human brain. 3. Characterize the effect of neuronal activity on glutamate uptake by GLT1 and expression of GLT1a and GLT1b in rat embryonic neurons in culture. These studies will have important consequences for our understanding of the pathogenesis of periventricular leukomalacia, bilirubin encephalopathy, hypoxia/ischemia, epilepsy, and schizophrenia, as well as for our understanding of the effects in children of drugs that are used to treat these conditions.