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。 巧合的是，谷氨酸转运蛋白似乎也是缺氧/缺血时积累的细胞外谷氨酸毒性浓度的来源。谷氨酸转运蛋白GLT 1（人类中的EAAT 2）似乎是前脑中最重要的。 虽然GLT 1被认为主要定位于星形胶质细胞，但我们最近发现了一种在神经元中表达的剪接变体（GLT 1b）。 此外，使用培养的胚胎大鼠神经元的研究表明，GLT 1转运活性和蛋白表达都受到神经元活性的调节。 在这个项目中，目标是表征EAAT 2在人脑中的发育和区域表达，并了解这种转运蛋白是如何调节的。 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可能是在正常情况下谷氨酸能机制起作用的位点，并且可能是在缺氧/缺血期间兴奋性毒性机制起作用的位点。激发该项目的假设是EAAT 2变体形式将在人脑中显示独立的发育和区域变化，并且它们的活性和表达将受到不同机制的调节。 该项目的具体目标是：1.表征EAAT 2 mRNA在人脑中的发育和区域表达。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的大鼠胚胎神经元中GLT 1摄取谷氨酸以及GLT 1a和GLT 1b表达的影响。这些研究将对我们理解脑室周围白质软化、胆红素脑病、缺氧/缺血、癫痫和精神分裂症的发病机制，以及我们理解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.