The Role of Cell-Type Specific Expression of GLT1 at Excitatory Synapses

描述（申请人提供）：谷氨酸转运体对兴奋性突触内和周围谷氨酸浓度的精确调节对兴奋性突触的正常功能至关重要。缺血性脑损伤是美国成年人死亡的第三大原因，在缺血性脑损伤期间，细胞外谷氨酸浓度升高，导致谷氨酸受体过度激活引起兴奋性毒性。本研究的长期目标是了解谷氨酸转运体如何正常调节突触和突触周围的谷氨酸浓度，以及缺血如何破坏谷氨酸稳态从而产生兴奋毒性。虽然已知谷氨酸转运蛋白在星形胶质细胞中表达，但兴奋性突触突触前末端表达的谷氨酸转运蛋白的身份尚不清楚，这代表了我们对兴奋性突触的认识和理解的主要空白。我们发现GLT1，以前被认为只在成熟大脑的星形胶质细胞中表达，在海马的轴突终末表达。该提议的一个主要假设是GLT1是主要的谷氨酸转运蛋白，在整个前脑的兴奋末梢表达。与在星形胶质细胞中表达的GLT1相比，轴突末端表达的GLT1的功能尚不清楚。我们假设神经元中GLT1的表达对兴奋性突触的正常功能很重要，通过防止溢出到突触周围的谷氨酸受体以及兴奋性突触之间的串扰。我们进一步假设，从兴奋性终末释放谷氨酸在缺血兴奋性毒性损伤的发病机制中起重要作用。我们已经产生了一个突变小鼠，其中GLT1基因的一个关键外显子两侧有loxP位点，允许使用Cre-loxP重组技术产生细胞类型特异性敲除。在这个项目中，我们将比较星形胶质细胞或神经元中GLT1缺失对正常条件下和兴奋性毒性损伤模型中谷氨酸稳态和突触功能的影响。具体目的是：1)对星形胶质细胞或神经元中GLT1缺失的小鼠系进行表型、形态学和生理学分析。2)表征不同细胞类型表达的GLT1在缺血性损伤发病机制中的作用。3)确定GLT1是否在海马外区域的兴奋性终末表达。GLT1在兴奋性末梢的表达对我们理解兴奋性突触传递、突触可塑性和缺血性损伤的生理学具有重要意义。利用将为该项目生产的小鼠系，我们希望对GLT1的神经元表达在兴奋性突触谷氨酸的正常和异常调节中的作用获得重要的见解。

DESCRIPTION (provided by applicant): The precise regulation by glutamate transporters of glutamate concentrations in and around excitatory synapses is critical for the normal function of excitatory synapses. During cerebral ischemic injury, the third leading cause of death of adults in the United States, extracellular glutamate concentration rises, leading to excitotoxicity caused by excess activation of glutamate receptors. The long-term goal of this research is to understand how glutamate transporters regulate synaptic and perisynaptic glutamate concentrations normally, and how ischemia disrupts glutamate homeostasis to produce excitotoxicity. Although glutamate transporters are well known to be expressed in astrocytes, the identity of the glutamate transporter expressed in the presynaptic terminal of excitatory synapses was unknown, representing a major gap in our knowledge and understanding of excitatory synapses. We discovered that GLT1, previously thought to be exclusively expressed in astrocytes in the mature brain, is expressed in axon terminals in the hippocampus. A major hypothesis of this proposal is that GLT1 is the major glutamate transporter expressed in excitatory terminals throughout the forebrain. The function of GLT1 expressed in axon terminals as opposed to GLT1 expressed in astrocytes is unclear. We hypothesize that expression of GLT1 in neurons is important for the normal function of excitatory synapses, by preventing spillover onto perisynaptic glutamate receptors as well as cross-talk between excitatory synapses. We further hypothesize that the release of glutamate from excitatory terminals is important in the pathogenesis of excitotoxic injury in ischemia. We have generated a mutant mouse in which a critical exon in the GLT1 gene has been flanked with loxP sites, allowing the use of Cre-loxP recombination technology to produce cell-type specific knockouts. In this project we will compare the effects of deletion of GLT1 in astrocytes or in neurons, on glutamate homeostasis and synaptic function under normal conditions and in models of excitotoxic injury. The specific aims are to: 1) Perform phenotypical, morphological, and physiological analysis of mouse lines in which GLT1 is deleted in astrocytes or neurons. 2) Characterize the role of GLT1 expressed in different cell types in the pathogenesis of ischemic injury. 3) Determine whether GLT1 is expressed in excitatory terminals in regions outside the hippocampus. The expression of GLT1 in excitatory terminals has important implications for our understanding of the physiology of excitatory synaptic transmission, synaptic plasticity, and ischemic injury. Using mouse lines that will be produced for this project, we hope to gain important insights into the role of neuronal expression of GLT1 into the normal and abnormal regulation of glutamate at the excitatory synapse. PUBLIC HEALTH RELEVANCE: The long-term goal of this research is to understand how glutamate transporters normally regulate glutamate concentrations at and around the excitatory synapse, and how ischemia disrupts this regulation to produce excitotoxicity-death of neurons caused by excess release of glutamate and activation of glutamate receptors. We discovered that the glutamate transporter GLT1 is expressed in excitatory axon terminals, but the function of glutamate transporters in excitatory terminals, as opposed to astrocytes, is unclear. In this project we make mouse lines in which GLT1 expression is deleted in neurons or in astrocytes to compare the effects of expression of GLT1 in these different locations on glutamate homeostasis and synaptic function under normal conditions and in models of excitotoxic injury.