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

GLT1 细胞类型特异性表达在兴奋性突触中的作用

• 批准号: 8070188
• 负责人: PAUL ALLEN ROSENBERG
• 金额: $ 55.59万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-30 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8070188
• 关键词: Role; Cell; Type; Specific; Expression

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.

描述（由申请人提供）：谷氨酸转运蛋白在兴奋性突触中和周围对谷氨酸浓度的精确调节对于兴奋性突触的正常功能至关重要。在脑缺血性损伤期间，美国成年人死亡的第三大原因，细胞外谷氨酸浓度上升，导致兴奋性毒性是由于谷氨酸受体过量激活而引起的。这项研究的长期目标是了解谷氨酸转运蛋白如何正常调节突触和谷氨酸的谷氨酸浓度，以及缺血如何破坏谷氨酸稳态以产生兴奋性毒性。尽管众所周知，谷氨酸转运蛋白在星形胶质细胞中表达，但在兴奋性突触突触突触突触突触突触突触突触中表达的谷氨酸转运蛋白的身份尚不清楚，这代表了我们的知识和对兴奋性突触的理解的主要差距。我们发现，以前认为在成熟大脑中的星形胶质细胞中仅表达的GLT1在海马轴突末端表达。该提议的一个主要假设是GLT1是在整个前脑的兴奋性末端表达的主要谷氨酸转运蛋白。与星形胶质细胞中表达的GLT1相比，在轴突末端表达的GLT1的功能尚不清楚。我们假设神经元中GLT1的表达对于兴奋性突触的正常功能很重要，它通过防止溢出到周突触性谷氨酸受体以及兴奋性突触之间的串扰。我们进一步假设，从兴奋性末端释放谷氨酸对缺血性兴奋性毒性损伤的发病机理很重要。我们已经产生了一种突变小鼠，其中GLT1基因中的临界外显子已与LOXP位点两侧，从而可以使用CRE-LoxP重组技术来产生细胞类型的特异性敲除。在该项目中，我们将比较星形胶质细胞或神经元中GLT1缺失的影响，对正常条件和兴奋性损伤模型中的谷氨酸稳态和突触功能。具体目的是：1）对在星形胶质细胞或神经元中删除GLT1的小鼠系进行表型，形态和生理分析。 2）表征在不同细胞类型中表达的GLT1在缺血性损伤发病机理中的作用。 3）确定GLT1是否在海马以外地区的兴奋性末端中表达。 GLT1在兴奋性终端中的表达对我们对兴奋性突触传播，突触可塑性和缺血性损伤的生理学的理解具有重要意义。使用将为该项目产生的小鼠系，我们希望能够对GLT1神经元表达在兴奋性突触中的正常和异常调节中的神经元表达的作用进行重要见解。 公共卫生相关性：这项研究的长期目标是了解谷氨酸转运蛋白通常如何调节兴奋性突触时及其周围的谷氨酸浓度，以及缺血如何破坏该调节以产生过量释放谷氨酸和谷氨酰胺受体激活引起的神经元兴奋性神经元的兴奋性死亡。我们发现，谷氨酸转运蛋白GLT1在兴奋性轴突末端表达，但是与星形胶质细胞相反，谷氨酸转运蛋白在兴奋性末端的功能尚不清楚。在该项目中，我们制作了在神经元或星形胶质细胞中删除GLT1表达的小鼠线，以比较在正常条件下以及兴奋性损伤模型中，在这些不同位置对谷氨酸稳态和突触功能的表达影响。