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: 8151071
• 负责人: PAUL ALLEN ROSENBERG
• 金额: $ 56.06万
• 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/8151071
• 关键词: Accounting; Adult; Astrocytes; Brain; Cause of Death; Cell membrane; Cerebrum; Cessation of life; Corpus striatum structure; Defect; Detection; Disease; Electrons; Excitatory Synapse; Exons; Genes; Genetic Recombination; Genotype; Glutamate Receptor; Glutamate Transporter; Glutamates; Goals; Health; Hippocampus (Brain); Homeostasis; Injury; Ion Transport; Ischemia; Knock-out; Knockout Mice; Knowledge; Learning; Location; Long-Term Potentiation; Memory; Messenger RNA; Microscopic; Modeling; Mus; Mutant Strains Mice; Neurons; Pathogenesis; Performance; Phenotype; Physiological; Physiology; Play; Presynaptic Terminals; Prosencephalon; Proteins; Publishing; Regulation; Research; Role; Seizures; Site; Synapses; Synaptic Cleft; Synaptic Transmission; Synaptic plasticity; Technology; Testing; United States; Work; behavior test; cell type; diaminobenzidine peroxidase; excitotoxicity; extracellular; falls; immunocytochemistry; insight; operation; prevent; public health relevance; receptor; research study; synaptic function; tool

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）：谷氨酸转运蛋白对兴奋性突触内部和周围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。脑缺血损伤是美国成年人死亡的第三大原因，在脑缺血损伤期间，细胞外谷氨酸浓度升高，导致谷氨酸受体过度激活而引起兴奋性毒性。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突触周围谷氨酸浓度，以及缺血如何破坏谷氨酸稳态以产生兴奋毒性。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中的重大差距。我们发现，以前认为 GLT1 只在成熟大脑的星形胶质细胞中表达，但现在却在海马体的轴突末端表达。该提议的一个主要假设是 GLT1 是在整个前脑兴奋性末梢表达的主要谷氨酸转运蛋白。与星形胶质细胞中表达的 GLT1 不同，轴突末梢中表达的 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，可以防止溢出到突触周围谷氨酸受体以及兴奋性突触之间的串扰。我们进一步假设兴奋性末梢释放谷氨酸对于缺血中兴奋性毒性损伤的发病机制很重要。我们培育了一种突变小鼠，其中 GLT1 基因中的关键外显子两侧带有 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。具体目标是： 1) 对星形胶质细胞或神经元中 GLT1 缺失的小鼠品系进行表型、形态学和生理学分析。 2)表征不同细胞类型中表达的GLT1在缺血性损伤发病机制中的作用。 3)确定GLT1是否在海马以外区域的兴奋性末梢表达。 GLT1 在兴奋性末梢的表达对于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。使用为本项目生产的小鼠品系，我们希望获得关于 GLT1 神经元表达在兴奋性突触谷氨酸正常和异常调节中的作用的重要见解。 公共健康相关性：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及其周围的谷氨酸浓度，以及缺血如何破坏这种调节，从而产生兴奋性毒性，即谷氨酸过量释放和谷氨酸受体激活引起的神经元死亡。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。在这个项目中，我们制作了在神经元或星形胶质细胞中删除 GLT1 表达的小鼠品系，以比较正常条件下和兴奋性毒性损伤模型中这些不同位置的 GLT1 表达对谷氨酸稳态和突触功能的影响。