Regulation of glutamate transport in astrocyte subtypes and in ALS

星形胶质细胞亚型和 ALS 中谷氨酸转运的调节

• 批准号: 9027947
• 负责人: Michael Byrne Robinson
• 金额: $ 48.56万
• 依托单位: CHILDREN'S HOSP OF PHILADELPHIA
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-15 至 2020-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9027947
• 关键词: Acute; Adult; Affect; Amyotrophic Lateral Sclerosis; Area; Astrocytes; Autopsy; Bacterial Artificial Chromosomes; Brain; Cell Death; Cells; Chronic; Data; Disease; Disease Progression; Elements; Endothelium; Family; Fluorescence-Activated Cell Sorting; Genes; Genetic; Genetic Transcription; Glutamate Receptor; Glutamates; Horns; Human; In Vitro; Length; Link; Mediating; Monitor; Motor Neurons; Mus; Neuraxis; Neurodegenerative Disorders; Neurologic; Neurons; Neurotransmitters; Pathogenesis; Pathologic; Pathology; Patients; Pattern; Population; Predisposition; Process; Promoter Regions; Property; Prosencephalon; Proteins; Regulation; Reporter; Rodent; Rodent Model; Shapes; Signal Transduction; Staging; Stimulus; Testing; Tissue Sample; Tissues; Toxic effect; Transcriptional Regulation; Transgenic Mice; Transgenic Organisms; Untranslated RNA; Ventral Horn of the Spinal Cord; base; chromatin immunoprecipitation; differential expression; excitotoxicity; extracellular; glutamatergic signaling; human tissue; in vivo; mRNA Expression; motor neuron degeneration; mouse model; mutant; neuron loss; notch protein; prevent; promoter; protein expression; public health relevance; synaptogenesis; transcription factor

 DESCRIPTION (provided by applicant): Glutamate (Glu) is the predominant excitatory neurotransmitter in the mammalian central nervous system (CNS). Excessive activation of glutamate receptors leads to excitotoxicity, which in turn contributes to cell death observed after acute neurologic insults and in chronic neurodegenerative diseases. A family of Na+-dependent transporters controls extracellular Glu and prevents excitotoxic activation of Glu receptors. The GLT1/EAAT2 subtype of transporter mediates the bulk of this activity in the forebrain, and it is almost exclusively expressed in astrocytes. The levels of GLT1 are decreased in several neurologic diseases, including amyotrophic lateral sclerosis (ALS). To study transcriptional regulation of GLT1 we generated a BAC-GLT1-eGFP transgenic mouse that utilizes a large bacterial artificial chromosome (BAC) to express eGFP under the control of the full length GLT1 promoter. To better understand which region of the GLT1 promoter is necessary and/or sufficient for astroglial GLT1 expression, we generated a family of promoter reporter mice that utilize increasing amounts of the 5' non-coding region of the GLT1 gene (2.5, 6.7, 7.9, and 8.3 kilobases) to express tdTomato. When we crossed these mice with the BAC-GLT1-eGFP mice to produce dual reporter mice we made two exciting and unexpected observations. First, the promoter region between 7.9 and 8.3 kb is required for specific expression of tdTomato in astroglia. This region contains a domain that is evolutionarily conserved from rodents to humans, suggesting that this domain is critical for selective in vivo astroglia expression of GLT1. Second, although tdTomato is only found in eGFP-expressing astroglia, not all eGFP-expressing astroglia express tdTomato; the tdTomato/eGFP (double+) astrocytes are enriched in regions where GLT1 selectively decreases in ALS. This suggests that the 8.3 kb portion of the GLT1 promoter is only sufficient to induce expression of GLT1 in a defined subset of astrocytes, providing evidence for the existence of distinct subtypes of astrocytes. Based on these and other preliminary data, we propose two specific aims: 1) We will test the hypothesis that subtypes of astrocytes use different extrinsic stimuli (neurons and endothelia) to activate different intrinsic signals (Pax6 and Notch with associated promoter elements) to control subtype-specific expression of GLT1. We will determine if this differential control of GLT1 generalizes to proteins that are differentially expressed in these subpopulations of astrocytes. We will confirm that these subpopulations of astrocytes are found in humans. 2) We will test the hypothesis that the subtypes of astroglia identified by the 8.3 kb promoter reporter mice are selectively affected in mouse models of ALS. We will confirm pathologic changes using human tissue. Finally, we will test the hypothesis that the subtype of astroglia identified by the 8.3 kb promoter reporter mice selectively contributes to the known non-cell autonomous motor neuron degeneration.

 描述（由申请人提供）：谷氨酸（Glu）是哺乳动物中枢神经系统（CNS）中主要的兴奋性神经递质。谷氨酸受体的过度激活导致兴奋性毒性，这反过来又有助于细胞死亡后观察到的 急性神经损伤和慢性神经变性疾病。Na+依赖性转运蛋白家族控制细胞外Glu并防止Glu受体的兴奋毒性激活。转运蛋白的GLT 1/EAAT 2亚型介导前脑中的大部分活性，并且它几乎仅在星形胶质细胞中表达。GLT 1的水平在几种神经系统疾病中降低，包括肌萎缩侧索硬化症（ALS）。为了研究GLT 1的转录调控，我们产生了BAC-GLT 1-eGFP转基因小鼠，其利用大的细菌人工染色体（BAC）在全长GLT 1启动子的控制下表达eGFP。为了更好地理解GLT 1启动子的哪个区域对于星形胶质细胞GLT 1表达是必需的和/或足够的，我们产生了一个启动子报告小鼠家族，其利用增加量的GLT 1基因的5'非编码区（2.5、6.7、7.9和8.3个酶）来表达tdTomato。当我们将这些小鼠与BAC-GLT 1-eGFP小鼠杂交以产生双报告小鼠时，我们得到了两个令人兴奋和意想不到的观察结果。首先，7.9和8.3 kb之间的启动子区域是tdTomato在星形胶质细胞中特异性表达所必需的。该区域包含从啮齿动物到人类进化上保守的结构域，表明该结构域对于GLT 1的选择性体内星形胶质细胞表达至关重要。 第二，虽然tdTomato仅在表达eGFP的星形胶质细胞中发现，但并非所有表达eGFP的星形胶质细胞都表达tdTomato; tdTomato/eGFP（双+）星形胶质细胞富集在ALS中GLT 1选择性减少的区域。这表明GLT 1启动子的8.3 kb部分仅足以诱导GLT 1在定义的星形胶质细胞亚群中的表达，为星形胶质细胞的不同亚型的存在提供了证据。基于这些和其他初步数据，我们提出了两个具体的目标：1）我们将测试星形胶质细胞亚型使用不同的外部刺激（神经元和内皮细胞）激活不同的内在信号（Pax 6和Notch与相关的启动子元件）来控制GLT 1亚型特异性表达的假设。我们将确定GLT 1的这种差异控制是否适用于在这些星形胶质细胞亚群中差异表达的蛋白质。我们将证实这些星形胶质细胞亚群存在于人类中。2)我们将测试的假设，即由8.3 kb启动子报告小鼠识别的星形胶质细胞的亚型选择性地影响ALS的小鼠模型。我们将使用人体组织来确认病理变化。最后，我们将检验由8.3kb的DNA鉴定的星形胶质细胞亚型 启动子报告小鼠选择性地促成已知的非细胞自主运动神经元变性。