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

基本信息

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
项目类别：
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

项目摘要

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（应用程序提供）：谷氨酸（GLU）是哺乳动物中枢神经系统（CNS）中的主要兴奋性神经递质。谷氨酸接收器的过度激活导致兴奋性毒性，这反过急性神经系统感染和慢性神经退行性疾病。 Na+依赖性转运蛋白的家族控制细胞外GLU并防止GLU受体的兴奋性激活。转运蛋白的GLT1/EAAT2亚型介导了前脑中的大部分活性，并且几乎仅在星形胶质细胞中表达。几种神经系统疾病（包括肌萎缩性侧索硬化症（ALS））的GLT1水平降低。为了研究GLT1的转录调控，我们产生了一种BAC-GLT1-EGFP转基因小鼠，该小鼠利用大型细菌人造染色体（BAC）在全长GLT1启动子的控制下表达EGFP。为了更好地理解GLT1启动子的哪个区域是必需的和/或足以用于星形胶质细胞GLT1表达，我们生成了一组启动子记者的小鼠家族，该小鼠利用GLT1基因的5'非编码区域（2.5、6.7、7.9和8.3 kb）的量增加量增加。当我们将这些小鼠与BAC-GLT1-EGFP小鼠越过这些小鼠以产生双重报道小鼠时，我们进行了两种令人兴奋的意外观察结果。首先，tdtomato在星形胶质细胞中的特异性表达需要7.9至8.3 kb之间的启动子区域。该区域包含一个从啮齿动物到人类的进化构成的结构域，这表明该域对于选择性的体内星形胶质细胞表达至关重要。其次，尽管仅在表达EGFP的星形胶质细胞中发现TDTOMATO，但并非所有表达EGFP的Astroglia Express Tdtomato; TDTOMATO/EGFP（双+）星形胶质细胞在ALS中有选择下降的区域富含。这表明GLT1启动子的8.3 Kb部分仅足以在星形胶质细胞的定义子集中诱导GLT1的表达，从而提供了存在星形胶质细胞不同亚型的证据。基于这些和其他初步数据，我们提出了两个具体的目的：1）我们将测试以下假设：星形胶质细胞亚型使用不同的外部刺激（神经元和内皮）激活不同的内部信号（具有相关启动子元素的PAX6和Notch）来控制GLTT1的子型特异性表达。我们将确定GLT1的这种差异控制是否普遍为星形胶质细胞亚群中不同表达的蛋白质。我们将确认在人类中发现了星形胶质细胞的这些亚群。 2）我们将检验以下假设：在ALS的小鼠模型中，由8.3 Kb启动子记者小鼠鉴定的星形胶质细胞的亚型有选择地影响。我们将使用人体组织证实病理变化。最后，我们将检验以下假设，即8.3 kb鉴定的星形胶质体的亚型启动子记者小鼠有选择地有助于已知的非电容自动运动神经元变性。