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Exosomes, MiRNAs and Astroglial Glutamate Transporter EAAT2/GLT1 Regulation

外泌体、miRNA 和星形胶质细胞谷氨酸转运蛋白 EAAT2/GLT1 调节

基本信息

批准号：
8821167
负责人：
Yongjie Yang
金额：
$ 24.75万
依托单位：
TUFTS UNIVERSITY BOSTON
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-09-01 至 2016-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8821167
关键词：
3&apos Untranslated Regions Adult Area Astrocytes Binding Binding Sites Biogenesis Biological Assay Brain Cells Communication Complex Development Electron Microscopy Excitatory Amino Acid Transporter 2 Fluorescence Genetic Glutamate Transporter Glutamates Immune In Vitro Injection of therapeutic agent Injury Knowledge Label Lipids Luciferases Malignant Neoplasms Mediating Membrane Messenger RNA MicroRNAs Modeling Molecular Motor Neurons Mus Neuraxis Neurons Pathway interactions Physiological Physiology Play Proteins Publishing Regulation Reporter Rodent Role Signal Pathway Signal Transduction Synapses Synaptic Transmission Therapeutic Tissues Transcript Transfection Transgenic Mice Translational Regulation Vesicle Virus Diseases analog base cell type excitotoxicity femoral nerve human DICER1 protein in vivo insight mRNA Expression nervous system disorder novel novel strategies protein expression public health relevance retrograde transport selective expression synaptic function uptake

项目摘要

DESCRIPTION (provided by applicant): The astroglial excitatory amino acid transporter 2 (EAAT2, rodent analog GLT1) is one of the most important functional synaptic proteins in astroglia and plays important physiological and pathological functions in CNS. The regulation of EAAT2/GLT1 expression has become an excellent model for understanding how neuronal signals coordinate astroglial function at synapses. Exosomes are a class of newly identified membrane vesicles (40-100nm) of endosomal origin that are secreted from cells. They contain various biomolecules, including proteins, lipids, mRNAs and microRNAs (miRNAs). Exosome-mediated communication and its physiological significance in the CNS are essentially unknown. Based on our recently published results, we hypothesize that exosome-mediated transfer of mir-124a from neurons to astroglia increases GLT1 protein expression by suppressing astroglial miRNAs that inhibit GLT1 mRNA 3' UTR function and reduce GLT1 protein expression in astroglia. In this application, we will focus on the following aims: 1) Identify astroglial miRNAs involved in exosome mir-124a-dependent GLT1 expression regulation in astroglia we will identify astroglial miRNAs that are significantly down-regulated by neuronal exosome treatment and miR-124a transfection using miRNA microarrays. We will then validate expression changes of identified astroglial miRNAs using QRT-PCR and determine whether these astroglial miRNAs directly inhibit GLT1 3' UTR function using a luciferase based assay. Finally, we will determine whether these identified astroglial miRNAs reduce GLT1 protein expression levels and GLT1-dependent glutamate uptake in astroglia. 2) Investigate the exosome and mir-124a transfer from neurons to astroglia in vivo we will investigate the in vivo exosome and mir-124 transfer from neurons to astroglia using genetic and labeling approaches. We will generate transgenic mice that selectively express eGFP-tagged CD63 (green fluorescence) in neurons to characterize the temporal and spatial profile of in vivo transfer of neuronal exosomes (indicated by the eGFP-labeled CD63) to astroglia in intact CNS. We will also selectively deliver Alexa 750 (far red fluorescence) labeled mir-124a to motor neurons by femoral nerve injection and retrograde transport, and then examine its transfer from motor neurons to astroglia in vivo. In summary, this project will identify specific miRNAs that modulate GLT1 protein expression. These newly identified miRNAs will provide novel approaches for modulating synaptic function and are promising targets for developing GLT1-based therapeutics for the treatment of neurological diseases/injuries. In addition, this study will characterize a novel exosome-mediated signaling pathway from neurons to astroglia that regulates astroglial GLT1 expression. This intercellular pathway provides new knowledge about how neuronal signals coordinate astroglial functions at synapses.

描述（申请人提供）：星形胶质细胞兴奋性氨基酸转运蛋白2（EAAT 2，啮齿动物类似物GLT 1）是星形胶质细胞中最重要的功能性突触蛋白之一，在CNS中发挥重要的生理和病理功能。EAAT 2/GLT 1表达的调节已经成为理解神经元信号如何在突触处协调星形胶质细胞功能的极好模型。外泌体是一类新发现的内体来源的膜囊泡（40- 100 nm），由细胞分泌。它们含有各种生物分子，包括蛋白质、脂质、mRNA和microRNA（miRNA）。外泌体介导的通讯及其在CNS中的生理意义基本上是未知的。基于我们最近发表的结果，我们假设外泌体介导的mir-124 a从神经元转移到星形胶质细胞通过抑制星形胶质细胞miRNA来增加GLT 1蛋白表达，所述星形胶质细胞miRNA抑制GLT 1 mRNA 3' UTR功能并减少星形胶质细胞中GLT 1蛋白表达。1）鉴定参与外泌体mir-124 a依赖性GLT 1表达调控的星形胶质细胞miRNAs我们将使用miRNA微阵列鉴定通过神经元外泌体处理和miR-124 a转染显著下调的星形胶质细胞miRNAs。然后，我们将使用QRT-PCR验证鉴定的星形胶质细胞miRNA的表达变化，并使用基于荧光素酶的测定来确定这些星形胶质细胞miRNA是否直接抑制GLT 1 3' UTR功能。最后，我们将确定这些鉴定的星形胶质细胞miRNAs是否降低星形胶质细胞中GLT 1蛋白表达水平和GLT 1依赖的谷氨酸摄取。2)研究神经元到星形胶质细胞的外泌体和mir-124 a的体内转移我们将使用遗传和标记方法研究神经元到星形胶质细胞的外泌体和mir-124的体内转移。我们将产生在神经元中选择性表达eGFP标记的CD 63（绿色荧光）的转基因小鼠，以表征神经元外泌体（由eGFP标记的CD 63指示）向完整CNS中的星形胶质细胞的体内转移的时间和空间分布。我们还将通过股神经注射和逆行运输选择性地将Alexa 750（远红荧光）标记的mir-124 a递送到运动神经元，然后在体内检测其从运动神经元到星形胶质细胞的转移。总之，本项目将鉴定调节GLT 1蛋白表达的特定miRNA。这些新鉴定的miRNAs将提供调节突触功能的新方法，并且是开发用于治疗神经疾病/损伤的基于GLT 1的疗法的有希望的靶点。此外，本研究将表征一种新的外泌体介导的信号通路，从神经元到星形胶质细胞，调节星形胶质细胞GLT 1的表达。这种细胞间通路为神经元信号如何在突触处协调星形胶质细胞功能提供了新的知识。