Exosomes, MiRNAs and Astroglial Glutamate Transporter EAAT2/GLT1 Regulation

描述(申请人提供)：星形胶质兴奋性氨基酸转运体2(EAAT2，啮齿动物类似物GLT1)是星形胶质细胞中最重要的功能突触蛋白之一，在中枢神经系统中发挥着重要的生理和病理功能。EAAT2/GLT1表达的调控已成为了解神经元信号如何协调突触星形胶质功能的一个很好的模型。Exosome是新近发现的一类新发现的膜小泡(40-100 nm)，起源于细胞内体，由细胞分泌。它们含有各种生物分子，包括蛋白质、脂类、mRNAs和microRNAs(MiRNAs)。外切体介导的通讯及其在中枢神经系统中的生理意义基本上是未知的。根据我们最近发表的结果，我们假设外切体介导的mir-124A从神经元到星形胶质细胞的转移通过抑制星形胶质细胞miRNAs而增加GLT1蛋白的表达，而miRNAs抑制了GLT1基因3‘非编码区的功能，并降低了星形胶质细胞中GLT1蛋白的表达。在这一应用中，我们将专注于以下目标：1)鉴定星形胶质细胞miRNAs参与依赖外切体mir-124A的星形胶质细胞GLT1表达调控。我们将利用miRNA微阵列鉴定神经元exosome处理和miR-124A转染显著下调的星形胶质细胞miRNAs。然后，我们将使用QRT-PCR来验证已鉴定的星形胶质细胞miRNAs的表达变化，并使用基于荧光素酶的检测来确定这些星形胶质细胞miRNAs是否直接抑制GLT1 3‘UTR功能。最后，我们将确定这些已识别的星形胶质miRNAs是否会降低星形胶质细胞中GLT1蛋白的表达水平和GLT1依赖的谷氨酸摄取。2)在活体内研究外切体和mir-124A从神经元到星形胶质细胞的转移。我们将用遗传学和标记的方法研究活体内外切体和mir-124从神经元到星形胶质细胞的转移。我们将建立转基因小鼠，选择性地在神经元中表达绿色荧光蛋白标记的CD63(绿色荧光)，以表征在体内将神经元外切体(由绿色荧光蛋白标记的CD63指示)转移到完整中枢神经系统星形胶质细胞的时间和空间分布。我们还将通过股神经注射和逆行转运的方法选择性地将Alexa 750(远红荧光)标记的mir-124A输送到运动神经元，然后在体内检测其从运动神经元到星形胶质细胞的转移。总而言之，该项目将识别调节GLT1蛋白表达的特定miRNAs。这些新发现的miRNAs将为调节突触功能提供新的途径，并有望成为开发基于GLT1的治疗神经疾病/损伤的靶点。此外，这项研究将描述一种新的从神经元到星形胶质细胞的外切体介导的信号通路，它调节星形胶质细胞GLT1的表达。这种细胞间通路为神经元信号如何协调突触的星形胶质功能提供了新的知识。

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.