Exosomes, MiRNAs and Astroglial Glutamate Transporter EAAT2/GLT1 Regulation

星形胶质兴奋性氨基酸转运蛋白2 （EAAT2，啮齿动物类似物GLT1）是星形胶质细胞中最重要的功能性突触蛋白之一，在中枢神经系统中具有重要的生理和病理功能。EAAT2/GLT1表达的调控已经成为理解神经元信号如何协调突触星形胶质细胞功能的一个很好的模型。外泌体是一类新发现的膜囊泡（40-100nm），起源于内体，由细胞分泌。它们含有多种生物分子，包括蛋白质、脂质、mrna和microRNAs （miRNAs）。外泌体介导的通讯及其在中枢神经系统中的生理意义基本上未知。基于我们最近发表的研究结果，我们假设外泌体介导的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中抑制GLT1 mRNA 3' UTR功能和降低GLT1蛋白表达的mirna来增加GLT1蛋白的表达。在本应用中，我们将关注以下目标：1)鉴定参与星形胶质细胞中外泌体miR-124a依赖性GLT1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-124a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miRNAs的表达变化，并使用基于荧光素酶的检测确定这些星形胶质细胞miRNAs是否直接抑制GLT1 3' UTR功能。最后，我们将确定这些鉴定的星形胶质细胞mirna是否会降低GLT1蛋白表达水平和星形胶质细胞中GLT1依赖的谷氨酸摄取。2)研究外泌体和mir-124a从神经元到星形胶质细胞的体内转移我们将使用遗传和标记方法研究体内外泌体和mir-124从神经元到星形胶质细胞的转移。我们将产生在神经元中选择性表达egfp标记的CD63（绿色荧光）的转基因小鼠，以表征完整中枢神经系统中神经元外泌体（由egfp标记的CD63指示）在体内转移到星形胶质细胞的时间和空间特征。我们还将通过股神经注射和逆行运输选择性地将Alexa 750（远红色荧光）标记的mir-124a传递到运动神经元，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，本项目将确定调节GLT1蛋白表达的特异性mirna。这些新发现的mirna将为调节突触功能提供新方法，并为开发基于glt1的治疗神经系统疾病/损伤的疗法提供有希望的靶点。此外，本研究将描述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GLT1表达。这种细胞间通路提供了关于神经元信号如何协调突触星形胶质细胞功能的新知识。

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.