Exosomal miRNA signaling in Cocaine Addiction

摘要 此应用程序响应PAR-15-284(细胞外小泡和物质滥用)。 可卡因自身给药显著降低谷氨酸转运体GLT1蛋白表达和 损害细胞外谷氨酸的摄取。谷氨酸转运体GLT1是生理上占优势的 哺乳动物中枢神经系统中的谷氨酸转运体。GLT1是选择性的和丰富的 在出生后发育的星形胶质细胞中表达。它们通常集中在血浆中 突触周围星形胶质突起的膜，在那里它们严格控制细胞外谷氨酸水平以 限制兴奋性突触的谷氨酸“溢出”/“溢出”。GLT1功能失调的机制 在可卡因(和其他成瘾物质)中，自我给药目前尚不清楚。外显体是一类 新发现的膜小泡(40-100 nm)起源于内体，由细胞分泌；它们包含 各种生物分子，包括蛋白质、脂类、mRNAs和microRNAs(MiRNAs)。Exosome介导的 神经元到神经胶质细胞的细胞间信号及其在中枢神经系统中的生理意义基本上是未知的。 根据我们之前发表的和其他的初步结果，我们假设外体-- 介导mir-124从神经元到星形胶质细胞的转移发生改变，这是GLT1异常调节的基础。 可卡因成瘾模型。在本申请中，我们将重点关注以下目标：1)研究外切体和 在可卡因成瘾模型中，我们将首先检查MIR-124从神经元到星形胶质细胞的转移 纹状体神经元培养的胞外体分泌动力学。我们还将用多巴胺培养CD63-eGFP/f 受体d1或d2(drd1或drd2)Cre小鼠，允许在d1+或d2+介质中选择性标记外切体 刺状神经元(MSN)。然后，我们将检查标记的外切体从D1+或D2+MSN转移到 可卡因成瘾不同阶段伏隔核内邻近星形胶质细胞的原位观察我们会 用mir-124原位杂交法检测可卡因NAC中星形胶质细胞mir-124水平的变化 模特。2)确定mir-124介导的GLT1上调是否可以减少可卡因复发- 相关的突触激活，我们将进行立体定向注射，将mir-124运送到NAC核心 可卡因自我给药并测试外源性递送的mir-124是否能够防止GLT1丢失和 减弱可卡因复吸时NAC内MSN上增强的突触激活。 综上所述，本研究将研究外切体和mi-124从神经元到 可卡因成瘾模型中的星形胶质细胞。这项研究将提供关于病理(生理)的新见解。 外切体介导的miRNA转移在哺乳动物中枢神经系统中的意义，特别是在理解如何 神经元对神经胶质信号的失调会导致药物成瘾和复发。最后，对MIR-124‘S进行了试验 可卡因复吸对突触激活的影响可能为干预可卡因提供新的途径 旧病复发。

Abstract This application is responsive to the PAR-15-284 (Extracellular Vesicles and Substance Abuse). Cocaine self-administration significantly reduces glutamate transporter GLT1 protein expression and impairs uptake of extracellular glutamate. Glutamate transporter GLT1 is the physiologically dominant glutamate transporter in the mammalian central nervous system (CNS). GLT1 is selectively and abundantly expressed in astrocytes after postnatal development. They are typically concentrated on the plasma membranes of peri-synaptic astroglial processes where they tightly control extracellular glutamate levels to limit the “spill-out”/“spill-over” of glutamate from excitatory synapses. The mechanisms for GLT1 dysregulation in cocaine (and other addictive substance) self-administration are currently unknown. 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 intercellular signaling from neuron to glia and its physiological significance in the CNS are essentially unknown. Based on our previously published and additional preliminary results, we hypothesize that exosome- mediated transfer of mir-124 from neurons to astrocytes is altered, which underlie GLT1 dysregulation in the cocaine addiction model. In this application, we will focus on the following aims: 1) Investigate exosome and mir-124 transfer from neurons to astrocytes in the cocaine addiction model we will first examine exosome secretion dynamics from striatum neuronal cultures. we will also breed CD63-eGFPf/f with dopamine receptor D1 or D2 (Drd1 or Drd2) Cre mice that allow selective labeling of exosomes in D1+ or D2+ medium spiny neurons (MSNs). We will then examine the transfer of labeled exosomes from D1+ or D2+ MSNs to neighboring astrocytes in nucleus accumbens (NAc) during different stages of cocaine addiction in situ. We will also examine changes of mir-124 levels in astrocytes by mir-124 in situ hybridization in NAc in the cocaine model. 2) Determine whether mir-124-mediated up-regulation of GLT1 attenuates cocaine relapse- associated synaptic activation we will perform stereotaxic injection to deliver mir-124 into NAc core during cocaine self-administration and test whether exogenously delivered mir-124 is able to prevent GLT1 loss and attenuate enhanced synaptic activation on MSNs in NAc during cocaine relapse. In summary, this study will investigate alterations of exosome and mi-124 transfer from neurons to astrocytes in the cocaine addiction model. This study will provide novel insights about the patho(physiological) significance of exosome-mediated miRNA transfer in mammalian CNS, especially in understanding how dysregulation of neuron to glial signaling contributes to drug addiction and relapse. Lastly, the test of mir-124's effects on cocaine relapse-associated synaptic activation may provide a new approach to intervene cocaine relapse.