Exosomal miRNA signaling in Cocaine Addiction

可卡因成瘾中的外泌体 miRNA 信号传导

• 批准号: 9307767
• 负责人: Yongjie Yang
• 金额: $ 20.63万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-01 至 2019-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9307767
• 关键词: Astrocytes; Attenuated; Brain; Cell membrane; Cells; Cocaine; Cocaine Dependence; Corpus striatum structure; Development; Dopamine D1 Receptor; Drug Addiction; Excitatory Amino Acid Transporter 2; Excitatory Synapse; Glutamate Receptor; Glutamate Transporter; Glutamates; Homeostasis; Human; Immune signaling; Impairment; In Situ; In Situ Hybridization; In Vitro; Injection of therapeutic agent; Label; Lipids; Malignant Neoplasms; Mediating; Membrane; Messenger RNA; MicroRNAs; Modeling; Motor Neurons; Mus; Neuraxis; Neuroglia; Neurons; Nucleus Accumbens; Pathway interactions; Physiological; Process; Proteins; Publishing; Relapse; Reporter; Self Administration; Signal Transduction; Structure; Substance abuse problem; Synapses; Synaptic plasticity; Testing; Up-Regulation; Vesicle; Virus Diseases; analog; base; cell type; cocaine relapse; drug relapse; drug seeking behavior; exosome; extracellular; extracellular vesicles; frontier; in vivo; insight; neurotransmission; novel; novel strategies; postnatal; prevent; protein expression; tool; uptake

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.

抽象的 该应用程序响应 PAR-15-284（细胞外囊泡和药物滥用）。 可卡因自我给药显着降低谷氨酸转运蛋白 GLT1 蛋白表达 损害细胞外谷氨酸的吸收。谷氨酸转运蛋白 GLT1 是生理上的主导蛋白 哺乳动物中枢神经系统（CNS）中的谷氨酸转运蛋白。 GLT1 具有选择性和丰富性 出生后发育后在星形胶质细胞中表达。它们通常集中在等离子体上 突触周围星形胶质细胞过程的膜，它们严格控制细胞外谷氨酸水平 限制兴奋性突触中谷氨酸的“溢出”/“溢出”。 GLT1 失调的机制 可卡因（和其他成瘾物质）的自我给药目前尚不清楚。外泌体是一类 新鉴定的细胞分泌的内体来源的膜囊泡（40-100nm）；它们包含 各种生物分子，包括蛋白质、脂质、mRNA 和 microRNA (miRNA)。外泌体介导 从神经元到神经胶质细胞的细胞间信号传导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额外的初步结果，我们假设外泌体- mir-124 介导的从神经元到星形胶质细胞的转移发生改变，这是 GLT1 失调的基础 可卡因成瘾模型。在此应用中，我们将重点关注以下目标：1）研究外泌体和 我们将首先检查可卡因成瘾模型中 mir-124 从神经元转移到星形胶质细胞 来自纹状体神经元培养物的外泌体分泌动力学。我们还将用多巴胺培育 CD63-eGFPf/f 受体 D1 或 D2（Drd1 或 Drd2）Cre 小鼠，允许在 D1+ 或 D2+ 培养基中选择性标记外泌体 棘神经元（MSN）。然后，我们将检查标记的外泌体从 D1+ 或 D2+ MSN 转移到 在可卡因成瘾的不同阶段，伏核（NAc）中邻近的星形胶质细胞原位。我们将 还通过可卡因中的 NAc 中的 mir-124 原位杂交检查星形胶质细胞中 mir-124 水平的变化 模型。 2) 确定 mir-124 介导的 GLT1 上调是否会减弱可卡因复吸 - 相关的突触激活，我们将进行立体定位注射，将 mir-124 递送到 NAc 核心中 可卡因自我给药并测试外源递送的 mir-124 是否能够防止 GLT1 丢失和 减弱可卡因复吸期间 NAc 中 MSN 上增强的突触激活。 总之，本研究将研究外泌体和 mi-124 从神经元转移到 可卡因成瘾模型中的星形胶质细胞。这项研究将为病理（生理）提供新的见解 外泌体介导的 miRNA 在哺乳动物中枢神经系统中转移的重要性，特别是在了解如何 神经元与神经胶质信号传导的失调会导致药物成瘾和复发。最后是mir-124的测试 对可卡因复发相关突触激活的影响可能提供干预可卡因的新方法 复发。