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.

摘要 此应用程序响应PAR-15-284（细胞外囊泡和物质滥用）。 辅酶A自身给药显著降低谷氨酸转运蛋白GLT 1蛋白表达， 损害细胞外谷氨酸的摄取。谷氨酸转运蛋白GLT 1是生理上占主导地位的 谷氨酸转运蛋白在哺乳动物中枢神经系统（CNS）。GLT 1选择性地和大量地 在出生后发育的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，在那里它们严格控制细胞外谷氨酸水平， 限制谷氨酸从兴奋性突触的“溢出”/“溢出”。GLT 1失调的机制 在可卡因（和其他成瘾物质）中自我给药目前是未知。外泌体是一类 新鉴定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（miRNAs）。外泌体介导 从神经元到神经胶质的细胞间信号传导及其在CNS中的生理学意义基本上是未知的。 基于我们先前发表的和额外的初步结果，我们假设外泌体- mir-124从神经元到星形胶质细胞的介导转移发生改变，这是GLT 1在星形胶质细胞中失调的基础。 可卡因成瘾模型在本申请中，我们将重点关注以下目标：1）研究外泌体， 在可卡因成瘾模型中，mir-124从神经元转移到星形胶质细胞，我们将首先检查 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养CD 63-eGFP f/f， 受体D1或D2（Drd 1或Drd 2）Cre小鼠，其允许在D1+或D2+培养基中选择性标记外泌体 多刺神经元（MSNs）。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，在原位，相邻的星形胶质细胞在脑桥核（NAc）。我们将 用原位杂交法检测可卡因对星形胶质细胞NAc中miR-124表达的影响 模型2)确定mir-124介导的GLT 1上调是否减弱可卡因复发- 我们将进行立体定位注射以将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是否能够预防GLT 1损失， 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提供新的见解，病理（生理） 外泌体介导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 神经元到神经胶质信号传导的失调有助于药物成瘾和复发。最后，对米尔-124的测试 对可卡因复发相关突触激活的影响可能为干预可卡因提供新的途径 复发