RNAi knockdown of Cav1.3 and addiction

Cav1.3 的 RNAi 敲低和成瘾

基本信息

批准号：
7586158
负责人：
Anjali M RAJADHYAKSHA
金额：
$ 21万
依托单位：
WEILL MEDICAL COLL OF CORNELL UNIV
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-04-01 至 2010-09-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7586158
关键词：
AMPA Receptors Addictive Behavior Address Amphetamines Behavior Behavioral Brain CREB1 gene Calcium Signaling Cells Clinical Cocaine Communities Corpus striatum structure Data Detection Dopamine Dopamine D2 Receptor Dorsal Down-Regulation Drug usage Fluorescence Future Gene Silencing Gene Targeting Glutamate Receptor Glutamates Green Fluorescent Proteins Habits Health Human Immunohistochemistry In Situ Hybridization Knock-out Laboratory Finding Lead Learning Link Long-Term Depression Measures Mediating Memory Messenger RNA Modeling Molecular Motor Activity Mus Neuronal Plasticity Neurons Neurotransmitters Pathway interactions Pharmaceutical Preparations Phenotype Phosphorylation Physiological Play Proteins Protocols documentation RNA RNA Interference Receptor Signaling Recombinants Rodent Rodent Model Role Signal Pathway Signal Transduction Site Specificity Synaptic plasticity Techniques Technology Testing Up-Regulation Ventral Tegmental Area Viral Viral Vector Virus Western Blotting Work addiction base behavioral sensitization cell type in vitro testing in vivo novel strategies protein expression psychostimulant relating to nervous system research study reward circuitry small hairpin RNA tool

项目摘要

DESCRIPTION (provided by applicant): The psychostimulants, amphetamine and cocaine are among the most reinforcing drugs that are abused by humans and a major health issue in the clinical and scientific communities. Repeated drug use causes long-lasting neuronal adaptations in the brain that leads to compulsive addictive behavior both in humans and in rodent models of addiction. However the precise mechanisms by which psychostimulants cause persistent alterations in the brain remain elusive. Calcium signaling plays a pivotal role in psychostimulant-mediated behavioral and molecular changes. Recent studies have highlighted the role of the Cav1.3 L-type Ca2+ channel (LTCC) and its molecular pathways in neuronal plasticity. Work from our lab finds that Cav1.3 LTCCs mediate several aspects of dopamine and glutamate signaling, primary neurotransmitters involved in psychostimulant action. We find that in amphetamine sensitized mice, Cav1.3 LTCCs mediate downregulation of amphetamine- induced phosphorylation of the GluR1 subunit of glutamate receptors via activation of the dopamine D2 long (D2L) receptor-signaling pathway in the dorsal striatum (dStr), a region involved in the habit-forming aspects of addiction. We further find that this adaptation occurs only following extended drug-free period and is a correlate of sensitized behavior. Hence in this proposal we aim to further explore the role of Cav1.3 LTCCs in upregulation of D2L signaling in the model of amphetamine-induced behavioral sensitization that shares many features of synaptic plasticity evident in models of learning and memory. However one of challenges in studying Cav1.3 LTCCs is the lack of subunit specific pharmacological agents. In this application we propose to use RNA interference (RNAi) technology, a powerful mechanism that allows sequence-specific knockdown of target genes in the brain with spatial and temporal specificity. In Specific Aim 1, we will generate recombinant adenoassociated viral (rAAV) vectors to deliver short hairpin RNA (shRNA) molecules specific for Cav1.3 into the ventral tegmental area (VTA), the primary neural site that initiates mechanisms that underlie psychostimulant-induced behaviors. shRNAs with high knockdown efficiency first tested in vitro will then be used in vivo in mouse VTA to specifically degrade Cav1.3 mRNA resulting in a spatial knockdown. In Specific Aim 2, VTA-specific Cav1.3 knockdown mice will be tested in an amphetamine behavioral sensitization protocol and the role of VTA Cav1.3 LTCCs in mediating adaptation of D2L and GluR1 signaling in the dStr will be examined. In Specific Aim 3, VTA cell-type specific phenotype of Cav1.3 knockdown will be characterized by examining phosphorylation of Cav1.3 targets, CREB and ERK. The RNAi approach will allow the elucidation of the regional and temporal specificity of Cav1.3 LTCCs in amphetamine-induced behavioral and molecular plasticity. Furthermore the tools generated here will allow the targeting of other intracellular molecules of the Cav1.3 LTCC pathway towards a better understanding of the mechanisms that lead to persistent alteration in behavior following psychostimulant exposure.

描述（由申请人提供）：心理刺激剂，苯丙胺和可卡因是人类滥用的最强化药物，也是临床和科学社区中的主要健康问题。重复使用药物会导致大脑中持久的神经元适应，从而导致人类和啮齿动物成瘾模型中的强迫性成瘾行为。但是，精神刺激物引起大脑持续改变的确切机制仍然难以捉摸。钙信号传导在精神刺激介导的行为和分子变化中起关键作用。最近的研究强调了CAV1.3 L型Ca2+通道（LTCC）及其分子途径在神经元可塑性中的作用。我们实验室的工作发现，CAV1.3 LTCC介导多巴胺和谷氨酸信号传导的几个方面，这是参与精神刺激作用的主要神经递质。我们发现，在苯丙胺敏化的小鼠中，CAV1.3 LTCCS通过激活多巴胺D2长（D2L）受体信号途径的激活，涉及谷氨酸受体的GLUR1亚基的苯丙胺诱导的磷酸化下调，该地区涉及到型成型的区域中，该区域涉及到型成型的地区。我们进一步发现，这种适应仅在无药物时期才会发生，并且是敏化行为的相关性。因此，在此提案中，我们旨在进一步探讨CAV1.3 LTCC在上调D2L信号传导中的作用，在苯丙胺诱导的行为敏化模型中，具有在学习和记忆模型中可见的突触可塑性的许多特征。但是，研究CAV1.3 LTCC的挑战之一是缺乏亚基特定的药理学剂。在此应用中，我们建议使用RNA干扰（RNAI）技术，这是一种强大的机制，可以具有空间和时间特异性的序列特异性敲低大脑中的靶基因。在特定的目标1中，我们将生成重组腺体相关的病毒（RAAV）向量，以传递针对CAV1.3特异性的短发蛋白RNA（SHRNA）分子进入腹侧段盖区域（VTA），这是一种启动的机制，该机制启动了精神刺激性诱导的行为。首先在体外测试的具有高敲低效率的shRNA将在小鼠VTA中使用，以特异性降解Cav1.3 mRNA，导致空间敲低。在特定的目标2中，将在苯丙胺行为敏化方案中测试VTA特异性CAV1.3敲低小鼠，并且将检查VTA CAV1.3 LTCC在介导D2L和GlUR1信号在DSTR中的适应性中的作用。在特定的目标3中，VTA细胞类型的Cav1.3敲低的特异性表型将以检查CAV1.3靶标，CREB和ERK的磷酸化来表征。 RNAi方法将允许在苯丙胺诱导的行为和分子可塑性中阐明CAV1.3 LTCC的区域和时间特异性。此外，此处生成的工具将允许靶向CAV1.3 LTCC途径的其他细胞内分子，以更好地理解导致精神刺激暴露后行为持续改变的机制。