RNAi knockdown of Cav1.3 and addiction

描述(申请人提供)：精神刺激剂、安非他明和可卡因是人类滥用的最具强化作用的药物之一，也是临床和科学界的一个主要健康问题。反复使用药物会导致大脑中持久的神经元适应，从而在人类和成瘾的啮齿动物模型中导致强迫性成瘾行为。然而，精神刺激剂导致大脑持续变化的确切机制仍然不清楚。钙信号在精神刺激剂介导的行为和分子变化中起着关键作用。最近的研究强调了Cav1.3 L型钙离子通道(LTCC)及其分子通路在神经元可塑性中的作用。我们实验室的工作发现，Cav1.3 LTCC介导了多巴胺和谷氨酸信号的几个方面，这是参与心理刺激作用的主要神经递质。我们发现，在苯丙胺致敏的小鼠中，Cav1.3 LTCC通过激活背侧纹状体(DSTR)中的多巴胺D2长(D2L)受体信号通路，介导了苯丙胺诱导的谷氨酸受体GluR1亚单位磷酸化的下调，背侧纹状体(DSTR)是参与成瘾成瘾的一个区域。我们进一步发现，这种适应只发生在延长的无药期之后，并且与致敏行为相关。因此，在这项建议中，我们旨在进一步探讨Cav1.3 LTCC在苯丙胺诱导的行为敏化模型中D2L信号上调中的作用，该模型具有许多在学习和记忆模型中明显的突触可塑性特征。然而，研究Cav1.3 LTCC的挑战之一是缺乏亚基特异性的药理学药物。在这项应用中，我们建议使用RNA干扰(RNAi)技术，这是一种强大的机制，允许序列特定的敲除大脑中具有空间和时间特异性的目标基因。在具体目标1中，我们将产生重组腺相关病毒(RAAV)载体，将针对Cav1.3的短发夹状RNA(ShRNA)分子运送到腹侧被盖区(VTA)，腹侧被盖区是启动精神刺激剂诱导行为的机制的主要神经部位。首先在体外测试具有高敲除效率的shRNAs，然后在体内使用小鼠VTA来特异性地降解Cav1.3 mRNA，从而导致空间敲除。在特定目标2中，VTA特异性Cav1.3基因敲除小鼠将在苯丙胺行为敏化方案中进行测试，并将检查VTA Cav1.3 LTCC在DSTR中介导D2L和GluR1信号适应中的作用。在特定目标3中，将通过检测Cav1.3靶标、CREB和ERK的磷酸化来表征Cav1.3基因敲除的VTA细胞型特定表型。RNAi方法将有助于阐明Cav1.3 LTCC在苯丙胺诱导的行为和分子可塑性中的区域和时间特异性。此外，这里生成的工具将允许靶向Cav1.3 LTCC途径的其他细胞内分子，从而更好地理解导致精神刺激剂暴露后行为持续改变的机制。

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.