Transcriptional Mechanisms of Addiction-Related Neural Plasticity

描述(由申请人提供)：确定调节大脑奖赏可塑性的关键分子仍然是当前药物滥用研究的重要目标。我们最近发现了MEF2转录因子作为可卡因诱导的突触和与反复暴露可卡因相关的行为可塑性的调节因子的关键作用。我们发现，增加MSN脊椎密度需要依赖可卡因对NAC中MEF2的抑制，而长期暴露可卡因后NAC中突触连接的增强代表了一种补偿机制，限制了与成瘾相关的不良适应行为反应，而不是支持它们。在这项授权中，我们将阐明可卡因和cAMP诱导的控制纹状体MEF2活性的信号事件，包括令人兴奋的IIa类组蛋白脱乙酰酶的新调控机制，这可能对药物成瘾的表观遗传调控具有重要意义。为此，我们提出如下建议：具体目标1：我们的初步发现表明，慢性可卡因暴露通过抑制MEF2的磷酸化的cAMP依赖的过程(P-S408/444)抑制MEF2的活性。为此，我们将验证可卡因和cAMP信号通过控制P-S408/444水平来调节MEF2活性的假设。在此过程中，我们还将表征可卡因暴露后P-MEF2在体内的空间和时间调节。为此，我们将使用现有的实验方法，如免疫组织化学、基于RNAi的蛋白质替换和转录报告分析，在体内和在培养的原代纹状体神经元中测试P-S408/444 MEF2对可卡因和cAMP依赖的MEF2调节的重要性。具体目的2：我们的初步研究结果表明，钙调素信号调节因子(RCS)的过度表达以一种Ser55依赖的方式(PKA位点)负向调节培养的纹状体神经元中MEF2的活性。为此，我们将验证P-S55 RCS是cAMP依赖的抑制基础转录和钙激活的MEF2依赖转录所必需的假设，并测试RCS在体内发挥作用以限制对可卡因的致敏行为反应的假设。为此，我们将使用已建立的实验方法分析现有的RCS基因敲除小鼠，以在体内测试RCS作为MEF2负调控因子和可卡因诱导的行为可塑性调节因子在体内培养的纹状体神经元的重要性。具体目的3：我们的初步研究表明，cAMP/PKA信号的激活通过在一个新的CDK5位点去磷酸化HDAC5来促进纹状体神经元中HDAC5的核输入。为此，我们将检验这一假设，即慢性可卡因暴露通过依赖PKA的HDAC5去磷酸化促进HDAC5核定位增强，从而减少转录反应并限制对重复暴露可卡因的敏化行为反应。我们将使用新的P-HDAC5抗体与接触可卡因的小鼠的NAC组织、HDAC5核质穿梭的机制分析以及表达HDAC5磷酸位点突变的小鼠的行为分析来测试这一想法。

DESCRIPTION (provided by applicant): Identifying key molecules that mediate brain reward plasticity remains an important goal of current drug abuse research. We recently identified a key role for MEF2 transcription factors as regulators of cocaine-induced synaptic and behavioral plasticity associated with repeated cocaine exposure. We find that cocaine-dependent inhibition of MEF2 in the NAc is required for increased MSN spine density, and that enhanced synaptic connectivity in the NAc after chronic cocaine exposure represents a compensatory mechanism that limits maladaptive behavioral responses associated with addiction, rather than supporting them. In this grant, we will elucidate cocaine- and cAMP-induced signaling events that control MEF2 activity in the striatum, including an exciting new regulatory mechanism for class IIa histone deacetylases that could have important implications for epigenetic regulation of drug addiction. To this end, we propose the following: Specific Aim 1: Our preliminary findings suggest that chronic cocaine exposure inhibits MEF2 activity by a cAMP-dependent process involving inhibitory phosphorylation of MEF2 (P-S408/444). In this aim, we will test the hypothesis that cocaine and cAMP signaling regulates MEF2 activity through control of P-S408/444 levels. In doing so, we will also characterize the spatial and temporal regulation of P-MEF2 in vivo after cocaine exposure. To these ends, we will use established experimental approaches, such as immunohistochemistry, RNAi-based protein replacement and transcriptional reporter assays, to test in vivo and in cultured primary striatal neurons the importance of P-S408/444 MEF2 for cocaine and cAMP-dependent regulation of MEF2. Specific Aim 2: Our preliminary findings revealed that overexpression of the regulator of calmodulin signaling (RCS) negatively regulates MEF2 activity in cultured striatal neurons in a Ser55 dependent manner (PKA site). In this aim, we will test the hypothesis that P-S55 RCS is required for cAMP-dependent inhibition of basal and calcium-activated MEF2-dependent transcription, and test the hypothesis that RCS functions in vivo to limit sensitized behavioral responses to cocaine. To this end, we will analyze existing RCS knockout mice using established experimental approaches to test in vivo, and in cultured striatal neurons, the importance of RCS as a MEF2 negative regulator and as a cocaine-induced regulator of behavioral plasticity in vivo. Specific Aim 3: Our preliminary studies indicate that activation of cAMP/PKA signaling promotes the nuclear import of HDAC5 in striatal neurons via dephosphorylation of HDAC5 at a novel Cdk5 site. In this aim, we will test the hypothesis that chronic cocaine exposure, via PKA-dependent dephosphorylation of HDAC5, promotes enhanced HDAC5 nuclear localization, which serves to reduce transcriptional responses and limit sensitized behavioral responses to repeated cocaine exposure. We will test this idea using novel P-HDAC5 antibodies with NAc tissues of cocaine exposed mice, mechanistic analysis of HDAC5 nucleocytoplasmic shuttling, and behavioral analysis of HDAC5 phospho-site mutant-expressing mice.