Transcriptional Mechanisms of Addiction-Related Neural Plasticity

描述（由申请人提供）：确定介导大脑奖励可塑性的关键分子仍然是当前药物滥用研究的重要目标。我们最近确定了MEF 2转录因子作为可卡因诱导的突触和行为可塑性的调节因子的关键作用，与重复可卡因暴露相关。我们发现，可卡因依赖性抑制MEF 2在NAc是必需的MSN棘密度增加，和增强突触连接在NAc慢性可卡因暴露后代表一种补偿机制，限制适应不良的行为反应与成瘾，而不是支持他们。在这项研究中，我们将阐明可卡因和cAMP诱导的控制纹状体MEF 2活性的信号传导事件，包括一种令人兴奋的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，我们提出以下建议：具体目标1：我们的初步研究结果表明，慢性可卡因暴露抑制MEF 2活性的cAMP依赖性过程，涉及抑制磷酸化MEF 2（P-S408/444）。在这个目标中，我们将测试可卡因和cAMP信号通过控制P-S408/444水平来调节MEF 2活性的假设。在这样做时，我们也将表征可卡因暴露后体内P-MEF 2的空间和时间调节。为此，我们将使用已建立的实验方法，如免疫组织化学，基于RNAi的蛋白质替代和转录报告基因测定，在体内和培养的原代纹状体神经元中测试P-S408/444 MEF 2对可卡因和cAMP依赖性调节MEF 2的重要性。 具体目标二：我们的初步研究结果表明，过度表达钙调素信号（RCS）的调节器负调控MEF 2的活性在培养的纹状体神经元中的Ser 55依赖性的方式（PKA网站）。在这个目标中，我们将测试的假设，P-S55 RCS是需要cAMP依赖性抑制的基础和钙激活的MEF 2依赖性转录，并测试的假设，RCS功能在体内限制致敏的行为反应可卡因。为此，我们将分析现有的RCS基因敲除小鼠使用已建立的实验方法来测试在体内，并在培养的纹状体神经元，RCS的重要性，作为一个MEF 2负调节和可卡因诱导的调节器的行为可塑性在体内。 具体目标3：我们的初步研究表明，cAMP/PKA信号的激活促进HDAC 5的纹状体神经元通过HDAC 5在一个新的Cdk 5位点的去磷酸化的核输入。在这个目标中，我们将测试的假设，慢性可卡因暴露，通过PKA依赖的HDAC 5去磷酸化，促进增强HDAC 5核定位，这有助于减少转录反应和限制敏感的行为反应，重复可卡因暴露。我们将使用新的P-HDAC 5抗体与可卡因暴露小鼠的NAc组织、HDAC 5核质穿梭的机制分析和HDAC 5磷酸化位点突变体表达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.