Transcriptional Mechanisms of Addiction-Related Neural Plasticity

成瘾相关神经可塑性的转录机制

• 批准号: 8623115
• 负责人: Christopher W Cowan
• 金额: $ 32万
• 依托单位: MCLEAN HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-03-01 至 2016-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8623115
• 关键词: Abstinence; Acute; Antibodies; Behavior; Behavioral; Biological Assay; Brain; Calcium; Calmodulin; Chronic; Cocaine; Corpus striatum structure; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Dendritic Spines; Dopamine; Dopamine D1 Receptor; Drug Addiction; Drug Controls; Drug abuse; Drug usage; Epigenetic Process; Event; Future; Gene Expression; Genes; Genetic Transcription; Goals; Grant; HDAC5 gene; Histone Deacetylase; Histones; Immunohistochemistry; Injection of therapeutic agent; Knockout Mice; Knowledge; Learning; Mediating; Mus; Neuronal Plasticity; Neurons; Nuclear; Nuclear Import; Phosphorylation; Phosphorylation Site; Phosphotransferases; Process; Protein Dephosphorylation; Proteins; RNA Interference; Regulation; Relative (related person); Reporter; Research; Rewards; Role; Signal Pathway; Signal Transduction; Site; Site-Directed Mutagenesis; Synapses; Testing; Tissues; Vertebral column; addiction; adverse outcome; base; calcineurin phosphatase; cocaine exposure; density; human disease; in vivo; insight; mutant; myocyte-specific enhancer-binding factor 2; new therapeutic target; novel; nucleocytoplasmic transport; overexpression; public health relevance; research study; response; transcription factor

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.

描述（由申请人提供）：识别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 MEF2 转录因子作为可卡因诱导的突触和与反复接触可卡因相关的行为可塑性的调节剂的关键作用。我们发现，NAc 中 MEF2 的可卡因依赖性抑制是增加 MSN 棘密度所必需的，并且长期接触可卡因后 NAc 中突触连接的增强代表了一种补偿机制，限制而不是支持与成瘾相关的适应不良行为反应。在这笔资助中，我们将阐明可卡因和 cAMP 诱导的控制纹状体中 MEF2 活性的信号事件，包括 IIa 类组蛋白脱乙酰酶的令人兴奋的新调节机制，该机制可能对药物成瘾的表观遗传调节产生重要影响。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，长期接触可卡因通过涉及 MEF2 抑制性磷酸化的 cAMP 依赖性过程抑制 MEF2 活性 (P-S408/444)。为此，我们将测试可卡因和 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，我们还将描述可卡因暴露后体内 P-MEF2 的空间和时间调节。为此，我们将使用已建立的实验方法，例如免疫组织化学、基于 RNAi 的蛋白质替代和转录报告测定法，在体内和培养的原代纹状体神经元中测试 P-S408/444 MEF2 对可卡因和 MEF2 的 cAMP 依赖性调节的重要性。 具体目标 2：我们的初步研究结果表明，钙调蛋白信号调节剂 (RCS) 的过度表达以 Ser55 依赖性方式（PKA 位点）负向调节培养的纹状体神经元中的 MEF2 活性。为此，我们将检验以下假设：P-S55 RCS 是 cAMP 依赖性抑制基础和钙激活 MEF2 依赖性转录所必需的，并检验 RCS 在体内发挥作用以限制对可卡因的敏化行为反应的假设。为此，我们将使用已建立的实验方法分析现有的 RCS 敲除小鼠，以在体内和培养的纹状体神经元中测试 RCS 作为 MEF2 负调节剂和可卡因诱导的体内行为可塑性调节剂的重要性。 具体目标 3：我们的初步研究表明，cAMP/PKA 信号传导的激活通过 HDAC5 在新的 Cdk5 位点的去磷酸化促进纹状体神经元中 HDAC5 的核输入。为此，我们将测试以下假设：慢性可卡因暴露通过 PKA 依赖性 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可卡因暴露的敏感行为反应。我们将使用新型 P-HDAC5 抗体与可卡因暴露小鼠的 NAc 组织、HDAC5 核质穿梭的机制分析以及 HDAC5 磷酸位点突变体表达小鼠的行为分析来测试这一想法。