RNA methylation in synaptic plasticity and drug-seeking

摘要 突触可塑性异常是药物成瘾的重要生物学机制。地区旷日持久的 突触的变化依赖于基因表达的变化，证据支持 表观遗传机制然而，组蛋白修饰，DNA甲基化或DNA羟甲基化影响 基因在转录水平的表达，这不仅是缓慢影响蛋白质合成，而且缺乏 突触特异性已发现RNA N6-甲基腺苷（m6 A）修饰显著影响RNA 拼接、导出、本地化、翻译效率和稳定性。所有这些都是制约翻译的关键因素 以及突触中潜在的局部翻译。我们的初步数据和已发表的研究都表明， m6 A依赖性通路的缺陷显著损害神经元功能，包括多巴胺信号传导 和多巴胺依赖性学习。因此，我们建议将重点放在m6 A修饰的mRNAs的作用， 突触可塑性和药物成瘾模型。我们将使用m6 A甲基转移酶胃L14 纹状体D1阳性神经元缺失。在目标1中，我们将检验以下假设： 突变小鼠具有受损的食欲巴甫洛夫学习和受损的可卡因线索诱导恢复 自我管理。在目标2中，我们将记录大脑切片，并测试突变小鼠是否有受损 皮质纹状体可塑性和可卡因引起的皮质纹状体突触强度变化减弱 exposure.在目标3中，我们将确定m6 A RNA甲基化如何调节突触蛋白的翻译。 对药物挑战的反应和神经元活动的其他变化。我们首先会检查是否接触可卡因 在体内影响m6 A RNA甲基化水平并鉴定下游靶标。其余的建议 实验将检测m6 A RNA甲基化对索马体蛋白翻译的影响程度， 树突，其中局部翻译受突触控制。我们将使用分离的神经元皮质-纹状体 来自胃L14敲除小鼠和对照小鼠的共培养物。

Abstract Aberrant synaptic plasticity is a key underlying biological mechanism of drug addiction. The long-lasting synaptic changes are dependent on changes in gene expression and evidence support the involvement of epigenetic mechanisms. However, histone modifications, DNA methylation or DNA hydroxymethylation affects gene expression at transcriptional level, which is not only slow in affecting protein synthesis but also lacks synaptic specificity. RNA N6-methyladenosine (m6A) modification has been found to significantly affect RNA splicing, export, localization, translation efficiency and stability. All there are key factors in regulating translation and potentially localized translation in synapses. Both our preliminary data and published studies suggest that deficiency in m6A dependent pathways significantly impairs neuronal function including dopamine signaling and dopamine dependent learning. Therefore we propose to focus on the role of m6A modification of mRNAs in synaptic plasticity and in drug addiction models. We will use mice with the m6A methyltransferase METTL14 deleted in D1 positive striatal neurons in the nucleus accumbens. In Aim 1, we will test the hypothesis that mutant mice have impaired appetitive Pavlovian learning and impaired cue-induced reinstatement of cocaine self-administration. In Aim 2, we will record from brain slices and test if mutant mice have impaired corticostriatal plasticity and diminished changes in corticostriatal synaptic strengths caused by cocaine exposure. In Aim 3, we will determine how m6A RNA methylation regulates synaptic protein translation in responses to drug challenges and other changes on neuronal activity. We will first examine if cocaine exposure in vivo affects level of m6A RNA methylation and identify downstream targets. The rest of the proposed experiments will examine the degree to which m6A RNA methylation affects protein translation in the soma vs. dendrites where local translation is under control of synapses. We will use dissociated neuronal cortical-striatal co-cultures from METTL14 knockout and control mice.