Contributions of aberrant synaptic protein monoaminylation to opiate use disorder

项目总结 鸦片类药物的使用、依赖和上瘾是持久的公共卫生问题，导致大量的经济损失。 和社会健康负担，以及越来越多的过量用药。吸毒成瘾的特征是 慢性复发性疾病。然而，到目前为止，仍然没有足够的数据来研究分子 持续阿片类药物诱导的神经生物学变化的潜在机制，导致缺乏 治疗和预防复发的有效治疗和干预措施。 长期以来，毒瘾一直被认为是多巴胺(DA)信号的紊乱。然而，治疗性的 针对受体介导的DA神经传递的干预尚未完全有效 治疗。因此，我们实验室的首要目标--也是这项应用的重点--一直是 调查与成瘾表型有关的多巴胺的新的、非规范的行为。我们的实验室 最近发现了大脑中DA的一个新的信号部分，称为多巴胺基化(DOP)，DA在其中扮演着 用于在底物蛋白质上建立翻译后修饰(PTM)的供体来源(例如， 组蛋白H3)通过转谷氨酰胺酶2(TGM2)酶转酰胺化。在我们实验室最近所做的努力中 无偏见地鉴定这些PTM的其他底物(现在集中在细胞核中的突触蛋白上 伏隔/NAC，一个关键的大脑奖赏区域)，我们开发了一种新的化学标记方法，当 与质谱学相结合，在大脑中发现了数百种多巴胺化蛋白质，两者都是 慢性海洛因后神经功能正常和对异常多巴胺信号的反应 大鼠自我给药(SA)。其中，gCaMKII：1)被发现仅在 位于其自身抑制螺旋[谷氨酰胺(Q)285]内的单个氨基酸残基，一个只存在两个氨基酸残基的位置 远离关键的苏氨酸(T)残基(287)的氨基酸，当苏氨酸(T)被磷酸化时，该残基引导钙调蛋白 (CaM)隔离；2)海洛因SA后其多巴胺基化上调，无论是在急性和 长期禁欲，但不是对自然回报的反应；以及3)是参与 介导长距离信号从突触到脑内的核，最终促进CREB的激活和 神经元的可塑性。因此，gCaMKII上的这种多巴胺基化事件可能代表了一种关键的收敛机制 将海洛因反应中的多巴胺能信号改变与CREB介导的转录异常联系起来。AS 因此，我们推测gCaMKIIQ285dop可能通过异常在海洛因复吸中发挥直接作用。 NAC中CREB信号的调制。在目标1中，我们将充分描述gCaMKIIQ285dop的时间效应 在海洛因SA的背景下，吸毒与复发的脆弱性。在目标2中，我们将探索gCaMKIIQ285dop的 对海洛因SA后CREB信号/转录的影响，可能导致复发易感性的事件。在……里面 目的3，我们将研究gCaMKIIQ285dop介导的CREB信号/转录在d1和d2中的作用 海洛因SA戒断过程中多巴胺受体表达的MSN对复吸行为的调节作用

PROJECT SUMMARY Opiate use, dependence and addiction represent enduring public health issues, resulting in substantial financial and societal health burdens, as well as increasing numbers of overdoses. Drug addiction is characterized as a chronic, relapsing disease. However, to date, there remains insufficient data examining the molecular mechanisms underlying persistent opiate-induced neurobiological changes, which has led to a scarcity of effective therapies and interventions to treat and prevent relapse. Drug addiction has long been thought of as a disorder of dopamine (DA) signaling. However, therapeutic interventions targeting receptor mediated DA neurotransmission have not yet resulted in fully efficacious treatments. Therefore, an overarching goal of our laboratories – and the focus of this application – has been to investigate novel, non-canonical actions of DA involved in mediating addiction phenotypes. Our laboratory recently identified a novel signaling moiety for DA in brain, termed dopaminylation (dop), whereby DA acts as a donor source for the establishment of post-translational modifications (PTM) on substrate proteins (e.g., histone H3) via transamidation by the Transglutaminase 2 (TGM2) enzyme. In more recent efforts by our lab to unbiasedly identify additional substrates of these PTMs (focused now on synaptic proteins in nucleus accumbens/NAc, a key brain reward region), we developed a novel chemical tagging approach that, when coupled to mass spectrometry, allowed for the discovery of hundreds of dopaminylated proteins in brain, both in the context of normal neural function and in response to aberrant dopamine signaling following chronic heroin self-administration (SA) in rats. Among them, gCaMKII: 1) was found to be robustly dopaminylated at only a single amino acid residue located within its autoinhibitory helix [glutamine (Q)285], a site that exists only two amino acids away from a critical threonine (T) residue (287), which when phosphorylated directs Calmodulin (CaM) sequestration; 2) is upregulated in its dopaminylation following heroin SA, both during acute and prolonged abstinence, but not in response to natural rewards; and 3) represents a critical substrate involved in mediating long range signals from the synapse to nucleus in brain, ultimately promoting CREB activation and neuronal plasticity. Thus, this dopaminylation event on gCaMKII may represent a critical convergent mechanism linking altered dopaminergic signaling in response to heroin to CREB mediated transcriptional abnormalities. As such, we hypothesize that gCaMKIIQ285dop may play a direct role in mediating heroin relapse via aberrant modulation of CREB signaling in NAc. In Aim 1, we will fully characterize gCaMKIIQ285dop’s temporal effects on drug taking vs. relapse vulnerability in the context of heroin SA. In Aim 2, we will explore gCaMKIIQ285dop’s effects on CREB signaling/transcription following heroin SA, events that may precipitate relapse vulnerability. In Aim 3, we will investigate roles for gCaMKIIQ285dop mediated CREB signaling/transcription in D1 vs. D2 dopamine receptor-expressing MSNs during abstinence from heroin SA in the regulation of relapse behaviors.