Identification of CaMKII as a novel substrate of dopaminylation following heroin self administration

CaMKII 鉴定为海洛因自我给药后多巴胺酰化的新型底物

• 批准号: 10462196
• 负责人: Andrew F Stewart
• 金额: $ 4.52万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-19 至 2024-01-18
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10462196
• 关键词: Abstinence; Acute; Affect; Alanine; Amino Acids; Back; Behavior; Behavior Control; Binding; Brain; Calcium; Calmodulin; Cell Culture Techniques; Cell Nucleus; Cells; Cessation of life; ChIP-seq; Chemicals; Chronic; Co-Immunoprecipitations; Complex; Corpus striatum structure; Coupled; Coupling; Cues; Custom; Cyclic AMP Response Element; Data; Disease; Dopamine; Enzymes; Epigenetic Process; Event; Functional disorder; Gene Expression Profile; Genetic Transcription; Glutamine; Heroin; Histone H3; Immunoprecipitation; In Vitro; Intervention; Laboratories; Learning; Link; Long-Term Potentiation; Mass Spectrum Analysis; Mediating; Memory; Modeling; Mutate; Neurons; Neurophysiology - biologic function; Nuclear; Nuclear Translocation; Nucleus Accumbens; Opioid; Pharmaceutical Preparations; Phosphorylation; Phosphotransferases; Play; Post-Translational Protein Processing; Proteins; Public Health; Rattus; Recombinants; Regulation; Relapse; Research; Rewards; Role; Saline; Self Administration; Signal Transduction; Site; Source; Substance Use Disorder; Substance abuse problem; Sucrose; Synapses; Synaptic plasticity; Testing; Threonine; Tissues; United States; Validation; Ventral Tegmental Area; Viral; Virus; Western Blotting; adeno-associated viral vector; behavioral outcome; calmodulin-dependent protein kinase II; drug of abuse; efficacious treatment; experimental study; heroin use; in vivo; knock-down; mesolimbic system; monoamine; mutant; neurobiological mechanism; new therapeutic target; novel; postsynaptic; receptor; response; socioeconomics; transcription factor; transcriptome sequencing; transglutaminase 2

Heroin use disorder (HUD) represents an enduring public health issue resulting in significant socioeconomic burdens to the United States, with domestic opiate-related deaths quadrupling from 1999 to 2017. Despite this the neurobiological mechanisms underlying HUD remain poorly understood. All drugs of abuse modulate dopaminergic signaling and have long been thought of as disorders of dopamine (DA) signaling. However, pharmacotherapeutic interventions targeting receptor mediated DA-signaling have not resulted in efficacious treatments. Our laboratory recently identified a novel signaling moiety for DA, termed dopaminylation, whereby DA itself acts as a post-translational modification (PTM) on substrate proteins via transamidation by the Transglutaminase 2 (TGM2) enzyme. I sought, then, to unbiasedly identify additional synaptic substrates of dopaminylation in vivo, utilizing a novel chemical tagging approach coupled to mass spectrometry. I identified 164 novel putative synaptic substrates of dopaminylation in Nucleus Accumbens (NAc), both in the context of normal neural function and in response to abstinence from chronic heroin self-administration. Following validation of a number of putative substrates, I turned my focus to gCaMKII as: 1) it is highly abundant 2) it is dopaminylated at a single amino acid residue, located within it’s autoinhibitory helix (glutamine [Q]285), a site that exists only two amino acids away from the critical threonine (T) residue 287. T287 is phosphorylated to direct Calmodulin (CaM) sequestration and subsequent nuclear translocation – thus, this site, represents an exciting ‘test’ case for establishing the importance of this signaling moiety in post synaptic plasticity 3) it is upregulated in its dopaminylation state following abstinence from heroin SA, an effect that persists to AD14 and 4)represents a critical substrate involved in mediating long range signals from the synapse to the nucleus in brain, ultimately promoting CREB activation. gCaMKII is a synaptic protein demonstrated to be necessary for learning/memory, late-long-term-potentiation (LTP) and excitation transcription coupling. CREB is an important transcription factor in brain implicated in all types of SUD, where dynamic alterations in activation have been observed to affect drug-related behaviors. I have additionally demonstrated that monoamine molecules can infiltrate striatal and cortical neurons in vitro, as well as that treatment of these neurons with DA induces gCaMKII dopaminylation. I also observed the dynamic regulation of gCaMKII/CaM localization and subsequent downstream CREB signaling in response to DA treatment in primary cortical-striatal neuronal culture. I therefore hypothesize that gCaMKIIQ285dop may represent a novel dopaminergic signaling moiety in brain, and may play a direct role in mediating heroin relapse behaviors via aberrant modulation of CREB signaling in NAc.

海洛因使用障碍(HUD)是一个持久的公共健康问题，导致重大的社会经济问题 给美国带来了负担，从1999年到2017年，国内与阿片类药物有关的死亡人数翻了两番。尽管如此 HUD背后的神经生物学机制仍然知之甚少。所有滥用药物都会调节 多巴胺能信号，长期以来一直被认为是多巴胺(DA)信号的障碍。然而， 针对受体介导的DA信号的药物治疗干预没有产生有效的结果 治疗。我们的实验室最近发现了DA的一个新的信号部分，称为多巴胺基化，由此 DA本身作为底物蛋白质的翻译后修饰(PTM)，通过底物蛋白的转酰胺化 转谷氨酰胺酶2(TGM2)。然后，我试图不偏不倚地辨认出其他突触底物 体内的多巴胺基化，利用一种新的化学标记方法与质谱学相结合。我确认了 164伏核(NAC)多巴胺基化的新突触底物，既在 神经功能正常，对慢性海洛因自我给药戒断的反应。跟随 验证了一些可能的底物后，我把注意力转向了gCaMKII，因为：1)它非常丰富 位于其自身抑制螺旋(谷氨酰胺[Q]285)内的单一氨基酸残基上的多巴胺。 它距离关键的苏氨酸(T)残基287只有两个氨基酸。T287被磷酸化为直接 钙调蛋白(CaM)的隔离和随后的核转位-因此，这个位置代表了一个令人兴奋的 确定这个信号部分在突触后可塑性中的重要性的测试案例3)它被上调 在海洛因SA戒断后的多巴胺化状态下，这种效应持续到AD14和4)代表 一种关键的底物，最终参与将长距离信号从突触传递到大脑中的核 促进CREB激活。GCaMKII是一种突触蛋白，被证明是学习/记忆所必需的， 晚期长时程增强(LTP)和激发转录偶联。CREB是一种重要的转录因子 在与所有类型的SUD有关的大脑中，已观察到激活的动态变化影响 与毒品有关的行为。我还证明了单胺分子可以渗透到纹状体和 体外培养的大脑皮层神经元，以及DA处理这些神经元可诱导gCaMKII多巴胺基化。我 还观察了gCaMKII/CaM定位和随后的下游CREB信号的动态调节 在原代培养的皮质-纹状体神经元中对DA处理的反应。因此，我假设 GCaMKIIQ285dop可能代表了脑内一种新的多巴胺能信号转导部分，并可能在 NAC中CREB信号的异常调节介导海洛因复吸行为。