Phosphoproteomics of Extinction and Reconsolidation of Drug Memories

描述（由申请人提供）：最近，研究人员一直在研究行为干预的使用，旨在破坏与药物相关的记忆来治疗成瘾。其中一种干预措施是暴露或消失疗法，即在没有药物的情况下反复出现与药物相关的线索，这样成瘾者就会知道，这些线索不再预测药物的可用性，他们就会停止产生渴望和复发。另一种干预是破坏记忆的再巩固，即先给出一个毒品线索，然后再进行干预，使成瘾者“忘记”这个线索与吸毒有关。这些方法在治疗其他精神疾病方面取得了一些成功。虽然这两个过程听起来很相似，但有证据表明它们实际上涉及不同的神经生物学过程。例如，增强消退的药物也可能增强而不是抑制再巩固。同样，抑制再巩固的药物也可能抑制灭绝。换句话说，同样的治疗可能会对药物记忆产生相反的影响，这取决于是否发生了消失或重新巩固。因此，有必要找到既能增强消退又能抑制再巩固的药物，这样就不会无意中增加复发的可能性。在本申请中，候选人将使用蛋白质组学技术进行研究，以识别在药物记忆重新巩固和消失过程中被差异激活和抑制的蛋白质。候选人在药物成瘾的行为药理学方面接受过大量训练。该候选人还发表了几篇研究药物记忆消失和再巩固的神经生物学的论文。然而，候选人几乎没有进行蛋白质组学研究所需的生化技术经验，因此该提案包括进行这些研究所需的蛋白质组学，生物化学和分子生物学的结构化培训的充足机会。候选人的短期职业目标是获得这些技术方面的培训，并成功管理一个独立的实验室。从长远来看，候选人计划在研究机构获得终身教授的职位，以建立一个基础广泛的转化实验室，专注于识别和验证成瘾的新治疗方法。耶鲁大学的训练环境非常好，无论是完成实验的能力，还是为候选人提供的训练机会。耶鲁大学拥有全国领先的蛋白质组学核心设施之一，以及可用于鉴定和量化蛋白质磷酸化差异的最新技术专家。这对于提出的实验至关重要，因为该假设的一个关键组成部分是，通过磷酸化状态的变化对蛋白质的差异激活将允许识别用于治疗成瘾的药物开发的新靶点。此外，在精神科候选人所在部门有几位专家可以提供生化和分子生物学技术方面的培训。耶鲁大学的培训提供了几个研究人员的优势，他们是在成瘾领域使用拟议技术的专家。候选人将由博士指导。Jane Taylor, Angus Nairn， Ralph DiLeone和Erol Gulcicek，他们都在研究中提供了丰富的专业知识。大学还提供完成这些实验所需的所有设备和实验室空间。最后，大学提供支持、资金和培训机会，例如研讨会和讲习班，以学习新技术并接受资助、实验室管理、指导和负责任的研究行为方面的培训。该候选人建议使用磷酸化蛋白质组学来鉴定新的信号级联，这些信号级联在药物提示记忆重新巩固和消失过程中被差异激活/抑制。一旦确定了靶点，它们将使用二次确认方法进行验证，包括多反应监测（MRM）和Western blotting。这些靶点将在行为动物模型中进一步验证，使用已知的药理学药物或通过病毒介导的基因转移进行RNA干扰。候选人还将通过Western blotting确定已知的被恐惧记忆的重新巩固和消除差异激活/抑制的特定蛋白质的活性是否也受到药物记忆的调节。然后，候选人将在动物模型中测试这些靶点的药理学或病毒操作，包括大麻素受体1激动剂和靶向抑制核因子κ B （NFkB）的病毒。这些实验将为候选人提供学习几种新技术的机会，并有可能获得有关药物记忆过程的新信息，这些信息可能导致未来作为独立科学家获得资助。拟议中的实验还可能导致开发出新的、更有效的成瘾治疗方法，从而长期减少对毒品的渴望和复发。候选人还将在培训期间花费大量时间学习管理独立实验室的重要技能，包括撰写拨款、管理预算、指导学生和技术人员以及负责任的研究行为培训。

DESCRIPTION (provided by applicant): Recently, researchers have been investigating the use of behavioral interventions aimed at disrupting drug- associated memories to treat addiction. One of these interventions is exposure or extinction therapy where cues associated with drugs are repeatedly presented without the drug, so that an addict learns that those cues no longer predict drug availability and they stop producing craving and relapse. Another intervention is to disrupt memory reconsolidation, where a drug cue is presented followed by an intervention designed to make the addict "forget" that the cue was associated with drug use. These methods have been used with some success to treat other psychiatric disorders. While both of these processes sound similar, there is evidence that they actually engage distinct neurobiological processes. For example, pharmacological agents that enhance extinction are also likely to enhance rather than inhibit reconsolidation. Likewise, agents that inhibit reconsolidation are also likely to inhibit extinction. In other words, the same treatment can potentially produce opposite effects on a drug memory depending on whether extinction or reconsolidation is occurring. Therefore, there is a need to find medications that can both enhance extinction and inhibit reconsolidation simultaneously, so that one does not unintentionally increase the likelihood of relapse. In this application, the candidate will conduct studies using proteomics technology to identify proteins that are differentially activated and inhibited by drug memory reconsolidation and extinction processes. The candidate has had substantial training in the behavioral pharmacology of drug addiction. The candidate has also published several papers investigating the neurobiology of both drug memory extinction and reconsolidation. However, the candidate has had little experience with the biochemical techniques required to do proteomic studies, so this proposal includes ample opportunity for structured training in proteomics, biochemistry, and molecular biology that are necessary to do these studies. The candidate's short-term career goal is to obtain training in these techniques and in the successful management of an independent laboratory. In the long-term, the candidate plans to obtain a position as a tenure-track professor at research institution to develop a broad-based, translational laboratory that will be focused on identifying and validating novel treatments for addiction. The training environment at Yale University is excellent both for the ability to complete the proposed experiments and for the training opportunities available to the candidate. Yale University has one of the leading proteomics core facilities in the nation, and experts in the latest technologies available for identifying and quantifying differences in protein phosphorylation. This is essential for the proposed experiments, as one critical component of the hypothesis is that differential activation of proteins by changes in phosphorylation state will allow the identification of novel targets for medication development to treat addiction. In addition, there are several experts within the candidate's division in the Department of Psychiatry who can provide training in biochemical and molecular biological techniques. Training at Yale University provides the advantage of several researchers in close proximity who are experts in the use of the proposed technologies in the field of addiction. The candidate will be mentored by Drs. Jane Taylor, Angus Nairn, Ralph DiLeone, and Erol Gulcicek who all provide a wealth of expertise in the research proposed. The university also provides all of the equipment and laboratory space necessary for completion of these experiments. Finally, the university provides the support, funding, and training opportunities, such as seminars and workshops, to learn new techniques and receive training in grantsmanship, lab management, mentoring, and the responsible conduct of research. The candidate proposes to use phospho-proteomics to identify novel signaling cascades that are differentially activated/inhibited by drug-cue memory reconsolidation and extinction processes. Once targets are identified, they will be validated using secondary confirmation methods including multiple reactions monitoring (MRM) and Western blotting. The targets will then be further validated in a behavioral animal model using known pharmacological agents or RNA interference via viral-mediated gene transfer. The candidate will also determine by Western blotting whether the activity of specific proteins known to be differentially activated/inhibited by reconsolidation and extinction of fear memories are also regulated by drug memories. Then, the candidate will test pharmacological or viral manipulations of these targets, which include cannabinoid receptor 1 agonists and viruses targeting inhibition of nuclear factor kappa B (NFkB) in the animal model. These experiments will provide the candidate with the opportunity to learn several new techniques and has the potential for obtaining novel information about drug memory processes that can lead to future grants as an independent scientist. The proposed experiments could also lead to the development of novel, more effective treatments for addiction that result in a long-term reduction in craving and relapse. The candidate will also spend significant time during the training period learning important skills for managing an independent laboratory, including grant writing, managing budgets, mentoring students and technicians, and training in the responsible conduct of research. PUBLIC HEALTH RELEVANCE: Addiction is a debilitating disease with significant costs to the individual and society. Successful treatments for addiction have been difficult to find, so some researchers are attempting behavioral therapies to reduce the craving caused by drug memories. The proposed studies will use proteomics to identify proteins that are activated by drug memories and will validate these proteins as targets for developing medications that could enhance the efficacy of behavioral therapies.