Neuronal subtype and circuit-specific epigenetic mechanisms in addiction

 描述(由申请人提供)：本提案请求支持一项全面的培训计划，该计划将使候选人Gregory C.Sartor博士能够扩展、发展和完善为富有成效的独立研究生涯所必需的技术技能。培训计划的目标是通过有监督的动手培训和授课课程获得先进的分子生物学技术，以便将复杂的方法纳入特定的目标，重点是阐明成瘾中细胞类型和电路特定的表观遗传机制。高级科学培训将集中在两个关键领域：1)从与成瘾相关的脑区分离神经元亚型的荧光激活细胞(FACS)，以及2)开发病毒介导的基因转移工具，以特定细胞类型和连接的方式操纵表观遗传靶标。职业发展活动将由Claes Wahlestedt博士指导，Kasahara博士和Gray博士提供合作支持，他们都在建议的方法方面拥有不重叠的专业知识，并在指导博士后实习生方面有出色的记录。研究部分将研究被称为BET溴域的乙酰赖氨酸阅读器蛋白，这是一种新兴的、可用药的表观遗传学靶点，目前正在进行多项临床试验。这些含溴结构域的蛋白质结合乙酰化组蛋白，并作为大分子复合体招募的支架，改变染色质的可及性和转录活性。我最近发现，在长期服用可卡因后，BET蛋白上调并招募到伏核(NAC)中BDNF的启动子区域。此外，我的数据显示，对这些BET蛋白(全身和NAC内)的药理抑制可以减弱对可卡因的行为反应。然而，最近的证据表明，可卡因诱导的行为和相关的染色质修饰在NAC中以细胞类型特有的方式受到调控。因此，为了充分了解这些蛋白质在复杂的成瘾行为中的作用，在这个解剖和功能不同的区域内对BET蛋白进行精确的检测是至关重要的。我的总体假设是，以细胞类型和投射依赖的方式调节表观遗传阅读器蛋白将减弱可卡因寻找行为。AIM 1将要求进行流式细胞仪培训，以确定短期和长期使用可卡因后BET mRNA/蛋白表达的细胞类型特定时间进程。AIM 2将要求熟练掌握病毒介导的基因转移技术，以便在寻找可卡因的行为中操纵NAC内特定细胞类型的BET蛋白。R00非依赖阶段建立在这一训练的基础上，以更具体地关注可卡因寻找行为期间BET蛋白的连接特定变化和操作。总之，这些创新研究将是第一次系统地探索复杂行为中细胞类型和连接特定方式的表观遗传学靶标。

 DESCRIPTION (provided by applicant): This proposal requests support for a comprehensive training plan that will enable the candidate, Dr. Gregory C. Sartor, to expand, develop, and refine technical skills that are necessary for a productive independent research career. The goal of the training plan is to acquire advanced molecular biology techniques through supervised hands-on training and didactic coursework in order to incorporate sophisticated methods into Specific Aims focused on elucidating cell type- and circuit-specific epigenetic mechanisms in addiction. The advanced scientific training will be focused on two key areas: 1) Fluorescence-activated cell sorting (FACS) of neuronal subtypes from addiction-related brain regions, and 2) developing viral-mediated gene transfer tools to manipulate epigenetic targets in a cell type- and connection-specific fashion. The career development activities will be mentored by Dr. Claes Wahlestedt with collaborative support from Drs. Kasahara and Gray, all of which have non-overlapping expertise in the methods proposed and have exceptional records of mentoring postdoctoral trainees. The research component will investigate acetyl lysine reader proteins, termed BET bromodomains, an emerging, drugable epigenetic target that is currently being studied in multiple clinical trials. These bromodomain-containing proteins bind acetylated histones and serve as a scaffold for the recruitment of macromolecular complexes that modify chromatin accessibility and transcriptional activity. I recently found that BET proteins are upregulated and recruited to the promoter region of Bdnf in the nucleus accumbens (NAc) following chronic cocaine administration. Furthermore, my data show that pharmacological inhibition of these BET proteins (systemic and intra-NAc) attenuates behavioral responses to cocaine. However, recent evidence indicates that cocaine-induced behaviors and associated chromatin modifications are regulated in a cell type-specific manner in the NAc. Therefore, precise examinations of BET proteins within this anatomically and functionally heterogeneous region are critically needed to fully understand the role of these proteins in complex addiction behaviors. My overall hypothesis is that modulation of epigenetic reader proteins in a cell type- and projection-dependent manner will attenuate cocaine seeking behaviors. Aim 1 will require training in FACS in order to determine the cell-type specific time course of BET mRNA/protein expression following short and long access to cocaine self-administration. Aim 2 will require proficiency in viral-mediated gene transfer techniques in order to manipulate BET proteins in specific cell types within the NAc during cocaine seeking behaviors. The R00 independent phase builds on this training to focus more specifically on connection-specific changes and manipulations of BET proteins during cocaine seeking behaviors. Together, these innovative studies will be the first to systemically explore epigenetic targets in cell type- and connection- specific manner during complex behaviors.