Neuronal subtype and circuit-specific epigenetic mechanisms in addiction

 描述（由申请人提供）：本提案要求支持一个全面的培训计划，使候选人，格雷戈里博士。Sartor，以扩大，发展和完善的技术技能，是必要的一个富有成效的独立研究生涯。培训计划的目标是通过有监督的实践培训和教学课程获得先进的分子生物学技术，以便将复杂的方法纳入专注于阐明成瘾细胞类型和回路特异性表观遗传机制的特定目标。高级科学培训将集中在两个关键领域：1）成瘾相关脑区神经元亚型的细胞活化细胞分选（FACS），以及2）开发病毒介导的基因转移工具，以细胞类型和连接特异性方式操纵表观遗传靶点。职业发展活动将由Claes Wahlestedt博士指导，并得到Kasahara博士和Gray博士的合作支持，他们在所提出的方法方面都具有非重叠的专业知识，并在指导博士后学员方面有着出色的记录。研究部分将研究乙酰赖氨酸阅读器蛋白，称为BET溴结构域，这是一种新兴的可药用表观遗传靶点，目前正在多项临床试验中进行研究。这些含溴结构域的蛋白结合乙酰化组蛋白，并作为一个骨架的大分子复合物，修改染色质的可及性和转录活性的招聘。我最近发现，BET蛋白上调，并招募到启动子区的BDNF在神经核（NAc）慢性可卡因管理。此外，我的数据表明，这些BET蛋白质（全身和内NAc）的药理学抑制减弱可卡因的行为反应。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中以细胞类型特异性的方式进行调节。因此，在这个解剖学上和功能上异质的区域内的BET蛋白的精确检查是非常需要的，以充分理解这些蛋白在复杂的成瘾行为中的作用。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求行为。目标1将需要在FACS中的训练，以确定在短期和长期获得可卡因自我施用后BET mRNA/蛋白质表达的细胞类型特异性时间过程。目标2将需要熟练掌握病毒介导的基因转移技术，以便在可卡因寻求行为期间操纵NAc内特定细胞类型中的BET蛋白。R00独立阶段建立在这种训练的基础上，更具体地关注可卡因寻求行为期间BET蛋白质的连接特异性变化和操纵。总之，这些创新的研究将是第一个系统地探索表观遗传目标在细胞类型和连接特定的方式在复杂的行为。

 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.