Small-molecule probes of chromatin-mediated neuroplasticity

描述(由申请人提供)：促进我们对神经可塑性的理解，并在此基础上开发新的治疗方法，对于改善神经系统疾病的治疗和预防至关重要。最近的分子、细胞和行为发现揭示了改变染色质结构的表观遗传机制在维持稳定的基因表达模式和改变与情绪和记忆形成相关的神经可塑性方面的重要性。然而，这些信号事件的动态性和组合性意味着我们对神经系统潜在分子机制的理解和能力仍然有限。为了克服这些限制，本提案中概述的研究的长期目标是系统地开发影响神经活动调节基因转录的染色质重塑复合体的选择性、脑穿透性、小分子探针(SMPS)。我们的总体假设是，通过选择性地靶向组蛋白脱乙酰酶(HDAC)和组蛋白乙酰转移酶(HAT)家族中特定成员的酶活性，有可能操纵组蛋白在某些即刻早期基因(IEGs)的启动子中的乙酰化状态，从而影响神经活性调节的基因转录和神经可塑性。为了开发严格检验这一假设所需的方法和SMP，拟议的研究将解决以下目标。在目标I中，将确定两种类型的SMPS的结构-活性-关系，它们通过影响某些HDAC和HAT亚型的活性来增强cAMP反应元件(CRE)介导的转录。作为一个子目标，使用亲和探针的蛋白质组谱将被用于确定两种类型SMPS靶向的染色质重塑复合体的成分。在AIM II中，将开发一种实时、自动的基于显微镜的成像分析方法，对表达IEG表达的遗传编码荧光报告的细菌人工染色体(BAC)转基因小鼠的培养神经元进行成像分析。作为一个次目标，本实验将与组蛋白修饰的免疫荧光检测法结合使用，以表征利用SMPS和RNAi介导的基因沉默来操纵HDAC/HAT-复合体活性对IEG表达的影响。在目标III中，将确定特定的HDAC抑制剂和HAT激活剂在小鼠海马区依赖记忆和抑郁样行为的行为测试中的作用，以及脑基因表达模式和组蛋白乙酰化的相应变化的测量。意义：我们预计这些多学科研究将为神经可塑性的分子机制以及这些机制与开发治疗记忆和情绪障碍的新疗法的相关性提供新的线索。公共卫生相关性：促进我们对大脑可塑性的理解，并在此基础上开发新的治疗方法，对于改善对无数中枢神经系统疾病的治疗和预防至关重要。这项工作将使用小分子作为探针，在生化和小鼠行为研究中表征基因表达在调节与情绪和记忆障碍相关的大脑可塑性方面所起的作用。这些多学科研究将为大脑可塑性的分子机制以及这些机制与开发治疗记忆和情绪障碍的新疗法的相关性提供新的线索。

DESCRIPTION (provided by applicant): Advancing our understanding of neuroplasticity and the development of novel therapeutics based upon this knowledge is critical in order to improve the treatment and prevention of nervous system disorders. Recent molecular, cellular, and behavioral findings have revealed the importance of epigenetic mechanisms that alter chromatin structure in maintaining stable patterns of gene expression and altering neuroplasticity associated with mood and memory formation. However, the dynamic and combinatorial nature of these signaling events has meant that the state of our understanding and ability to manipulate the underlying molecular mechanisms in the nervous system remains limited. To overcome these limitations, the long-term goals of the studies outlined in this proposal are to systematically develop selective, brain-penetrant, small-molecule probes (SMPs) of chromatin-remodeling complexes that affect neural activity-regulated gene transcription. Our overall hypothesis is that by selectively targeting the enzymatic activity of specific members of the histone deacetylase (HDAC) and histone acetyltransferase (HAT) families that it will be possible manipulate the acetylation state of histones in the promoters of certain immediate early genes (IEGs) thereby affecting neural- activity-regulated gene transcription and neuroplasticity. To develop the methods and SMPs necessary to rigorously test this hypothesis the proposed studies will address the following aims. In Aim I, the structure- activity-relationships of two types of SMPs that enhance cAMP response element (CRE)-mediated transcription through affecting the activity of certain HDAC and HAT isoforms will be determined. As a sub-aim, proteomic profiling using affinity probes will be used to determine the components of the chromatin-remodeling complexes targeted by both types of SMPs. In Aim II, a real time, automated microscopy-based imaging assay of cultured neurons from bacterial artificial chromosome (BAC)-transgenic mice expressing a genetically encoded fluorescent reporter of IEG expression, will be developed. As a sub-aim, this assay will be used in combination with immunofluorescent detection of histone-modifications to characterize the effect of manipulating HDAC/HAT-complex activities on IEG expression using SMPs and RNAi-mediated gene silencing. In Aim III, the effect of specific HDAC inhibitors and HAT activators in mouse behavioral tests of hippocampal-dependent memory and depression-like behavior will be determined along with measurements of corresponding changes in brain gene expression patterns and histone acetylation. Significance: We anticipate these multidisciplinary studies will shed new light on molecular mechanisms of neuroplasticity and the relevance of these mechanisms to the development of novel therapeutics for memory and mood disorders. PUBLIC HEALTH RELEVANCE: Advancing our understanding of brain plasticity and the development of novel therapeutics based upon this knowledge is critical in order to improve the treatment and prevention of a myriad of central nervous system disorders. This work will characterize the role that gene expression plains in mediating aspects of brain plasticity relevant to mood and memory disorders using small molecules as probes in biochemical and mouse behavioral studies. These multidisciplinary studies will shed new light on molecular mechanisms of brain plasticity and the relevance of these mechanisms to the development of novel therapeutics for their treatment of memory and mood disorders.