Histone deacetylases - therapeutic targets for functional restoration after strok

描述（由申请人提供）：发现改善缺血性卒中结局的干预措施已被证明具有挑战性，目前的治疗仅限于溶栓、阿司匹林和卒中单元管理。一种理想的中风治疗方法将最大限度地减少对成熟神经元和白色物质的损伤，并最大限度地从内源性祖细胞产生新的神经元。我们自己和其他人最近在体外和中风动物模型中的发现表明，组蛋白去乙酰化酶（HDAC）抑制剂符合这些标准。给药保护分离的神经元免于诱导的凋亡性细胞死亡和白色物质免于氧-葡萄糖剥夺，导致脑缺血后改善的组织学和功能结果，并且似乎驱动多能神经祖细胞的“神经元”分化。因此，我们假设HDAC抑制中风可能有急性效应（改善白色物质兴奋性毒性），并在中期和长期，通过减少细胞凋亡和促进再生增加的好处。由于许多HDAC抑制剂已经被FDA批准（伏立诺他），或者由于其他原因在临床试验中进展良好（MS-275），因此可以加速将这些药物中的一种或多种重新用于中风。此外，新的特异性抑制剂的开发正在进行中，特别是针对I类HDAC的抑制剂，这是本文关注的焦点。目的1中所述的研究旨在使用体外和离体制剂来鉴定解释这些抑制剂的有益作用的特定HDAC，其中用shRNA敲除个体HDAC，然后使转导的细胞/组织经受氧-葡萄糖剥夺。在确定了特定的HDAC后，我们将在目标2中探索负责HDAC抑制的有益作用的潜在底物和机制。最后，在最后一个目标中，我们考虑用MS-275（一种I类HDAC抑制剂）治疗小鼠是否保持解剖完整性并促进短暂脑缺血（大脑中动脉闭塞）的长期功能（运动、感觉、认知）恢复，以及功能恢复是否与“神经元发生”的程度相关。两个最近开发的转基因系，p53+/+和p53 -/- mitoCFP小鼠，将采用，使我们可以监测缺血性病理CFP+纤维束，评估药物相关的变化，线粒体频率和分布在神经元细胞体及其过程中，并确定是否HDAC抑制最终涉及的目标，除了神经元p53。 公共卫生相关性：该提案是为了响应PA-08-099卒中后功能恢复机制而提交的，“这是一个促进研究的资助机会，旨在了解导致功能恢复的大脑修复过程，以开发优化现有实践的方法，并开发改善卒中后结局的新方法”。目前，没有药物可用于增强人类中风后的神经保护和恢复功能。实验动物研究表明，广泛抑制组蛋白去乙酰化酶（HDAC）的活动在脑损伤后的结果在神经元的保护，也促进新的神经元的产生。本提案中概述的研究目的是确定涉及哪些HDAC，以便选择针对这些特定酶的特定药物。将对选定的药物进行测试，以揭示它们对中风损伤小鼠恢复的功能益处。

DESCRIPTION (provided by applicant): Finding interventions that improve outcome from ischemic stroke has proved to be challenging and current treatment is limited to thrombolysis, aspirin, and management in a stroke unit. An ideal stroke therapeutic would minimize damage to mature neurons and white matter, and also maximize the generation of new neurons from endogenous progenitors. Recent findings of our own and of others, both in vitro and in animal models of stroke, suggest that histone deacetylase (HDAC) inhibitors meet these criteria. Drug administration protects isolated neurons from induced apoptotic cell death and white matter from oxygen-glucose deprivation, results in improved histologic and functional outcomes following cerebral ischemia, and appears to drive 'neuronal' differentiation of multipotent neural progenitor cells. Therefore, we hypothesize that HDAC inhibition in stroke may have acute effects (to ameliorate white matter excitotoxicity), and in the intermediate and longer term, added benefit by reducing apoptosis and encouraging regeneration. As a number of HDAC inhibitors are either already approved by the FDA (vorinostat), or well-advanced in clinical trials (MS-275) for other reasons, then re- purposing one or more of these drugs for stroke could be expedited. In addition, development of new and specific inhibitors is ongoing, especially against the Class I HDACs which are the focus of interest here. Studies described in Aim 1 are designed to identify the specific HDACs which account for the beneficial actions of these inhibitors, using in vitro and ex vivo preparations in which individual HDACs are knocked down with shRNA and transduced cells/tissues then subjected to oxygen-glucose deprivation. Having identified specific HDACs, we shall explore in Aim 2 the potential substrates and mechanisms responsible for the beneficial actions of HDAC inhibition. Finally, in the last aim we consider whether treatment of mice with MS-275, a Class I HDAC inhibitor, preserves anatomic integrity and promotes long term functional (motor, sensory, cognitive) recovery from transient cerebral ischemia (middle cerebral artery occlusion), and whether restoration of function correlates with the extent of 'neuronogenesis'. Two recently developed transgenic lines, p53+/+ and p53 -/- mitoCFP mice, will be employed so that we can monitor ischemic pathology in CFP+ fiber tracts, assess drug-associated changes in mitochondrial frequency and distribution within neuronal cell bodies and their processes, and also determine whether HDAC inhibition ultimately involves targets in addition to neuronal p53. PUBLIC HEALTH RELEVANCE: This proposal is submitted in response to PA-08-099 Mechanisms of functional recovery after stroke, "a funding opportunity to promote research to understand the processes of brain repair that lead to functional recovery in order to develop methods to optimize existing practices and to develop new approaches to improve post-stroke outcomes". Currently, there is no drug available to enhance neuroprotection and restore function following human stroke. Experimental animal studies suggest that broad inhibition of histone deacetylase (HDAC) activities in the brain following injury results in neuronal protection and also promotes the generation of new neurons. The purpose of the studies outlined in this proposal is to identify which HDACs are involved in order to select specific drugs to target these particular enzymes. Selected drugs will be tested to reveal what functional benefits they confer on the recovery of mice from stroke injury.