Histone deacetylases - therapeutic targets for functional restoration after strok

描述(由申请人提供)：找到改善缺血性中风预后的干预措施已被证明是具有挑战性的，目前的治疗仅限于溶栓、阿司匹林和卒中单元的治疗。理想的中风疗法将最大限度地减少对成熟神经元和白质的损害，并最大限度地从内源性祖细胞产生新的神经元。我们自己和其他人在体外和中风动物模型中的最新发现表明，组蛋白脱乙酰酶(HDAC)抑制剂符合这些标准。给药保护分离的神经元免受诱导的细胞凋亡，保护脑白质免受缺氧-葡萄糖剥夺的影响，改善脑缺血后的组织学和功能结果，并似乎推动多潜能神经前体细胞的“神经元”分化。因此，我们假设在中风中抑制HDAC可能有急性影响(改善脑白质兴奋性毒性)，从中长期来看，通过减少细胞凋亡和鼓励再生而增加益处。由于许多HDAC抑制剂要么已经得到FDA(Vorinostat)的批准，要么由于其他原因在临床试验中处于良好的先进状态(MS-275)，那么这些药物中的一种或多种用于中风的重新用途可能会加快。此外，新的特定抑制剂的开发正在进行中，特别是针对I类HDAC，这是这里感兴趣的焦点。目标1中描述的研究旨在使用体外和体外准备方法确定说明这些抑制剂有益作用的特定HDAC，在这些准备中，单个HDAC被shRNA击倒，转导的细胞/组织然后受到氧-葡萄糖剥夺。在确定了特定的HDAC之后，我们将在目标2中探索对HDAC抑制的有益作用负责的潜在底物和机制。最后，在最后一个目标中，我们考虑了使用I类HDAC抑制剂MS-275治疗小鼠是否保持了解剖完整性，并促进了从短暂性脑缺血(大脑中动脉闭塞)中恢复长期功能(运动、感觉、认知)，以及功能的恢复是否与“神经发生”的程度相关。最近开发的两个转基因系，P53/和P53-/-mitoCFP小鼠，将被用于监测CFP纤维束的缺血病理，评估药物相关的线粒体频率和分布在神经元细胞体及其突起中的变化，并确定HDAC抑制最终是否涉及神经元P53以外的靶点。 公共卫生相关性：本提案是针对PA-08-099卒中后功能恢复机制提出的，“这是一个资助机会，可以促进研究，了解导致功能恢复的大脑修复过程，以便开发方法来优化现有做法，并开发新的方法来改善卒中后的结果”。目前，还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，脑损伤后广泛抑制组蛋白脱乙酰酶(HDAC)活性可起到神经元保护作用，并促进新神经元的生成。这项提案中概述的研究的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。选定的药物将进行测试，以揭示它们对小鼠从中风损伤中恢复有什么功能上的好处。

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.