Histone deacetylases - therapeutic targets for functional restoration after strok

描述（由申请人提供）：寻找改善缺血性卒中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、阿司匹林和卒中单位的管理。理想的中风治疗方法是将对成熟神经元和白质的损伤降到最低，并最大限度地从内源性祖细胞中产生新神经元。我们自己和其他人最近的发现，无论是在体外还是在中风的动物模型中，都表明组蛋白去乙酰化酶（HDAC）抑制剂符合这些标准。给药可保护分离的神经元免受诱导的凋亡细胞死亡和白质免受氧糖剥夺，改善脑缺血后的组织学和功能结果，并似乎可驱动多能神经祖细胞的“神经元”分化。因此，我们假设HDAC抑制在中风中可能具有急性作用（改善白质兴奋性毒性），并且在中期和长期内，通过减少细胞凋亡和促进再生而增加益处。由于许多HDAC抑制剂要么已经获得FDA批准（伏立诺他），要么由于其他原因在临床试验中取得进展（MS-275），那么重新利用一种或多种这些药物治疗中风可能会加快。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，这是我们感兴趣的焦点。在Aim 1中描述的研究旨在识别特定的hdac，这些hdac可以解释这些抑制剂的有益作用，使用体外和离体制剂，其中单个hdac被shRNA敲除，然后转导细胞/组织，然后进行氧葡萄糖剥夺。在确定了特定的HDAC后，我们将在Aim 2中探讨抑制HDAC有益作用的潜在底物和机制。最后，在最后一个目标中，我们考虑用一类HDAC抑制剂MS-275治疗小鼠是否能保持解剖完整性，促进短暂性脑缺血（大脑中动脉闭塞）后的长期功能（运动、感觉、认知）恢复，以及功能恢复是否与“神经发生”的程度相关。我们将使用两种最近开发的转基因细胞系p53+/+和p53 -/- mitoCFP小鼠，以便监测CFP+纤维束的缺血病理，评估与药物相关的线粒体频率和分布在神经元细胞体及其过程中的变化，并确定HDAC抑制最终是否涉及神经元p53以外的靶点。

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.