Novel Targets for Stroke Intervention - Gene Discovery for Modulators of Infarction

闭塞供应大脑的血管会导致缺血性中风和 脑梗塞--脑组织不可逆转的死亡。导致中风的危险因素，特别是涉及脂质的因素 新陈代谢，构成了目前降低中风风险的治疗方法的基础。然而，尽管几十年来 对脑梗塞期间发生的分子事件的研究，这些发现转化为 用于治疗中风结果(脑组织死亡)的“可用药”靶点一直令人相当失望。小说 将需要采取方法来确定新的、更具生理相关性的目标。科学的 我们建议的前提是，自然发生的等位基因变异是 这些神经保护性基因变异将提供一条新的途径 朝着中风治疗的新目标迈进。然而，基因作图方法在脑梗塞面积方面的应用 人类(例如，缺血性中风患者的脑梗塞体积的GWA)是固有的问题 闭塞血管的范围和位置的广泛变化，特别是时间上的变化 在第一次发现的症状和医疗干预之间的窗口。到目前为止，我们还没有找到发布的 对缺血性卒中患者的脑梗塞体积进行评估。马尔丘克实验室已经采取了另一种选择，正向遗传 发现调控脑梗塞的新基因的途径。我们已经通过外科手术封闭了中脑的远端 在超过35个近交系小鼠的大脑动脉中发现，脑梗塞体积相差50倍以上。 这些强大且高度可重复性的心肌梗死面积差异至少比我们所看到的大10倍。 但重要的是，这是由小鼠的自然等位基因变异引起的 基因组。我们已经绘制了其中几个遗传基因座的图谱，目标1和2的目标是识别 这些新基因调节着梗塞的大小。然而，这种基因发现方法需要在体内进行 在数以千计的成年动物身上进行外科检测。我们需要一个更具可伸缩性但在生理上更相关的 筛选平台，将这种方法转化为全基因组范围的规模。劳合社实验室率先推出了 模拟氧/葡萄糖卒中的脑梗塞发现平台的研制 在体外脑组织外植体中的剥夺(OGD；一种具有良好特征的缺血损伤模型)。不像 分离的神经元在培养中，脑片外植体保留了完整器官的复杂的多细胞性质， 从而保留并代表在脑组织中发生的复杂的细胞间相互作用 脑梗塞。在AIMS 1和AIMS 2中，这一体外OGD平台将用于确定病因 我们先前定位的基因座中的基因。我们在这些目标中获得的经验将导致目标3，在那里 体外OGD分析将用于直接定位和识别新的脑梗塞基因，使用 协同杂交的遗传作图资源群体。我们的研究利用了 由联合PIS开发的创新方法，以实施识别新药的新战略 治疗缺血性中风的靶点。

Occlusion of the blood vessels supplying the brain leads to ischemic stroke and infarction—irreversible death of brain tissue. Risk factors causing stroke, especially those involving lipid metabolism, form the basis of current therapies to reduce stroke risk. However, despite decades of research on the molecular events occurring during infarction, the translation of these discoveries to “druggable” targets to treat stroke outcome (death of brain tissue) has been quite disappointing. Novel approaches will be required to identify new and more physiologically relevant targets. The scientific premise of our proposal is that naturally occurring allelic variation underlies the profound differences in seen in stroke outcomes and that these neuro-protective gene variants would provide a novel path towards new targets for stroke treatment. However, genetic mapping approaches for infarct size in the human (e.g., GWAS of infarct volume among ischemic stroke patients) are intrinsically problematic due to wide variation in the extent and location of the occluded vessel, and especially, variation in the time window between first recognized symptoms and medical intervention. To date, we can find no published GWAS for infarct volume in ischemic stroke. The Marchuk lab has taken an alternative, forward genetic approach to discover novel genes modulating infarction. We have surgically occluded the distal middle cerebral artery in over 35 inbred mouse strains and found that infarct volume differs more than 50-fold. These robust and highly reproducible differences in infarct size are at least 10-fold larger than that seen in any engineered mouse lines but, importantly, are caused by natural allelic variation in the mouse genome. We have mapped several of these genetic loci and the goals of Aims 1 and 2 are to identify these novel genes regulating in infarct size. However, this gene discovery approach has required in vivo surgical assays in thousands of adult animals. We need a more scalable yet physiologically relevant screening platform to transform this approach to full genome-wide scale. The Lo lab has pioneered the development of such a discovery platform for cerebral infarction, simulating stroke by Oxygen/Glucose Deprivation (OGD; a well-characterized model for ischemic injury) in ex vivo brain tissue explants. Unlike isolated neurons in culture, brain slice explants retain the complex multicellular nature of the intact organ, and thus retain and represent the complex intercellular interactions occurring in brain tissue during cerebral infarction. In Aims 1 and 2, this ex vivo OGD platform will be used to identify the causative genes in our previously mapped loci. Our experience gained in these aims will lead to Aim 3, where the ex vivo OGD assay will be used to directly map and identify novel cerebral infarction genes, using the genetic mapping resource population of the Collaborative Cross. Our study takes advantage of innovative approaches developed by the co-PIs to implement a novel strategy for identifying novel drug targets to treat ischemic stroke.