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

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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.