Objectives: The overall goal of this proposal is to determine the importance of adult neurogenesis to the recovery process following stroke. Adult-born neurons are a potential substrate for repair after focal ischemic injury, and it is well established that neural stem cells (NSCs) proliferate and their progeny migrate to sites of injury after experimental stroke. Accumulating evidence indicates that large numbers of adult-generated neurons migrate to the injured striatum and cortex following stroke, but few of these neurons integrate and survive for prolonged periods. Data are conflicting, moreover, as to the phenotypes of the neurons that do survive and the extent to which they integrate into brain circuitry. Determination of cell fate or functional integration is difficult because the tools used to label these cells have been limited. Research Plan: In the proposed studies, innovative transgenic mouse models and retroviral techniques will be used to specifically label or ablate adult-generated neurons in the setting of experimental stroke. In addition, our preliminary data from a Bax knockout mouse line defective in a critical programmed cell death pathway for adult NSCs suggest that more newborn neurons survive after excitotoxic striatal injury in these mice. Using these genetic approaches, we will test our central hypothesis that adult-generated neurons contribute to repair after focal ischemia via neuronal replacement; moreover, inhibiting this process will be detrimental to stroke recovery and, conversely, stimulating the survival of adult NSC progeny will improve recovery. Studies in Specific Aim 1 will characterize the long-term survival, phenotypes and integration of adult-born neurons after focal ischemic injury. Experiments in Specific Aim 2 will determine if adult neurogenesis is critical to the repair process by ablating newly dividing cells following stroke.
In Aim 3, we will use conditional Bax null mice to examine whether loss of the pro-apoptotic protein Bax selectively in adult-generated neurons leads to their increased survival and integration, and improved functional recovery after focal ischemic injury. Methods: We will use the transient middle cerebral artery occlusion model in adult mice.
Aim 1 will be accomplished by using retroviral labeling of dividing cells and conditional transgenic NSC reporter mice to track the fate of adult-generated neurons.
In Aim 2, we will ablate adult-generated neurons using a conditional transgenic mouse model that expresses a pharmacologically activated suicide gene selectively in NSCs. The methods for Aim 3 will involve crossing a neuroblast-specific cre driver mouse line (doublecortin-CreERT2) with a floxed Bax line to delete Bax in neuronal precursors generated after stroke.
For Aims 2 and 3, we will determine the influence of experimental manipulations on infarct size histologically, and animals will be behaviorally tested to determine if suppressing neurogenesis impairs or stimulating neurogenesis improves, respectively, motor skill recovery after focal ischemic injury. Clinical Relevance: No regenerative therapies exist for stroke. Progress in these aims will increase our understanding of reparative processes in the brain following focal ischemic injury and offers the potential to contribute to novel regenerative therapies for stroke, a disorder that leads to a substantial disease burden in the Veteran population.
Project Narrative Stroke is a common and potentially devastating neurological disorder with no proven regenerative therapies. Neural stem cells (NSCs) exist in the adult brain and give rise to new nerve cells after stroke, but most fail to survive and rewire the brain. Using novel mouse models, this proposal aims to determine whether the new nerve cells born in the adult brain after stroke actually rewire and contribute to repair, and if stimulating this process will improve stroke recovery. Progress in this area offers advances toward novel restorative therapies for stroke and other brain insults.
