Stroke causes cell death but also birth and migration of new neurons within sites of ischemic damage. The cellular environment that induces neuronal regeneration and migration after stroke has not been defined. We have used a model of long-distance migration of newly born neurons from the subventricular zone to cortex after stroke to define the cellular cues that induce neuronal regeneration after CNS injury. Mitotic, genetic, and viral labeling and chemokine/growth factor gain-and loss-of-function studies show that stroke induces neurogenesis from a GFAP-expressing progenitor cell in the subventricular zone and migration of newly born neurons into a unique neurovascular niche in peri-infarct cortex. Within this neurovascular niche, newly born, immature neurons closely associate with the remodeling vasculature. Neurogenesis and angiogenesis are causally linked through vascular production of stromal-derived factor 1 (SDF1) and angiopoietin 1 (Ang1). Furthermore, SDF1 and Ang1 promote post-stroke neuroblast migration and behavioral recovery. These experiments define a novel brain environment for neuronal regeneration after stroke and identify molecular mechanisms that are shared between angiogenesis and neurogenesis during functional recovery from brain injury.

Erythropoietin (EPO) is the principal growth factor regulating the production of red blood cells. Recent studies demonstrated that exogenous EPO acts as a neuroprotectant and regulates neurogenesis. Using a genetic approach, we evaluate the roles of endogenous EPO and its classical receptor (EPOR) in mammalian neurogenesis. We demonstrate severe and identical embryonic neurogenesis defects in animals null for either the Epo or EpoR gene, suggesting that the classical EPOR is essential for EPO action during embryonic neurogenesis. Furthermore, by generating conditional EpoR knock-down animals, we demonstrate that brain-specific deletion of EpoR leads to significantly reduced cell proliferation in the subventricular zone and impaired post-stroke neurogenesis. EpoR conditional knockdown leads to a specific deficit in post-stroke neurogenesis through impaired migration of neuroblasts to the peri-infarct cortex. Our results suggest that both EPO and EPOR are essential for early embryonic neural development and that the classical EPOR is important for adult neurogenesis and for migration of regenerating neurons during post-injury recovery.

Here we show that conditional deletion of Pten in a subpopulation of adult neural stem cells in the subependymal zone (SEZ) leads to persistently enhanced neural stem cell self-renewal without sign of exhaustion. These Pten null SEZ-born neural stem cells and progenies can follow the endogenous migration, differentiation, and integration pathways and contribute to constitutive neurogenesis in the olfactory bulb. As a result, Pten deleted animals have increased olfactory bulb mass and enhanced olfactory function. Pten null cells in the olfactory bulb can establish normal connections with peripheral olfactory epithelium and help olfactory bulb recovery from acute damage. Following a focal stroke, Pten null progenitors give rise to greater numbers of neuroblasts that migrate to peri-infarct cortex. However, in contrast to the olfactory bulb, no significant long-term survival and integration can be observed, indicating that additional factors are necessary for long-term survival of newly born neurons after stroke. These data suggest that manipulating PTEN-controlled signaling pathways may be a useful step in facilitating endogenous neural stem/progenitor expansion for the treatment of disorders or lesions in regions associated with constitutive neurogenesis.

Stroke induces proliferation of newly born neurons in the subventricular zone, migration of these immature neurons away from the SVZ, and localization within peri-infarct tissues. These 3 processes of proliferation, migration, and localization constitute distinct spatial and temporal zones within poststroke neurogenesis with distinct molecular and cell-cell signaling environments. Immature neurons migrate after stroke in close association with blood vessels and astrocytic processes, in a process that involves matrix metalloproteinases. This poststroke migration shares similar features with normal neuroblast migration in the rostral migratory stream. Immature neurons localize in the peri-infarct cortex in a neurovascular niche where neurogenesis is causally linked to angiogenesis through the vascular factors SDF-1 and angiopoietin-1. Other vascular and neuronal growth factors have also been linked to poststroke neuroblast localization in peri-infarct tissue, including erythropoietin. Most data on poststroke neurogenesis derive from laboratory rodents, which may have an abnormal or blunted degree of neurogenesis and neuroplasticity compared to normal, wild rodents. This will likely affect translational application of the principles of poststroke neurogenesis from mouse to man.

A fundamental organizing feature of the retina is the presence of regularly spaced distributions of neurons, yet we have little knowledge of how this patterning emerges during development. Among these retinal mosaics, the spatial organization of the dopaminergic amacrine cells is unique: using nearest-neighbor and Vornoi domain analysis, we found that the dopaminergic amacrine cells were neither randomly distributed, nor did they achieve the regularity documented for other retinal cell types. Autocorrelation analysis revealed the presence of an exclusion zone surrounding individual dopaminergic amacrine cells and modeling studies confirmed this organization, as the mosaic could be simulated by a minimal distance spacing rule defined by a broad set of parameters. Experimental studies determined the relative contributions of tangential dispersion, fate determination, and cell death in the establishment of this exclusion zone. Clonal boundary analysis and simulations of proximity-driven movement discount tangential dispersion, while data from bcl-2 overexpressing mice rule out feedback-inhibitory fate-deterministic accounts. Cell death, by contrast, appears to eliminate dopaminergic amacrine cells that are within close proximity, thereby establishing the exclusion zone surrounding individual cells and in turn creating their mosaic regularity.

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Stroke induces neural stem cells to differentiate into immature neuroblasts, migrate from the subventricular zone into the area of damage, and differentiate into neurons. These findings suggest that the adult brain has a limited process of neuronal regeneration after stroke. Neural stem cells depend on a biochemical niche that is linked to the vascular system and utilize vascular endothelial growth factor (VEGF) and erythropoietin (Epo) for neuronal differentiation. Together, VEGF and Epo are under the control of the hypoxia inducible transcription factor (Hif-1). Hif-1 is induced after focal ischemia and the Hif-1 target genes VEGF and Epo are increased near the area of injury after stroke. This data shows that the neural stem cells utilize the Hif system, and that Hif signaling is induced by stroke. These findings have lead to the hypothesis for my project: that HIF-1a, VEGF, and Epo are important for subventricular-derived neuroblast migration to areas of injury following a focal stroke. The specific aims for this project are to determine the colocalization of the Hif system with migrating neuroblasts after stroke. I will characterize the expression patterns of HIF1, VEGF, Epo and EpoR in association with migration of neuroblasts to peri-cortex. Focal ischemia will be induced with permanent distal MCA ligation. Animals will be sacrificed, and analyzed immunohistochemically for the pattern and of HIF-1, VEGF, Epo and EpoR. Hif-1, VEGF, Epo and EpoR staining will be performed with polysialic neural cell-adhesion molecule staining in confocal immunofluorescence to co-localize components of the Hif system with migrating neuroblasts.

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