Prevention and acute tissue salvage have been the traditional treatment strategies for stroke, the third leading cause of death in the United States. New discoveries have created the possibility of a third treatment arm, regeneration. These discoveries show that stroke induces neural progenitor cell (NPC) proliferation in selected regions of injured brain, albeit with incomplete cell replacement. The factors that regulate these NPCs are incompletely understood. Ischemia may induce recapitulation of highly conserved developmental pathways that once regulated embryonic proliferation, differentiation and integration. In particular, the phylogenetically conserved sonic hedgehog (Shh) pathway may play a modulating role in ischemia-induced proliferation and differentiation/survival of NPCs. The goal of this project is to test the hypothesis that the sonic hedgehog pathway is essential for ischemia-induced proliferation and/or differentiation of hippocampal neural progenitor cells. Preliminary data show that ischemia up-regulates Shh within the hippocampus, and that hypoxia causes similar changes in embryonic NPCs in vitro. Furthermore, inhibiting Shh blocks the ischemia/hypoxia-induced proliferation of hippocampal and embryonic NPCs.
Aim 1 will test the hypothesis that NPCs isolated from adult hippocampus will respond to hypoxia in a manner reminiscent of embryonic NPCs. We believe it is important to understand the significance of Shh's impact on adult NPCs given that stroke disproportionately impacts adults, and adult and embryonic NPCs probably differ.
Aim 2 will expand on preliminary data suggesting that hypoxia-inducible factor 1 alpha (HIF1-alpha) up-regulates Shh and test the hypothesis that HIF1alpha mediates hypoxia-stimulated Shh transcription. Results of Aim 2 begin the important steps towards understanding the mechanism of Shh up-regulation.
Aim 3 a will test the hypothesis that smoothened (Smo), the protein transducer of the pathway, modulates the effect on hypoxia-induced proliferation of NPC cultures. We propose to promote a gain of function of Smo with the small molecule stimulator, leiosamine, or Smo over-expression. We propose to induce loss of function of Smo with the small molecule inhibitor, cyclopamine, or Smo RNAi.
Aim 3 b investigates Smo gain or loss of function in an in vivo stroke model to test the relevancy of Smo modulation on NPC response of proliferation and differentiation. My long-term goal is to understand the complex cellular environment that regulates NPC proliferation and differentiation in ischemic stroke. Understanding such processes may assist in identifying and tailoring novel post-stroke therapeutic modalities. While working toward this goal, with mentor guidance, I hope to obtain experience in the most up-to-date approaches in cellular and molecular neurobiology as well as general laboratory management skills that will provide a foundation for creating an independent laboratory.
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