Ciliary neurotrophic factor (CNTF) in the subventricular zone (SVZ) of the forebrain promotes neurogenesis in adult mice. We found that CNTF is normally repressed in the CNS by cell contact and increases greatly after stroke-induced neuron loss. This helped us to discover a new focal adhesion kinase (FAK)-JNK signaling pathway that is activated by ligand binding to DvE5 integrin in glia and potently inhibit their CNTF expression. Importantly, systemic treatment with pharmacological FAK inhibitors stimulates SVZ CNTF and neurogenesis. Our data show that stroke-induced neurogenesis is mediated entirely by stroke-induced CNTF, which may be counteracted by stroke-induced LIF and inflammation over the first week. In vitro, vitronectin represses CNTF and stimulates LIF, perhaps representing a vascular-derived regulator of neurogenesis. Here we will define these pathways in vivo, using a combination of available transgenic mice and pharmacological drugs in adult mice. Specifically, Aim 1a will determine whether vitronectin represses CNTF expression through the DvE5 integrin-FAK pathway in SVZ astrocytes under normal conditions. This will be done by intracerebral rescue experiments in vitronectin-/- mice, injecting integrin blocking antibodies against Dv and E5 integrin subunits, using E5 integrin-/- and inducible astroglial FAK-/- mice and defining effects on progenitors vs. neural stem cells (NSCs).
Aim 1 b will test whether this is mediated by JNK and/or other pathways, using intracerebral injections of pharmacological inhibitors and JNK-/- mice, while measuring effects on CNTF expression and neurogenesis.
Aim 2 a will determine whether stroke-induced leakage of blood-derived vitronectin causes increased LIF expression to stimulate NSC self-renewal. This will be done in a middle cerebral artery occlusion (MCAO) model by i.v. injections of vitronectin in vitronectin-/- mice.
Aim 2 b will determine whether the same integrin-FAK pathways play a role after MCAO, and whether CNTF and LIF can be regulated by FAK inhibitors.
Aim 2 c will define whether LIF is involved in repressing neurogenesis or is preconditioning the SVZ for later CNTF-induced neurogenesis.
Aim 3 a. Wwill test whether systemic FAK inhibitor treatment is more effective in inducing SVZ CNTF and neurogenesis when started after inflammation has resolved following MCAO, or Aim 3b when given intermittently. FAK acts via a different mechanism than the D2 dopamine receptor, which we showed increases SVZ neurogenesis by increasing astroglial CNTF. Therefore, Aim 3c will combine FAK inhibitor and D2 agonist to maximize CNTF expression and neurogenesis after MCAO, and determine whether this allows more new neurons to populate the injured tissue. The potential effects of these treatments on sensorimotor function will be assessed by behavioral tests.
This grant examines the role of our newly discovered integrin signaling pathway in reducing CNTF, one of the important molecules involved in stem cell proliferation and new cell production in the normal adult brain and after stroke. We previously found that stroke causes an increase in CNTF and this so-called neurogenesis, and that we can increase both by using pharmacological drugs which block this integrin pathway. Using both genetic approaches and pharmacological drugs, we expect to identify new treatments to maximize neurogenesis for replacing lost cells or protecting surviving ones that will hopefully extend beyond stroke to treatments of other neurological disease.
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