Date of Award
Doctor of Philosophy (PhD)
Jeffrey L. Spees
Stroke or cerebral vascular accident (CVA) is the 4th leading cause of mortality and the principle cause of long-term disability in the United States. Unfortunately, current reperfusion-based treatments (e.g. thrombolysis, tPA) cannot be administered to the majority of patients presenting with ischemic stroke. Accordingly, new treatments for ischemic stroke are desperately needed.
Reactive astrocytes perform key roles in tissue repair and remodeling following stroke such as preservation and repair of the blood-brain barrier, modulation of immune cell invasion, glutamate uptake and neuroprotection, and glial scar formation. The proliferative subpopulation of reactive astrocytes found immediately adjacent to the infarct core after stroke (known as the peri-infarct area) is particularly important for protecting the brain parenchyma from ischemic damage and inflammation. Defining the signaling network that controls reactive astrocyte formation and function has potential to provide new treatment strategies for patients ineligible for reperfusion therapy.
Notch1 signaling is required for the proliferation of peri-infarct reactive astrocytes after stroke. To identify downstream targets and potential functional effectors of Notch1 signaling in reactive astrocytes, we developed an ex vivo forward signaling screen. To generate large quantities of adult reactive astrocytes, we employed adult Reactive astrocyte-derived Neural Stem Cells (Rad-NSCs) isolated from the peri-infarct area of mice after stroke. Astrocytes re-differentiated from Rad-NSCs (AstroRad-NSC) were then exposed to immobilized Jagged-1, a Notch1 ligand. In response to Jagged-1, many genes involved in reactive astrocyte-mediated tissue protection, metabolic regulation, angiogenesis and glial scar formation were up-regulated. Of special interest, several genes for proteins that regulate with glutamate uptake and metabolism were increased by Jagged-1/Notch signaling, including the glial-specific GLutamate-ASpartate Transporter (GLAST). With loss-of-function experiments, we determined that deletion of Notch1 decreased GLAST transcript and protein levels in cultured AstroRad-NSC. Furthermore, we isolated reactive astrocytes directly from cerebral cortex after stroke and confirmed the effects of Notch1 on GLAST in vivo. Our results suggest that treatments designed to stimulate Notch1 signaling after stroke may promote glutamate uptake, thereby decreasing excitotoxicity and neuronal cell death.
Binding of Endothelin peptides to the type B Endothelin receptor (ETBR) has been shown to alter cell proliferation. Investigating a possible relationship between Jagged-1/Notch1 and Endothelin signaling in reactive astrocytes, we determined that Notch1 signaling regulated ETBR indirectly, by activating STAT3, an unidentified transcriptional activator of ETBR. Using inducible transgenic astrocyte-specific conditional knockout (cKO) mice (GFAP-ETBR-cKO), we found that specific deletion of ETBR in reactive astrocytes phenocopied the defect in reactive astrocyte proliferation observed in our previous work with GFAP-Notch1-cKO mice. Notably, the Notch1-STAT3-ETBR axis we identified is likely to control reactive astrocyte proliferation in most, if not all, forms of CNS injury.
The experimental results presented in this doctoral dissertation provide novel insight into signaling mechanisms that may someday be exploited to improve care for patients with stroke and other forms of CNS injury or disease.
Number of Pages
LeComte, Matthew David, "Direct And Indirect Targets Of Jagged1/notch1 Signaling In Reactive Astrocytes." (2014). Graduate College Dissertations and Theses. 271.