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Dhivyaa Alagappan
Interdisciplinary Ph.D. Program

B. Tech., 2002, Anna University, India

Thesis Advisor: Steven W. Levison, Ph.D.
Department of Neurology and Neuroscience

Cancer Center, G level Conference Room (G-1196)

Friday, March 14, 2008
3:00 p.m.


Brain injury, as a result of an hypoxic/ischemic (H/I) insult, remains a major cause of neurologic disability in the newborn and affects up to 2-3 per 1000 term births and a higher percentage of premature infants. The surviving infants develop neurological and psychological problems ranging from classical cerebral palsy to mild learning deficits that may not appear until later in life. Presently, there are no treatments to prevent this brain injury or therapeutic regimens to stimulate brain regeneration and repair. The discovery that the brain retains a population of neural stem cells that persists through adult life that have the capacity to form multiple brain cell types upon stimulation offers the promise of regeneration and repair after brain injury from an endogenous source. Therefore, the rationale for our research has been to identify mechanisms that regulate the expansion of these endogenous neural stem cells and progenitors (NSPs) present in the subventricular zone (SVZ) of the postnatal brain for regeneration. The goals of this investigation were to test the hypotheses that: 1) Brain injury expands the numbers of NSPs in the SVZ by enhancing their responsiveness to epidermal growth factor (EGF); 2) Early growth response 1 (Egr-1) is a critical regulator of EGF-mediated expansion of the precursors within the SVZ during recovery from brain injury; 3) IGF-1R signaling is necessary for the EGF-mediated expansion of SVZ NSPs following neonatal H/I.
Using rat and murine models of neonatal hypoxia/ischemia, we show that the injury induces quiescent SVZ precursors to enter the cell cycle and it reduces their cell cycle time, which is EGF-dependent, and FGF-2 independent. EGF receptor (EGF-R) expression within the damaged SVZ increased three-fold at the mRNA. Flow cytometric analyses revealed increased EGF-R expression on both lineage marker positive progenitors and on putative neural stem cells along with increased EGF-R expression per cell. Pharmacologically inhibiting the EGF-R using a highly-specific quinazoline (PD153035) reduced the expansion of NSPs following injury.
EGF-R induction after injury could be recapitulated in vitro in the absence of a brain cell niche by exposing NSPs to a combination of hypoxia (2% O2) and hypoglycemia (3 mM glucose) whereas neither condition alone was sufficient. This combined insult stimulated the zinc finger transcription factor Egr-1 to accumulate in the cell nucleus resulting in an increase in Egr-1 bound to DNA-elements on the EGF-R promoter sequence. Confirming the role of this stress response gene protein in regulating EGF-R expression, suppressing Egr-1 expression using silencing RNAs targeted against Egr-1 dramatically decreased the expression of the EGF-R within NSPs and abrogated the increase in EGF-R after hypoxia/hypoglycemia.
Signaling through the IGF receptor was also necessary for the EGF-mediated NSP expansion following neonatal H/I. The conventional growth conditions for NSPs use superphysiological levels of insulin [25g/ml] and EGF. Placing NSPs into cell culture medium with a physiological concentration of insulin [25ng/ml] (which is insufficient to stimulate the IGF-1 type 1 receptor) compromised their ability to form neurospheres and severely curtailed their ability to proliferate as indicated by a dramatic decrease in the size of the spheres. This reduced growth capacity could not be attributed simply to failure to survive, as NSPs maintained in low insulin media and EGF for 7 days retained the capacity to form spheres when transferred to high insulin medium containing EGF. Not only did the IGF-1R appear to be required for the normal proliferation of NSPs, but superphysiological concentrations of insulin were also required for the expansion of NSPs after neonatal H/I. Moreover, FGF-2 addition could not compensate for the presumed reduction in IGF-1R signaling.
On the basis of these observations, we conclude that H/I causes the accumulation of Egr-1, which in turn enhances the expression of EGF-R in NSPs. As a consequence of increased EGF-R, together with IGF-1R, signaling quiescent stem cells are recruited to enter the cell cycle and as a consequence of enhanced EGF-R signaling, the rate at which they traverse the cell cycle is increased. These processes increase the size of the neural precursor pool available for regenerating the cells lost as a consequence of injury.

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