Glucocorticoids exacerbate hypoxia induced neuronal death in the developing rat cortex through regulation of the pro-apoptotic gene Bnip3

Abstract

Dexamethasone (DEX) is a synthetic glucocorticoid that is prescribed to treat premature infants with bronchopulmonary dysplasia. However, glucocorticoids exacerbate metabolic insults, such as hypoxia-ischemia, in the rat brain. Furthermore, DEX, acting independent of a secondary insult, induces apoptosis in the developing rat cortex and hippocampus. In the adult rat hippocampus, DEX-induced apoptosis is attributed to Bcl-2 family member regulation, but in the developing brain the putative effector proteins for DEX-mediated apoptosis have yet to be identified. Bnip3 is a pro-apoptotic Bcl-2 family member that is upregulated in the mature rat brain during hypoxia-ischemia. Since Bnip3 is a hypoxic responsive gene, the experiments in this dissertation investigate Bnip3 as a putative effector protein for glucocorticoid exacerbation of hypoxic insults in the developing rat cortex. In chapter 3, I determined Bnip3 localization and mRNA ontogeny in the postnatal rat brain to identify brain regions and ages that may be susceptible to hypoxic insults. Bnip3 mRNA was localized to the neonatal cortex and hippocampus. Furthermore, Bnip3 mRNA levels were found to be greatest at postnatal day 6.5 in the female anterior and posterior cingulate cortices and hippocampus. Conversely, in the male brain Bnip3 mRNA was only increased in the anterior cingulate cortex and this was at postnatal day 6.5. In chapter 4, I subsequently investigated glucocorticoid and hypoxia interactions of Bnip3 mRNA regulation. I found that Bnip3 mRNA expression was increased in neonatal rat pups treated with DEX. An increase in Bnip3 mRNA was also measured in primary cortical neurons 72 hours after treatment with RU28362, a glucocorticoid receptor selective agonist. In cortical neurons, hypoxia increased Bnip3 mRNA expression and this was exacerbated with RU28362 treatment. This treatment paradigm also corresponded to decreased cell viability. Glucocorticoid and hypoxia regulation of the Bnip3 promoter was found to be mediated by a glucocorticoid response element and hypoxic response element. Finally, Bnip3 overexpression in cortical neurons increased cell death. This effect was eliminated when the Bnip3 transmembrane domain was removed. Cumulatively, these studies identify a novel pathway in the developing cortex through which glucocorticoids may exacerbate a metabolic insult, such as hypoxia.