Neonatal Bilirubin Neurotoxicity and P-Glycoprotein

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DESCRIPTION (provided by applicant): P-glycoprotein (Pgp), an ATP-binding cassette efflux pump expressed on endothelial cells and astrocytes of the bloodbrain barrier (BBB), may serve an important role in limiting the passage of bilirubin into the central nervous system (CNS). Studies performed during years 01-04 have demonstrated: i) Pgp mediated bilirubin transport in vitro; ii) bilirubin stimulated Pgp ATPase activity; iii) limited Pgp expression in the immature murine and human CNS; and iv) a marked early postnatal increase in BBB Pgp. These novel observations raise the distinct possibility that human BBB Pgp attenuates CNS bilirubin content, a phenomenon we have shown in Pgp sufficient as compared with Pgp deficient null mutant transgenic mice (167), thereby diminishing the risk for kernicterus. Preliminary studies in our lab show that Pgp i) confers cellular resistance against bilirubin-induced apoptosis, and ii) is expressed in selected cells of the CNS parenchyma in addition to those of the BBB in developing human neonates. Taken together, these findings suggest that Pgp may confer protection against bilirubin induced cell injury both at the BBB and within the CNS parenchyma. In the current proposal, we will extend our previous observations and recent preliminary findings by testing three hypotheses: 1) Pgp acts to inhibit cellular apoptosis induced by bilirubin in vitro and in vivo; 2) Pgp is expressed in cells of the CNS parenchyma as well as cells of the BBB in humans; and 3) CNS Pgp expression is upregulated by postnatal hyperbilirubinemia and antenatal maternal vitamin A treatment in Gunn rat pups, and that combined antenatal vitamin A treatment with postnatal hyperbilirubinemia will be protective against bilirubin-induced apoptosis in vivo. We will use cell lines (Caco-2, neuroblastoma, bovine brain capillary endothelial and LLC-PK1 cells tranfected with the gene for human or murine Pgp) and the Gunn rat model of neonatal jaundice to characterize the role of Pgp in protecting against bilirubin-induced apoptosis, the apoptotic pathways involved, and the contribution of apoptosis to cell death. The prevalence of apoptosis in kernicterus in human neonates, as well as the ontogeny and regional localization of human CNS Pgp expression will be assessed in archival postmortem tissue. The information obtained will provide novel insights regarding the role CNS Pgp plays in conferring resistance against kernicterus and serve as an impetus towards developing modalities that enhance CNS Pgp expression in neonates thereby affording newborns additional protection against kernicterus - a chronically disabling neurologic condition that remains of marked clinical importance both in the United States and abroad.