Neonatal Bilirubin Neurotoxicity and P-Glycoprotein

描述(申请人提供)：P-糖蛋白(PGP)，一种表达在血脑屏障(BBB)内皮细胞和星形胶质细胞上的三磷酸腺苷结合盒外排泵，可能在限制胆红素进入中枢神经系统(CNS)方面发挥重要作用。01-04年的研究表明：1)Pgp介导的体外胆红素转运；ii)胆红素刺激Pgp ATPase活性；iii)Pgp在未成熟的小鼠和人中枢神经系统中的表达受限；以及iv)出生后早期BBB Pgp显著增加。这些新的观察结果提出了一种明显的可能性，即人的BBB PGP降低了中枢神经系统胆红素的含量，与PGP缺失的零突变转基因小鼠(167)相比，PGP中的这一现象是足够的，从而降低了核心黄鼠病的风险。我们实验室的初步研究表明，PGP I)赋予细胞对胆红素诱导的细胞凋亡的抵抗力，II)除在发育中的新生儿的血脑屏障中表达外，还在中枢神经系统实质的特定细胞中表达。综上所述，这些发现表明，PGP可能在血脑屏障和中枢神经系统实质内对胆红素诱导的细胞损伤具有保护作用。在目前的提案中，我们将扩展我们先前的观察和最近的初步发现，验证三个假设：1)PGP在体外和体内均可抑制胆红素诱导的细胞凋亡；2)PGP在人类中枢神经系统实质细胞和血脑屏障细胞中均有表达；3)出生后高胆红素血症和产前维生素A治疗可上调Gunn大鼠幼鼠中枢Pgp的表达，而产前维生素A治疗和出生后高胆红素血症联合应用将在体内对抗胆红素诱导的细胞凋亡。我们将使用Caco-2、神经母细胞瘤、牛脑毛细血管内皮细胞和转人或小鼠Pgp基因的LLC-PK1细胞和Gunn大鼠新生黄疸模型来研究Pgp对胆红素诱导的细胞凋亡的保护作用、涉及的凋亡途径以及细胞死亡的作用。人类新生儿核黄体细胞凋亡的发生率，以及人类中枢神经系统Pgp表达的个体发育和区域定位将在档案尸检组织中进行评估。所获得的信息将为CNS PGP在增强对核黄体炎的抵抗力方面所起的作用提供新的见解，并将推动开发增强新生儿CNS Pgp表达的方法，从而为新生儿提供针对核黄体病的额外保护。核黄体症是一种长期致残的神经疾病，在美国和国外仍具有显著的临床重要性。

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.