The Role of Lipid Rafts in Billirubin Neurotoxicity

 说明(申请人提供)：胆红素是血红素的分解产物，是一种已知的神经毒物。在美国，几乎所有妊娠35周的早产儿都会发生高胆红素血症。在早产儿中，胆红素在什么水平会导致神经发育损害尚不清楚。胆红素升高到未结合的游离胆红素(Bf)浓度超过血浆结合能力的水平。然后，Bf穿过血脑屏障，与其靶标结合，包括神经元的磷脂。一旦Bf穿过血脑屏障，就会发生胆红素引起的神经功能障碍的临床综合征。急性症状包括尖叫、癫痫、低张或高张，而慢性症状包括运动和平衡障碍。尽管高胆红素血症的发病率很高，但Bf对神经功能的影响机制尚不清楚。Bf与神经元磷脂的亲和力提示脂筏可能是高胆红素血症的主要靶点。脂筏是含有胆固醇和磷脂鞘磷脂的细胞膜的特殊动态微区。它们促进蛋白质之间的相互作用，促进信号传递和蛋白质运输。例如, 脂筏在L1细胞黏附分子(L1)介导的突起生长中起重要作用，L1是小脑发育的关键步骤，依赖于信号转导和蛋白质转运。我们的初步结果表明，胆红素通过L1抑制脂筏功能 记者。此外，我们先前的工作表明，补充胆碱可以减少脂筏功能障碍，胆碱是磷脂酰胆碱和鞘磷脂的前体。我们的新假设是：1)游离未结合胆红素结合并导致脂筏功能障碍，导致小脑功能障碍；2)补充胆碱将减少这些影响。我们将使用已建立的出生后第6天杂合子Gunn大鼠小脑颗粒神经元培养模型和磺胺二甲氧嘧啶处理纯合子Gunn大鼠作为活体大鼠模型来解决这些假设。我们将使用GABAA受体L1、βⅢ微管蛋白、Flotilin和小窝蛋白作为报告蛋白，在体外和体内测试小脑颗粒神经元的脂筏功能，测定小脑重量和测试行为结果：恒定和加速旋转棒试验。我们将通过两个具体的目标来验证我们的假设：第一个目标是确定胆红素是否在脂筏中积聚，以及它对脂筏功能和细胞凋亡的影响。胆碱的作用 对胆红素这些影响的补充将被确定。第二个目的是测定胆红素对小脑重量和小脑功能的影响，以及胆碱预处理的影响。

 DESCRIPTION (provided by applicant): Bilirubin, the breakdown product of heme, is a known neurotoxicant. In the United States, hyperbilirubinemia occurs in almost all preterm neonates <35 weeks gestation. In preterm infants, it is unknown at what level bilirubin causes neurodevelopmental harm. Bilirubin rises to a level where the unconjugated free bilirubin (Bf) concentration exceeds the plasma's capacity to bind it. Then, Bf crosses the blood brain barrier and binds to its targets including the phospholipids of neurons. Once Bf crosses the blood brain barrier, the clinical syndrome of bilirubin-induced neurologic dysfunction can occur. Acute symptoms include high pitched cry, seizures, and hypo- or hypertonicity, whereas chronic symptoms include disturbed movement and balance. Despite the high prevalence of hyperbilirubinemia, the mechanisms underlying the effects of Bf on neuronal function are poorly understood. The affinity of Bf with neuronal phospholipids suggests that lipid rafts might be a major target in hyperbilirubinemia. Lipid rafts are specialized dynamic microdomains of the plasma membrane containing cholesterol as well as the phospholipid sphingomyelin. They promote protein-protein interactions and facilitate signaling and protein trafficking. For example, lipid rafts play a major role on L1 cell adhesion molecule (L1) mediated neurite outgrowth, a crucial step in cerebellar development that depends on both signal transduction and protein trafficking. Our preliminary results show that bilirubin inhibits lipid raft function using L1 as a reporter. In addition, our prior work shows that lipid raft dysfunction can be reduced with supplementation of choline, a precursor of phosphatidylcholine and sphingomyelin. Our novel hypotheses are that 1) free unconjugated bilirubin binds to and causes lipid raft dysfunction, resulting in cerebellar dysfunction; and 2) supplementation with choline will reduce these effects. We will address these hypotheses using both the well established model of cultured cerebellar granule neurons from postnatal day 6 heterozygous Gunn rat pups and the sulfadimethoxine treated homozygous Gunn rat as an in vivo rat pup model. We will use L1, the GABAA receptor, β III tubulin, flotilin and caveolin as reporter proteins to test lipid raft functon in cerebellar granule neurons (CGN) in vitro and in Gunn rat pups in vivo, determine cerebellar weight and test behavioral outcomes: both the constant and accelerating rotarod test. We will test our hypotheses using two specific aims: The first aim will determine if bilirubin accumulates within lipid rafts and its effects on functions of lipid rafts and apoptosis. The effect of choline supplementation on these effects of bilirubin will be determined. In the second aim, the effect of bilirubin on cerebellar weight and cerebellar function will be measured, and the effect of pretreatment with choline determined.