The Role of Lipid Rafts in Billirubin Neurotoxicity

脂筏在胆红素神经毒性中的作用

• 批准号: 9112521
• 负责人: CYNTHIA FRANCES BEARER
• 金额: $ 23.26万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-04-01 至 2018-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9112521
• 关键词: Abnormal coordination; Acute; Address; Affect; Affinity; Albumins; Apoptosis; Basal Ganglia; Betaine; Bilirubin; Binding; Biochemical; Biological Assay; Blood - brain barrier anatomy; Brain region; Caveolins; Cell membrane; Cerebellar Diseases; Cerebellum; Cholesterol; Choline; Chronic; Clinical; Crying; Cytoplasmic Granules; DNA Methylation; Data; Development; Dietary Intervention; Equilibrium; Experimental Models; Exposure to; Foundations; Functional disorder; Funding; Ganglioside GM1; Gene Expression; Glucuronic Acids; Gunn Rats; Hearing problem; Heme; High Prevalence; Hyperbilirubinemia; In Vitro; Injection of therapeutic agent; Intervention; Kernicterus; Knowledge; Laminin; Lead; Lecithin; MAPK3 gene; Measures; Mediating; Membrane Microdomains; Methionine; Mitogen-Activated Protein Kinases; Modeling; Motor; Movement; Movement Disorders; Neural Cell Adhesion Molecule L1; Neuraxis; Neurites; Neurodevelopmental Deficit; Neurologic Dysfunctions; Neurons; Neurotoxins; Pathway interactions; Phospholipids; Phototherapy; Plasma; Play; Pregnancy; Premature Infant; Proteins; RNA methylation; Rattus; Receptor Signaling; Reporter; Research; Role; Rotarod Performance Test; Seizures; Severities; Signal Pathway; Signal Transduction; Signaling Protein; Small Interfering RNA; Sphingomyelins; Sum; Symptoms; Syndrome; Testing; Therapeutic; Tubulin; United States; Weight; Work; acute symptom; behavioral outcome; brain cell; cell type; choline supplementation; comparative effectiveness; equilibration disorder; high risk; histone methylation; in vivo; knockout animal; neurotoxicity; novel; persistent symptom; postnatal; preterm newborn; prevent; protein protein interaction; protein transport; public health relevance; pup; ranpirnase; receptor; total measurement Bilirubin

 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.

 描述（由申请人提供）：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周的早产儿都会出现高胆红素血症。在早产儿中，尚不清楚胆红素达到什么水平会导致神经发育损害。胆红素升高到未结合的游离胆红素 (Bf) 浓度超过血浆结合胆红素的能力的水平。然后，Bf 穿过血脑屏障并与其目标结合，包括神经元的磷脂。 Bf一旦穿过血脑屏障，就会出现胆红素引起的神经功能障碍的临床综合征。急性症状包括高声哭泣、癫痫发作、肌张力低下或肌张力过高，而慢性症状包括运动和平衡障碍。尽管高胆红素血症的患病率很高，但 Bf 对神经元功能影响的机制尚不清楚。 Bf 与神经元磷脂的亲和力表明脂筏可能是高胆红素血症的主要目标。脂筏是质膜的专门动态微域，含有胆固醇和磷脂鞘磷脂。它们促进蛋白质-蛋白质相互作用并促进信号传导和蛋白质运输。例如， 脂筏在 L1 细胞粘附分子 (L1) 介导的神经突生长中发挥重要作用，这是小脑发育中的关键步骤，依赖于信号转导和蛋白质运输。我们的初步结果表明胆红素利用 L1 作为抑制脂筏功能 记者。此外，我们之前的工作表明，补充胆碱（磷脂酰胆碱和鞘磷脂的前体）可以减少脂筏功能障碍。我们的新假设是：1）游离非结合胆红素结合并引起脂筏功能障碍，导致小脑功能障碍； 2）补充胆碱会减少这些影响。我们将使用来自出生后第 6 天的杂合 Gunn 大鼠幼崽的已建立的小脑颗粒神经元培养模型和磺胺二甲氧嘧啶处理的纯合 Gunn 大鼠作为体内大鼠幼崽模型来解决这些假设。我们将使用 L1、GABAA 受体、β III 微管蛋白、flotilin 和 Caveolin 作为报告蛋白，在体外测试小脑颗粒神经元 (CGN) 和体内 Gunn 幼鼠的脂筏功能，确定小脑重量并测试行为结果：恒定和加速旋转测试。我们将使用两个具体目标来检验我们的假设：第一个目标将确定胆红素是否在脂筏内积聚及其对脂筏功能和细胞凋亡的影响。胆碱的作用 将确定补充胆红素对这些作用的影响。第二个目标是测量胆红素对小脑重量和小脑功能的影响，并确定胆碱预处理的效果。