Modeling Progressive Familial Intrahepatic Cholestasis Type I Caused by ATP8B1 deficiency

项目摘要/摘要 ATP8B1/FIC1属于P型三磷酸腺苷酶的P4亚家族。在肝脏中，它被定位于 以维持其脂质不对称，这对胆汁的正常功能是必不可少的。 盐分运输船。在人类中，ATP8B1突变导致罕见的慢性肝内胆汁淤积症 常染色体隐性遗传。最严重的ATP8B1缺乏症，进行性家族性肝内 I型胆汁淤积症(PFIC1)，以儿童早期血清GGT正常的胆汁淤积和瘙痒为特征 这可能会发展为终末期肝病。由于现有实验模型的限制，ATP8B1 我们对ATP8B1缺乏如何导致胆汁淤积的了解还不完全， 阻碍了对PFIC1的有效治疗的发展。我们的长期目标是了解该病的病因 慢性肝内胆汁淤积症，提高患者的诊疗水平。该AP的总体目标是- 目的是建立一种新的斑马鱼ATP8B1缺乏症模型，以更好地了解其发病机制 治疗胆汁淤积症，发现新的治疗策略。我们的初步研究表明，Atp8b1- 缺乏的斑马鱼会出现胆汁淤积，并在两周大时死亡，这提供了概念证据 斑马鱼可以用来模拟PFIC1。在这个应用中，我们建议进行进一步的表型分析 对atp8b1的种系突变斑马鱼进行研究，并建立体内研究atp8b1功能的方法。我们将决定 用斑马鱼验证患者变异并测试药物干预的效果的可行性 胆汁淤积症。其基本原理是，开发更多的ATP8B1缺乏症动物模型将补充 现有的体外和啮齿动物模型，为我们对ATP8B1功能的理解带来了新的认识 ATP8B1缺乏引起胆汁淤积的机制。在目标1中，我们将描述胆汁淤积的特征- 斑马鱼Atp8b1基因缺失引起的IC表型我们将追踪荧光的亚细胞分布 野生型和atp8b1突变幼虫肝脏和肠道中的脂类物质了解ATP8B1如何调节 体内脂类的运输。在目标2中，我们将确定使用斑马鱼来验证生物 ATP8B1患者突变体的功能。我们将在斑马鱼中引入两个常见的ATP8B1错义变体 并确定它们是否会改变ATP8B1的表达并导致胆汁淤积。我们会测试是否用4- 苯丁酸拯救了这些动物。本申请中提出的研究具有很高的创新性-- 因为它建立了一种新的ATP8B1缺乏症斑马鱼模型，为研究提供了独特的优势 胆汁动态平衡和胆汁淤积表型。我们还将为未来的药物筛查开发一条流水线，以确定 治疗由ATP8B1缺乏引起的胆汁淤积的化合物。这项研究也具有重要意义，因为 新的体内模型有望促进我们对ATP8B1在胆汁动态平衡和 PFIC1的发病机制。这项研究的结果将为具有高度竞争力的 R01应用，以进一步探讨疾病机制和药物发现。

PROJECT SUMMARY/ABSTRACT ATP8B1/FIC1 belongs to the P4 subfamily of P-type adenosine triphosphatases. In the liver, it is localized on the bile canalicular membrane to maintain its lipid asymmetry that is essential for proper function of the bile salt transporters. In humans, mutations in ATP8B1 result in rare forms of chronic intrahepatic cholestasis with autosomal recessive inheritance. The most severe form of ATP8B1 deficiency, progressive familial intrahepatic cholestasis type I (PFIC1), is characterized by normal serum GGT cholestasis and pruritus in early childhood that can progress into end-stage liver disease. Due to the limitations of existing experimental models, ATP8B1 is understudied and our understanding of how ATP8B1 deficiency results in cholestasis remains incomplete, precluding the development of effective therapy for PFIC1. Our long-term goal is to understand the etiology of chronic intrahepatic cholestasis and improve patient diagnosis and treatment. The overall objective for this ap- plication is to establish a novel zebrafish model for ATP8B1 deficiency to better understand the pathogenesis of cholestasis and discover new therapeutic strategies. Our preliminary study demonstrated that Atp8b1- deficient zebrafish developed cholestasis and were lethal by 2 weeks of age, providing proof of concept that zebrafish can be used to model PFIC1. In this application, we propose to conduct further phenotypic analysis of atp8b1 germline mutant zebrafish and develop assays to study ATP8B1 function in vivo. We will determine the feasibility of using zebrafish to validate patient variants and test the effect of pharmaceutical intervention on cholestasis. The rationale is that developing additional animal models of ATP8B1 deficiency will complement the existing in vitro and rodent models and bring new knowledge into our understanding of ATP8B1 function and the mechanisms of cholestasis caused by ATP8B1 deficiency. In Aim 1, we will characterize the cholestat- ic phenotypes caused by Atp8b1 deficiency in zebrafish. We will track the subcellular distribution of fluorescent lipids in the liver and intestine of wildtype and atp8b1 mutant larvae to understand how ATP8B1 regulates the transport of lipids in vivo. In Aim 2, we will determine the feasibility of using zebrafish to validate the biological function of ATP8B1 patient variants. We will introduce two common ATP8B1 missense variants into zebrafish and determine if they change ATP8B1 expression and cause cholestasis. We will test if treatment with 4- phenylbutyric acid rescues these animals. The research proposed in this application is highly innovative be- cause it establishes a novel zebrafish model of ATP8B1 deficiency that offers unique advantages for studying bile homeostasis and cholestatic phenotypes. We will also develop a pipeline for future drug screen to identify compounds that rescue cholestasis caused by ATP8B1 deficiency. The study is also significant because this new in vivo model is expected to advance our understanding of ATP8B1 function in bile homeostasis and pathogenesis of PFIC1. The results from the study will provide critical preliminary data for a highly competitive R01 application to further pursue disease mechanisms and drug discovery.