Modeling Progressive Familial Intrahepatic Cholestasis Type I Caused by ATP8B1 deficiency

ATP8B1 缺乏引起的进行性家族性肝内胆汁淤积 I 型建模

基本信息

批准号：
10722357
负责人：
Chunyue Yin
金额：
$ 16.05万
依托单位：
CINCINNATI CHILDRENS HOSP MED CTR
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-09-01 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10722357
关键词：
ATP8B1 gene Acids Adenosine Adopted Adult Age Allografting Animal Model Animals Bile fluid Biochemical Biological Assay Biological Process CRISPR/Cas technology Cell Line Cell membrane Childhood Cholestasis Chronic Complement Cystic Fibrosis Transmembrane Conductance Regulator Data Development Diagnosis Diarrhea Disease Drug Screening Dyes Ensure Enterohepatic Circulation Etiology Excretory function Exhibits Experimental Models Extrahepatic Future Goals Health Hepatomegaly Histologic Homeostasis Human Hydrophobicity Image Impairment In Vitro Intervention Intestines Intrahepatic Cholestasis Knock-out Knowledge Larva Life Lipids Liver Medical Membrane Membrane Fluidity Methods Mission Modeling Mus Mutation Neonatal Outcome Pathogenesis Patient-Focused Outcomes Patients Pharmacologic Substance Phenotype Phospholipids Progressive intrahepatic cholestasis Proteins Pruritus Public Health Pump Receptor Signaling Refractory Research Resistance Rodent Model Serum Study models Symptoms Testing United States National Institutes of Health Validation Variant Zebrafish analog autosome bile salts disability drug discovery early childhood early onset effective therapy end stage liver disease graft failure hepatocellular injury improved in vivo in vivo Model innovation insertion/deletion mutation lipid transport liver transplantation member mutant new therapeutic target novel novel therapeutic intervention novel therapeutics preclinical study receptor expression screening sensor

项目摘要

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.

项目摘要/摘要 ATP8B1/FIC1属于P型腺苷三磷酸酶的P4亚家族。在肝脏中，它本地化在胆管上膜上保持其脂质不对称性，这对于胆汁的正常功能至关重要盐转运蛋白。在人类中，ATP8B1中的突变导致少数形式的慢性肝内胆汁淤积常染色体隐性继承。 ATP8B1缺乏，进行性家庭肝内的最严重形式胆汁淤积I型（PFIC1）的特征是童年时期正常血清GGT胆汁淤积和瘙痒。这可以发展成为末期肝病。由于现有实验模型的局限性ATP8B1 被研究不足，我们对ATP8B1缺乏如何导致胆汁淤积的理解仍然不完整，排除PFIC1有效疗法的开发。我们的长期目标是了解慢性肝内胆汁淤积并改善患者的诊断和治疗。这个ap-的总体目标 Plication是为ATP8B1缺乏症建立一种新型的斑马鱼模型，以更好地了解发病机理胆汁淤积并发现新的治疗策略。我们的初步研究表明ATP8B1- 缺乏斑马鱼发展了胆汁淤积，到2周大，并提供了致命的概念证明斑马鱼可用于建模PFIC1。在此应用中，我们建议进行进一步的表型分析 ATP8B1种系突变体斑马鱼的作品，并开发了在体内研究ATP8B1功能的测定法。我们将确定使用斑马鱼验证患者变体的可行性并测试药物干预对胆汁淤积。理由是，开发ATP8B1缺乏症的其他动物模型将补充现有的体外和啮齿动物模型，并将新知识带入我们对ATP8B1功能的理解以及由ATP8B1缺乏引起的胆汁淤积机理。在AIM 1中，我们将表征胆汁降着斑马鱼中ATP8B1缺乏引起的IC表型。我们将跟踪荧光的亚细胞分布 Wildtype和Atp8b1突变幼虫的肝脏和肠中的脂质，以了解ATP8B1如何调节体内脂质的运输。在AIM 2中，我们将确定使用斑马鱼验证生物学的可行性 ATP8B1患者变体的功能。我们将向斑马鱼介绍两个常见的ATP8B1错义变体并确定它们是否改变ATP8B1表达并引起胆汁淤积。我们将测试4-- 苯基丁酸营救了这些动物。在本应用程序中提出的研究是高度创新的因为它建立了一种新型的斑马鱼ATP8B1缺乏症模型，该模型为研究提供了独特的优势胆汁稳态和胆固醇表型。我们还将开发一条管道，以识别未来的药物屏幕可以营救由ATP8B1缺乏引起的胆汁淤积的化合物。这项研究也很重要，因为这预计新的体内模型将提高我们对胆稳态中ATP8B1功能的理解 PFIC1的发病机理。研究的结果将为高度竞争性提供关键的初步数据 R01应用进一步追求疾病机制和药物发现。