Niemann-Pick C liver-specific proteostasis and pathology

摘要 C型尼曼-皮克病是一种致命的常染色体隐性遗传性神经内脏脂肪沉积病 影响所有年龄段的人。大约85%的患者有肝肿大，可以发展为肝脏脂肪变性， 肝硬变、肝细胞癌和肝功能衰竭。肝脏缺陷对患有以下疾病的患者尤其有害 新生儿发病，其中10%在6个月大时死于肝功能衰竭。尽管肝脏是重要的贡献者 对于疾病，细胞驱动因素和病理生理学还不完全清楚。尼曼-皮克C是由 晚期内质体/溶酶体胆固醇输出蛋白NPC1中300多个功能点突变。 我们之前已经证明，这些突变中最常见的I1061T主要是通过 FAM134B依赖的ER选择性自噬(ER吞噬)，但有必要了解这是如何 通路对其他致病突变起作用。此外，初步数据表明，大脑和 肝脏表达两种不同的FAM134B亚型，可能具有不同的功能。因此，有一个 需要了解Niemann-Pick C肝脏的蛋白平衡和发病机制。解决这些问题的下一步是 需求是追求这一应用的总体目标：(I)确定介导组织特异性的效应器 NPC1蛋白稳定和(Ii)定义不同细胞类型对Niemann-Pick C肝脏病理的贡献。这里 我们将检验我们的中心假设，即细胞类型特定的通路调节NPC1蛋白稳定和驱动 尼曼-皮克C级肝脏病理检查。我们将使用诱导的肝细胞和神经元来验证我们的假设。 含有一组Niemann-Pick C致病突变的同基因人类IPSCs。我们将利用 生化和遗传学分析以确定NPC1蛋白平衡和FAM134B异构体的程度 功能依赖于组织(目标1)。此外，我们将利用NPC1 loxP小鼠来删除NPC1 在全球范围内，在Kupffer细胞/巨噬细胞或肝细胞中研究这些细胞如何对肝脏病理起作用， 功能和炎症(目标2)。这些研究有望确定新的途径，显著 通过许多致病突变导致Niemann-Pick C相关肝病的表现。 这将为未来的发现努力确定和测试新的治疗策略奠定基础，这些策略可以纠正 肝脏和大脑都有。我们这个项目的基本原理是Kupffer细胞/巨噬细胞对 组织特异性蛋白平衡和肝细胞类型对疾病病理学的影响将提供一个强有力的科学框架 开发新的肝靶向Niemann-Pick C疗法。此外，我们还概述了职业发展计划，以 利用导师、技术培训、研讨会、肝脏会议和 R01授予写作新兵训练营。密歇根大学已经承诺提供支持和设施，以允许Dr。 舒尔茨完成了拟议的研究，并参加了他们广泛的培训研讨会。完成 拟议的5年研究和培训计划将为舒尔茨博士的独立研究生涯做好准备，并 协助从NIDDK获得R01。

ABSTRACT Niemann-Pick disease type C is an invariably fatal autosomal recessive neurovisceral lipid storage disease affecting all ages. Approximately 85% of patients have hepatomegaly which can develop into hepatic steatosis, cirrhosis, hepatocellular carcinoma, and liver failure. Liver defects are especially detrimental in patients with neonatal onset, with 10% dying from liver failure by 6 months of age. Although the liver is a significant contributor to disease, the cellular drivers and pathophysiology are incompletely understood. Niemann-Pick C is caused by over 300 loss-of-function point mutations in the late endosomal/lysosomal cholesterol-exporting protein NPC1. We have previously shown that the most common of these mutations, I1061T, is primarily degraded by FAM134B-dependent ER-selective autophagy (ER-phagy), but there is a crucial need to understand how this pathway works for other disease-causing mutations. Furthermore, preliminary data indicates that the brain and liver express two different isoforms of FAM134B with potentially divergent functions. Consequently, there is a need to understand Niemann-Pick C liver proteostasis and pathogenesis. The next step in addressing these needs is to pursue the overall objectives of this application: (i) determine the effectors mediating tissue-specific NPC1 proteostasis and (ii) define the contribution of different cell types to Niemann-Pick C liver pathology. Here we will test our central hypothesis is that cell type-specific pathways regulate NPC1 proteostasis and drive Niemann-Pick C liver pathology. We will test our hypothesis using induced hepatocytes and neurons from isogenic human iPSCs containing a panel of Niemann-Pick C disease-causing mutations. We will leverage biochemical and genetic assays to establish the extent to which NPC1 proteostasis and FAM134B isoform function are tissue-dependent (Aim 1). Additionally, we will take advantage of Npc1 loxP mice to delete Npc1 globally, in Kupffer cells/macrophages, or hepatocytes to study how these cells contribute to liver pathology, function, and inflammation (Aim 2). These studies are expected to identify novel pathways that significantly contribute to manifestations of Niemann-Pick C-related liver disease across many disease-causing mutations. This will set the stage for the future discovery efforts to identify and test new therapeutic strategies which correct both liver and brain. Our rationale for this project is that defining influence of Kupffer cells/macrophages on tissue-specific proteostasis and liver cell types on disease pathology will provide a strong scientific framework to develop new liver targeted Niemann-Pick C therapeutics. In addition, we outline a career development plan to increase liver biology knowledge by leveraging mentorship, technical training, seminars, liver conferences, and R01 grant writing boot camps. The University of Michigan has committed its support and facilities to allow Dr. Schultz to complete the proposed research and participate in their extensive training seminars. Completion of the proposed 5-year research and training plans will prepare Dr. Schultz for an independent research career and assist in securing an R01 from NIDDK.