Niemann-Pick C liver-specific proteostasis and pathology

摘要 C型尼曼-匹克病是一种常染色体隐性遗传性神经内脏脂质沉积病 影响所有年龄。大约85%的患者有肝肿大，可发展为肝脂肪变性， 肝硬化、肝细胞癌和肝衰竭。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。虽然肝脏是一个重要的贡献者 对于疾病，细胞驱动因素和病理生理学还不完全了解。尼曼-匹克C是由 晚期内体/溶酶体胆固醇输出蛋白NPC 1中超过300个功能丧失点突变。 我们以前已经证明，这些突变中最常见的I1061 T主要是由 FAM 134 B依赖的ER选择性自噬（ER-吞噬），但有一个关键需要了解这是如何 其他致病突变也是如此。此外，初步数据表明，大脑和 肝脏表达具有潜在不同功能的两种不同的FAM 134 B同种型。因此，有一个 需要了解Niemann-Pick C肝蛋白质稳态和发病机制。解决这些问题的下一步是 所需的是追求本申请的总体目标：（i）确定介导组织特异性免疫应答的效应物。 NPC 1蛋白质稳态和（ii）定义不同细胞类型对尼曼-皮克C肝脏病理学的贡献。这里 我们将测试我们的中心假设，即细胞类型特异性途径调节NPC 1蛋白质稳态并驱动NPC 1蛋白质稳态。 尼曼匹克C肝脏病理学。我们将使用诱导的肝细胞和神经元来验证我们的假设。 在一个实施方案中，本发明涉及含有一组尼曼-匹克C致病突变的同基因人iPSC。我们将利用 生物化学和遗传测定以确定NPC 1蛋白质抑制和FAM 134 B同种型 功能依赖于组织（目标1）。此外，我们将利用Npc 1 loxP小鼠来删除Npc 1， 在全球范围内，在枯否细胞/巨噬细胞或肝细胞中研究这些细胞如何促进肝脏病理学， 功能和炎症（目标2）。这些研究有望发现新的途径， 在许多致病突变中，导致尼曼-匹克C相关肝病的表现。 这将为未来的发现工作奠定基础，以确定和测试新的治疗策略， 肝脏和大脑我们对这个项目的基本原理是，确定库普弗细胞/巨噬细胞对 组织特异性蛋白质稳态和肝细胞类型对疾病病理学的影响将提供强有力的科学框架， 开发新的肝脏靶向尼曼-匹克C疗法。此外，我们还制定了职业发展计划， 通过利用导师、技术培训、研讨会、肝脏会议来增加肝脏生物学知识， 授予写作靴子训练营。密歇根大学已承诺提供支持和设施，让博士。 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， 协助从NIDDK获得R 01。

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.