Investigation of the mechanism by which NPC1 dysfunction leads to liver disease

描述（由申请方提供）：C型尼曼匹克病（NPC）是一种常染色体隐性遗传性胆固醇沉积病，其特征为胆固醇和鞘糖脂的溶酶体储存。NPC患者患有胆汁淤积、长期黄疸和肝脾肿大。NPC 1蛋白功能障碍导致肝脏疾病的机制尚不清楚。我们开发了一种独特的小鼠模型来研究NPC 1肝病。用NPC 1特异性反义寡核苷酸处理小鼠导致NPC 1蛋白表达的肝脏特异性和可逆性敲低。我们的目标是确定溶酶体脂质沉积导致肝脏疾病的机制。我们的假设是NPC肝病是由大量的脂质储存引发的，这导致溶酶体不稳定和促凋亡蛋白酶和脂质的释放。肝细胞凋亡然后通过TNF α途径传播。我们预计，如果NPC 1在敲除小鼠中重新表达，那么纤维化的肝脏将恢复。由于我们的NPC 1敲低模型是可逆的，因此我们有能力研究疾病消退。具体目的#1 -确定NPC小鼠肝细胞中的溶酶体脂质储存是否导致溶酶体破裂并释放其内容物以激活细胞凋亡。我们的初步结果表明，NPC 1敲低导致从溶酶体释放组织蛋白酶。我们将确定组织蛋白酶敲低是否减少NPC肝细胞凋亡。具体目标#2 -确定NPC肝病是否通过TNFa途径传播。我们的初步结果表明，NPC 1敲低在TNF α缺陷型小鼠导致不太严重的疾病表型。我们将使用TNF 1敲除小鼠、TNF α-受体的肝特异性敲除、TNF-RI和TNF α途径的抑制剂来检验这一假设。具体目标#3 -确定NPC 1敲除小鼠模型中NPC 1蛋白重新表达后NPC肝病逆转的过程。我们的初步结果表明，NPC 1的重新表达导致肝损伤和炎症减少。我们将确定NPC 1再表达完全逆转肝病表型的程度。公共卫生相关性：我们的研究将确定启动和传播尼曼-匹克C型肝病的信号，为设计治疗策略提供关键信息。我们还将确定NPC肝病可以逆转的程度，以实现NPC治疗的最大益处。

DESCRIPTION (provided by applicant): Niemann Pick disease type C (NPC) is an autosomal recessive lipidosis that is characterized by lysosomal storage of cholesterol and glycosphingolipids. NPC patients suffer from cholestasis, prolonged jaundice, and hepatosplenomegaly. The mechanism by which NPC1 protein dysfunction leads to liver disease is unknown. We have developed a unique mouse model with which to study NPC1 liver disease. Treatment of mice with a NPC1 specific antisense oligonucleotide leads to liver specific and reversible knockdown of NPC1 protein expression. Our goal is to determine the mechanism by which lysosomal lipid storage leads to liver disease. Our hypothesis is that NPC liver disease is initiated by the massive lipid storage, which leads to lysosome destabilization and release of pro-apoptotic proteases and lipids. Hepatocyte apoptosis is then propagated through the TNFa pathway. We expect that if NPC1 were re-expressed in the knockdown mouse, then the fibrotic liver would recover. Because our model of NPC1 knockdown is reversible, we have the ability to study disease regression. Specific Aim #1 - To determine if the lysosomal lipid storage in NPC mouse hepatocytes leads to rupture of lysosomes and release of their contents to activate apoptosis. Our preliminary results suggest that NPC1 knockdown leads to release of cathepsins from lysosomes. We will determine if cathepsin knockdown reduces NPC hepatocyte apoptosis. Specific Aim #2 - To determine if NPC liver disease is propagated through the TNFa pathway. Our preliminary results indicate that NPC1 knockdown in TNFa-deficient mice leads to a less severe disease phenotype. We will test this hypothesis using TNF1 knockout mice, liver- specific knockdown of the TNFa-receptor, TNF-RI, and an inhibitor of the TNFa pathway. Specific Aim #3 - To determine the course of reversal of NPC liver disease upon re-expression of the NPC1 protein in the NPC1 knockdown mouse model. Our preliminary results indicate that NPC1 re-expression leads to reduced liver injury and inflammation. We will determine the extent to which NPC1 re-expression fully reverses the hepatic disease phenotype. PUBLIC HEALTH RELEVANCE: Our studies will identify the signals that initiate and propagate Niemann-Pick C liver disease, providing information that is critical for designing therapeutic strategies. We will also determine the extent to which NPC liver disease can be reversed in order to achieve maximal benefit from an NPC therapy.