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

描述(申请人提供)：Niemann Pick病C型(NPC)是一种常染色体隐性遗传性脂肪沉积症，其特征是溶酶体储存胆固醇和糖鞘糖脂。鼻咽癌患者有胆汁淤积、黄疸延长和肝脾肿大等症状。NPC1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，用来研究NPC1肝病。用NPC1特异性反义寡核苷酸治疗小鼠，可导致肝脏特异性和可逆的NPC1蛋白表达下调。我们的目标是确定溶酶体脂肪储存导致肝脏疾病的机制。我们的假设是，鼻咽癌肝病是由大量的脂肪储存引起的，导致溶酶体失稳和促凋亡的蛋白酶和脂类的释放。然后，肝细胞凋亡通过TNFa途径传播。我们预计，如果NPC1在基因敲除的小鼠中重新表达，那么纤维化的肝脏将会恢复。因为我们的NPC1基因敲除模型是可逆的，所以我们有能力研究疾病回归。具体目的#1-确定NPC小鼠肝细胞中溶酶体脂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，NPC1基因敲除导致组织蛋白从溶酶体中释放。我们将确定组织蛋白酶基因敲除是否减少鼻咽癌肝细胞的凋亡。具体目标#2-确定鼻咽癌肝病是否通过TNFa途径传播。我们的初步结果表明，在TNFa缺乏的小鼠中，NPC1基因敲除导致了一种不那么严重的疾病表型。我们将使用TNF1基因敲除的小鼠、肝脏特异性的TNFa受体、肿瘤坏死因子-RI和TNFa途径的抑制剂来验证这一假设。具体目的#3-在NPC1基因敲除小鼠模型中，确定NPC1蛋白重新表达后鼻咽癌肝病的逆转过程。我们的初步结果表明，NPC1的重新表达可以减少肝脏损伤和炎症。我们将确定NPC1重新表达在多大程度上完全逆转肝病的表型。公共卫生相关性：我们的研究将确定启动和传播尼曼-皮克C肝病的信号，提供对设计治疗策略至关重要的信息。我们还将确定鼻咽癌肝病可以被逆转的程度，以便从鼻咽癌治疗中获得最大好处。

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.