Rescuing Niemann-Pick C Disease: Pathways of Liver and Brain Degeneration

描述（由申请人提供）：尼曼匹克C型（NPC）病是一种致命的儿科疾病。这种疾病是由两个基因中的任何一个突变引起的，NPC1编码一种13跨膜结构域的固醇结合蛋白，NPC2编码一种可溶性固醇结合蛋白。任何一个基因的缺失都会导致细胞器运输异常和溶酶体内游离胆固醇的积累。NPC患者表现为肝肿大，进行性认知和运动功能丧失，伴有小脑大量浦肯野神经元（PN）死亡。鼻咽癌没有有效的治疗方法。具体目标1：确定为什么浦肯野神经元在NPC疾病中死亡，以及在什么阶段疾病可以被阻止或逆转。我们将在Npc1-/-背景下为特定类型的细胞（如神经元、星形胶质细胞或肝细胞）提供功能性标记的Npc1蛋白，以确定每种细胞类型如何导致疾病。我们的转基因是用tet技术设计的，允许细胞特异性和时间调节标记的Npc1的产生。具体目标2：了解鼻咽癌疾病如何影响浦肯野神经元的细胞内运输。Npc1的缺失在成纤维细胞中引起显著的细胞内运输缺陷。为了了解pn是如何受到影响的，我们将培养它们，并跟踪NGF和其他标记有量子点的分子的贩运。我们将使用便携式双光子显微镜结合细胞器染料来表征野生型和Npc1-/-小鼠的活星形胶质细胞和PNs中的囊泡运动。在我们新工程小鼠的活脑中，我们将分析PNs、其他神经元和星形胶质细胞中npc1阳性细胞器的运动，以确定哪些变化可能导致细胞死亡。特异性目的3：确定炎症是否保护或导致鼻咽癌细胞死亡。我们将在Npc-/-小鼠的神经元、肝细胞和炎症细胞中产生功能性Npc1，并观察哪一种在预防炎症方面最有效。我们将使用减少炎症的小鼠突变与Npc1-/-联合使用，观察PN存活和肝脏病理是改善还是恶化。我们将用氯代酸填充脂质体消融肝巨噬细胞，并评估巨噬细胞在NPC肝损伤中的作用。特异性目标4：发现自噬是否保护或导致鼻咽癌细胞死亡。我们将把NPC小鼠与不能触发自噬的突变体进行杂交：Toll样受体-7和beclin-1缺陷小鼠。相反，我们将测试在神经元中过度表达Beclin1并因此增强自噬的小鼠，或通过Beclin1病毒感染或雷帕霉素治疗增强自噬。溶酶体储存障碍如NPC包括近60种不同的情况，其中大多数损害肝脏和/或大脑功能。包括鼻咽癌在内的一些疾病与阿尔茨海默病有相似之处。我们使用了一种新的方法来改造小鼠，这将使我们能够了解不同的细胞类型和过程是如何导致疾病的。了解炎症和自噬在NPC神经退行性变中的作用，对阻止或逆转疾病进展的治疗干预具有直接意义。

DESCRIPTION (provided by applicant): Niemann Pick type C (NPC) disease is a fatal pediatric disorder. The disease is due to mutations in either of two genes, NPC1, which encodes a 13 transmembrane domain sterol-binding protein, and NPC2, which encodes a soluble sterol-binding protein. Loss of either gene causes aberrant organelle trafficking and accumulation of free cholesterol within lysosomes. NPC patients suffer from hepatomegaly and progressive cognitive and locomotion losses, with massive Purkinje neuron (PN) death in the cerebellum. There is no effective treatment for NPC. Specific Aim 1: Determine why Purkinje neurons die in NPC disease and at what stages the disease can be arrested or reversed. We will provide a functional tagged- Npc1 protein to specific classes of cells, eg neurons, astrocyte, or liver cells, in the Npc1-/- background, to define how each cell type contributes to disease. Our transgenes are engineered with Tet-technology to allow cell-specific and temporal regulation of tagged Npc1 production. Specific Aim 2: Learn how NPC disease affects intracellular trafficking in Purkinje neurons. Loss of Npc1 causes striking intracellular trafficking defects in fibroblasts. To learn how PNs are affected, we will culture them and track the trafficking of NGF and other molecules labeled with quantum dots. We will use a portable two-photon microscope in combination with organelle dyes to characterize vesicular movements in living astrocytes and PNs of wild-type and Npc1-/- mice. In living brains of our newly engineered mice we will analyze movements of Npc1-positive organelles in PNs, other neurons, and astrocytes to determine what changes may cause cell death. Specific Aim 3: Determine whether inflammation protects from, or causes, NPC cell death. We will produce functional Npc1 in neurons, hepatocytes, and inflammatory cells, in otherwise Npc-/- mice, and see which of these is most effective in preventing inflammation. We will use mouse mutations that reduce inflammation in combination with Npc1-/-, and see whether PN survival and liver pathology are improved or worsened. We will ablate hepatic macrophages with chlodronate-filled liposomes and assess the role of macrophages in NPC liver damage. Specific Aim 4: Discover whether autophagy protects from, or causes, NPC cell death. We will cross NPC mice with mutants that cannot trigger autophagy: Toll like receptor-7 and beclin-1 deficient mice. Conversely we will test mice that have over-expressing Beclin1 in neurons and consequent have heightened autophagy, or enhance autophagy with Beclin1 virus infections or rapamycin treatments. Lysosome storage disorders like NPC encompass nearly 60 different conditions, most of which damage liver and/or brain function. Some, including NPC, have similarities to Alzheimer disease. We have used a novel approach to engineer mice that will allow us to learn how different cell types and processes contribute to disease. Learning the roles of inflammation and autophagy in NPC neurodegeneration has direct implications for therapeutic interventions that will arrest or reverse disease progression.