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

描述（由申请方提供）：C型尼曼匹克（NPC）病是一种致死性儿科疾病。这种疾病是由于两种基因中的任何一种突变引起的，即编码13个跨膜结构域的固醇结合蛋白的NPC1和编码可溶性固醇结合蛋白的NPC2。任何一个基因的缺失都会导致细胞器的异常运输和游离胆固醇在溶酶体内的积累。NPC患者患有肝肿大和进行性认知和运动丧失，小脑中有大量浦肯野神经元（PN）死亡。目前尚无有效的治疗方法。具体目标1：确定浦肯野神经元在NPC疾病中死亡的原因，以及疾病在什么阶段可以被阻止或逆转。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。我们的转基因工程与Tet-technology允许细胞特异性和时间调节标记Npc1生产。具体目标2：了解NPC疾病如何影响浦肯野神经元的细胞内运输。Npc1的缺失导致成纤维细胞中显著的细胞内运输缺陷。为了了解PNs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，以表征野生型和Npc 1-/-小鼠的活星形胶质细胞和PN中的囊泡运动。在我们新工程小鼠的活体大脑中，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。具体目标3：确定炎症是否保护或导致NPC细胞死亡。我们将在神经元，肝细胞和炎症细胞中产生功能性Npc 1，在其他Npc-/-小鼠中，并观察其中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-/-结合，并观察PN存活率和肝脏病理学是否改善或恶化。我们将用阿曲膦酸盐填充的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。具体目标4：发现自噬是否保护或导致NPC细胞死亡。我们将NPC小鼠与不能触发自噬的突变体杂交：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Beclin 1的小鼠，从而增强自噬，或通过Beclin 1病毒感染或雷帕霉素治疗增强自噬。像NPC这样的溶酶体储存障碍包括近60种不同的疾病，其中大多数损害肝脏和/或大脑功能。包括NPC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如何导致疾病。了解炎症和自噬在NPC神经变性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.