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

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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.