Reversal of Pathologic Lipid Accumulation in NPC1-Deficient Neurons by Drug-Promoted Release of LAMP1-Coated Lamellar Inclusions.

UNLABELLED: Aging and pathologic conditions cause intracellular aggregation of macromolecules and the dysfunction and degeneration of neurons, but the mechanisms are largely unknown. Prime examples are lysosomal storage disorders such as Niemann-Pick type C (NPC) disease, where defects in the endosomal-lysosomal protein NPC1 or NPC2 cause intracellular accumulation of unesterified cholesterol and other lipids leading to neurodegeneration and fatal neurovisceral symptoms. Here, we investigated the impact of NPC1 deficiency on rodent neurons using pharmacologic and genetic models of the disease. Improved ultrastructural detection of lipids and correlative light and electron microscopy identified lamellar inclusions as the subcellular site of cholesterol accumulation in neurons with impaired NPC1 activity. Immunogold labeling combined with transmission electron microscopy revealed the presence of CD63 on internal lamellae and of LAMP1 on the membrane surrounding the inclusions, indicating their origins from intraluminal vesicles of late endosomes and of a lysosomal compartment, respectively. Lamellar inclusions contained cell-intrinsic cholesterol and surface-labeled GM1, indicating the incorporation of plasma membrane components. Scanning electron microscopy revealed that the therapeutic drug candidate β-cyclodextrin induces the subplasmalemmal location of lamellar inclusions and their subsequent release to the extracellular space. In parallel, β-cyclodextrin mediated the NPC1-independent redistribution of cholesterol within neurons and thereby abolished a deleterious cycle of enhanced cholesterol synthesis and its intracellular accumulation, which was indicated by neuron-specific transcript analysis. Our study provides new mechanistic insight into the pathologic aggregation of macromolecules in neurons and suggests exocytosis as cellular target for its therapeutic reversal. SIGNIFICANCE STATEMENT: Many neurodegenerative diseases involve pathologic accumulation of molecules within neurons, but the subcellular location and the cellular impact are often unknown and therapeutic approaches lacking. We investigated these questions in the lysosomal storage disorder Niemann-Pick type C (NPC), where a defect in intracellular cholesterol transport causes loss of neurons and fatal neurovisceral symptoms. Here, we identify lamellar inclusions as the subcellular site of lipid accumulation in neurons, we uncover a vicious cycle of cholesterol synthesis and accretion, which may cause gradual neurodegeneration, and we reveal how β-cyclodextrin, a potential therapeutic drug, reverts these changes. Our study provides new mechanistic insight in NPC disease and uncovers new targets for therapeutic approaches.