Dataset Information

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

ABSTRACT: 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 ?-cyclodextrin induces the subplasmalemmal location of lamellar inclusions and their subsequent release to the extracellular space. In parallel, ?-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 ?-cyclodextrin, a potential therapeutic drug, reverts these changes. Our study provides new mechanistic insight in NPC disease and uncovers new targets for therapeutic approaches.

SUBMITTER: Demais V

PROVIDER: S-EPMC6601882 | biostudies-literature | 2016 Jul

REPOSITORIES: biostudies-literature

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

Demais Valérie V Barthélémy Amélie A Perraut Martine M Ungerer Nicole N Keime Céline C Reibel Sophie S Pfrieger Frank W FW

The Journal of neuroscience : the official journal of the Society for Neuroscience 20160701 30

<h4>Unlabelled</h4>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 investig ...[more]

PMID: 27466344

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Project description:The aberrant accumulation of alpha-synuclein (?-syn) is believed to contribute to the onset and pathogenesis of Parkinson's disease (PD). The autophagy-lysosome pathway (ALP) is responsible for the high capacity clearance of ?-syn. ALP dysfunction is documented in PD and pre-clinical evidence suggests that inhibiting the ALP promotes the pathological accumulation of ?-syn. We previously identified the pathological accumulation of ?-syn in the brains of mice deficient for the soluble lysosomal enzyme alpha-Galactosidase A (?-Gal A), a member of the glycosphingolipid metabolism pathway. In the present study, we quantified ?-Gal A activity and levels of its glycosphingolipid metabolites in postmortem temporal cortex specimens from control individuals and in PD individuals staged with respect to ?-syn containing Lewy body pathology. In late-state PD temporal cortex we observed significant decreases in ?-Gal A activity and the 46kDa "active" species of ?-Gal A as determined respectively by fluorometric activity assay and western blot analysis. These decreases in ?-Gal A activity/levels correlated significantly with increased ?-syn phosphorylated at serine 129 (p129S-?-syn) that was maximal in late-stage PD temporal cortex. Mass spectrometric analysis of 29 different isoforms of globotriaosylceramide (Gb3), a substrate of ?-Gal A indicated no significant differences with respect to different stages of PD temporal cortex. However, significant correlations were observed between increased levels of several Gb3 isoforms and with decreased ?-Gal A activity and/or increased p129S-?-syn. Deacylated Gb3 (globotriaosylsphingosine or lyso-Gb3) was also analyzed in PD brain tissue but was below the limit of detection of 20pmol/g. Analysis of other lysosomal enzymes revealed a significant decrease in activity for the lysosomal aspartic acid protease cathepsin D but not for glucocerebrosidase (GCase) or cathepsin B in late-stage PD temporal cortex. However, a significant correlation was observed between decreasing GCase activity and increasing p129S-?-syn. Together our findings indicate ?-Gal A deficiency in late-stage PD brain that correlates significantly with the pathological accumulation of ?-syn, and further suggest the potential for ?-Gal A and its glycosphingolipid substrates as putative biomarkers for PD.

Project description:Background & Aims:Microvillus inclusion disease (MVID) is a congenital intestinal malabsorption disorder caused by defective apical vesicular transport. Existing cellular models do not fully recapitulate this heterogeneous pathology. The aim of this study was to characterize 3-dimensional intestinal organoids that continuously generate polarized absorptive cells as an accessible and relevant model to investigate MVID. Methods:Intestinal organoids from Munc18-2/Stxbp2-null mice that are deficient for apical vesicular transport were subjected to enterocyte-specific differentiation protocols. Lentiviral rescue experiments were performed using human MUNC18-2 variants. Apical trafficking and microvillus formation were characterized by confocal and transmission electron microscopy. Spinning disc time-lapse microscopy was used to document the lifecycle of microvillus inclusions. Results:Loss of Munc18-2/Stxbp2 recapitulated the pathologic features observed in patients with MUNC18-2 deficiency. The defects were fully restored by transgenic wild-type human MUNC18-2 protein, but not the patient variant (P477L). Importantly, we discovered that the MVID phenotype was correlated with the degree of enterocyte differentiation: secretory vesicles accumulated already in crypt progenitors, while differentiated enterocytes showed an apical tubulovesicular network and enlarged lysosomes. Upon prolonged enterocyte differentiation, cytoplasmic F-actin-positive foci were observed that further progressed into classic microvillus inclusions. Time-lapse microscopy showed their dynamic formation by intracellular maturation or invagination of the apical or basolateral plasma membrane. Conclusions:We show that prolonged enterocyte-specific differentiation is required to recapitulate the entire spectrum of MVID. Primary organoids can provide a powerful model for this heterogeneous pathology. Formation of microvillus inclusions from multiple membrane sources showed an unexpected dynamic of the enterocyte brush border.

Dataset Information

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

Publications

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

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