Tonic prime-boost of STING signalling mediates Niemann-Pick disease type C.

The classic mode of STING activation is through binding the cyclic dinucleotide 2'3'-cyclic GMP-AMP (cGAMP), produced by the DNA sensor cyclic GMP-AMP synthase (cGAS), which is important for the innate immune response to microbial infection and autoimmune disease. Modes of STING activation that are independent of cGAS are much less well understood. Here, through a spatiotemporally resolved proximity labelling screen followed by quantitative proteomics, we identify the lysosomal membrane protein Niemann-Pick type C1 (NPC1) as a cofactor in the trafficking of STING. NPC1 interacts with STING and recruits it to the lysosome for degradation in both human and mouse cells. Notably, we find that knockout of Npc1 'primes' STING signalling by physically linking or 'tethering' STING to SREBP2 trafficking. Loss of NPC1 protein also 'boosts' STING signalling by blocking lysosomal degradation. Both priming and boosting of STING signalling are required for severe neurological disease in the Npc1-/- mouse. Genetic deletion of Sting1 (the gene that encodes STING) or Irf3, but not that of Cgas, significantly reduced the activation of microglia and relieved the loss of Purkinje neurons in the cerebellum of Npc1-/- mice, leading to improved motor function. Our study identifies a cGAS- and cGAMP-independent mode of STING activation that affects neuropathology and provides a therapeutic target for the treatment of Niemann-Pick disease type C.

In Vitro and In Vivo Evaluation of 6-O-α-Maltosyl-ß-Cyclodextrin as a Potential Therapeutic Agent Against Niemann-Pick Disease Type C.

Niemann-Pick disease Type C (NPC) is a rare lysosomal storage disease characterized by the dysfunction of intracellular cholesterol trafficking with progressive neurodegeneration and hepatomegaly. We evaluated the potential of 6-O-α-maltosyl-ß-cyclodextrin (G2-ß-CD) as a drug candidate against NPC. The physicochemical properties of G2-ß-CD as an injectable agent were assessed, and molecular interactions between G2-ß-CD and free cholesterol were studied by solubility analysis and two-dimensional proton nuclear magnetic resonance spectroscopy. The efficacy of G2-ß-CD against NPC was evaluated using Npc1 deficient Chinese hamster ovary (CHO) cells and Npc1 deficient mice. G2-ß-CD in aqueous solution showed relatively low viscosity and surface activity; characteristics suitable for developing injectable formulations. G2-ß-CD formed higher-order inclusion complexes with free cholesterol. G2-ß-CD attenuated dysfunction of intercellular cholesterol trafficking and lysosome volume in Npc1 deficient CHO cells in a concentration dependent manner. Weekly subcutaneous injections of G2-ß-CD (2.9 mmol/kg) ameliorated abnormal cholesterol metabolism, hepatocytomegaly, and elevated serum transaminases in Npc1 deficient mice. In addition, a single cerebroventricular injection of G2-ß-CD (21.4 µmol/kg) prevented Purkinje cell loss in the cerebellum, body weight loss, and motor dysfunction in Npc1 deficient mice. In summary, G2-ß-CD possesses characteristics favorable for injectable formulations and has therapeutic potential against in vitro and in vivo NPC models.

Niemann-Pick C1-deficient mice lacking sterol O-acyltransferase 2 have less hepatic cholesterol entrapment and improved liver function.

Cholesteryl esters are generated at multiple sites in the body by sterol O-acyltransferase (SOAT) 1 or SOAT2 in various cell types and lecithin cholesterol acyltransferase in plasma. Esterified cholesterol and triacylglycerol contained in lipoproteins cleared from the circulation via receptor-mediated or bulk-phase endocytosis are hydrolyzed by lysosomal acid lipase within the late endosomal/lysosomal (E/L) compartment. Then, through the successive actions of Niemann-Pick C (NPC) 2 and NPC 1, unesterified cholesterol (UC) is exported from the E/L compartment to the cytosol. Mutations in either NPC1 or NPC2 lead to continuing entrapment of UC in all organs, resulting in multisystem disease, which includes hepatic dysfunction and in some cases liver failure. These studies investigated primarily whether elimination of SOAT2 in NPC1-deficient mice impacted hepatic UC sequestration, inflammation, and transaminase activities. Measurements were made in 7-wk-old mice fed a low-cholesterol chow diet or one enriched with cholesterol starting 2 wk before study. In the chow-fed mice, NPC1:SOAT2 double knockouts, compared with their littermates lacking only NPC1, had 20% less liver mass, 28% lower hepatic UC concentrations, and plasma alanine aminotransferase and aspartate aminotransferase activities that were decreased by 48% and 36%, respectively. mRNA expression levels for several markers of inflammation were all significantly lower in the NPC1 mutants lacking SOAT2. The existence of a new class of potent and selective SOAT2 inhibitors provides an opportunity for exploring if suppression of this enzyme could potentially become an adjunctive therapy for liver disease in NPC1 deficiency. NEW & NOTEWORTHY In Niemann-Pick type C1 (NPC1) disease, the entrapment of unesterified cholesterol (UC) in the endosomal/lysosomal compartment of all cells causes multiorgan disease, including neurodegeneration, pulmonary dysfunction, and liver failure. Some of this sequestered UC entered cells initially in the esterified form. When sterol O-acyltransferase 2, a cholesterol esterifying enzyme present in enterocytes and hepatocytes, is eliminated in NPC1-deficient mice, there is a reduction in their hepatomegaly, hepatic UC content, and cellular injury.

Lipid-mediated motor-adaptor sequestration impairs axonal lysosome delivery leading to autophagic stress and dystrophy in Niemann-Pick type C.

Niemann-Pick disease type C (NPC) is a neurodegenerative lysosomal storage disorder characterized by lipid accumulation in endolysosomes. An early pathologic hallmark is axonal dystrophy occurring at presymptomatic stages in NPC mice. However, the mechanisms underlying this pathologic change remain obscure. Here, we demonstrate that endocytic-autophagic organelles accumulate in NPC dystrophic axons. Using super-resolution and live-neuron imaging, we reveal that elevated cholesterol on NPC lysosome membranes sequesters kinesin-1 and Arl8 independent of SKIP and Arl8-GTPase activity, resulting in impaired lysosome transport into axons, contributing to axonal autophagosome accumulation. Pharmacologic reduction of lysosomal membrane cholesterol with 2-hydroxypropyl-ß-cyclodextrin (HPCD) or elevated Arl8b expression rescues lysosome transport, thereby reducing axonal autophagic stress and neuron death in NPC. These findings demonstrate a pathological mechanism by which altered membrane lipid composition impairs lysosome delivery into axons and provide biological insights into the translational application of HPCD in restoring axonal homeostasis at early stages of NPC disease.

Cerebellar Long Noncoding RNA Expression Profile in a Niemann-Pick C Disease Mouse Model.

Niemann-Pick type C (NP-C) disease is a neurodegenerative lysosomal storage disorder primarily caused by mutations in NPC1. However, its pathogenesis remains poorly understood. While mounting evidence has demonstrated the involvement of long noncoding RNAs (lncRNAs) in the pathogenesis of neurodegenerative disorders, the lncRNA expression profile in NP-C has not been determined. Here, we used RNA-seq analysis to determine lncRNA and mRNA expression profiles of the cerebella of NPC1-/- mice. We found that 272 lncRNAs and 856 mRNAs were significantly dysregulated in NPC1-/- mice relative to controls (≥ 2.0-fold, p < 0.05). Quantitative real-time PCR (qRT-PCR) was utilized to validate the expression of selected lncRNAs and mRNAs. Next, a lncRNA-mRNA coexpression network was employed to examine the potential roles of the differentially expressed (DE) lncRNAs. Functional analysis revealed that mRNAs coexpressed with lncRNAs are mainly linked to immune system-related processes and neuroinflammation. Moreover, knockdown of the lncRNA H19 ameliorated changes in ROS levels and cell viability and suppressed the lipopolysaccharide (LPS)-induced inflammatory response in vitro. Our findings indicate that dysregulated lncRNA expression patterns are associated with NP-C pathogenesis and offer insight into the development of novel therapeutics based on lncRNAs.

Toolbox Accelerating Glycomics (TAG): Glycan Annotation from MALDI-TOF MS Spectra and Mapping Expression Variation to Biosynthetic Pathways.

Glycans present extraordinary structural diversity commensurate with their involvement in numerous fundamental cellular processes including growth, differentiation, and morphogenesis. Unlike linear DNA and protein sequences, glycans have heterogeneous structures that differ in composition, branching, linkage, and anomericity. These differences pose a challenge to developing useful software for glycomic analysis. To overcome this problem, we developed the novel Toolbox Accelerating Glycomics (TAG) program. TAG consists of three units: 'TAG List' creates a glycan list that is used for database searching in TAG Expression; 'TAG Expression' automatically annotates and quantifies glycan signals and draws graphs; and 'TAG Pathway' maps the obtained expression information to biosynthetic pathways. Herein, we discuss the concepts, outline the TAG process, and demonstrate its potential using glycomic expression profile data from Chinese hamster ovary (CHO) cells and mutants lacking a functional Npc1 gene (Npc1 knockout (KO) CHO cells). TAG not only drastically reduced the amount of time and labor needed for glycomic analysis but also detected and quantified more glycans than manual analysis. Although this study was limited to the analysis of N-glycans and free oligosaccharides, the glycomic platform will be expanded to facilitate the analysis of O-glycans and glycans of glycosphingolipids.

The Effects of Extracellular Serum Concentration on APP Processing in Npc1-Deficient APP-Overexpressing N2a Cells.

Amyloid precursor protein (APP) is cleaved by a set of proteases including α-/ß-/γ- and recently identified η-secretases, generating C-terminal fragments (CTFs) of varying lengths and amyloid ß (Aß) peptides, which are considered to play a pivotal role in Alzheimer's disease (AD) pathogenesis. Cellular cholesterol content/distribution can regulate the production/clearance of APP metabolites and hence modify AD pathology. To determine the functional relation between endosomal-lysosomal (EL) cholesterol sequestration and APP metabolism, we used our recently developed mouse N2a-ANPC cells that overexpress Swedish mutant human APP in the absence of cholesterol-trafficking Niemann-Pick type C1 (Npc1) protein. Here, we report that neither increased levels nor EL cholesterol sequestration altered APP holoprotein levels but caused the intracellular accumulation of APP α-/ß-/η-CTFs and Aß1-40/42 peptides. The levels of APP-cleaved products increased as a function of extracellular serum concentration in N2a-ANPC cells, which are more vulnerable to death than the control cells. Additionally, we show that pH of the lysosomal vesicles in N2a-ANPC cells shifted to a less acidic range with increasing serum concentrations, thus making them less efficient functionally. Interestingly, the addition of cholesterol to the culture media not only increased the levels of cellular cholesterol and APP-cleaved products but also rendered the cells more vulnerable to toxicity. Collectively, our results suggest that extracellular cholesterol concentration in serum under conditions of Npc1 deficiency can influence intracellular cholesterol content/distribution and lysosomal efficacy, triggering the accumulation of toxic APP-cleaved products, eventually leading to cell death.