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.

Familial Alzheimer's disease associated with heterozygous NPC1 mutation.

INTRODUCTION: NPC1 mutations are responsible for Niemann-Pick disease type C (NPC), a rare autosomal recessive neurodegenerative disease. Patients harbouring heterozygous NPC1 mutations may rarely show parkinsonism or dementia. Here, we describe for the first time a large family with an apparently autosomal dominant late-onset Alzheimer's disease (AD) harbouring a novel heterozygous NPC1 mutation. METHODS: All the five living siblings belonging to the family were evaluated. We performed clinical evaluation, neuropsychological tests, assessment of cerebrospinal fluid markers of amyloid deposition, tau pathology and neurodegeneration (ATN), structural neuroimaging and brain amyloid-positron emission tomography. Oxysterol serum levels were also tested. A wide next-generation sequencing panel of genes associated with neurodegenerative diseases and a whole exome sequencing analysis were performed. RESULTS: We detected the novel heterozygous c.3034G>T (p.Gly1012Cys) mutation in NPC1, shared by all the siblings. No other point mutations or deletions in NPC1 or NPC2 were found. In four siblings, a diagnosis of late-onset AD was defined according to clinical characterisation and ATN biomarkers (A+, T+, N+) and serum oxysterol analysis showed increased 7-ketocholesterol and cholestane-3ß,5α,6ß-triol. DISCUSSION: We describe a novel NPC1 heterozygous mutation harboured by different members of a family with autosomal dominant late-onset amnesic AD without NPC-associated features. A missense mutation in homozygous state in the same aminoacidic position has been previously reported in a patient with NPC with severe phenotype. The alteration of serum oxysterols in our family corroborates the pathogenic role of our NPC1 mutation. Our work, illustrating clinical and biochemical disease hallmarks associated with NPC1 heterozygosity in patients affected by AD, provides relevant insights into the pathogenetic mechanisms underlying this possible novel association.

Importance of the biochemical investigations for the functional characterization of a NPC1 variant identified by exome sequencing.

Niemann-Pick disease type C (NPC) is one of the lysosomal storage disorders. It is caused by biallelic pathogenic variants in NPC1 or NPC2, which results in a defective cholesterol trafficking inside the late endosome and lysosome. There is a high clinical variability in the age of presentation and the phenotype of this disorder making the diagnosis challenging. Here, we report a patient with an infantile onset global developmental delay, microcephaly and dysmorphic features, homozygous for c.3560C>T (p.A1187V) variant in NPC1. His plasma oxysterol levels were normal on two occasions. His lyso-sphingomyelin-509 (lyso-SM 509) and urinary bile acid levels were normal. Based on the phenotype and biochemical features, the diagnosis of NPC was excluded in this patient. We emphasize the importance of functional characterization in the classification of novel variants to prevent a misdiagnosis. Matching the phenotype and biochemical evidence with the molecular genomic tests is crucial for the confirmation of genetic diagnoses.

Novel compound heterozygous mutations of the NPC1 gene associated with Niemann-pick disease type C: a case report and review of the literature.

BACKGROUND: Niemann-Pick Disease type C is a fatal autosomal recessive lipid storage disorder caused by NPC1 or NPC2 gene mutations and characterized by progressive, disabling neurological deterioration and hepatosplenomegaly. Herein, we identified a novel compound heterozygous mutations of the NPC1 gene in a Chinese pedigree. CASE PRESENTATION: This paper describes an 11-year-old boy with aggravated walking instability and slurring of speech who presented as Niemann-Pick Disease type C. He had the maternally inherited c.3452 C > T (p. Ala1151Val) mutation and the paternally inherited c.3557G > A (p. Arg1186His) mutation using next-generation sequencing. The c.3452 C > T (p. Ala1151Val) mutation has not previously been reported. CONCLUSIONS: This study predicted that the c.3452 C > T (p. Ala1151Val) mutation is pathogenic. This data enriches the NPC1 gene variation spectrum and provides a basis for familial genetic counseling and prenatal diagnosis.

Npc1 gene mutation abnormally activates the classical Wnt signalling pathway in mouse kidneys and promotes renal fibrosis.

Niemann-Pick disease type C1 (NPC1) is a lysosomal lipid storage disease caused by NPC1 gene mutation. Our previous study found that, compared with wild-type (Npc1+/+ ) mice, the renal volume and weight of Npc1 gene mutant (Npc1-/- ) mice were significantly reduced. We speculate that Npc1 gene mutations may affect the basic structure of the kidneys of Npc1-/- mice, and thus affect their function. Therefore, we randomly selected postnatal Day 28 (P28) and P56 Npc1+/+ and Npc1-/- mice, and observed the renal structure and pathological changes by haematoxylin-eosin staining. The level of renal fibrosis was detected by immunofluorescence histochemical techniques, and western blotting was used to detect the expression levels of apoptosis-related proteins and canonical Wnt signalling pathway related proteins. The results showed that compared with Npc1+/+ mice, the kidneys of P28 and P56 Npc1-/- mice underwent apoptosis and fibrosis; furthermore, there were obvious vacuoles in the cytoplasm of renal tubular epithelial cells of P56 Npc1-/- mice, the cell bodies were loose and foam-like, and the canonical Wnt signalling pathway was abnormally activated. These results showed that Npc1 gene mutation can cause pathological changes in the kidneys of mice. As age increased, vacuoles developed in the cytoplasm of renal tubular epithelial cells, and apoptosis of renal cells, abnormal activation of the Wnt signalling pathway, and promotion of renal fibrosis increased.

Individualized management of genetic diversity in Niemann-Pick C1 through modulation of the Hsp70 chaperone system.

Genetic diversity provides a rich repository for understanding the role of proteostasis in the management of the protein fold in human biology. Failure in proteostasis can trigger multiple disease states, affecting both human health and lifespan. Niemann-Pick C1 (NPC1) disease is a rare genetic disorder triggered by mutations in NPC1, a multi-spanning transmembrane protein that is trafficked through the exocytic pathway to late endosomes (LE) and lysosomes (Ly) (LE/Ly) to globally manage cholesterol homeostasis. Defects triggered by >300 NPC1 variants found in the human population inhibit export of NPC1 protein from the endoplasmic reticulum (ER) and/or function in downstream LE/Ly, leading to cholesterol accumulation and onset of neurodegeneration in childhood. We now show that the allosteric inhibitor JG98, that targets the cytosolic Hsp70 chaperone/co-chaperone complex, can significantly improve the trafficking and post-ER protein level of diverse NPC1 variants. Using a new approach to model genetic diversity in human disease, referred to as variation spatial profiling, we show quantitatively how JG98 alters the Hsp70 chaperone/co-chaperone system to adjust the spatial covariance (SCV) tolerance and set-points on an amino acid residue-by-residue basis in NPC1 to differentially regulate variant trafficking, stability, and cholesterol homeostasis, results consistent with the role of BCL2-associated athanogene family co-chaperones in managing the folding status of NPC1 variants. We propose that targeting the cytosolic Hsp70 system by allosteric regulation of its chaperone/co-chaperone based client relationships can be used to adjust the SCV tolerance of proteostasis buffering capacity to provide an approach to mitigate systemic and neurological disease in the NPC1 population.

Loading of cell cultures with cholesterol-dextran particles as a new functional test for Niemann-Pick type C disease.

Deuterium-labeled cholesterol-dextran particles (d4-CholDex), prepared by co-precipitation, were internalized by cultured human skin fibroblasts and HEK293 cells. Subcellular particles from d4-CholDex-treated HEK293 cells were fractionated on iodixanol gradients. More than 60% of d4-cholesterol (d4-UC) in the gradient co-fractionated with lysosomal markers and NPC1. This and formation of d4-cholesteryl esters (d4-CE) in the cells suggests that d4-CholDex is lysosomally processed. In accordance with these findings, we observed an increase in lysosomal cholesterol content by fluorescence microscopy in CholDex-loaded cells. Fibroblast cultures including 13 NPC1-deficient, four heterozygous and six control lines were treated with d4-CholDex at final d4-UC concentration of 0.05 mg/ml (127.98 µmol/L) for 3 h and chased for 48 h in medium without d4-CholDex. Concentrations of d4-UC and d4-CE in harvested cells were measured by tandem mass spectrometry (MS/MS). d4-UC/d4-CE ratios were elevated in NP-C lines compared to controls (n = 6, mean = 4.36, range = 1.89-8.91), with the highest ratios in severe NP-C1 phenotypes and the lowest in adolescent/adult type patients. There were overlaps between NP-C1 forms: early infantile (n = 1, mean = 48.6), late infantile (n = 4, mean = 36.3, range = 20.6-54.0), juvenile (n = 5, mean = 24.7, range = 13.4-38.3), adolescent/adult (n = 3, mean = 14.5, range = 11.7-19.8). The ratios in NP-C1 heterozygotes were mildly elevated (n = 4, mean = 16.4, range = 14.9-17.4) and comparable to patients with adolescent/adult NP-C1. The test can be useful in evaluation of suspected NP-C patients with inconclusive results of biomarker or molecular tests. Its advantages include standardized preparation of particles with longer shelf life at 4 °C, quantitative results, and no requirement for radioactive chemicals.

Whole-exome sequencing analysis to identify novel potential pathogenetic NPC1 mutations in two Chinese families with Niemann-Pick disease type C.

BACKGROUND: Niemann-Pick disease type C (NPC) is an autosomal recessive lipid storage disorder, affecting the nervous system and the internal organs. It is characterized by the presence of foam cells in bone marrow, liver, and spleen biopsies. Although many mutations in NPC1 have been identified to be related to disease onset, the relationship between genotype and phenotype remains unclear. To elucidate the genetic heterogeneity of NPC, we described the clinical manifestations and possible genetic pathogenesis of two patients from unrelated families with NPC. METHODS: DNA was extracted from the peripheral blood of the two patients and their families and from healthy individuals. Whole-exome sequencing followed by Sanger sequencing was performed to verify the mutations identified in their families. RESULTS: We identified four mutations in NPC1 in the two patients from different families: c.1290delC (p.F431Lfs*18)/c.2807G > A(p.G936D) in family A and c.3604_3605insA (p.I1202Nfs*56)/c.881 + 3A > G in family B from their parents. Bioinformatics analysis predicted these mutations to be deleterious, suggesting that mutations in exons are highly conservative. The patient in family A presented with a developmental delay that was different from the typical symptoms of developmental regression in family B. CONCLUSION: Our study identified three novel mutations and one known mutation in NPC1 and evaluated their pathogenicity, enriching the NPC1 mutation and phenotype spectrum and providing a new basis for the genetic and prenatal diagnosis of this disease.

Targeting defective sphingosine kinase 1 in Niemann-Pick type C disease with an activator mitigates cholesterol accumulation.

Niemann-Pick type C (NPC) disease is a lysosomal storage disorder arising from mutations in the cholesterol-trafficking protein NPC1 (95%) or NPC2 (5%). These mutations result in accumulation of low-density lipoprotein-derived cholesterol in late endosomes/lysosomes, disruption of endocytic trafficking, and stalled autophagic flux. Additionally, NPC disease results in sphingolipid accumulation, yet it is unique among the sphingolipidoses because of the absence of mutations in the enzymes responsible for sphingolipid degradation. In this work, we examined the cause for sphingosine and sphingolipid accumulation in multiple cellular models of NPC disease and observed that the activity of sphingosine kinase 1 (SphK1), one of the two isoenzymes that phosphorylate sphingoid bases, was markedly reduced in both NPC1 mutant and NPC1 knockout cells. Conversely, SphK1 inhibition with the isotype-specific inhibitor SK1-I in WT cells induced accumulation of cholesterol and reduced cholesterol esterification. Of note, a novel SphK1 activator (SK1-A) that we have characterized decreased sphingoid base and complex sphingolipid accumulation and ameliorated autophagic defects in both NPC1 mutant and NPC1 knockout cells. Remarkably, in these cells, SK1-A also reduced cholesterol accumulation and increased cholesterol ester formation. Our results indicate that a SphK1 activator rescues aberrant cholesterol and sphingolipid storage and trafficking in NPC1 mutant cells. These observations highlight a previously unknown link between SphK1 activity, NPC1, and cholesterol trafficking and metabolism.

Glial contribution to cyclodextrin-mediated reversal of cholesterol accumulation in murine NPC1-deficient neurons in vivo.

Niemann-Pick type C disease is a rare and fatal lysosomal storage disorder presenting severe neurovisceral symptoms. Disease-causing mutations in genes encoding either NPC1 or NPC2 protein provoke accumulation of cholesterol and other lipids in specific structures of the endosomal-lysosomal system and degeneration of specific cells, notably neurons in the central nervous system (CNS). 2-hydroxypropyl-beta-cyclodextrin (CD) emerged as potential therapeutic approach based on animal studies and clinical data, but the mechanism of action in neurons has remained unclear. To address this topic in vivo, we took advantage of the retina as highly accessible part of the CNS and intravitreal injections as mode of drug administration. Coupling CD to gold nanoparticles allowed us to trace its intracellular location. We report that CD enters the endosomal-lysosomal system of neurons in vivo and enables the release of lipid-laden lamellar inclusions, which are then removed from the extracellular space by specific types of glial cells. Our data suggest that CD induces a concerted action of neurons and glial cells to restore lipid homeostasis in the central nervous system.

A Virion-Based Assay for Glycoprotein Thermostability Reveals Key Determinants of Filovirus Entry and Its Inhibition.

Ebola virus (EBOV) entry into cells is mediated by its spike glycoprotein (GP). Following attachment and internalization, virions traffic to late endosomes where GP is cleaved by host cysteine proteases. Cleaved GP then binds its cellular receptor, Niemann-Pick C1. In response to an unknown cellular trigger, GP undergoes conformational rearrangements that drive fusion of viral and endosomal membranes. The temperature-dependent stability (thermostability) of the prefusion conformers of class I viral fusion glycoproteins, including those of filovirus GPs, has provided insights into their propensity to undergo fusion-related rearrangements. However, previously described assays have relied on soluble glycoprotein ectodomains. Here, we developed a simple enzyme-linked immunosorbent assay (ELISA)-based assay that uses the temperature-dependent loss of conformational epitopes to measure thermostability of GP embedded in viral membranes. The base and glycan cap subdomains of all filovirus GPs tested suffered a concerted loss of prefusion conformation at elevated temperatures but did so at different temperature ranges, indicating virus-specific differences in thermostability. Despite these differences, all of these GPs displayed reduced thermostability upon cleavage to GP conformers (GPCL). Surprisingly, acid pH enhanced, rather than decreased, GP thermostability, suggesting it could enhance viral survival in hostile endo/lysosomal compartments. Finally, we confirmed and extended previous findings that some small-molecule inhibitors of filovirus entry destabilize EBOV GP and uncovered evidence that the most potent inhibitors act through multiple mechanisms. We establish the epitope-loss ELISA as a useful tool for studies of filovirus entry, engineering of GP variants with enhanced stability for use in vaccine development, and discovery of new stability-modulating antivirals.IMPORTANCE The development of Ebola virus countermeasures is challenged by our limited understanding of cell entry, especially at the step of membrane fusion. The surface-exposed viral protein, GP, mediates membrane fusion and undergoes major structural rearrangements during this process. The stability of GP at elevated temperatures (thermostability) can provide insights into its capacity to undergo these rearrangements. Here, we describe a new assay that uses GP-specific antibodies to measure GP thermostability under a variety of conditions relevant to viral entry. We show that proteolytic cleavage and acid pH have significant effects on GP thermostability that shed light on their respective roles in viral entry. We also show that the assay can be used to study how small-molecule entry inhibitors affect GP stability. This work provides a simple and readily accessible assay to engineer stabilized GP variants for antiviral vaccines and to discover and improve drugs that act by modulating GP stability.

Hepatocellular carcinoma as a complication of Niemann-Pick disease type C1.

Niemann-Pick disease type C (NPC) is a rare and fatal lysosomal storage disorder characterized by neurodegeneration and hepatic involvement. Mutations in either NPC1 or NPC2, two genes encoding lysosomal proteins, lead to an intracellular accumulation of unesterified cholesterol and sphingolipids in late endosomes/lysosomes. Early cholestatic disease is considered a hallmark of patients with early disease onset. This can potentially result in liver failure shortly after birth or subclinical hepatic inflammation. Previous reports suggest an association between NPC and hepatocellular carcinoma, a cancer that is rare during childhood. We present a 12-year-old male with a known diagnosis of NPC1 disease who was found to have a stage III hepatocellular carcinoma, underwent surgical resection with adjuvant chemotherapy, and subsequently died from metastatic disease. This report provides evidence of an increased risk of hepatocellular carcinoma in NPC patients, suggesting a need for screening in this patient population.

Treatment trials in Niemann-Pick type C disease.

Niemann-Pick type C (NPC) disease is a genetically determined neurodegenerative metabolic disease. It belongs to the lysosomal storage diseases and its main cause is impaired cholesterol transport in late endosomes or lysosomes. It is an autosomal recessive inherited disease that results from mutations in the NPC1 or NPC2 genes. The treatment efforts are focused on the slowing its progression. The only registered drug, devoted for NPC patients is Miglustat. Effective treatment is still under development. NPC disease mainly affects the nervous system, and the crossing of the blood-brain barrier by medicines is still a challenge, therefore the combination therapies of several compounds are increasingly being worked on. The aim of this paper is to present the possibilities in treatment of Niemann-Pick type C disease. The discussed research results relate to animal studies.

Impaired Retromer Function in Niemann-Pick Type C Disease Is Dependent on Intracellular Cholesterol Accumulation.

Niemann-Pick type C disease (NPC) is a rare inherited neurodegenerative disorder characterized by an accumulation of intracellular cholesterol within late endosomes and lysosomes due to NPC1 or NPC2 dysfunction. In this work, we tested the hypothesis that retromer impairment may be involved in the pathogenesis of NPC and may contribute to increased amyloidogenic processing of APP and enhanced BACE1-mediated proteolysis observed in NPC disease. Using NPC1-null cells, primary mouse NPC1-deficient neurons and NPC1-deficient mice (BALB/cNctr-Npc1m1N), we show that retromer function is impaired in NPC. This is manifested by altered transport of the retromer core components Vps26, Vps35 and/or retromer receptor sorLA and by retromer accumulation in neuronal processes, such as within axonal swellings. Changes in retromer distribution in NPC1 mouse brains were observed already at the presymptomatic stage (at 4-weeks of age), indicating that the retromer defect occurs early in the course of NPC disease and may contribute to downstream pathological processes. Furthermore, we show that cholesterol depletion in NPC1-null cells and in NPC1 mouse brains reverts retromer dysfunction, suggesting that retromer impairment in NPC is mechanistically dependent on cholesterol accumulation. Thus, we characterized retromer dysfunction in NPC and propose that the rescue of retromer impairment may represent a novel therapeutic approach against NPC.

Patient-Specific iPSC-Derived Neural Differentiated and Hepatocyte-like Cells, Carrying the Compound Heterozygous Mutation p.V1023Sfs*15/p.G992R, Present the "Variant" Biochemical Phenotype of Niemann-Pick Type C1 Disease.

Niemann-Pick disease type C1 (NP-C1) is a rare lysosomal storage disorder caused by autosomal recessive mutations in the NPC1 gene. Patients display a wide spectrum on the clinical as well as on the molecular level, wherein a so-called "variant" biochemical phenotype can be observed. Here, we report an in vitro analysis of fibroblasts obtained from an NP-C1 patient carrying the undescribed compound heterozygous mutation p.V1023Sfs*15/p.G992R. Since NP-C1 is a neurovisceral disease and the patient suffers from severe neurological as well as hepatic symptoms, we extended our study to neural differentiated and hepatocyte-like cells derived from patient-specific induced pluripotent stem cells. We detected slightly increased intracellular cholesterol levels compared to the control cell line in fibroblasts, neural differentiated and hepatocyte-like cells, suggesting a "variant" biochemical phenotype. Furthermore, the total NPC1 protein, as well as post-ER glycoforms of the NPC1 protein, tended to be reduced. In addition, colocalization analysis revealed a mild reduction of the NPC1 protein in the lysosomes. The patient was diagnosed with NP-C1 at the age of 34 years, after an initial misdiagnosis of schizophrenia. After years of mild and unspecific symptoms, such as difficulties in coordination and concentration, symptoms progressed and the patient finally presented with ataxia, dysarthria, dysphagia, vertical supranuclear gaze palsy, and hepatosplenomegaly. Genetic testing finally pointed towards an NP-C1 diagnosis, revealing the so-far undescribed compound heterozygous mutation p.V1023Sfs*15/p.G992R in the NPC1 gene. In light of these findings, this case provides support for the p.G992R mutation being causative for a "variant" biochemical phenotype leading to an adult-onset type of NP-C1 disease.

Microglia activation in Niemann-Pick disease, type C1 is amendable to therapeutic intervention.

Niemann-Pick disease, type C1 (NPC1) is a neurodegenerative disorder with limited treatment options. NPC1 is associated with neuroinflammation; however, attempts to therapeutically target neuroinflammation in NPC1 have had mixed success. We show here that NPC1 neuroinflammation is characterized by an atypical microglia activation phenotype. Specifically, Npc1-/- microglia demonstrated altered morphology, reduced levels of lineage markers and a shift toward glycolytic metabolism. Treatment with 2-hydroxypropyl-ß-cyclodextrin (HPßCD), a drug currently being studied in a phase 2b/3 clinical trial, reversed all microglia-associated defects in Npc1-/- animals. In addition, impairing microglia mediated neuroinflammation by genetic deletion of IRF8 led to decreased symptoms and increased lifespan. We identified CD22 as a marker of dysregulated microglia in Npc1 mutant mice and subsequently demonstrated that elevated cerebrospinal fluid levels of CD22 in NPC1 patients responds to HPßCD administration. Collectively, these data provide the first in-depth analysis of microglia function in NPC1 and suggest possible new therapeutic approaches.

Pharmacokinetics and distribution of 2-hydroxypropyl-ß-cyclodextrin following a single intrathecal dose to cats.

2-Hydroxypropyl-ß-cyclodextrin (HP-ß-CD) is an experimental therapy for Niemann-Pick disease type C (NPC) that reduced neuronal cholesterol and ganglioside storage, reduced Purkinje cell death, and increased lifespan in npc1-/- mice and NPC1 cats. In this study, tissue distribution was investigated in normal cats that received a single 120-mg dose of [14 C]-HP-ß-CD (approximately 200 µCi/cat) via the cerebellomedullary cistern (CBMC) and lumbar cistern. One cat was euthanized at each of various time points up to 24 hours postdose for subsequent processing and quantitative whole-body autoradiographic analysis. HP-ß-CD-derived radioactivity absorbed from the CBMC was widely distributed to cat tissues; most tissues were observed to have reached their highest concentration at 1 hour postdose. HP-ß-CD-derived radioactivity penetrated into the deeper parts of the central nervous system with the highest concentration at 4 hours (403 µg Eq/g or 0.28 mM) and remained high (49.7 µg Eq/g or 0.03 mM) at 24 hours. The relatively long half-life (11-30 hours) in cerebral ventricles and the subarachnoid space surrounding the brain and spinal cord might contribute to the efficacy of HP-ß-CD in NPC1 cats. Other tissues with high concentrations of radioactivity were nasal turbinates, pituitary gland, and urinary bladder, while relatively low concentrations were observed in blood and bile.

Ultrastructure of spinal anterior horn cells in human Niemann-Pick type C (NPC) patient and mouse model of NPC with retroposon insertion in NPC1 genes.

Niemann-Pick disease type C (NPC) is a neurovisceral lipid-storage disease. Although NPC patients show lipid storage in anterior horn cells of the spinal cord, little information is available regarding the electron microscopic analyses of the morphologies of intra-endosomal lipid like-materials in the anterior horn cells of NPC patients. In this study, we elucidated the intra-endosomal ultrastructures in spinal anterior horn cells in an NPC patient, as well as in mutant BALB/c NPC1-/- mice with a retroposon insertion in the NPC1 gene. These morphologies were classified into four types: vesicle, multiple concentric sphere (MCS), membrane, and rose flower. The percentages of the composition in the NPC patient and NPC1-/- mice were: vesicle (55.5% and 14.9%), MCS (15.7% and 3.4%), membrane (23.6% and 57.1%), and rose flower (5.2% and 24.6%), respectively. Formation of the intra-endosomal structures could proceed as follows: (i) a vesicle or MCS buds off the endosome into the lumen; (ii) when a vesicle breaks down, a membrane is formed; and (iii) after an MCS breaks down, a rose flower structure is formed. Our new finding in this study is that ultrastructural morphology is the same between the NPC patient and NPC1-/- mice, although there are differences in the composition.

N-glycome of the Lysosomal Glycocalyx is Altered in Niemann-Pick Type C Disease (NPC) Model Cells.

Increasing evidence implicates lysosomal dysfunction in the pathogenesis of neurodegenerative diseases, including the rare inherited lysosomal storage disorders (LSDs) and the most common neurodegenerative diseases, such as Alzheimer's and Parkinson's disease (AD and PD). Although the triggers of the lysosomal impairment may involve the accumulated macromolecules or dysfunction of the lysosomal enzymes, the role of the lysosomal glycocalyx in the lysosomal (dys)function has not been studied. The goal of this work was to analyze whether there are changes in the lysosomal glycocalyx in a cellular model of a LSD Niemann-Pick type C disease (NPC). Using the ferrofluid nanoparticles we isolated lysosomal organelles from NPC1-null and CHOwt cells. The magnetically isolated lysosomal fractions were enriched with the lysosomal marker protein LAMP1 and showed the key features of NPC disease: 3-fold higher cholesterol content and 4-5 fold enlarged size of the particles compared with the lysosomal fractions of wt cells. These lysosomal fractions were further processed to isolate lysosomal membrane proteins using Triton X-114 and their N-glycome was analyzed by HILIC-UPLC. N-glycans presented in each chromatographic peak were elucidated using MALDI-TOF/TOF-MS. We detected changes in the N-glycosylation pattern of the lysosomal glycocalyx of NPC1-null versus wt cells which involved high-mannose and sialylated N-glycans. To the best of our knowledge this study is the first to report N-glycome profiling of the lysosomal glycocalyx in NPC disease cellular model and the first to report the specific changes in the lysosomal glycocalyx in NPC1-null cells. We speculate that changes in the lysosomal glycocalyx may contribute to lysosomal (dys)function. Further glycome profiling of the lysosomal glycocalyx in other LSDs as well as the most common neurodegenerative diseases, such as AD and PD, is necessary to better understand the role of the lysosomal glycocalyx and to reveal its potential contribution in lysosomal dysfunction leading to neurodegeneration.

Cholesterol Transport in Wild-Type NPC1 and P691S: Molecular Dynamics Simulations Reveal Changes in Dynamical Behavior.

The Niemann-Pick C1 (NPC1) protein is the main protein involved in NPC disease, a fatal lysosomal lipid storage disease. NPC1, containing 1278 amino acids, is comprised of three lumenal domains (N-terminal, middle lumenal, C-terminal) and a transmembrane (TM) domain that contains a five helix bundle referred to as the sterol-sensing domain (SSD). The exact purpose of the SSD is not known, but it is believed that the SSD may bind cholesterol, either as a part of the lipid trafficking pathway or as part of a signaling mechanism. A recent cryo-EM structure has revealed an itraconazole binding site (IBS) in the SSD of human NPC1. Using this structural data, we constructed a model of cholesterol-bound wild-type (WT) and mutant P691S and performed molecular dynamics (MD) simulations of each cholesterol-bound protein. For WT NPC1, cholesterol migrates laterally, in the direction of the lipid bilayer. In the case of P691S, cholesterol is observed for the first time to migrate away from the SSD toward the N-terminal domain via a putative tunnel that connects the IBS with the lumenal domains. Structural features of the IBS are analyzed to identify the causes for different dynamical behavior between cholesterol-bound WT and cholesterol-bound P691S. The side chain of Ser691 in the P691S mutant introduces a hydrogen bond network that is not present in the WT protein. This change is likely responsible for the altered dynamical behavior observed in the P691S mutant and helps explain the disrupted cholesterol trafficking behavior observed in experiments.