NPC1 Expression Research Articles

Increased NPC1 and STARD3 expression in placentas from women with Maternal Supraphysiological Hypercholesterolemia (MSPH) promotes lysosomal and mitochondrial dysfunction.

Increased NPC1 and STARD3 expression in placentas from women with Maternal Supraphysiological Hypercholesterolemia (MSPH) promotes lysosomal and mitochondrial dysfunction.

Lysosomal cholesterol accumulation in aged astrocytes impairs cholesterol delivery to neurons and can be rescued by cannabinoids.

Cholesterol is crucial for the proper functioning of eukaryotic cells, especially neurons, which rely on cholesterol to maintain their complex structure and facilitate synaptic transmission. However, brain cells are isolated from peripheral cholesterol by the blood-brain barrier and mature neurons primarily uptake the cholesterol synthesized by astrocytes for proper function. This study aimed to investigate the effect of aging on cholesterol trafficking in astrocytes and its delivery to neurons. We found that aged astrocytes accumulated high levels of cholesterol in the lysosomal compartment, and this cholesterol buildup can be attributed to the simultaneous occurrence of two events: decreased levels of the ABCA1 transporter, which impairs ApoE-cholesterol export from astrocytes, and reduced expression of NPC1, which hinders cholesterol release from lysosomes. We show that these two events are accompanied by increased microR-33 in aged astrocytes, which targets ABCA1 and NPC1. In addition, we demonstrate that the microR-33 increase is triggered by oxidative stress, one of the hallmarks of aging. By coculture experiments, we show that cholesterol accumulation in astrocytes impairs the cholesterol delivery from astrocytes to neurons. Remarkably, we found that this altered transport of cholesterol could be alleviated through treatment with endocannabinoids as well as cannabidiol or CBD. Finally, according to data demonstrating that aged astrocytes develop an A1 phenotype, we found that cholesterol buildup is also observed in reactive C3+ astrocytes. Given that reduced neuronal cholesterol affects synaptic plasticity, the ability of cannabinoids to restore cholesterol transport from aged astrocytes to neurons holds significant implications in aging and inflammation.

The Aging Lacrimal Gland of Female C57BL/6J Mice Exhibits Multinucleate Macrophage Infiltration Associated With Lipid Dysregulation.

Loss of function of the lacrimal gland (LG), which produces the aqueous tear film, is implicated in age-related dry eye. To better understand this deterioration, we evaluated changes in lipid metabolism and inflammation in LGs from an aging model. LG sections from female C57BL/6J mice of different ages (young, 2-3 months; intermediate, 10-14 months; old, ≥24 months) were stained with Oil Red-O or Toluidine blue to detect lipids. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis and western blotting of LG lysates determined differences in the expression of genes and proteins related to lipid metabolism. A photobleaching protocol to quench age-related autofluorescence was used in LG sections to evaluate changes in immunofluorescence associated with NPC1, NPC2, CTSL, and macrophages (F4/80, CD11b) with age using confocal fluorescence microscopy. Old LGs showed increased lipids prominent in basal aggregates in acinar cells and in extra-acinar sites. LG gene expression of Npc1, Npc2, Lipa, and Mcoln2, encoding proteins involved in lipid metabolism, was increased with age. NPC1 was also significantly increased in old LGs by western blotting. In photobleached LG sections, confocal fluorescence microscopy imaging of NPC1, NPC2, and CTSL immunofluorescence showed age-associated enrichment in macrophages labeled to detect F4/80. Although mononuclear macrophages were detectable in LG at all ages, this novel multinucleate macrophage population containing NPC1, NPC2, and CTSL and enriched in F4/80 and some CD11b was increased with age at extra-acinar sites. Lipid-metabolizing proteins enriched in F4/80-positive multinucleated macrophages are increased in old LGs adjacent to sites of lipid deposition in acini.

Abstract 160: Macrophage-specific Ablation Of Bmal1 Regulates Cholesterol Transport And Enhances Atherosclerosis

We have previously shown that global and liver-specific Bmal1 deficient mice develop enhanced atherosclerosis. Macrophages play a critical role in atherosclerosis, yet the role of macrophage Bmal1 in cholesterol uptake, intracellular transport and atherosclerosis remains unknown. Here, we used global and macrophage-specific Bmal1 deficient mice to clarify the role of macrophage Bmal1 in atherosclerosis. We found that macrophage-specific Bmal1 deficient mice show increased atherosclerosis. Mechanistically, Bmal1 deficient macrophages exhibited: (1) increased expression of Lox-1 and Cd36 and uptake of oxLDL; (2) decreased expression of Abca1 and Abcg1 and reduced cholesterol efflux, and reverse cholesterol transport; and (3) diminished export of free cholesterol from lysosomes due to reduced expression of Npc1 and Npc2. Molecular studies showed that Bmal1 directly regulates Npc1 and Npc2 by binding to E-boxes in their promoters, whereas it indirectly regulates Abca1 and Abcg1 by regulating ZNF202. Furthermore, we provide evidence that Bmal1 interacts with both Clock and Npas2 to regulate ZNF202, Npc1 and Npc2. Our data demonstrate that macrophage Bmal1 is a key regulator of oxLDL uptake, lysosomal cholesterol export, and cholesterol efflux, and, therefore, plays a crucial role in the prevention of atherosclerosis.

Targeting NPC1 in Renal Cell Carcinoma

Rapidly proliferating cancer cells have a greater requirement for cholesterol than normal cells. Tumor cells are largely dependent on exogenous lipids given that their growth requirements are not fully met by endogenous pathways. Our current study shows that ccRCC cells have redundant mechanisms of cholesterol acquisition. We demonstrate that all major lipoproteins (i.e., LDL, HDL, and VLDL) have a comparable ability to support the growth of ccRCC cells and are equally effective in counteracting the antitumor activities of TKIs. The intracellular trafficking of exogenous lipoprotein-derived cholesterol appears to be distinct from the movement of endogenously synthesized cholesterol. De novo synthetized cholesterol is transported from the endoplasmic reticulum directly to the plasma membrane and to the acyl-CoA: cholesterol acyltransferase, whereas lipoprotein-derived cholesterol is distributed through the NPC1-dependent endosomal trafficking system. Expression of NPC1 is increased in ccRCC at mRNA and protein levels, and high expression of NPC1 is associated with poor prognosis. Our current findings show that ccRCC cells are particularly sensitive to the inhibition of endolysosomal cholesterol export and underline the therapeutic potential of targeting NPC1 in ccRCC.

Impact of miR-29c-3p in the Nucleus Accumbens on Methamphetamine-Induced Behavioral Sensitization and Neuroplasticity-Related Proteins.

Methamphetamine (METH) abuse inflicts both physical and psychological harm. While our previous research has established the regulatory role of miR-29c-3p in behavior sensitization, the underlying mechanisms and target genes remain incompletely understood. In this study, we employed the isobaric tags for relative and absolute quantitation (iTRAQ) technique in conjunction with Ingenuity pathway analysis (IPA) to probe the putative molecular mechanisms of METH sensitization through miR-29c-3p inhibition. Through a microinjection of AAV-anti-miR-29c-3p into the nucleus accumbens (NAc) of mice, we observed the attenuation of METH-induced locomotor effects. Subsequent iTRAQ analysis identified 70 differentially expressed proteins (DEPs), with 22 up-regulated potential target proteins identified through miR-29c-3p target gene prediction and IPA analysis. Our focus extended to the number of neuronal branches, the excitatory synapse count, and locomotion-related pathways. Notably, GPR37, NPC1, and IREB2 emerged as potential target molecules for miR-29c-3p regulation, suggesting their involvement in the modulation of METH sensitization. Quantitative PCR confirmed the METH-induced aberrant expression of Gpr37, Npc1, and Ireb2 in the NAc of mice. Specifically, the over-expression of miR-29c-3p led to a significant reduction in the mRNA level of Gpr37, while the inhibition of miR-29c-3p resulted in a significant increase in the mRNA level of Gpr37, consistent with the regulatory principle of miRNAs modulating target gene expression. This suggests that miR-29c-3p potentially influences METH sensitization through its regulation of neuroplasticity. Our research indicates that miR-29c-3p plays a crucial role in regulating METH-induced sensitization, and it identified the potential molecular of miR-29c-3p in regulating METH-induced sensitization.

Expression of components involved in cholesterol homeostasis maintenance during experimental autoimmune encephalomyelitis in rat spinal cord.

Dysregulations in cholesterol homeostasis contribute to the pathogenesis of multiple sclerosis (MS) and its best described animal model, experimental autoimmune encephalomyelitis (EAE). Cholesterol is an important component of myelin, which is necessary for signal transmission between neurons. Demyelination leads to the formation of oxysterols, degradation products of cholesterol that are ligands for nuclear liver X receptors (LXRs). Genes regulated by LXRs are involved in cholesterol efflux, absorption, transport, and excretion, which we investigated in this study. In this study, we detected changes in gene expression of Srebf1, Ldlr, Soat1, Abca1, Lrp1, and Npc1, all of which are important in the regulation of cholesterol homeostasis, during the course of EAE in male and female rats. In particular, differential expression of Srebf1, Ldlr, and Soat1 was observed in the spinal cord of male and female rats during EAE. Moreover, these genes are altered during EAE. In contrast, the expression of Abca1 and Lrp1 was significantly affected only by sex. In male animals, the expression of Npc1 is conspicuously reduced in EAE pathology. Thus, our study confirms the involvement of enzymes of cholesterol metabolism in the pathophysiology of EAE, with sex and disease progression affecting the expression of these genes. These findings may improve the understanding of neurodegenerative diseases associated with impaired lipid metabolism in the brain, such as MS/EAE.

Application prospect of replacement of fish meal with spray-dried egg meal in diets for swimming crab (Portunus trituberculatus)

An 8-week feeding trial was conducted to evaluate the effects of replacing fish meal with spray-dried egg meal (SEM) on growth performance, anti-oxidative capacity, mTOR pathway, and cholesterol metabolism in juvenile Portunus trituberculatus. The results showed that the highest percent weight gain was observed in crabs fed diet with 10% replacement of fish meal with SEM. Essential amino acids in the muscle and hepatopancreas significantly decreased with the increase of dietary replacement levels of fish meal with SEM. The lowest content of MDA in hepatopancreas was occurred at those fed diet with 40% replacement of fish meal with SEM, the activities of T-AOC, GSH-PX and content of GSH in hepatopancreas significantly increased with dietary SEM replacement fish meal increasing from 10% to 50%. The expression of akt, eif4e1a, eif4e2, eif4e3, and 4ebp1 significantly up-regulated, however, the expression of tor was showed significantly linear down-regulated, and the highest expression of akt, eif4e2 and 4ebp1 were found in crabs fed diet with 20% replacement of fish meal with SEM, the highest expression of eif4e1a was observed in those fed diet with 10% replacement of fish meal with SEM, the highest expression of eif4e3 was occurred in crabs fed diet with 30% replacement of fish meal with SEM. In cholesterol metabolism, the expression of abcg1 and lrp2 significantly down-regulated and the expression of npc1 significantly up-regulated with increase of replacement level of fish meal with SEM. The content of T-CHO in hemolymph and hepatopancreas significantly linear increased. In conclusion, based on quadratic broken-line regression analysis of PWG against the dietary replacement of fish meal with SEM, the optimal replacement level of fish meal with SEM was estimated to be 15.94% for juvenile swimming crab.

Exploring the diagnostic value, prognostic value, and biological functions of NPC gene family members in hepatocellular carcinoma based on a multi-omics analysis.

Liver cancer is a cunning malignancy with a high incidence and mortality rate among cancers worldwide. The NPC gene family members (NPCs: NPC1, NPC2, and NPC1L1) are closely linked to the development of multiple cancers, but their role in liver cancer remains unclear. As a result, we must investigate their functions in liver hepatocellular carcinoma (LIHC). NPCs were significantly differentially expressed between normal and LIHC tissues, with a high mutation frequency in LIHC. The ROC curve analysis revealed that NPC1/NPC2 had high diagnostic and prognostic values in LIHC. NPC1 expression was also found to be negatively correlated with its methylation level. The differentially expressed genes between high and low NPC1 expression groups in LIHC were mainly related to channel activity, transporter complexes, and plasma membrane adhesion molecules. Additionally, NPC1 expression was significantly associated with multiple immune cells and immunization checkpoints. It was hypothesized that a TUG1/SNHG4-miR-148a-3p-NPC1 regulatory axis is associated with hepatocarcinogenesis. Finally, the protein expression of NPC1 in LIHC tissues and paraneoplastic tissues was detected, and NPC1-knockdown HepG2 cells (NPC1KO) inhibited the proliferation, migration, and invasion. This study helped to identify new prognostic markers and potential immunotherapeutic targets for LIHC and revealed the molecular mechanisms underlying NPC1 regulation in LIHC. The NPCs play a key role in the prognosis and diagnosis of LIHC and may be an important indicator for LIHC prognosis and diagnosis; NPC1 might be a potential therapeutic target in LIHC.

Adenosine A2A Receptor Activation Regulates Niemann-Pick C1 Expression and Localization in Macrophages.

Adenosine plays an important role in modulating immune cell function, particularly T cells and myeloid cells, such as macrophages and dendritic cells. Cell surface adenosine A2A receptors (A2AR) regulate the production of pro-inflammatory cytokines and chemokines, as well as the proliferation, differentiation, and migration of immune cells. In the present study, we expanded the A2AR interactome and provided evidence for the interaction between the receptor and the Niemann-Pick type C intracellular cholesterol transporter 1 (NPC1) protein. The NPC1 protein was identified to interact with the C-terminal tail of A2AR in RAW 264.7 and IPMФ cells by two independent and parallel proteomic approaches. The interaction between the NPC1 protein and the full-length A2AR was further validated in HEK-293 cells that permanently express the receptor and RAW264.7 cells that endogenously express A2AR. A2AR activation reduces the expression of NPC1 mRNA and protein density in LPS-activated mouse IPMФ cells. Additionally, stimulation of A2AR negatively regulates the cell surface expression of NPC1 in LPS-stimulated macrophages. Furthermore, stimulation of A2AR also altered the density of lysosome-associated membrane protein 2 (LAMP2) and early endosome antigen 1 (EEA1), two endosomal markers associated with the NPC1 protein. Collectively, these results suggested a putative A2AR-mediated regulation of NPC1 protein function in macrophages, potentially relevant for the Niemann-Pick type C disease when mutations in NPC1 protein result in the accumulation of cholesterol and other lipids in lysosomes.

Discovery of sterically-hindered phenol compounds with potent cytoprotective activities against ox-LDL–induced retinal pigment epithelial cell death as a potential pharmacotherapy

Late-stage dry age-related macular degeneration (AMD) or geographic atrophy (GA) is an irreversible blinding condition characterized by degeneration of retinal pigment epithelium (RPE) and the associated photoreceptors. Clinical and genetic evidence supports a role for dysfunctional lipid processing and accumulation of harmful oxidized lipids in the pathogenesis of GA. Using an oxidized low-density lipoprotein (ox-LDL)-induced RPE death assay, we screened and identified sterically-hindered phenol compounds with potent protective activities for RPE. The phenol-containing PPARγ agonist, troglitazone, protected against ox-LDL–induced RPE cell death, whereas other more potent PPARγ agonists did not protect RPE cells. Knockdown of PPARγ did not affect the protective activity of troglitazone in RPE, confirming the protective function is not due to the thiazolidine (TZD) group of troglitazone. Prototypical hindered phenol trolox and its analogs potently protected against ox-LDL–induced RPE cell death whereas potent antioxidants without the phenol group failed to protect RPE. Hindered phenols preserved lysosomal integrity against ox-LDL–induced damage and FITC-labeled trolox was localized to the lysosomes in RPE cells. Analogs of trolox inhibited reactive oxygen species (ROS) formation induced by ox-LDL uptake in a dose-dependent fashion and were effective at sub-micromolar concentrations. Treatment with trolox analog 2,2,5,7,8-pentamethyl-6-chromanol (PMC) significantly induced the expression of the lysosomal protein NPC-1 and reduced intracellular cholesterol level upon ox-LDL uptake. Our data indicate that the lysosomal-localized hindered phenols are uniquely potent in protecting the RPE against the toxic effects of ox-LDL, and may represent a novel pharmacotherapy to preserve the vision in patients with GA.

SAT-145 Cholesterol Uptake as a Critical Vulnerability in Triple Negative Breast Cancer

Triple Negative Breast Cancer (TNBC) is an aggressive subtype of cancer with poor prognosis due to high metastatic potential and lack of targeted therapies. Normal epithelial cells express the microRNA-200c (miR-200c), a potent suppressor of epithelial-to-mesenchymal transition (EMT). However, miR-200c is silenced or lost in TNBC, allowing a de-differentiated, non-epithelial phenotype and aberrant expression of genes conferring invasive and chemoresistant characteristics. Recent literature has demonstrated that EMT promotes altered tumor cell metabolism, creating novel vulnerabilities that can be exploited therapeutically. In addition to driving global metabolic changes, miR-200c-induced reversal of EMT alters key cholesterol metabolism genes that support the uptake of dietary cholesterol from the bloodstream. Intracellular cholesterol homeostasis is critical for cell survival and is carefully regulated, but how these homeostatic mechanisms adapt during tumor progression is poorly understood. Based on preliminary data, I hypothesize that TNBCs depend on exogenous cholesterol uptake and availability to maintain cell viability and an invasive phenotype. This work aims to identify novel cholesterol-related targets in breast cancer and delineate mechanisms regulating cholesterol homeostasis in normal and cancer physiology.Restoration of miR-200c in TNBC leads to alteration of the cholesterol uptake components low- and very-low-density-lipoprotein receptors LDLR and VLDLR, through direct and indirect mechanisms previously unexplored in cancer. miR-200c further inhibits Niemann-Pick Type C (NPC1), a lysosomal protein necessary for utilization of exogenous cholesterol. Interestingly, expression of NPC1 in TNBC correlates with a unique inability of cells to proliferate in the absence of exogenous LDL supply, suggesting defects in de novo cholesterol biosynthesis. Further, NPC1 inhibition leads to cell death in TNBC but not more epithelial-like breast cancers. Whether this cell death is due to disruption in critical cholesterol supply or due to defective lysosome dysfunction is currently being investigated. Overall, this work suggests a role of NPC1 in cancer cell metastasis that has not been previously explored, and identifies cholesterol uptake as a targetable dependency in TNBC.

Transcript, protein, metabolite and cellular studies in skin fibroblasts demonstrate variable pathogenic impacts of NPC1 mutations

BackgroundNiemann-Pick type C (NP-C) is a rare neurovisceral genetic disorder caused by mutations in the NPC1 or the NPC2 gene. NPC1 is a multipass-transmembrane protein essential for egress of cholesterol from late endosomes/lysosomes. To evaluate impacts of NPC1 mutations, we examined fibroblast cultures from 26 NP-C1 patients with clinical phenotypes ranging from infantile to adult neurologic onset forms. The cells were tested with multiple assays including NPC1 mRNA expression levels and allele expression ratios, assessment of NPC1 promoter haplotypes, NPC1 protein levels, cellular cholesterol staining, localization of the mutant NPC1 proteins to lysosomes, and cholesterol/cholesteryl ester ratios. These results were correlated with phenotypes of the individual patients.ResultsOverall we identified 5 variant promoter haplotypes. Three of them showed reporter activity decreased down to 70% of the control sequence. None of the haplotypes were consistently associated with more severe clinical presentation of NP-C. Levels of transcripts carrying null NPC1 alleles were profoundly lower than levels of the missense variants. Low levels of the mutant NPC1 protein were identified in most samples. The protein localised to lysosomes in cultures expressing medium to normal NPC1 levels. Fibroblasts from patients with severe infantile phenotypes had higher cholesterol levels and higher cholesterol/cholesteryl ester ratios. On the contrary, cell lines from patients with juvenile and adolescent/adult phenotypes showed values comparable to controls.ConclusionNo single assay fully correlated with the disease severity. However, low residual levels of NPC1 protein and high cholesterol/cholesteryl ester ratios associated with severe disease. The results suggest not only low NPC1 expression due to non-sense mediated decay or low mutant protein stability, but also dysfunction of the stable mutant NPC1 as contributors to the intracellular lipid transport defect.

Implication of protein glycosylation impairment in cellular cholesterol accumulation caused by Presenilin deficiency

BackgroundMajor genetic causes of familial Alzheimer disease (FAD) are mutations in genes encoding presenilin (PS) 1 and 2, respectively. PS1 and PS2 are transmembrane proteins known to constitute the catalytic subunits of the γ‐secretase complex, a membrane protease involved in proteolytic processing of amyloid precursor protein (APP) that gives rise to amyloid β‐protein (Aβ) secretion. In addiction, PSs are known to be involved in cellular cholesterol metabolism, which is fundamental for physiological cellular functions. Alterations in cholesterol metabolism could be implicated in AD development, indicated by the genetic risk factor apolipoprotein E (APOE). However, the molecular mechanisms how PSs play a role in cellular cholesterol metabolism is still not fully clarified. In this study, we characterized cholesterol metabolism in the cells deficient in either PS1 or PS2.MethodsWe have studied PS1‐ or PS2‐knock out (KO) MEFs by mass spectrometry (MS) for sterols including cholesterol and by microscopy with a cholesterol binding compound, filipin. Expression level of proteins involved in cellular cholesterol metabolism was analyzed by Western blotting (WB).ResultsCellular level of cholesterol as well as its precursors, desmosterol and lathosterol, were elevated in PS1‐ and PS2‐KO as compared to WT MEFs. Fluorescence microscopy also revealed intracellular cholesterol accumulation in PS1 and PS2 KO cells. Interestingly, the subcellular distribution of cholesterol was different between PS1‐ and PS2‐KO MEFs. We further analyzed expression of proteins involved in cellular cholesterol metabolism, including NPC1, an intracellular cholesterol transporter whose deficiency leads to lysosomal cholesterol storage and neurodegeneration in Niemann‐Pick disease type C. The deficiency of PS1‐ and PS2‐KO MEFs was associated with altered glycosylation of NPC1. The glycosylation and expression of other membrane proteins, such as N‐cadherin, LAMP2, and LRP1 was also dependent on the PS genotype. To investigate the role of protein glycosylation in cellular cholesterol metabolism, we chronically treated wild‐type MEFs with several inhibitors of protein glycosylation targeting different steps of glycosylation processes. The inhibitors differentially affected protein glycosylation pattern and subcellular cholesterol distribution. Importantly, some of the inhibitors partially mimicked the cellular cholesterol accumulation as well as the protein glycosylation pattern observed in PS1‐ and PS2‐KO MEFs.Discussion/ConclusionsThese results suggest that deficiency of PS1 or PS2 differentially affect cellular cholesterol metabolism via impairment of protein glycolsylation. The exact mechanisms of altered expression of NPC1 as well as γ‐secretase activity in the impairment of cholesterol metabolism and protein glycosylation remain to be elucidated. It will be interesting to further dissect the role of protein glycosylation and cholesterol mismetabolism in the pathogenesis of AD.Support or Funding InformationGerman Research Foundation (DFG) (Project number: 348207315, Naoto Oikawa)

Complement Component C3 Participates in Early Stages of Niemann-Pick C Mouse Liver Damage.

Niemann–Pick type C (NPC), a lysosomal storage disorder, is mainly caused by mutations in the NPC1 gene. Niemann–Pick type C patients and mice show intracellular cholesterol accumulation leading to hepatic failure with increased inflammatory response. The complement cascade, which belongs to the innate immunity response, recognizes danger signals from injured tissues. We aimed to determine whether there is activation of the complement system in the liver of the NPC mouse and to assess the relationship between C3 activation, a final component of the pathway, and NPC liver pathology. Niemann–Pick type C mice showed high levels of C3 staining in the liver which unexpectedly decreased with aging. Using an inducible NPC1 hepatocyte rescue mouse model, we restored NPC1 expression for a short time in young mice. We found C3 positive cells only in non-rescued cells, suggesting that C3 activation in NPC cells is reversible. Then, we studied the effect of C3 ablation on NPC liver damage at two postnatal time points, P56 and P72. Deletion of C3 reduced the presence of hepatic CD68-positive cells at postnatal day 56 and prevented the increase of transaminase levels in the blood of NPC mice. These positive effects were abrogated at P72, indicating that the complement cascade participates only during the early stages of liver damage in NPC mice, and that its inhibition may serve as a new potential therapeutic strategy for the disease.

Suppression of Notch Signaling Stimulates Progesterone Synthesis by Enhancing the Expression of NR5A2 and NR2F2 in Porcine Granulosa Cells

The conserved Notch pathway is reported to be involved in progesterone synthesis and secretion; however, the exact effects remain controversial. To determine the role and potential mechanisms of the Notch signaling pathway in progesterone biosynthesis in porcine granulosa cells (pGCs), we first used a pharmacological γ-secretase inhibitor, N-(N-(3,5-difluorophenacetyl-l-alanyl))-S-phenylglycine t-butyl ester (DAPT), to block the Notch pathway in cultured pGCs and then evaluated the expression of genes in the progesterone biosynthesis pathway and key transcription factors (TFs) regulating steroidogenesis. We found that DAPT dose- and time-dependently increased progesterone secretion. The expression of steroidogenic proteins NPC1 and StAR and two TFs, NR5A2 and NR2F2, was significantly upregulated, while the expression of HSD3B was significantly downregulated. Furthermore, knockdown of both NR5A2 and NR2F2 with specific siRNAs blocked the upregulatory effects of DAPT on progesterone secretion and reversed the effects of DAPT on the expression of NPC1, StAR, and HSD3B. Moreover, knockdown of NR5A2 and NR2F2 stimulated the expression of Notch3. In conclusion, the inhibition of Notch signaling stimulated progesterone secretion by enhancing the expression of NPC1 and StAR, and the two TFs NR5A2 and NR2F2 acted as downstream TFs of Notch signaling in regulating progesterone synthesis.

STARD1 and NPC1 expression as pathological markers associated with astrogliosis in post-mortem brains from patients with Alzheimer's disease and Down syndrome.

Alzheimer´s disease (AD) is a progressive neurodegenerative disorder of complex etiology, while Down syndrome (DS) is considered a genetically determined form of AD. Alterations in cholesterol homeostasis have been linked to AD although the role in this association is not well understood. Increased expression of STARD1 and NPC1, which are involved in intracellular cholesterol trafficking, has been reported in experimental AD models but not in patients with AD. Here we analyzed endolysosomal/mitochondrial cholesterol homeostasis, expression of NPC1 and STARD1 and correlation with pathological markers of AD in cortex and hippocampus from post-mortem brains from patients with AD and DS. NPC1 expression was observed in hippocampus from patients with AD and DS. Moreover, STARD1 expression increased in hippocampus and cortex from patients with AD and DS, respectively, and its immunoreactivity discriminated controls from AD or DS with a better accuracy than Aβ42. Hippocampal areas stained with the recombinant GST-PFO probe showed increased mitochondrial cholesterol within astrocytes of brains from patients with AD and DS-brains compared to controls. Lysosomal cholesterol accumulation within hippocampal astrocytes was higher in DS than in AD. These data revealed increased intracellular cholesterol loading in hippocampus from patient with AD and DS and suggest that STARD1 could be a potential pre-clinical marker associated with early stages of AD pathology.

Baculovirus Utilizes Cholesterol Transporter NIEMANN-Pick C1 for Host Cell Entry.

The dual roles of baculovirus for the control of natural insect populations as an insecticide, and as a tool for foreign gene expression and delivery, have called for a comprehensive understanding of the molecular mechanisms governing viral infection. Here, we demonstrate that the Bombyx mori Niemann-Pick C1 (BmNPC1) is essential for baculovirus infection in insect cells. Both pretreatment of B. mori embryonic cells (BmE) with NPC1 antagonists (imipramine or U18666A) and down-regulation of NPC1 expression resulted in a significant reduction in baculovirus BmNPV (B. mori nuclear polyhedrosis virus) infectivity. Disruption of BmNPC1 could decrease viral entry (2 hpi) rather than reduce the viral binding to the BmE cells. Furthermore, our results showed that NPC1 domain C binds directly and specifically to the viral glycoprotein GP64, which is responsible for both receptor binding and fusion. Antibody blocking assay also revealed that the domain C specific polyclonal antibody inhibited BmNPV infection, indicating that NPC1 domain C most likely plays a role during viral fusion in endosomal compartments. Our results, combined with previous studies identifying an essential role of human NPC1 (hNPC1) in filovirus infection, suggest that the glycoprotein of several enveloped viruses possess a shared strategy of exploiting host NPC1 proteins during virus intracellular entry events.

Abstract 877: Sigma 2 ligands kill pancreatic cancer cells via disruption of lysosomal cholesterol transport

Abstract Introduction: The sigma 2 receptor is overexpressed in pancreatic cancer and has been an attractive target for development of cancer selective therapies. However, the mechanism of sigma 2 mediated cancer cell death remains elusive. The sigma 2 receptor has been recently identified as TMEM97, a lysosomal/endoplasmic reticulum protein involved in regulation of cholesterol export in conjunction with NPC1. Methods: Uptake of SW43 in pancreatic cancer cells was visualized using a NBD fluorophore conjugated ligand. Intracellular cholesterol staining was visualized using filipin staining. Protein expression was quantified by both Western blot and RT qPCR. Cell death was assayed in triplicate using Titer-Glo viability assay. Results: We hypothesized that sigma 2 ligands may kill pancreatic cancer cells through disruption of lysosomal cholesterol export. Using ASPC1 pancreatic cancer cells, we first demonstrate that sigma 2 ligand SW43 is rapidly taken up and targets to endo-lysosomal compartments, and that treatment with SW43 causes a rapid and dose dependent sequestration of cholesterol in lysosomal compartments as visualized by filipin cholesterol staining. This correlated to an increase in TMEM97 and corresponding decrease in NPC1 expression following SW43 treatment. We next demonstrate that this cholesterol sequestration phenotype is required for cell death, and might be related to the free amine side chain of SW43, as its N-phthalimide derivative fails to induce both lysosomal cholesterol sequestration and cell death despite high doses of ligand used. Lysosomal cholesterol sequestration from sigma 2 ligand treatment sensitizes cells to further targeting of cholesterol metabolism. Using low doses of simvastatin and SW43, we demonstrate that targeting both cholesterol export and cholesterol synthesis displays significant synergism in killing ASPC1 pancreatic cancer cells. Conclusions: These findings provide further insight into how the sigma 2 receptor/ligand interaction mediates cancer cell death and provides the basis for rationally combined targeted therapy regimens to be tested in vivo. This work was supported by NIH R01CA163764 and NIH T32CA009621-26A1 and the SUS Foundation Resident Research Award. Citation Format: Linda X. Jin, Suwanna Vangveravong, Narendra V. Sankpal, Darren R. Cullinan, Peter Goedegebuure, Dirk Spitzer, William G. Hawkins. Sigma 2 ligands kill pancreatic cancer cells via disruption of lysosomal cholesterol transport [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 877.

Effect of probucol on expression of Niemann-Pick C1 protein and cholesterol efflux in RAW264.7 macrophages

Objective To observe the effects of the serum from the mice pretreated with probucol on cellular cholesterol efflux and the impact of different concentrations of probucol on the expression of Niemann-Pick C1 protein (NPC1) in macrophages. Methods Sixteen male C57BL/6 mice were randomly divided into two groups and fed on either vehicle or 0.5% w/w probucol for 4 weeks respectively. The serum was collected and the lipids profile was detected by enzymatic method. Raw264.7 macrophages were loaded with acetylated low-density lipoprotein and labeled with 3H-cholesterol, then the efflux of cholesterol was quantitated with the serum mentioned above as the acceptors. Reverse transcription polymerase chain reaction and Western blotting were used to evaluate the mRNA and protein expression of NPC1. Results More cholesterol efflux from the cultured macrophages was mediated by the serum from the mice treated with probucol for 4 weeks than those from the control mice [(36.30±0.02)% vs. (21.20±0.04)%, P=0.003]. Different concentrations of probucol (0, 10, 20, 50, and 100 μmol/L) dose-dependently upregulated the mRNA [(0.75±0.03), (0.94±0.04), (1.18±0.06), (1.22±0.03) vs. (0.52±0.04), P=0.036, P=0.021, P=0.045, P=0.037] and protein [(0.68±0.05), (0.85±0.02), (1.16±0.04), (1.19±0.05) vs. (0.49±0.02), P=0.011, P=0.040, P=0.034, P=0.015] expression of NPC1 in macrophages, and promoted cholesterol efflux [(46.10±0.03)%, (51.40±0.05)%, (55.30±0.06)%, (57.80±0.08)% vs. (36.30±0.02)%]. Conclusion The serum from the mice treated with probucol markedly promoted cholesterol efflux from the cultured macrophages. Probucol promoted efflux from the macrophages dose-dependently probably through increasing the expression of NPC1. Key words: Probucol; Niemann-Pick C1 protein; Macrophages; Cholesterol