Intracellular Cholesterol Trafficking Research Articles

Mutant induced neurons and humanized mice enable identification of Niemann-Pick C1 proteostatic therapies.

Therapeutics that rescue folding, trafficking, and function of disease-causing missense mutants are sought for a host of human diseases, but efforts to leverage model systems to test emerging strategies have met with limited success. Such is the case for Niemann-Pick type C1 disease, a lysosomal disorder characterized by impaired intracellular cholesterol trafficking, progressive neurodegeneration, and early death. NPC1, a multipass transmembrane glycoprotein, is synthesized in the endoplasmic reticulum and traffics to late endosomes/lysosomes, but this process is often disrupted in disease. We sought to identify small molecules that promote folding and enable lysosomal localization and functional recovery of mutant NPC1. We leveraged a panel of isogenic human induced neurons expressing distinct NPC1 missense mutations. We used this panel to rescreen compounds that were reported previously to correct NPC1 folding and trafficking. We established mo56-hydroxycholesterol (mo56Hc) as a potent pharmacological chaperone for several NPC1 mutants. Furthermore, we generated mice expressing human I1061T NPC1, a common mutation in patients. We demonstrated that this model exhibited disease phenotypes and recapitulated the protein trafficking defects, lipid storage, and response to mo56Hc exhibited by human cells expressing I1061T NPC1. These tools established a paradigm for testing and validation of proteostatic therapeutics as an important step towards the development of disease-modifying therapies.

Cytotoxic sigma-2 ligands trigger cancer cell death via cholesterol-induced-ER-stress

Sigma-2-ligands (S2L) are characterized by high binding affinities to their cognate sigma-2 receptor, overexpressed in rapidly proliferating tumor cells. As such, S2L were developed as imaging probes (ISO1) or as cancer therapeutics, alone (SV119 [C6], SW43 [C10]) and as delivery vehicles for cytotoxic drug cargoes (C6-Erastin, C10-SMAC). However, the exact mechanism of S2L-induced cytotoxicity remains to be fully elucidated. A series of high-affinity S2L were evaluated regarding their cytotoxicity profiles across cancer cell lines. While C6 and C10 displayed distinct cytotoxicities, C0 and ISO1 were essentially non-toxic. Confocal microscopy and lipidomics analysis in cellular and mouse models revealed that C10 induced increases in intralysosomal free cholesterol and in cholesterol esters, suggestive of unaltered intracellular cholesterol trafficking. Cytotoxicity was caused by cholesterol excess, a phenomenon that contrasts the effects of NPC1 inhibition. RNA-sequencing revealed gene clusters involved in cholesterol homeostasis and ER stress response exclusively by cytotoxic S2L. ER stress markers were confirmed by qPCR and their targeted modulation inhibited or enhanced cytotoxicity of C10 in a predicted manner. Moreover, C10 increased sterol regulatory element-binding protein 2 (SREBP2) and low-density lipoprotein receptor (LDLR), both found to be pro-survival factors activated by ER stress. Furthermore, inhibition of downstream processes of the adaptive response to S2L with simvastatin resulted in synergistic treatment outcomes in combination with C10. Of note, the S2L conjugates retained the ER stress response of the parental ligands, indicative of cholesterol homeostasis being involved in the overall cytotoxicity of the drug conjugates. Based on these findings, we conclude that S2L-mediated cell death is due to free cholesterol accumulation that leads to ER stress. Consequently, the cytotoxic profiles of S2L drug conjugates are proposed to be enhanced via concurrent ER stress inducers or simvastatin, strategies that could be instrumental on the path toward tumor eradication.

Co-Expression of Niemann-Pick Type C1-Like1 (NPC1L1) with ACE2 Receptor Synergistically Enhances SARS-CoV-2 Entry and Fusion.

The entry of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) into human embryonic kidney (HEK293T) cells has been shown to be a cholesterol-rich, lipid raft-dependent process. In this study, we investigated if the presence of a cholesterol uptake receptor Niemann-pick type c1-like1 (NPC1L1) impacts SARS-CoV-2 cell entry. Initially, we utilized reporter-based pseudovirus cell entry assays and a spike (S) glycoprotein-mediated cell-to-cell fusion assay. Using Chinese hamster ovary (CHO-K1) cells, which lack endogenous receptors for SARS-CoV-2 entry, our data showed that the co-expression of NPC1L1 together with the ACE2 receptor synergistically increased SARS-CoV-2 pseudovirus entry even more than the cells expressing ACE-2 receptor alone. Similar results were also found with the HEK293T cells endogenously expressing the ACE2 receptor. Co-cultures of effector cells expressing S glycoprotein together with target cells co-expressing ACE-2 receptor with NPC1L1 significantly promoted quantitative cell-to-cell fusion, including syncytia formation. Finally, we substantiated that an elevated expression of NPC1L1 enhanced entry, whereas the depletion of NPC1L1 resulted in a diminished SARS-CoV-2 entry in HEK293T-ACE2 cells using authentic SARS-CoV-2 virus in contrast to their respective control cells. Collectively, these findings underscore the pivotal role of NPC1L1 in facilitating the cellular entry of SARS-CoV-2. Importance: Niemann-Pick type C1-like1 (NPC1L1) is an endosomal membrane protein that regulates intracellular cholesterol trafficking. This protein has been demonstrated to play a crucial role in the life cycle of several clinically important viruses. Although SARS-CoV-2 exploits cholesterol-rich lipid rafts as part of its viral entry process, the role of NPC1L1 in SARS-CoV-2 entry remains unclear. Our research represents the first-ever demonstration of NPC1L1's involvement in facilitating SARS-CoV-2 entry. The observed role of NPC1L1 in human kidney cells is not only highly intriguing but also quite relevant. This relevance stems from the fact that NPC1L1 exhibits high expression levels in several organs, including the kidneys, and the fact that kidney damages are reported during severe cases of SARS-CoV-2. These findings may help us understand the new functions and mechanisms of NPC1L1 and could contribute to the identification of new antiviral targets.

Fatty acid-binding proteins 3, 7, and 8 bind cholesterol and facilitate its egress from lysosomes.

Cholesterol from low-density lipoprotein (LDL) can be transported to many organelle membranes by non-vesicular mechanisms involving sterol transfer proteins (STPs). Fatty acid-binding protein (FABP) 7 was identified in our previous study searching for new regulators of intracellular cholesterol trafficking. Whether FABP7 is a bona fide STP remains unknown. Here, we found that FABP7 deficiency resulted in the accumulation of LDL-derived cholesterol in lysosomes and reduced cholesterol levels on the plasma membrane. A crystal structure of human FABP7 protein in complex with cholesterol was resolved at 2.7 Å resolution. In vitro, FABP7 efficiently transported the cholesterol analog dehydroergosterol between the liposomes. Further, the silencing of FABP3 and 8, which belong to the same family as FABP7, caused robust cholesterol accumulation in lysosomes. These two FABP proteins could transport dehydroergosterol in vitro as well. Collectively, our results suggest that FABP3, 7, and 8 are a new class of STPs mediating cholesterol egress from lysosomes.

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.

GULP1 deficiency reduces adipogenesis and glucose uptake via downregulation of PPAR signaling and disturbing of insulin/ERK signaling in 3T3-L1 cells.

Obesity and metabolic disorders caused by alterations in lipid metabolism are major health issues in developed, affluent societies. Adipose tissue is the only organ that stores lipids and prevents lipotoxicity in other organs. Mature adipocytes can affect themselves and distant metabolism-related tissues by producing various adipokines, including adiponectin and leptin. The engulfment adaptor phosphotyrosine-binding domain-containing 1 (GULP1) regulates intracellular trafficking of glycosphingolipids and cholesterol, suggesting its close association with lipid metabolism. However, the role of GULP1 in adipocytes remains unknown. Therefore, this study aimed to investigate the function of GULP1 in adipogenesis, glucose uptake, and the insulin signaling pathway in adipocytes. A 3T3-L1 cell line with Gulp1 knockdown (shGulp1) and a 3T3-L1 control group (U6) were established. Changes in shGulp1 cells due to GULP1 deficiency were examined and compared to those in U6 cells using microarray analysis. Glucose uptake was monitored via insulin stimulation in shGulp1 and U6 cells using a 2-NBDG glucose uptake assay, and the insulin signaling pathway was investigated by western blot analysis. Adipogenesis was significantly delayed, lipid metabolism was altered, and several adipogenesis-related genes were downregulated in shGulp1 cells compared to those in U6 cells. Microarray analysis revealed significant inhibition of peroxisome proliferator-activated receptor signaling in shGulp1 cells compared with U6 cells. The production and secretion of adiponectin as well as the expression of adiponectin receptor were decreased in shGulp1 cells. In particular, compared with U6 cells, glucose uptake via insulin stimulation was significantly decreased in shGulp1 cells through the disturbance of ERK1/2 phosphorylation. This is the first study to identify the role of GULP1 in adipogenesis and insulin-stimulated glucose uptake by adipocytes, thereby providing new insights into the differentiation and functions of adipocytes and the metabolism of lipids and glucose, which can help better understand metabolic diseases.

Dual-monomer solvatochromic probe system (DSPS) for effectively differentiating lipid raft cholesterol and active membrane cholesterol in the inner-leaflet plasma membrane.

Monitoring active membrane cholesterol and lipid raft cholesterol in the inner leaflet of the plasma membrane is significant for understanding the membrane function and cellular physiopathological processes. Limited by existing methods, it is difficult to differentiate active membrane cholesterol and lipid raft cholesterol. A novel dual-monomer solvatochromic probe system (DSPS) that targets two types of cholesterol was developed. Acrylodan-BG/SNAP-D4 composed of SNAP-D4 cholesterol-recognizing monomers and solvatochromic acrylodan-BG-sensing monomers exhibits excellent cholesterol detecting properties in terms of selectivity, accuracy, convenience and economic benefits. Cell imaging revealed that lipid raft cholesterol emitted blue fluorescence, whereas active membrane cholesterol (which partially bobbed in aqueous cytosol) displayed green fluorescence; both the fluorescence emissions increased or decreased in a cholesterol-dependent manner. This system provides a new technology for the determination of two types of cholesterol, which is beneficial for the further study of membrane function, intracellular cholesterol trafficking, and cell signaling.

Mitochondrial Functionality Is Regulated by Alkylphospholipids in Human Colon Cancer Cells.

Alkylphospholipids (APLs) have been studied as anticancer drugs that interfere with biological membranes without targeting DNA. Although their mechanism of action is not fully elucidated yet, it is known that they disrupt the intracellular trafficking of cholesterol and its metabolism. Here, we analyzed whether APLs could also interfere with mitochondrial function. For this purpose, we used HT29 colorectal cancer cells, derived from a primary tumor, and SW620 colorectal cancer cells, derived from a metastasis site. After treatment with the APLs miltefosine and perifosine, we analyzed various mitochondrial parameters, including mitochondrial mass, cardiolipin content, mitochondrial membrane potential, H2O2 production, the levels of oxidative phosphorylation (OXPHOS) complexes, metabolic enzymes activity, the oxygen consumption rate, and the levels of apoptosis and autophagy markers. APLs, especially perifosine, increased mitochondrial mass while OXPHOS complexes levels were decreased without affecting the total oxygen consumption rate. Additionally, we observed an increase in pyruvate dehydrogenase (PDH) and isocitrate dehydrogenase (IDH) levels and a decrease in lactate dehydrogenase (LDH) activity, suggesting a metabolic rewiring induced by perifosine. These alterations led to higher mitochondrial membrane potential, which was potentiated by decreased uncoupling protein 2 (UCP2) levels and increased reactive oxygen species (ROS) production. Consequently, perifosine induced an imbalance in mitochondrial function, resulting in higher ROS production that ultimately impacted cellular viability.

Cyclodextrin-Mediated Cholesterol Depletion Induces Adiponectin Secretion in 3T3-L1 Adipocytes.

Adipocytes store a significant amount of cholesterol and triglycerides. However, whether cholesterol modulates adipocyte function remains largely unknown. We modulated the cholesterol level in adipocytes to examine its effect on the secretion of adiponectin, an important hormone specifically secreted by adipocytes. Treating differentiated 3T3-L1 adipocytes with 4 mM methyl-β-cyclodextrin (MβCD), a molecule with a high affinity for cholesterol, rapidly depleted cholesterol in adipocytes. Interestingly, MβCD treatment increased adiponectin in the medium without affecting its intracellular level, suggesting a modulation of secretion. By contrast, cholesterol addition did not affect adiponectin secretion, suggesting that cholesterol-depletion-induced intracellular cholesterol trafficking, but not reduced cholesterol level, accounted for MβCD-induced adiponectin secretion. MβCD-induced adiponectin secretion was reduced after 10 μg/mL U18666A treatment that suppressed cholesterol transport out of late endosomes/lysosomes. Depleting Niemann-Pick type C1 (NPC1) or NPC2 proteins, which mediate endosomal/lysosomal cholesterol export, consistently reduced MβCD-induced adiponectin secretion. Furthermore, treatment with 1 μM bafilomycin A1, which neutralized acidic endosomes/lysosomes, also attenuated MβCD-induced adiponectin secretion. Finally, MβCD treatment redistributed cellular adiponectin to lower-density fractions in sucrose gradient fractionation. Our results show that MβCD-mediated cholesterol depletion elevates the secretion of adiponectin, highlighting the involvement of endosomes and lysosomes in adiponectin secretion in adipocytes.

Different solubilizing ability of cyclodextrin derivatives for cholesterol in Niemann-Pick disease type C treatment.

Niemann-Pick disease type C (NPC) is a fatal neurodegenerative disorder caused by abnormal intracellular cholesterol trafficking. Cyclodextrins (CDs), the most promising therapeutic candidates for NPC, but with concerns about ototoxicity, are cyclic oligosaccharides with dual functions of unesterified cholesterol (UC) shuttle and sink that catalytically enhance the bidirectional flux and net efflux of UC, respectively, between the cell membrane and the extracellular acceptors. However, the properties of CDs that regulate these functions and how they could be used to improve treatments for NPC are unclear. We estimated CD-UC complexation for nine CD derivatives derived from native α-, β-, and γ-CD with different cavity sizes, using solubility and molecular docking analyses. The stoichiometry and complexation ability of the resulting complexes were investigated in relation to the therapeutic effectiveness and toxicity of each CD derivative in NPC experimental models. We found that shuttle and sink activities of CDs are dependent on cavity size-dependent stoichiometry and substituent-associated stability of CD-UC complexation. The ability of CD derivatives to form 1:1 and 2:1 complexes with UC were correlated with their ability to normalize intracellular cholesterol trafficking serving as shuttle and with their cytotoxicity associated with cellular UC efflux acting as sink, respectively, in NPC model cells. Notably, the ability of CD derivatives to form an inclusion complex with UC was responsible for not only efficacy but ototoxicity, while a representative derivative without this ability negligibly affected auditory function, underscoring its preventability. Our findings highlight the importance of strategies for optimizing the molecular structure of CDs to overcome this functional dilemma in the treatment of NPC.

Intracellular Cholesterol Trafficking.

Cholesterol is an essential lipid species of mammalian cells. Cells acquire it through synthesis in the endoplasmic reticulum (ER) and uptake from lipoprotein particles. Newly synthesized cholesterol is efficiently distributed from the ER to other organelles via lipid-binding/transfer proteins concentrated at membrane contact sites (MCSs) to reach the trans-Golgi network, endosomes, and plasma membrane. Lipoprotein-derived cholesterol is exported from the plasma membrane and endosomal compartments via a combination of vesicle/tubule-mediated membrane transport and transfer through MCSs. In this review, we provide an overview of intracellular cholesterol trafficking pathways, including cholesterol flux from the ER to other membranes, cholesterol uptake from lipoprotein donors and transport from the plasma membrane to the ER, cellular cholesterol efflux to lipoprotein acceptors, as well as lipoprotein cholesterol secretion from enterocytes, hepatocytes, and astrocytes. We also briefly discuss human diseases caused by defects in these processes and therapeutic strategies available in such conditions.

Species-specific differences in NPC1 protein trafficking govern therapeutic response in Niemann-Pick type C disease.

The folding and trafficking of transmembrane glycoproteins are essential for cellular homeostasis and are compromised in many diseases. In Niemann-Pick type C disease, a lysosomal disorder characterized by impaired intracellular cholesterol trafficking, the transmembrane glycoprotein NPC1 misfolds due to disease-causing missense mutations. While mutant NPC1 has emerged as a robust target for proteostasis modulators, drug development efforts have been unsuccessful in mouse models. Here, we demonstrated unexpected differences in trafficking through the medial Golgi between mouse and human I1061T-NPC1, a common disease-causing mutant. We established that these distinctions are governed by differences in the NPC1 protein sequence rather than by variations in the endoplasmic reticulum-folding environment. Moreover, we demonstrated direct effects of mutant protein trafficking on the response to small molecules that modulate the endoplasmic reticulum-folding environment by affecting Ca++ concentration. Finally, we developed a panel of isogenic human NPC1 iNeurons expressing WT, I1061T-, and R934L-NPC1 and demonstrated their utility in testing these candidate therapeutics. Our findings identify important rules governing mutant NPC1's response to proteostatic modulators and highlight the importance of species- and mutation-specific responses for therapy development.

Intravenous 2-hydroxypropyl-β-cyclodextrin (Trappsol® Cyclo™) demonstrates biological activity and impacts cholesterol metabolism in the central nervous system and peripheral tissues in adult subjects with Niemann-Pick Disease Type C1: Results of a phase 1 trial.

Niemann-Pick Disease Type C1 (NPC1) is a disorder of intracellular cholesterol and lipid trafficking that leads to the accumulation of cholesterol and lipids in the late endosomal/lysosomal compartment, resulting in systemic manifestations (including hepatosplenomegaly and lung infiltration) and neurodegeneration. Preclinical studies have demonstrated that systemically administered 2-hydroxypropyl-β-cyclodextrin (HPβCD; Trappsol® Cyclo™) restores cholesterol metabolism and homeostasis in peripheral organs and tissues and in the central nervous system (CNS). Here, we assessed the safety, pharmacokinetics, and pharmacodynamics of HPβCD in peripheral tissues and the CNS in adult subjects with NPC1. A Phase 1, randomized, double-blind, parallel group study enrolled 13 subjects with NPC1 who received either 1500 mg/kg or 2500 mg/kg HPβCD intravenously every 2 weeks for a total of 7 doses (14 weeks). Subjects were 18 years or older, with a confirmed diagnosis of NPC1 and evidence of systemic involvement on clinical assessment. Pharmacokinetic evaluations in plasma and cerebrospinal fluid (CSF) were performed at the first and seventh infusions. Pharmacodynamic assessments included biomarkers of systemic cholesterol synthesis (serum lathosterol) and degradation (serum 4β-hydroxycholesterol), secondary sphingomyelin storage (plasma lysosphingomyelin-509, now more accurately referred to as N-palmitoyl-O-phosphocholineserine [PPCS]), and CNS-specific biomarkers of neurodegeneration (CSF total Tau) and cholesterol metabolism (serum 24(S)-hydroxycholesterol [24(S)-HC]). Safety monitoring included assessments of liver and kidney function, infusion related adverse events, and hearing evaluations. Ten subjects completed the study, with 6 at the 1500 mg/kg dose and 4 at the 2500 mg/kg dose. One subject withdrew following the first infusion after experiencing hypersensitivity pneumonitis, and 2 subjects withdrew after meeting a stopping rule related to hearing loss. Overall, HPβCD had an acceptable safety profile. The observed pharmacokinetic profile of HPβCD was similar following the first and seventh infusions, with a plasma half-life of 2 h, a maximum concentration reached at 6 to 8 h, and no evidence of accumulation. Serum biomarkers of cholesterol metabolism showed reduced synthesis and increased degradation. Compared to Baseline, filipin staining of liver tissue showed significant reductions of trapped unesterified cholesterol at both dose levels at Week 14. Plasma PPCS levels were also reduced. HPβCD was detected at low concentrations in the CSF (maximum, 33 μM) at both dose levels and persisted longer in CSF than in plasma. Total Tau levels in CSF decreased in most subjects. Serum levels of 24(S)-HC, a cholesterol metabolite from the CNS that is exported across the blood-brain barrier and into the circulation, decreased after both the first and seventh doses. Hence, pharmacodynamic assessments in both peripheral and CNS-related tissue show target engagement. While not the aim of the study, subjects reported favorable impacts on their quality of life. The plasma pharmacokinetics and pharmacodynamics of HPβCD administered at two intravenous dose levels to subjects with NPC1 were comparable to those observed in preclinical models. HPβCD cleared cholesterol from the liver and improved peripheral biomarkers of cholesterol homeostasis. At low CSF concentrations, HPβCD appeared to be pharmacologically active in the CNS based on the increased efflux of 24(S)-HC and reduction in CSF total Tau, a biomarker of CNS neurodegeneration. These data support the initiation of longer-term clinical trials to evaluate the safety and efficacy of intravenous HPβCD in subjects with NPC1. (ClinicalTrials.gov numbers: present trial, NCT02939547; open-label extension of the present trial, NCT03893071; global pivotal trial, NCT04860960).

Mice deficient in ER protein seipin have reduced adrenal cholesteryl ester lipid droplet formation and utilization

Cholesteryl ester (CE)-rich lipid droplets (LDs) accumulate in steroidogenic tissues under physiological conditions and constitute an important source of cholesterol as the precursor for the synthesis of all steroid hormones. The mechanisms specifically involved in CE-rich LD formation have not been directly studied and are assumed by most to occur in a fashion analogous to triacylglycerol-rich LDs. Seipin is an endoplasmic reticulum protein that forms oligomeric complexes at endoplasmic reticulum-LD contact sites, and seipin deficiency results in severe alterations in LD maturation and morphology as seen in Berardinelli-Seip congenital lipodystrophy type 2. While seipin is critical for triacylglycerol-rich LD formation, no studies have directly addressed whether seipin is important for CE-rich LD biogenesis. To address this issue, mice with deficient expression of seipin specifically in adrenal, testis, and ovary, steroidogenic tissues that accumulate CE-rich LDs under normal physiological conditions, were generated. We found that the steroidogenic-specific seipin-deficient mice displayed a marked reduction in LD and CE accumulation in the adrenals, demonstrating the pivotal role of seipin in CE-rich LD accumulation/formation. Moreover, the reduction in CE-rich LDs was associated with significant defects in adrenal and gonadal steroid hormone production that could not be completely reversed by addition of exogenous lipoprotein cholesterol. We conclude that seipin has a heretofore unappreciated role in intracellular cholesterol trafficking.

Fine-tuned cholesterol solubilizer, mono-6-O-α-D-maltosyl-γ-cyclodextrin, ameliorates experimental Niemann–Pick disease type C without hearing loss

Niemann–Pick disease type C (NPC) is a fatal disorder with abnormal intracellular cholesterol trafficking resulting in neurodegeneration and hepatosplenomegaly. A cyclic heptasaccharide with different degrees of substitution of 2-hydroxypropyl groups, 2-hydroxypropyl-β-cyclodextrin (HP-β-CD), acts as a strong cholesterol solubilizer and is under investigation for treating this disease in clinical trials, but its physicochemical properties and ototoxicity remain a concern. Here, we evaluated the potential of mono-6-O-α-maltosyl-γ-CD (G2-γ-CD), a single-maltose-branched cyclic octasaccharide with a larger cavity than HP-β-CD, for treating NPC. We identified that G2-γ-CD ameliorated NPC manifestations in model mice and showed lower ototoxicity in mice than HP-β-CD. To investigate the molecular mechanisms of action behind the differential ototoxicity of these CDs, we performed cholesterol solubility analysis, proton nuclear magnetic resonance spectroscopy, and molecular modeling, and estimated that the cholesterol inclusion mode of G2-γ-CD maintained solely the 1:1 inclusion complex, whereas that of HP-β-CD shifted to the highly-soluble 2:1 complex at higher concentrations. We predicted the associations of these differential complexations of CDs with cholesterol with the profile of disease attenuation and of the auditory cell toxicity using specific cell models. We proposed that G2-γ-CD can serve as a fine-tuned cholesterol solubilizer for treating NPC, being highly biocompatible and physicochemically suitable for clinical application.

Redirecting Imipramine against Bluetongue Virus Infection: Insights from a Genome-wide Haploid Screening Study.

Bluetongue virus (BTV), an arbovirus of ruminants, is a causative agent of numerous epidemics around the world. Due to the emergence of novel reassortant BTV strains and new outbreaks, there is an unmet need for efficacious antivirals. In this study, we used an improved haploid screening platform to identify the relevant host factors for BTV infection. Our screening tool identified and validated the host factor Niemann–Pick C1 (NPC1), a lysosomal membrane protein that is involved in lysosomal cholesterol transport, as a critical factor in BTV infection. This finding prompted us to investigate the possibility of testing imipramine, an antidepressant drug known to inhibit NPC1 function by interfering with intracellular cholesterol trafficking. In this study, we evaluated the sensitivity of BTV to imipramine using in vitro assays. Our results demonstrate that imipramine pretreatment inhibited in vitro replication and progeny release of BTV-4, BTV-8, and BTV-16. Collectively, our findings highlight the importance of NPC1 for BTV infection and recommend the reprofiling of imipramine as a potential antiviral drug against BTV.

Effect of Rosa canina Methanol Extract on Membrane Trafficking in Different Niemann‐Pick C1 Phenotypes

Niemann Pick type C (NPC) disease is an autosomal recessive lysosomal storage disorder that is caused by mutations in Niemann‐Pick C1 (NPC1) or Niemann‐Pick C2 (NPC2) genes ultimately leading to progressive neurological deterioration. 95% of NPC cases arise from mutations in the NPC1 gene. NPC1 protein mediates the intracellular cholesterol trafficking and plays a crucial role in maintaining lipid homeostasis. Mutations in NPC1 are associated with defective cholesterol mobilization and an accumulation of unesterified cholesterol and subsequently sphingomyelin and glycosphingolipids in the late endo‐lysosomes. N‐butyldeoxynojirimycin, known as Miglustat, is one of the approved therapeutic options. It is an inhibitor of sphingolipid synthesis and is used in substrate reduction therapy. Nevertheless, the efficiency of Miglustat varies among NPC patients depending on the type of the mutation and moreover its accompanied by several side effects, such as diarrhea. The focus of this study is to find an alternative therapy that presents less side effects. Rosa canina L. methanol extract (RCME) has been shown to improve the protein trafficking and maintain cholesterol homeostasis. Here, we used skin‐derived fibroblasts from patients harbouring homozygote or compound heterozygote mutations to assess the effects of RCME on the trafficking of NPC1 and cellular cholesterol contents as compared to wild type cells. For this purpose, the cells were treated for 24 h with RCME (100 µg/ml) with and without Miglustat (100 µM) and the trafficking of NPC1 between the endoplasmic reticulum (ER) and the Golgi was examined by endoglycosidase H treatment. This treatment, which discriminates between mannose‐rich N‐linked glycosylated proteins and mature complex glycosylated forms, demonstrated a significant enhancement of the trafficking of a NPC1 mutants between the ER and the Golgi upon RCME treatment as compared to Miglustat. This trafficking behaviour was observed with those NPC1 mutants that are not entirely blocked in the ER and exit the ER at a slow rate. Markedly, the high cholesterol levels measured in the patients’ fibroblasts are substantially reduced upon RCME treatment, while almost similar cholesterol levels were maintained in the wild type cells. Impaired cellular cholesterol trafficking is associated with partial distortion of the lipid membrane composition, particularly of the cholesterol‐ and sphingolipids‐enriched membrane microdomains or lipid rafts (LRs). Our data show that RCME and also Miglustat maintained a normal LRs distribution as assessed by the LR marker flotillin 2.We conclude that RCME possess the capacity to reverse adverse effects caused by mutations in the NPC1gene including impaired NPC1 trafficking, elevated cholesterol levels and lipid raft alterations. All this together render the constituents of RCME, including quercetin, a promising agents in NPC therapy.

Intracellular Cholesterol Synthesis and Transport.

Cholesterol homeostasis is related to multiple diseases in humans, including cardiovascular disease, cancer, and neurodegenerative and hepatic diseases. The cholesterol levels in cells are balanced dynamically by uptake, biosynthesis, transport, distribution, esterification, and export. In this review, we focus on de novo cholesterol synthesis, cholesterol synthesis regulation, and intracellular cholesterol trafficking. In addition, the progression of lipid transfer proteins (LTPs) at multiple contact sites between organelles is considered.

The Cerebellum in Niemann-Pick C1 Disease: Mouse Versus Man.

Selective neuronal vulnerability is common to most degenerative disorders, including Niemann-Pick C (NPC), a rare genetic disease with altered intracellular trafficking of cholesterol. Purkinje cell dysfunction and loss are responsible for cerebellar ataxia, which is among the prevailing neurological signs of the NPC disease. In this review, we focus on some questions that are still unresolved. First, we frame the cerebellar vulnerability in the context of the extended postnatal time length by which the development of this structure is completed in mammals. In line with this thought, the much later development of cerebellar symptoms in humans is due to the later development and/or maturation of the cerebellum. Hence, the occurrence of developmental events under a protracted condition of defective intracellular cholesterol mobilization hits the functional maturation of the various cell types generating the ground of increased vulnerability. This is particularly consistent with the high cholesterol demand required for cell proliferation, migration, differentiation, and synapse formation/remodeling. Other major questions we address are why the progression of Purkinje cells loss is always from the anterior to the posterior lobes and why cerebellar defects persist in the mouse model even when genetic manipulations can lead to nearly normal survival.

Itraconazole Inhibits Intracellular Cholesterol Trafficking and Decreases Phosphatidylserine Level in Cervical Cancer Cells.

Itraconazole shows anticancer activity in various types of cancer but its underlying mechanism is unclear. We investigated the effect of itraconazole on membrane-associated lipids. To investigate the influences of itraconazole on cholesterol trafficking, cervical cancer CaSki cells were cultured with itraconazole and analyzed by Filipin staining followed by confocal microscopy. Effect on the glycerophospholipid profiles was analyzed by liquid chromatography/mass spectrometry (LC/MS). After itraconazole treatment, Filipin staining revealed cholesterol accumulation in the intracellular compartments, which was similar to the distribution after treatment of U18666A (cholesterol transport inhibitor). LC/MS analysis showed a significant decrease in phosphatidylserine levels and an increase in lysophosphatidylcholine levels in CaSki cells. Itraconazole inhibited cholesterol trafficking and altered the phospholipid composition. Alterations in the cell membrane can potentiate the anticancer activity of itraconazole.