Dataset Information

Differential Effects of 2-Hydroxypropyl-Cyclodextrins on Lipid Accumulation in Npc1-Null Cells.

ABSTRACT: Niemann-Pick disease type C (NPC) is an autosomal recessive disorder characterized by abnormal accumulation of free cholesterol and sphingolipids in lysosomes. The iminosugar miglustat, which inhibits hexosylceramide synthesis, is used for NPC treatment, and 2-hydroxypropyl-?-cyclodextrin (HP-?-CD), a cyclic oligosaccharide derivative, is being developed to treat NPC. Moreover, therapeutic potential of 2-hydroxypropyl-?-cyclodextrin (HP-?-CD) was shown in NPC models, although its mechanism of action remains unclear. Here, we investigated the effects of HP-?-CD, HP-?-CD, and their homolog 2-hydroxypropyl-?-cyclodextrin (HP-?-CD) on lipid accumulation in Npc1-null Chinese hamster ovary (CHO) cells compared with those of miglustat. HP-?-CD and HP-?-CD, unlike HP-?-CD, reduced intracellular free cholesterol levels and normalized the lysosome changes in Npc1-null cells but not in wild-type CHO cells. In contrast, miglustat did not normalize intracellular free cholesterol accumulation or lysosome changes in Npc1-null cells. However, miglustat decreased the levels of hexosylceramide and tended to increase those of sphingomyelins in line with its action as a glucosylceramide synthase inhibitor in both Npc1-null and wild-type CHO cells. Interestingly, HP-?-CD and HP-?-CD, unlike HP-?-CD, reduced sphingomyelins in Npc1-null, but not wild-type, cells. In conclusion, HP-?-CD and HP-?-CD reduce the accumulation of sphingolipids, mainly sphingomyelins, and free cholesterol as well as lysosome changes in Npc1-null, but not in wild-type, CHO cells.

SUBMITTER: Hoque S

PROVIDER: S-EPMC7038050 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Differential Effects of 2-Hydroxypropyl-Cyclodextrins on Lipid Accumulation in <i>Npc1</i>-Null Cells.

Hoque Sanzana S Kondo Yuki Y Sakata Nodoka N Yamada Yusei Y Fukaura Madoka M Higashi Taishi T Motoyama Keiichi K Arima Hidetoshi H Higaki Katsumi K Hayashi Akio A Komiya Takaki T Ishitsuka Yoichi Y Irie Tetsumi T

International journal of molecular sciences 20200130 3

Niemann-Pick disease type C (NPC) is an autosomal recessive disorder characterized by abnormal accumulation of free cholesterol and sphingolipids in lysosomes. The iminosugar miglustat, which inhibits hexosylceramide synthesis, is used for NPC treatment, and 2-hydroxypropyl-β-cyclodextrin (HP-β-CD), a cyclic oligosaccharide derivative, is being developed to treat NPC. Moreover, therapeutic potential of 2-hydroxypropyl-γ-cyclodextrin (HP-γ-CD) was shown in NPC models, although its mechanism of ac ...[more]

PMID: 32019132

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Project description:Niemann-Pick type C (NPC) disease is a fatal hereditary neurodegenerative disorder characterized by a massive accumulation of cholesterol in lysosomes and late endosomes due to a defect in intracellular cholesterol trafficking. Dysfunction in intracellular cholesterol trafficking is responsible for about 50 rare inherited lysosomal storage disorders including NPC. The lysosomal proteins NPC1 and NPC2 play a crucial role in trafficking of cholesterol from late endosomes and lysosomes to other cellular compartments. However, the detailed mechanisms of cholesterol trafficking at the late endosomes/lysosomes (LE/LY) are poorly understood. Studies showed that 2-hydroxypropyl-β-cyclodextrin (HPβCD) alleviates the cholesterol accumulation defect in animal model and has been approved for a phase 2b/3 clinical trial for NPC. HPβCD is known to bind cholesterol; however, the mechanisms how HPβCD mediates the exit of cholesterol from the LE/LY compartments are still unknown. Further, another cyclodextrin (CD) derivative, 2-hydroxypropyl-γ-cyclodextrin (HPγCD), was shown to reduce intracellular cholesterol accumulation in NPC patient cells and NPC mice model. Herein, we identified a number of candidate proteins differentially expressed in NPC patient-derived cells compared to cells derived from a healthy donor using a proteomic approach. Interestingly, both HPβCD and HPγCD treatments modulated the expression of most of these NPC-specific proteins. Data showed that treatment with both CDs induces the expression of the lysosome-associated membrane protein 1 (LAMP-1) in NPC patient-derived cells. Remarkably, LAMP-1 overexpression in HeLa cells rescued U18666A-induced cholesterol accumulation suggesting a role of LAMP-1 in cholesterol trafficking. We propose that HPβCD and HPγCD facilitate cholesterol export from the LE/LY compartments via the LAMP-1 protein, which may play a crucial role in cholesterol trafficking at the LE/LY compartments when there is no functional NPC1 protein. Together, this study uncovers new cellular mechanisms for cholesterol trafficking, which will contribute to development of novel therapeutic approaches for lysosomal storage diseases.

Project description:The rising risks of food microbial contamination and foodborne pathogens resistance have prompted an increasing interest in natural antimicrobials as promising alternatives to synthetic antimicrobials. Essential oils (EOs) obtained from natural sources have shown promising anticancer, antimicrobial, and antioxidant activities. EOs extracted from the resins of Pistacia lentiscus var. Chia are widely utilized for the treatment of skin inflammations, gastrointestinal disorders, respiratory infections, wound healing, and cancers. The therapeutic benefits of P. lentiscusessential oils (PO) are limited by their low solubility, poor bioavailability, and high volatility. Nanoencapsulation of PO can improve their physicochemical properties and consequently their therapeutic efficacy while overcoming their undesirable side effects. Hence, PO was extracted from the resins of P. lentiscusvia hydrodistillation. Then, PO was encapsulated into (2-hydroxypropyl)-beta-cyclodextrin (HPβCD) via freeze-drying. The obtained inclusion complexes (PO-ICs) appeared as round vesicles (22.62 to 63.19 nm) forming several agglomerations (180 to 350 nm), as detected by UHR-TEM, with remarkable entrapment efficiency (89.59 ± 1.47%) and a PDI of 0.1475 ± 0.0005. Furthermore, the encapsulation and stability of PO-ICs were confirmed via FE-SEM, 1H NMR, 2D HNMR (NOESY), FT-IR, UHR-TEM, and DSC. DSC revealed a higher thermal stability of the PO-ICs, reaching 351.0 °C. PO-ICs exerted substantial antibacterial activity against Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli as compared to free PO. PO-ICs showed significant enhancement in the antibacterial activity of the encapsulated PO against S. aureus with an MIC90 of 2.84 mg/mL and against P. aeruginosa with MIC90 of 3.62 mg/mL and MIC50 of 0.56 mg/mL. In addition, PO-ICs showed greater antimicrobial activity against E. coli by 6-fold with an MIC90 of 0.89 mg/mL, compared to free PO, which showed an MIC90 of 5.38 mg/mL. In conclusion, the encapsulation of PO into HPβCD enhanced its aqueous solubility, stability, and penetration ability, resulting in a significantly higher antibacterial activity.

Project description:UnlabelledAging and pathologic conditions cause intracellular aggregation of macromolecules and the dysfunction and degeneration of neurons, but the mechanisms are largely unknown. Prime examples are lysosomal storage disorders such as Niemann-Pick type C (NPC) disease, where defects in the endosomal-lysosomal protein NPC1 or NPC2 cause intracellular accumulation of unesterified cholesterol and other lipids leading to neurodegeneration and fatal neurovisceral symptoms. Here, we investigated the impact of NPC1 deficiency on rodent neurons using pharmacologic and genetic models of the disease. Improved ultrastructural detection of lipids and correlative light and electron microscopy identified lamellar inclusions as the subcellular site of cholesterol accumulation in neurons with impaired NPC1 activity. Immunogold labeling combined with transmission electron microscopy revealed the presence of CD63 on internal lamellae and of LAMP1 on the membrane surrounding the inclusions, indicating their origins from intraluminal vesicles of late endosomes and of a lysosomal compartment, respectively. Lamellar inclusions contained cell-intrinsic cholesterol and surface-labeled GM1, indicating the incorporation of plasma membrane components. Scanning electron microscopy revealed that the therapeutic drug candidate β-cyclodextrin induces the subplasmalemmal location of lamellar inclusions and their subsequent release to the extracellular space. In parallel, β-cyclodextrin mediated the NPC1-independent redistribution of cholesterol within neurons and thereby abolished a deleterious cycle of enhanced cholesterol synthesis and its intracellular accumulation, which was indicated by neuron-specific transcript analysis. Our study provides new mechanistic insight into the pathologic aggregation of macromolecules in neurons and suggests exocytosis as cellular target for its therapeutic reversal.Significance statementMany neurodegenerative diseases involve pathologic accumulation of molecules within neurons, but the subcellular location and the cellular impact are often unknown and therapeutic approaches lacking. We investigated these questions in the lysosomal storage disorder Niemann-Pick type C (NPC), where a defect in intracellular cholesterol transport causes loss of neurons and fatal neurovisceral symptoms. Here, we identify lamellar inclusions as the subcellular site of lipid accumulation in neurons, we uncover a vicious cycle of cholesterol synthesis and accretion, which may cause gradual neurodegeneration, and we reveal how β-cyclodextrin, a potential therapeutic drug, reverts these changes. Our study provides new mechanistic insight in NPC disease and uncovers new targets for therapeutic approaches.

Project description:2-Hydroxypropyl-beta-cyclodextrin (HPβCD) has gained recent attention as a potential therapeutic intervention in the treatment of the rare autosomal-recessive, neurodegenerative lysosomal storage disorder Niemann-Pick Disease Type C1 (NPC1). Notably, HPβCD formulations are not comprised of a single molecular species, but instead are complex mixtures of species with differing degrees of hydroxypropylation of the cyclodextrin ring. The degree of substitution is a critical aspect of the complex mixture as it influences binding to other molecules and thus could potentially modulate biological effects. VTS-270 (Kleptose HPB) and Trappsol® Cyclo™ are HPβCD products under investigation as novel treatments for NPC1. The purpose of the present work is to compare these two different products; analyses were based on ion distribution and abundance profiles using mass spectrometry methodology as a means for assessing key molecular distinctions between products. The method incorporated electrospray ionization and analysis with a linear low-field ion mobility quadrupole time-of-flight instrument. We observed that the number of hydroxypropyl groups (the degrees of substitution) are substantially different between the two products and greater in Trappsol Cyclo than in VTS-270. The principal ions of both samples are ammonium adducts. Isotope clusters for each of the major ions show doubly charged homodimers of the ammonium adducts. In addition, both products show doubly charged homodimers from adduction of both a proton and ammonium. Doubly charged heterodimers are also present, but are more intense in Trappsol Cyclo than in VTS-270. Based on the analytical differences observed between VTS-270 and Trappsol Cyclo with respect to the degree of substitution, the composition and fingerprint of the complex mixture, and the impurity profiles, these products cannot be considered to be the same; the potential biological and clinical implications of these differences are not presently known.

Project description:Natural antimicrobials have recently gained increasing interest over synthetic antimicrobials to overcome foodborne pathogens and food microbial contamination. Essential oils (EOs) obtained from Boswellia sacra resins (BO) were utilized for respiratory disorders, rheumatoid arthritis, malignant tumors, and viral infections. Like other EOs, the therapeutic potential of BO is hindered by its low solubility and bioavailability, poor stability, and high volatility. Several studies have shown excellent physicochemical properties and outstanding therapeutic capabilities of EOs encapsulated into various nanocarriers. This study extracted BO from B. sacra resins via hydrodistillation and encapsulated it into hydroxypropyl-beta-cyclodextrins (HPβCD) using the freeze-drying method. The developed inclusion complexes of BO (BO-ICs) had high encapsulation efficiency (96.79 ± 1.17%) and a polydispersity index of 0.1045 ± 0.0006. BO-ICs showed presumably spherical vesicles (38.5 to 59.9 nm) forming multiple agglomerations (136.9 to 336.8 nm), as determined by UHR-TEM. Also, the formation and stability of BO-ICs were investigated using DSC, FTIR, FE-SEM, UHR-TEM, 1H NMR, and 2D HNMR (NOESY). BO-ICs showed greater thermal stability (362.7 °C). Moreover, compared to free BO, a remarkable enhancement in the antimicrobial activities of BO-ICs was shown against three different bacteria: Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa. BO-ICs displayed significant antibacterial activity against Pseudomonas aeruginosa with an MIC90 of 3.93 mg mL-1 and an MIC50 of 0.57 mg mL-1. Also, BO-ICs showed an increase in BO activity against Escherichia coli with an MIC95 of 3.97 mg mL-1, compared to free BO, which failed to show an MIC95. Additionally, BO-ICs showed a more significant activity against Staphylococcus aureus with an MIC95 of 3.92 mg mL-1. BO encapsulation showed significantly improved antimicrobial activities owing to the better stability, bioavailability, and penetration ability imparted by encapsulation into HPβCD.

Project description:Silymarin (Sy) shows limited water solubility and poor oral bioavailability. Water-soluble hydroxypropyl (HPBCD) and randomly methylated (RAMEB) ?-cyclodextrins were designed to enhance anti-fibrotic efficiency of silymarin in CCl4-induced liver fibrosis in mice. Experimental fibrosis was induced by intraperitoneal injection with 2 ml/kg CCl4 (20% v/v) twice a week, for 7 weeks. Mice were orally treated with 50 mg/kg of Sy-HPBCD, Sy-RAMEB and free silymarin. For assessment of the spontaneous reversion of fibrosis, CCl4 treated animals were investigated after 2 weeks of recovery time. The CCl4 administration increased hepatic oxidative stress, augmented the expression of transforming growth factor-?1 (TGF-?1) and Smad 2/3, and decreased Smad 7 expression. Furthermore, increased ?-smooth muscle actin (?-SMA) expression indicated activation of hepatic stellate cells (HSCs), while up-regulation of collagen I (Col I) and matrix metalloproteinases (MMPs) expression led to an altered extracellular matrix enriched in collagen, confirmed as well by trichrome staining and electron microscopy analysis. Treatment with Sy-HPBCD and Sy-RAMEB significantly reduced liver injury, attenuating oxidative stress, restoring antioxidant balance in the hepatic tissue, and significantly decreasing collagen deposits in the liver. The levels of pro-fibrogenic markers' expression were also significantly down-regulated, whereas in the group for spontaneous regression of fibrosis, they remained significantly higher, even at 2 weeks after CCl4 administration was discontinued. The recovery was significantly lower for free silymarin group compared to silymarin/? cyclodextrins co-treatments. Sy-HPBCD was found to be the most potent anti-fibrotic complex. We demonstrated that Sy-HPBCD and Sy-RAMEB complexes decreased extracellular matrix accumulation by inhibiting HSC activation and diminished the oxidative damage. This might occur via the inhibition of TGF-?1/Smad signal transduction and MMP/tissue inhibitor of MMPs (TIMP) rebalance, by blocking the synthesis of Col I and decreasing collagen deposition. These results suggest that complexation of silymarin with HPBCD or RAMEB represent viable options for the its oral delivery, of the flavonoid as a potential therapeutic entity candidate, with applications in the treatment of liver fibrosis.

Dataset Information

Differential Effects of 2-Hydroxypropyl-Cyclodextrins on Lipid Accumulation in Npc1-Null Cells.

Publications

Differential Effects of 2-Hydroxypropyl-Cyclodextrins on Lipid Accumulation in <i>Npc1</i>-Null Cells.

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