Dataset Information

Synthesis, characterization, and evaluation of pluronic-based ?-cyclodextrin polyrotaxanes for mobilization of accumulated cholesterol from Niemann-Pick type C fibroblasts.

ABSTRACT: Several lines of evidence suggest that ?-cyclodextrin (?-CD) derivatives initiate the efflux of accumulated, unesterified cholesterol from the late endosomal/lysosomal compartment in Niemann Pick C (NPC) disease models. Unfortunately, repeated injections or continuous infusions of current ?-CD therapies are required to sustain suppression of symptoms and prolong life. In an effort to make CD treatment a more viable option by boosting efficacy and improving pharmacokinetics, a library of Pluronic surfactant-based ?-CD polyrotaxanes has been developed using biocompatible poly(ethylene glycol) (PEG)-polypropylene glycol (PPG)-PEG triblock copolymers. These compounds carry multiple copies of ?-CD as shown by (1)H NMR, 2D nuclear Overhouser effect spectroscopy, gel permeation chromatography/multiangle light scattering, analytical ultracentrifugation analysis, matrix assisted laser desorption/ionization mass spectrometry, and diffusion-ordered spectroscopy. Analyses of free ?-cyclodextrin contamination in the compounds were made by reverse phase high pressure liquid chromatography and hydrophilic interaction liquid chromatography. Dethreading kinetics were studied by reverse phase high pressure liquid chromatography, UV/vis, and (1)H NMR analysis. Filipin staining studies using npc2(-/-) fibroblasts show significant reversal of cholesterol accumulation after treatment with polyrotaxane compounds. The rate and efficacy of reversal is similar to that achieved by equivalent amounts of monomeric ?-CD alone.

SUBMITTER: Collins CJ

PROVIDER: S-EPMC4319568 | biostudies-literature | 2013 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Synthesis, characterization, and evaluation of pluronic-based β-cyclodextrin polyrotaxanes for mobilization of accumulated cholesterol from Niemann-Pick type C fibroblasts.

Collins Christopher J CJ McCauliff Leslie A LA Hyun Seok-Hee SH Zhang Zhaorui Z Paul Lake N LN Kulkarni Aditya A Zick Klaus K Wirth Mary M Storch Judith J Thompson David H DH

Biochemistry 20130430 19

Several lines of evidence suggest that β-cyclodextrin (β-CD) derivatives initiate the efflux of accumulated, unesterified cholesterol from the late endosomal/lysosomal compartment in Niemann Pick C (NPC) disease models. Unfortunately, repeated injections or continuous infusions of current β-CD therapies are required to sustain suppression of symptoms and prolong life. In an effort to make CD treatment a more viable option by boosting efficacy and improving pharmacokinetics, a library of Pluronic ...[more]

PMID: 23560535

Similar Datasets

Project description:BackgroundNiemann-Pick type C (NPC) disease is a fatal neurodegenerative disorder caused most commonly by a defect in the NPC1 protein and characterized by widespread intracellular accumulation of unesterified cholesterol and glycosphingolipids (GSLs). While current treatment therapies are limited, a few drugs tested in Npc1(-/-) mice have shown partial benefit. During a combination treatment trial using two such compounds, N-butyldeoxynojirimycin (NB-DNJ) and allopregnanolone, we noted increased lifespan for Npc1(-/-) mice receiving only 2-hydroxypropyl-beta-cyclodextrin (CD), the vehicle for allopregnanolone. This finding suggested that administration of CD alone, but with greater frequency, might provide additional benefit.Methodology/principal findingsAdministration of CD to Npc1(-/-) mice beginning at either P7 or P21 and continuing every other day delayed clinical onset, reduced intraneuronal cholesterol and GSL storage as well as free sphingosine accumulation, reduced markers of neurodegeneration, and led to longer survival than any previous treatment regime. We reasoned that other lysosomal diseases characterized by cholesterol and GSL accumulation, including NPC disease due to NPC2 deficiency, GM1 gangliosidosis and mucopolysaccharidosis (MPS) type IIIA, might likewise benefit from CD treatment. Treated Npc2(-/-) mice showed benefits similar to NPC1 disease, however, mice with GM1 gangliosidosis or MPS IIIA failed to show reduction in storage.Conclusions/significanceTreatment with CD delayed clinical disease onset, reduced intraneuronal storage and secondary markers of neurodegeneration, and significantly increased lifespan of both Npc1(-/-) and Npc2(-/-) mice. In contrast, CD failed to ameliorate cholesterol or glycosphingolipid storage in GM1 gangliosidosis and MPS IIIA disease. Understanding the mechanism(s) by which CD leads to reduced neuronal storage may provide important new opportunities for treatment of NPC and related neurodegenerative diseases characterized by cholesterol dyshomeostasis.

Project description:BACKGROUND: Niemann-Pick disease type C (NPC) is caused by defects in cholesterol efflux from lysosomes due to mutations of genes coding for NPC1 and NPC2 proteins. As a result, massive accumulation of unesterified cholesterol in late endosomes/lysosomes is observed. At the level of the organism these cholesterol metabolism disorders are manifested by progressive neurodegeneration and hepatosplenomegaly. Until now filipin staining of cholesterol deposits in cells has been widely used for NPC diagnostics. In this report we present an alternative method for cholesterol visualization and estimation using a cholesterol-binding bacterial toxin, perfringolysin O. METHODS: To detect cholesterol deposits, a recombinant probe, perfringolysin O fused with glutathione S-transferase (GST-PFO) was prepared. GST-PFO followed by labeled antibodies or streptavidin was applied for immunofluorescence and immunoelectron microscopy to analyze cholesterol distribution in cells derived from NPC patients. The identity of GST-PFO-positive structures was revealed by a quantitative analysis of their colocalization with several organelle markers. Cellular ELISA using GST-PFO was developed to estimate the level of unesterified cholesterol in NPC cells. RESULTS: GST-PFO recognized cholesterol with high sensitivity and selectivity, as demonstrated by a protein/lipid overlay assay and surface plasmon resonance analysis. When applied to stain NPC cells, GST-PFO decorated abundant deposits of cholesterol in intracellular vesicles that colocalized with filipin-positive structures. These cholesterol deposits were resistant to 0.05%-0.2% Triton X-100 used for cells permeabilization in the staining procedure. GST-PFO-stained organelles were identified as late endosomes/lysosomes based on their colocalization with LAMP-1 and lysobisphosphatidic acid. On the other hand, GST-PFO did not colocalize with markers of the Golgi apparatus, endoplasmic reticulum, peroxisomes or with actin filaments. Only negligible GST-PFO staining was seen in fibroblasts of healthy individuals. When applied to cellular ELISA, GST-PFO followed by anti-GST-peroxidase allowed a semiquantitative analysis of cholesterol level in cells of NPC patients. Binding of GST-PFO to NPC cells was nearly abolished after extraction of cholesterol with methyl-?-cyclodextrin. CONCLUSIONS: Our data indicate that a recombinant protein GST-PFO can be used to detect cholesterol accumulated in NPC cells by immunofluorescence and cellular ELISA. GST-PFO can be a convenient and reliable probe for revealing cholesterol deposits in cells and can be useful in diagnostics of NPC disease.

Dataset Information

Synthesis, characterization, and evaluation of pluronic-based ?-cyclodextrin polyrotaxanes for mobilization of accumulated cholesterol from Niemann-Pick type C fibroblasts.

Publications

Synthesis, characterization, and evaluation of pluronic-based β-cyclodextrin polyrotaxanes for mobilization of accumulated cholesterol from Niemann-Pick type C fibroblasts.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets