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Previous Article | Next Article 
Journal of Virology, October 2007, p. 11195-11207, Vol. 81, No. 20
0022-538X/07/$08.00+0 doi:10.1128/JVI.02559-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
School of Biomolecular and Biomedical Science, University College Dublin, Belfield Campus, Dublin 4, Ireland,1 Institut de Génétique Humaine, CNRS U.P.R. 1142, 141, rue de la Cardonille, 34396 Montpellier Cedex 5, France,2 Case Western Reserve, University School of Medicine, Cleveland, Ohio3
Received 20 November 2006/ Accepted 17 July 2007
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-cyclodextrin, which possessed less antiprion activity, had an IC50 of 750 µM. This report presents cyclodextrins as a new class of antiprion compound. For decades, the pharmaceutical industry has successfully used cyclodextrins for their complex-forming ability; this ability is due to the structural orientation of the glucopyranose units, which generate a hydrophobic cavity that can facilitate the encapsulation of hydrophobic moieties. Consequently, cyclodextrins could be ideal candidates for the treatment of prion diseases. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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PrPC and PrPSc have the same amino acid sequence but differ dramatically in conformation. PrPC consists of 42% -helix and 3% ß-sheet, while PrPSc possesses a much higher ß-sheet content of 43% and a lower -helix content of 30% (41, 52). The conversion of PrPC to the pathogenic isoform is a critical event. It is believed that the interaction of endogenous PrPC substrate with the pathogenic PrPSc template causes PrPC to unfold and refold as the ß-sheet-rich isoform PrPSc. This step starts a chain reaction, where each newly converted PrPSc molecule interacts with more PrPC molecules, fueling the formation of PrPSc (17, 48).
Lipid rafts, which are found in the plasma membrane, are also known as detergent-resistant microdomains (DRMs) (9, 38, 63) and are enriched in cholesterol and sphingolipids. The formation of rafts is dependent on cholesterol (51), which is believed to function as a spacer between hydrocarbon chains of sphingolipids, thereby holding the raft assembly together (56). The extraction of cholesterol from membranes leads to the dissociation of proteins from rafts (15, 25, 53). These rafts are important in many cell processes, such as membrane sorting and trafficking and signal transduction (23). As has been reported for other GPI-anchored proteins, both PrPC and PrPSc have been found to be associated with DRMs, and DRMs have been hypothesized to be involved in both the function of PrPC (34) and the conversion of PrPC to the pathogenic isoform (37, 38, 63). Moreover, in addition to the GPI anchor, PrP presents a sphingolipid binding domain that allows the interaction of the protein with the polar head groups of the sphingolipids (28, 64). A number of studies have provided evidence that in cell culture, conversion requires association with rafts (22, 63, 68), which may explain why cholesterol extraction modifies PrPSc formation in cell culture (63).
To date, there is no known cure for TSEs, as standard approaches to treating the diseases have proved ineffective. A range of compounds have been tested in the search for treatments for TSEs, and these compounds have been directed against targets such as preventing a rise in calcium, preventing apoptosis, directly interfering with conversion, and altering prion trafficking and dominant negative inhibition (2, 11, 12, 14, 19, 24, 29, 30, 35, 43-45, 46, 47, 54). Over the last several years, a wide variety of compounds have been tested for their effect in the treatment of TSEs, but unfortunately, none have been successful at completely eliminating the disease when given either immediately before, during, or after disease onset. Limitations on success have also related to the toxicity of antiprion compounds to humans and/or an inability to cross the blood-brain barrier (BBB), where most of the PrPSc accumulates.
Prion diseases share neuropathological characteristics with other neurodegenerative diseases, such as Parkinson's disease and Alzheimer's disease (AD). These characteristics include intracellular and extracellular aggregates of proteinaceous fibrils resulting from irregular protein-protein interactions (65). In a search for a new antiprion compound, ß-cyclodextrin (ß-CD), which reduces the toxic effects of an AD-associated protein (ß-amyloid [Aß] [amino acids 1 to 40]) in cell culture (10), was tested here. The formation of ß-sheet fibrils is a critical event in AD, and ß-CD, by attenuating fibrillization of the toxic peptide, appears to inhibit the toxic effects of Aß (10). The CDs are macrocyclic, nonreducing maltooligosaccharides made from -1,4-linked glucose units (58). -, ß-, and 
-CDs, composed of 6, 7, and 8 glucosyl units, respectively, are the parent CDs (61) and have attracted interest as natural complexing agents and as vehicles to increase drug delivery, bioavailability, and solubility (18, 58, 66).
In this report, the ability of noncytotoxic concentrations of ß-CD and methyl-ß-CD (Mß-CD) to clear PrPSc from ScN2a cells following 2 weeks of treatment is demonstrated. Of the naturally occurring CDs, ß-CD, with a half-maximal inhibitory concentration (IC50) of 75 µM, was much more efficient at clearing infection than was -CD or -CD, with IC50s of 750 µM and 1,150 µM, respectively. The antiprion activity of ß-CDs was dependent not only on the ring size of the CD but also on the cyclic nature of the molecule. The antiprion activity of the CDs tested related to their ability to sequester and move molecules. ß-CD modified the location of both PrPC and PrPSc, but it did so differently, separating the isoforms into different lipid domains. Such separations have the potential to restrict the conversion process. ß-CD also bound to PrPC and interfered with abnormal prion conversion. These data indicate that the antiprion action of ß-CDs is derived from a combination of mechanisms.
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MATERIALS AND METHODS |
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-, ß-, and -CDs; Mß-CD; maltohexaose (G6); maltoheptaose (G7); and water-soluble cholesterol complex were purchased from Sigma-Aldrich. Cell culture reagents, such as OptiMEM, fetal bovine serum, glutamine, and penicillin-streptomycin, were Gibco products supplied by Biosciences. Secondary antibody anti-mouse peroxidase conjugate and cholera toxin B subunit type Inaba 569B peroxidase conjugate were purchased from Calbiochem. Epoxy-activated Sepharose 6B was obtained from Amersham Pharmacia. Pefabloc was purchased from Boehringer Manheim, and G418 was purchased from Promega; all other reagents were purchased from Sigma-Aldrich. The primary antibody 3F4 (1:10,000 dilution) used in this study is characterized as recognizing the peptide epitope comprising amino acids 109 to 112 of human PrP and was purchased from Signet Laboratories. The primary antibody 8H4 used in this study is characterized as recognizing the peptide epitope comprising amino acids 175 to 185 of PrP and was described previously (27). Cell culture and effect of compounds on PrPC, PrPSc, and cholesterol levels. Neuroblastoma cells (N2a) transfected with mouse PrP (N2a58) and N2a58 cells infected with the 22L scrapie strain (N2a22L), as reported previously (39), were obtained from S. Lehmann (Montpelier, France). To obtain cells producing high levels of PrPSc, the N2a22L cell line was subcloned and is referred to as N2a22L20 in this study. 3F4-tagged mouse PrPC (MoPrPC) cDNA in the pcDNA3 vector was obtained from S. Lehmann (Montpelier, France). N2a cells were transfected with the cDNA using FuGENE transfection reagent according to the manufacturer's instructions. Stably transfected cells were screened and maintained using G418 resistance. The N2a22L, N2a22L20, N2a58, and 3F4MoPrPC N2a cells were cultured in OptiMEM supplemented with 10% fetal bovine serum, penicillin-streptomycin, 2 mM glutamine, and 300 µg/ml Geneticin (G418 sulfate) in a humidified atmosphere.
On passage, cells were treated with the compounds indicated in the text at the concentrations and times indicated unless otherwise stated. All compounds were solublized in Millipore water and were sterilized by filtering through a 0.2-µm filter prior to their being added to the medium. Cells were lysed in cold lysis buffer (0.5% [wt/vol] sodium deoxycholate, 0.5% [vol/vol] Triton X-100, 150 mM NaCl, and 50 mM Tris, pH 7.5) and analyzed for PrPC or PrPSc. The total protein concentration was measured using the bicinchoninic acid protein assay kit (Sigma).
Total cholesterol was measured by the method of Mizoguchi et al. (32). Twenty-five microliters of sample and 150 µl reagent solution [0.05 M MES (morpholineethanesulfonic acid) buffer, pH 6.1, 0.5 U ml–1 cholesterol oxidase, 4 U ml–1 cholesterol esterase, 0.2 mM 4-aminoantipyrine, 1 mM N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethoxyaniline sodium salt, and 4 U ml–1 horseradish peroxidase] were added and incubated at 37°C for 15 min, and cholesterol was measured at 600 nm.
Western blotting and densitometry. For PrPC analysis, lysates were adjusted to 18 µg of protein in gel loading buffer and boiled for 5 min prior to being loaded onto 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, followed by Western blotting. For PrPSc analysis, lysates were prepared to 200 µg for N2a22L cells and 50 µg for N2a22L20 cells and then digested with 16 µg of proteinase K (pK)/mg of protein at 37°C for 15 min. One millimolar Pefabloc was then added, and samples were incubated on ice for 5 min, followed by centrifugation at 14,000 rpm for 45 min. The resulting pellets were then resuspended in loading buffer, boiled for 5 min, and loaded onto 12% SDS-PAGE, followed by Western blotting using standard techniques. PrPC and PrPSc were detected by incubating immunoblots with antibody 8H4 (3F4-tagged MoPrPC was detected with antibody 3F4), followed by a horseradish peroxidase secondary antibody, and developed by enhanced chemiluminescence. Densitometry was performed with Bio-Rad Imager analysis software.
Sucrose density gradients. N2a22L20 cells from 3-by-60-mm dishes were lysed in 400 µl of ice-cold sucrose gradient (SG) lysis buffer (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA and 1% Triton X-100) on ice for 30 min. Lysates were then spun at 4,850 rpm for 10 min, and the 400-µl supernatant was adjusted to 40% (wt/vol) sucrose prepared in SG lysis buffer without detergent. One milliliter 25% (wt/vol) sucrose and 0.5 ml 5% (wt/vol) sucrose, both in SG lysis buffer without detergent, were then added. The gradient was then spun at 37,000 rpm for 18 h at 4°C. Fractions (10 x 230 µl) were collected from the top of the tube and processed for PrPC, PrPSc, and ganglioside GM1, a marker of DRM, as follows.
For PrPC detection, 100 µl of each fraction was diluted with 300 µl TN (10 mM Tris, pH 7.8, and 150 mM NaCl) and then made to 1% (wt/vol) Sarkosyl (to precipitate PrPSc) and incubated on ice for 30 min. Fractions were then spun at 45,000 rpm for 2 h at 4°C, and the supernatants were methanol (MeOH) precipitated. The precipitated pellet was resuspended in gel loading buffer, and PrPC was visualized by using 12% SDS-PAGE and Western blotting with 8H4 antibody. For PrPSc detection, 120 µl from each protein fraction was obtained and protein was determined using the bicinchoninic acid protein assay kit and treated with 16 µg pK/mg protein for 15 min at 37°C, followed by centrifugation at 70,000 rpm for 45 min. PrPSc was visualized by using SDS-PAGE and Western blotting with 8H4 antibody. For GM1 detection, 10 µl from each fraction was dotted onto a nitrocellulose membrane, which was then blocked in 3% bovine serum albumin for 3 h and incubated with cholera toxin B (1:4,000 dilution) for 1 h. Following four 10-min washes in TTBS (0.1 M Tris, 0.154 NaCl, pH 7.5, containing 0.3% [vol/vol] Tween 20), blots were developed by enhanced chemiluminescence.
Triton X-100 solubility assay. Cells were washed twice with ice-cold phosphate-buffered saline and were then left on ice for 15 min. The cells were then scraped into ice-cold buffer containing 1% Triton X-100, 150 mM NaCl, and 10 mM Tris-HCl, pH 7.8, and left on ice for 15 min. The lysate was then spun at 39,000 rpm in the ultracentrifuge Sorvall Discovery N12SE Micro Ultra at 2°C for 30 min. The pellet (insoluble protein) was resuspended in 1x loading buffer, and the supernatant (soluble protein) was methanol precipitated prior to centrifugation at 14,000 rpm for 10 min. The resulting pellet was resuspended in 1x loading buffer, and PrPC in the soluble and insoluble fractions was analyzed by 12% SDS-PAGE and immunoblotting with antibody 8H4.
Affinity chromatography and PrP binding. Freeze-dried epoxy-activated Sepharose 6B was reconstituted in water and activated with 0.1 M NaOH according to the manufacturer's instructions (Phamacia). The gel was incubated with or without 0.02 M ß-CD in 0.1 M NaOH for 16 h at 40°C with shaking. The gel was then washed in sequence with 0.1 M NaOH, distilled water, 0.1 M NaOH, and finally 0.1 M acetate buffer, pH 4.0. Both the control gel (without ß-CD) and the ß-CD-complexed gel were blocked with 0.2 M glycine, pH 8.0, for 4 h at 40°C. The gel was then washed with 0.1 M NaOH, followed by 0.1 M acetate buffer, pH 4.0.
For the binding of PrP to affinity gels containing ß-CD, gel made as described above with ß-CD incorporated was washed three times with 0.5 M HEPES, pH 7.5, and was resuspended in this buffer. Eight hundred micrograms of N2a cells transfected with 3F4 MoPrPC lysed in cold lysis buffer (0.5% [wt/vol] sodium deoxycholate, 0.5% [vol/vol] Triton X-100, 150 mM NaCl, and 50 mM Tris, pH 7.5) was then added to 800 µg of gel and mixed by inversion over a 15-min period. The mixture of gel and lysate was spun down at 1,000 rpm for 1 min, and the supernatant was removed and MeOH precipitated (S1). The gel was then washed four times with 500 µl of 0.5 M HEPES, pH 7.5, with a 15-min mixing period between each wash. A total of 500 µl of 0.5 M HEPES, pH 7.5, containing 500 µM ß-CD was then added, and the solution was mixed as before for 15 min and then spun at 1,000 rpm for 1 min. The supernatant was removed and MeOH precipitated (eluent 1 [E1]). A total of 500 µl of 0.5 M HEPES, pH 7.5, containing 1 mM ß-CD, was then added to the gel, and the solution was mixed as before for 15 min. It was then spun, and the supernatant was removed and MeOH precipitated (E2). The remaining 800 µl of gel was boiled in 400 µl of 1x loading buffer for 10 min. The mixture was spun at 1,000 rpm for 1 min, and the supernatant was taken and analyzed for PrPC (E3).
Conversion reaction. Two-by-60-mm dishes of N2a22L20 and 3F4 MoPrPC N2a cells rinsed twice with ice-cold phosphate-buffer saline were scraped into 300 µl of ice-cold conversion buffer (0.1 M MES, pH 6.2, and 1 µM CaCl2) and subjected to two freeze-thaw cycles and vortexed. The suspension was then incubated at 37°C for 72 h as indicated in the text. Two volumes of ice-cold lysis buffer (0.5% [wt/vol] sodium deoxycholate, 0.5% [vol/vol] Triton X-100, 150 mM NaCl, and 50 mM Tris, pH 7.5, 1 µg/ml pepstatin A, 1 µg/ml leupeptin, and 2 mM EDTA) was added, and samples were MeOH precipitated. The pellet was resuspended in 50 µl of ice-cold lysis buffer, and samples were then assayed for insolubility by centrifuging them at 70,000 rpm for 40 min at 4°C. The pellet (insoluble fraction) was resuspended in loading buffer. The supernatant (soluble fraction) was precipitated in MeOH. All samples were analyzed by SDS-12% PAGE and immunoblotting with antibody 3F4.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To examine the effect of ß-CD on scrapie, ß-CD was added to the N2a22L cell line at 100 µM and 500 µM at the time of passage and the cells were cultured in the presence of the compound for periods of 1 and 2 weeks. Cells were then lysed (Fig. 1A) and analyzed for PrPSc content by immunoblotting with 8H4 antibody. After 1 week of treatment, 500 µM ß-CD reduced PrPSc to 30% of the control level (Fig. 1A, lane 3, and B). Following 2 weeks of treatment, 100 µM ß-CD reduced PrPSc levels to 20% (Fig. 1A, lane 6, and B) and complete clearance was achieved with 500 µM (Fig. 1A, lane 7, and B). A total of 500 µM ß-CD also cleared PrPSc after 2 weeks of treatment in N2a22L20, which possessed six times more PrPSc than N2a22L did (Fig. 1C, lane 4). Clearance was measured as the disappearance of the proteinase-resistant core of PrP, amino acids 27 to 30, relative to the amount in the untreated control, which was taken as 100%, and clearance was comparable to that of the known antiprion compound Congo red (Fig. 1A, lane 5, and C, lane 3).
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CDs have higher antiprion activity than their linear counterparts.
The effect of other members of the natural CD family, -, ß-, and -CDs (Fig. 2A), were examined for potential antiprion activity. The N2a22L cell line was treated with 500 µM of -, ß-, and -CD for a period of 2 weeks, after which cells were analyzed for PrPSc levels by immunoblotting with 8H4 antibody (Fig. 2B, lanes 4 to 6). Of the CDs tested here, only ß-CD (Fig. 2B, lane 5) completely cured infection, whereas -CD and -CD demonstrated less antiprion activity (Fig. 2B, lanes 4 and 6). The structure-antiprion activity relationship of CDs was examined further by comparing their antiprion activity with that of their linear counterparts. The linear counterparts for ß-CD and -CD were maltoheptaose (G7) and maltohexaose (G6), respectively (Fig. 2A). The corresponding linear sugar for -CD, maltooctaose, was not commercially available. Once again, the compounds were added at 500 µM to the cells at the time of passage and the cells were cultured in this manner for a period of 2 weeks, after which the cells were lysed and analyzed for PrPSc levels. As G6 and G7 displayed less antiprion activity than - and ß-CD, respectively, the importance of the cyclic nature of the CDs for enhancing antiprion activity is demonstrated. G7 showed some ability to reduce PrPSc levels, but it was not able to reduce infection like ß-CD. CDs differ in size with -CD possessing 6, ß-CD possessing 7, and -CD possessing 8 glucopyranose units (Fig. 2A), leading to differences in the cavity sizes of the compounds (8 Å for -CD, 6 Å for ß-CD, and 4.5 Å for -CD) (66). These data indicate that the 6-Å size of ß-CD is important for antiprion activity, and this indication is further emphasized by comparing the IC50 values of ß-CD, -CD, and -CD for clearing infection in N2a22L.
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-, and -CD by adding a range of concentrations of the compounds to the cells at the time of passage over a period of 2 weeks, after which the cells were lysed and analyzed for PrPSc. ß-CD with an IC50 of 75 µM (Fig. 3A) was 10 times more efficient at clearing infection than was -CD, which had an IC50 of 750 µM (Fig. 3B). -CD had an IC50 of 1,150 µM (Fig. 3C).
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-CD for a period of 2 weeks to investigate whether the compounds had an effect on PrPC levels (Fig. 4A and B). Cells were then lysed and analyzed for PrPC levels by immunoblotting with antibody 8H4. ß-CD did not have a significant effect on PrPC levels compared to the control (Fig. 4A, lane 2, and B). On the other hand, -CD, which is 10 times less efficient than ß-CD as an antiprion compound, lowered PrPC levels by 30% (Fig. 4A, lane 3, and B).
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Effect of ß-CD on PrPC, PrPSc and GM1 association with DRMs. CDs are powerful extractors of cholesterol, but of the CDs, ß-CD had the highest capacity to sequester lipids (16, 40, 57). The accumulation of proteins in rafts is altered or abolished upon cholesterol depletion (46). This holds true for PrPC, and altering cholesterol levels interferes with PrPSc production in cell culture (63). So, to investigate how ß-CD acts against PrPSc, its effect on the floatation properties of PrPC and PrPSc was determined. Like most GPI-anchored proteins, PrPC and PrPSc are insoluble in cold Triton X-100 (9, 38, 63). This is due to their association with membrane domains or rafts that are enriched in cholesterol, sphingolipids, and glycosphingolipids; these domains are also referred to as DRMs (4, 8). The association of PrPC with such domains is linked with the conversion to PrPSc (37, 38, 63). The solubility of DRMs is thought to be associated with the way their lipid content affects their rigidity and, consequently, the inability of cold Triton X-100 to penetrate and solubilize (9). Consequently, PrPC and PrPSc float in buoyant fractions in density gradients and float in similar fractions to the raft resident, cell surface ganglioside GM1 (38).
To examine for the effect of ß-CD on floatation properties, floatation gradients were carried out with the N2a22L20 cell line. Cells were lysed after 3 days of growth, as for SGs, and the gradients were applied for 18 h at 37,000 rpm. Fractions were then processed for PrPC, PrPSc, and GM1 content as described in Materials and Methods and analyzed by immunoblotting with 8H4 antibody for PrPC and PrPSc and by dot blot analysis with CTXB for GM1. PrPC was separated from PrPSc by Sarkosyl treatment. Naslavsky et al. (38) reported that this methodology does not affect PrPC floatation properties. In the absence of ß-CD, as expected, PrPC, PrPSc and the raft marker, GM1, floated in light fractions (Fig. 5). To examine for the effect of ß-CD on the floatation characteristics, N2a22L20 cells were treated with ß-CD (500 µM) for 3 days, and floatation gradients were carried out as before. In keeping with the hypothesis that the removal of cholesterol increases the solubility of raft domains and consequently decreases the floatation of the associated GPI-anchored proteins, treatment with ß-CD caused PrPSc and the GM1 marker to float in heavier fractions. PrPSc peaked in fraction 4 prior to the addition of ß-CD and between fractions 5 to 8 afterwards (Fig. 5B, compare upper panel, lane 4, and lower panel, lanes 5 to 8, and E). The GM1 marker similarly shifted from fraction 5 to 6 under ß-CD treatment (Fig. 5C, compare lane 5, upper panel, and lane 6, lower panel, and F). Unexpectedly, however, upon treatment with ß-CD, PrPC floated in lighter fractions (fractions 3 and 4) (Fig. 5A, lower panel, lanes 3 and 4, and D) to the control (fraction 5) (Fig. 5A, upper panel, lane 5, and D). It would appear that ß-CD acts through modification of the lipid raft domain, but surprisingly, the two isoforms reacted differently, with PrPC floating in more insoluble fractions than PrPSc. This separation of the localization of the isoforms could have the potential to restrict conversion. However, although the major portions of PrPC and PrPSc have been separated, an overlap in gradients that could allow for a low level of conversion to occur does exist.
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CDs affect cholesterol levels but do not move PrPC to soluble Triton X-100 domains. CDs are known for their ability to complex with and sequester cholesterol. To examine this ability in N2a22L20 cells, cells were incubated for 1 h in the presence of increasing concentrations of either ß-CD or Mß-CD and their effects on total cholesterol levels were determined. The ability of chol-Mß-CD to reintroduce cholesterol was also examined. After the hour of treatment with the CDs, the medium was replaced, and cells were incubated for a further hour in the presence of chol-Mß-CD (Fig. 7A). Both CDs lowered cholesterol levels; however, Mß-CD was more efficient. At 0.5, 1, and 10 mM, ß-CD lowered levels to 97, 91, and 23% of those of the control, whereas Mß-CD lowered levels to 79, 63, and 25% (Fig. 7A). Results are in line with those reported previously (1). Chol-Mß-CD introduced 40% extra cholesterol into control cells and reintroduced cholesterol into CD-treated cells. Cells that had been treated with 0.5, 1, and 10 mM ß-CD had levels increased by chol-Mß-CD to 110, 128, and 81.8%, respectively, of those of the untreated control. For cells that had been treated with 0.5, 1, and 10 mM Mß-CD, the levels were brought to 118, 100, and 53%, respectively, relative to levels of the untreated control (Fig. 7A).
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ß-CD binding to PrPC. With CDs moving PrPC to domains of lower density in sucrose density experiments, an effect that chol-Mß-CD could not inhibit, it is possible that CDs (through cholesterol movement) could facilitate the generation of some altered density compartments to which PrPC, and not PrPSc, is directed. Alternatively, PrPC may be directed by these compounds to naturally existing high-lipid domains as a consequence of drug presence. Due to their hydrophobic cores, CDs possess the ability to encapsulate hydrophobic moieties. If CDs bound PrP, this binding could aid in the antiprion mechanism. Consequently, the ability of ß-CD to bind PrPC was determined.
ß-CD was bound to epoxy-activated Sepharose 6B as described in Materials and Methods. N2a cells expressing 3F4-tagged MoPrPC were lysed in cold lysis buffer (0.5% [wt/vol] sodium deoxycholate, 0.5% [vol/vol] Triton X-100, 150 mM NaCl and 50 mM Tris, pH 7.5). The lysate was then added to gel containing ß-CD that had been equilibrated to pH 7.5 with 0.5 M HEPES buffer, and the binding process was carried out as described in Materials and Methods. Samples were analyzed by 12% SDS-PAGE and immunoblotting with 3F4 antibody. When binding was carried out with ß-CD incorporated into the gel, approximately 50% of the 3F4-tagged PrPC bound (Fig. 8A, lane 2, and B) and no PrPC was eluted off in subsequent washes with 0.5 M HEPES buffer, pH 7.5, alone (data not shown). Depending on the strength of binding, it is sometimes possible to elute a bound protein with the complexing agent (ß-CD) in solution. However, neither 500 µM ß-CD nor 1 mM ß-CD in solution could elute PrPC from the gel (Fig. 8A, lanes 3 and 4, and B). Some protein-to-ligand complexes require more stringent methods for elution; therefore, the gel with PrPC bound was boiled in loading buffer, and 12% of the eluate was analyzed for PrPC content. A 100% elution of bound PrPC occurred under these conditions (Fig. 8A, lane 5, and B). On the other hand, when binding was carried out without ß-CD incorporated into gel, PrPC did not bind (data not shown).
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DISCUSSION |
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-1-4 bonds, and due to the structural orientation of the glucopyranose units, a hydrophobic cavity, which can facilitate the encapsulation of hydrophobic moieties, is generated (58, 61). This report demonstrates that CDs also possess antiprion activity, which appeared to be due to their ability to sequester compounds.
The modulation of cholesterol levels with Mß-CD on its own has been shown to alter the susceptibility of cells to Aß toxicity in PC12 cells (3) and, when Mß-CD is combined with lovastatin, to modify Aß production in hippocampal cells (57). In this report, the capacity of ß-CD to clear PrPSc from infected cell culture is demonstrated. The antiprion activity of ß-CD was time dependent, with PrPSc clearance in N2a22L cells occurring in 2 weeks with 500 µM ß-CD. Structural studies revealed the cyclic nature of the CDs to be important to the antiprion activity, and this is reflected in the IC50 for ß-CD (75 µM), which was 10 times lower than that for -CD (750 µM). The linear form of ß-CD also reduced PrPSc levels, but as it could not clear PrPSc, we infer that cyclicization of the molecule is significant for activity.
The antiprion action of ß-CD did not appear to relate to a reduction in the substrate PrPC within the cell for conversion. However, Mß-CD (which is also reported here, for the first time, to have antiprion activity) has recently been reported to facilitate the shedding of PrPC from neuronal cells (42). This shedding of PrPC could have the potential to prevent conversion by reducing the access of PrPSc to PrPC. This possibility would be in line with the report stating that the release of PrP from the plasma membrane by phosphatidylinositol-specific phospholipase C (13) prevents PrPSc formation. Additionally, although it has been reported that known antiprion compounds pass through endosomes and lysosomes and act at these compartments to interfere with PrPSc production (60, 62), ß-CD does not appear to act through these compartments, as the lysomotropic agent NH4Cl could not inhibit the action of ß-CD.
It is more likely that the antiprion activity of CDs relates to their ability to sequester and move molecules. CDs are known for their ability to remove cholesterol from cell membranes (40, 57), and as cholesterol levels affect PrPSc production (63), it is possible that their action may be through an effect on the lipid raft environment where both PrPC and PrPSc reside. Rafts are composed of sphingolipids and cholesterol, and the latter is thought to act as a spacer between the sphingolipids, keeping the lipid raft domain together (55). These domains house a number of protein networks, including an array of GPI-anchored proteins, and the ability of the domains to concentrate proteins/molecules may facilitate protein-to-protein interactions, including the conversion of PrPC to PrPSc (38, 63). Cholesterol extraction with Mß-CD has been reported to dissociate proteins from rafts and to move proteins from Triton X-100 insoluble to soluble compartments (46). The modulation of the raft environment has also been shown by others to interfere with the PrP conversion. However, reports on this are conflicting. Lovastatin, an inhibitor of 3-hydroxy-3-methylglutaryl-co-enzyme A reductase, which reduces cholesterol levels, reduced prion infection in cell culture (63). Likewise, squalestatin, an inhibitor of cholesterol production, also cured prion-infected neurons (5). Both squalestatin and lovastatin moved PrPC from Triton X-100 insoluble fractions to soluble fractions on extraction at 4°C (5, 63). Mß-CD did not do this. However, the depletion of sphingolipid with fumonisin B actually increased the formation of PrPSc in neuroblastoma cells and had no effect on the floatation properties of either PrPC or PrPSc (37), suggesting that the modulation of cholesterol and sphingolipids have different effects on the conversion.
Of the natural CDs, ß-CD presented with the highest antiprion activity. This result was not surprising, as the ability of CDs to extract cholesterol is highly dependent on CD size, with ß-CD being more effective than either - or -CD (33). However, in light of the report of fumonisin B, ß-CD's antiprion action, rather than proprion action, was unexpected. CDs are powerful cholesterol effluxors, but whereas -CD preferentially sequesters phospholipids, ß-CD preferentially extracts shingomyelin (33). Of course ß-CD and fumonisin B are most probably acting very differently; however, Naslavsky et al. (37) could not rule out whether the effect of fumonisin B on PrPSc related to the level of sphingolipids in rafts or the head group identity.
Unlike fumonisin B, ß-CD affected the floatation properties of both PrPC and PrPSc. Prior to treatment with ß-CD, PrPC, PrPSc, and the DRM marker GM1 all floated in buoyant fractions (Fig. 5A to F), which is in agreement with the results of other reports (38). However, on ß-CD treatment, PrPC and PrPSc were resolved into separate fractions (Fig. 5). PrPSc moved to the less buoyant fractions along with the GM1 marker. This result could be expected as an effect of cholesterol extraction on the location of GPI-anchored proteins, but PrPC moved to more buoyant fractions. This physical separation of PrPC and PrPSc could be sufficient to hinder the conversion process. However, a slight overlap in gradients still existed after treatment, and this overlap could still have allowed some conversion. In keeping with these data, Mß-CD, which had higher antiprion activity than that of ß-CD and had the highest ability to remove cellular cholesterol, failed to move PrPC to domains that were Triton X-100 soluble, but a higher percentage of PrPC was located in domains that were Triton X-100 insoluble after treatment. On extraction of neuroblastoma cells at 37°C prior to density floatation gradients, Naslavsky et al. (38) identified that in certain cell lines and in scrapie brain homogenate, PrPC and PrPSc could be resolved into different fractions, indicating that the two isoforms reside in rafts of different contents. This separation was seen only in certain clones, and the researchers proposed that the lipid content of cellular membranes differed between cell types. It is possible that ß-CD affects the lipid content of the rafts hosting PrPC differently than those hosting PrPSc, with PrPC residing in domains of higher lipid content after ß-CD treatment. Cholesterol can transfer between different lipid vesicles, and CDs have been reported, by binding cholesterol in water-soluble complexes, to donate cholesterol to cellular membranes (16, 26) and in these locations PrPC would reside.
As CDs have the capacity to sequester (33) and move hydrophobic molecules, the possibility that CDs act via a combination of mechanisms cannot be ruled out. ß-CD binds PrPC in affinity chromatography, and although this binding would not be specific to PrPC, as CDs can bind to a range of proteins (21), this binding may play a part in its action. CDs have also been reported to modify protein conformation (18). CDs interfere with insulin self-association (67) and the toxicity of Aß (10). Qin et al. (50) demonstrated the ability of ß-CD to inhibit the Aß peptide 12-28 ß-sheet conformational change. Interestingly, in keeping with our antiprion data that are specific for ß-CD, only ß-CD, and not -CD or -CD, possessed the ability to inhibit the structural conversion of Aß. It could be hypothesized that ß-CD may interact with the hydrophobic central core of PrP; such an interaction could lead to the ability of ß-CD to interfere with the abnormal conversion.
The successfulness of antiprion compounds is dependent on a number of factors, including their ability to pass the BBB. CDs have been used for a long time to increase the solubility of compounds and to aid drug delivery. They are biocompatible, show resistance to human enzymes, have low toxicity in humans, and do not elicit an immune response (18). One millimolar of ß-CD has been reported to pass the BBB without affecting the tight junctions; a tight junction breakdown has only been observed to start at 5 mM ß-CD (33). Consequently, ß-CD's antiprion IC50 of 75 µM falls well below the toxic level reported for this compound. CDs are a new class of antiprion compounds, and their usage has the potential to open up a number of avenues in the search for novel therapeutics in TSEs.
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FOOTNOTES |
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Published ahead of print on 15 August 2007.
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