Treatment of neutral glycosphingolipid lysosomal storage diseases via inhibition of the ABC drug transporter, MDR1 Cyclosporin A can lower serum and liver globotriaosyl ceramide levels in the Fabry mouse model Michael Mattocks1, Maria Bagovich1, Maria De Rosa1,4, Steve Bond2, Beth Binnington1, Vanessa I Rasaiah2, Jeffrey Medin2,3 and Clifford Lingwood1,4,5 Research Institute, The Hospital for Sick Children, Toronto, Canada Ontario Cancer Institute, University Health Network, Toronto, Canada Department of Medical Biophysics, University of Toronto, Canada Department of Laboratory Medicine and Pathology, University of Toronto, Canada Department of Biochemistry, University of Toronto, Canada Keywords enzyme replacement therapy; Gaucher disease; a-galactosidase; glucosyl ceramide translocase; HUS model Correspondence C Lingwood, Research Institute, The Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada Fax: +416 813 5993 Tel: +416 813 5998 E-mail: cling@sickkids.ca (Received 20 January 2006, revised March 2006, accepted 10 March 2006) doi:10.1111/j.1742-4658.2006.05223.x We have shown that the ABC transporter, multiple drug resistance protein (MDR1, P-glycoprotein) translocates glucosyl ceramide from the cytosolic to the luminal Golgi surface for neutral, but not acidic, glycosphingolipid (GSL) synthesis Here we show that the MDR1 inhibitor, cyclosporin A (CsA) can deplete Gaucher lymphoid cell lines of accumulated glucosyl ceramide and Fabry cell lines of globotriaosyl ceramide (Gb3), by preventing de novo synthesis In the Fabry mouse model, Gb3 is increased in the heart, liver, spleen, brain and kidney The lack of renal glomerular Gb3 is retained, but the number of verotoxin (VT1)-staining renal tubules, and VT1 tubular targeting in vivo, is markedly increased in Fabry mice Adult Fabry mice were treated with a-galactosidase (enzymereplacement therapy, ERT) to eliminate serum Gb3 and lower Gb3 levels in some tissues Serum Gb3 was monitored using a VT1 ELISA during a post-ERT recovery phase ± biweekly intra peritoneal CsA After weeks, tissue Gb3 content and localization were determined using VT1 ⁄ TLC overlay and histochemistry Serum Gb3 recovered to lower levels after CsA treatment Gb3 was undetected in wild-type liver, and the levels of Gb3 (but not gangliosides) in Fabry mouse liver were significantly depleted by CsA treatment VT1 liver histochemistry showed Gb3 accumulated in Kupffer cells, endothelial cell subsets within the central and portal vein and within the portal triad Hepatic venule endothelial and Kupffer cell VT1 staining was considerably reduced by in vivo CsA treatment We conclude that MDR1 inhibition warrants consideration as a novel adjunct treatment for neutral GSL storage diseases The lysosomal storage diseases (LSD) are genetic deficiencies in glycoconjugate catabolism, each due to a lack of a specific lysosomal sugar hydrolase or its acti- vator protein [1] The (mainly neurological) symptoms are due to the intracellular accumulation of the enzyme substrate In the ‘glycosphingolipidoses’, this Abbreviations BSA, bovine serum albumin; CsA, cyclosporin A; ERT, enzyme replacement therapy; Gb3, globotriaosyl ceramide; GlcCer, glucosyl ceramide; GSL, glycosphingolipid; HUS, hemolytic uremic syndrome; LacCer, lactosyl ceramide; LSD, lysosomal storage disease; MDR1, multiple drug resistance protein (P-glycoprotein); NGS, normal goat serum; VT1, verotoxin 1; TLC, thin layer chromatogram 2064 FEBS Journal 273 (2006) 2064–2075 ª 2006 The Authors Journal compilation ª 2006 FEBS M Mattocks et al accumulation results in the formation of lipid inclusions and multilamellar structures which prevent normal cell function Symptoms depend on the enzyme, age of onset and residual enzyme activity [1] Because only  10% residual enzyme activity may be sufficient to avert clinical symptoms, exogenous enzyme-replacement therapy (ERT) has been developed, particularly in the two neutral GSL storage diseases, Gaucher (glucosyl ceramide accumulates) and Fabry (globotriaosyl ceramide, Gb3, accumulates) [2–4] a-Galactosidase administered to Fabry patients is able to reduce serum levels of Gb3 by 50% [5], liver Gb3 and by inference, kidney Gb3 levels [5,6] In the Fabry mouse model, in which the a-galactosidase is abscent [7], elevated serum Gb3 levels (serum Gb3 is undetectable in normal mice) can be deleted by ERT, but tissue Gb3 is more refractory This may be due, in part, to the direct access of the enzyme to the serum substrate To digest accumulated Gb3 in tissue, the enzyme must be taken up by cells within the tissue and targeted intracellularly to the lysosome This is achieved, in vitro at least, via the mannose phosphate receptor pathway and replacement a-galactosidase is phosphomannosylated to promote such uptake [8] Within the Fabry mouse tissues, liver Gb3 is most susceptible to a-galactosidase therapy Although some lowering of spleen and heart Gb3 is seen, renal Gb3 is more resistant [9] In the Fabry mouse, there is no gross pathology (although a thrombotic deficiency has recently been found) [10], but in Fabry disease, the primary pathology is in the kidney [1], the major site of Gb3 synthesis in man [11], and in the heart, possibly due to the association of Gb3 synthesis with the microvasculature GSL synthesis in man and mouse are distinct, particularly in the kidney, where Gb3 can be found in the human, but not murine, glomerulus [12–14] Despite its clinical success, the extraordinary cost of ERT has limited patient access and promoted the development of alternative strategies Gene therapy is a candidate strategy for Fabry which may eventually prove the most satisfactory [15] The third approach has been to develop procedures to restrict the synthesis of Gb3 Two strategies have been developed Both have focused on inhibitors of glucosyl ceramide synthase This enzyme is the first glycosyl transferase required for the synthesis of most GSLs, including Gb3 (in Fabry disease) and of course, GlcCer (in Gaucher disease) By inhibiting this enzyme, the synthesis of most GSLs (and all gangliosides) is prevented The glucosyl ceramide synthase substrate mimic, d,l-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), or its derivatives with improved selectivity [16], provide one approach [17,18] Imino sugar-based glucosyl ceramide MDR1 inhibition and GSL storage disease synthase inhibitors, such as N-butyldeoxynojirimycin, have proven effective in animal storage disease models [19] and in clinical trials for Gaucher disease [20,21] Such imino sugars, however, also inhibit glucosidase processing of N-linked high mannose oligosaccharides [22] and glycogen breakdown [23] Unlike all other GSLs, GlcCer is made on the outer leaflet of the Golgi bilayer [24] and must be ‘flipped’ into the lumen to access the glycosyltransferases for further carbohydrate elongation Multiple drug resistance protein (MDR1) can function as a glycolipid flippase [25,26] We showed MDR1 to be responsible for this translocation in the majority of cultured cells [27,28] The conversion of ceramide to GlcCer and other GSLs has been associated with drug resistance, as a means to avoid ceramide-induced cell death [29,30], although this has been questioned [31] MDR1mediated GlcCer translocation into the Golgi could be a component of such resistance However, we found that MDR1-translocated GlcCer is used only for neutral GSL synthesis [28] because inhibition of MDR1 does not affect cellular ganglioside synthesis This provides a degree of selectivity not available in the other approaches to substrate reduction therapy as a clinical management for Fabry disease In addition, the longterm clinical experience with drugs that modulate MDR1 in cancer, and, for cyclosporin A (CsA), immunosuppression, would provide significant advantage, in terms of defined toxicity and dosage Although MDR1 expression varies within tissues, expression in the kidney, and liver [32,33], sites of Gb3 accumulation in the Fabry mouse, make this a feasible approach In order to begin to address this potential, we determined the effect of CsA on Gb3 synthesis in the Fabry mouse model Our studies have further delineated the abnormal Gb3 synthesis in this model and have shown that the inhibition of MDR1 is a viable potential approach to the reduction of Gb3 in both the serum and certain tissues of this model Results MDR1 inhibition in LSD cell lines Epstein Barr virus (EBV) transformed B-cell lines from Gaucher and Fabry LSD patients were cultured with lm CsA for four days The GSL fractions were purified and separated by thin layer chromatography (TLC) Figure 1A shows the accumulation of glucosyl ceramide (GlcCer) was prevented in CsA-treated Gaucher B lymphoblasts Three cell lines were tested GlcCer accumulated in each, but in one cell line, lactosyl ceramide accumulated also (Fig 1A, lane 6) In each FEBS Journal 273 (2006) 2064–2075 ª 2006 The Authors Journal compilation ª 2006 FEBS 2065 MDR1 inhibition and GSL storage disease A M Mattocks et al B C D E F GlcCer GalCer GM2 LacCer GM1 Gb3 Gb4 Gb5 Fig Effect of cyclosporin A (CsA) on cultured Gaucher and Fabry B-cell line glycosphingolipids (GSLs) The neutral GSL fraction (from · 106 cells per lane) was separated by thin layer chromatogram (TLC) (C ⁄ M ⁄ W 65 : 25 : v ⁄ v ⁄ v) The doublets corresponding to GlcCer and Gb3 are shown by arrows (A) Gaucher lymphoblastoid cell lines, detected using orcinol spray Lane 1, GSL standards, GlcCer, GalCer, LacCer, Gb3, Gb4, Gb5 (Forssman) as indicated Lanes 2, 4, 6, Neutral GSLs of untreated 5072, 5410, 5831 Gaucher cell lines Lanes 3, 5, Neutral GSLs of CsA-treated 5072, 5410, 5831 cell lines (B–F) Fabry lymphoblastoid cell line Cells were grown with 14C-serine 14C-Radiolabeled GSLs were detected by phosphoimaging (B) Orcinol detection of total neutral GSL fraction, (C) VT1 overlay of panel B to detect Gb3 only, (D) 14C-metabolic radiolabeled GSL phosphoimage of panel B Lane 1, GSL standards as in (A, lane 1); lane 2, untreated cells; lane 3, CsA-treated cells The 14C-radiolabeled species below Gb3 were not characterized (E) The ganglioside fraction from14C-labeled Fabry cells was separated by TLC (C ⁄ M ⁄ W 60 : 25 : 10 0.2 M CaCl2 v ⁄ v ⁄ v) and detected using orcinol (GM3), or (F) phosphoimaging of the 14C-metabolic labeled species Lane 1, ganglioside standards GM2 and GM1 as indicated; lane 2, untreated cells; lane 3, CsA-treated cells The accumulated lymphoid GlcCer in Gaucher cells was eliminated by CsA The extent to which more complex neutral GSLs were reduced varied between cell lines CsA treatment of Fabry cells significantly reduced the Gb3 and neutral GSL content without effect on the ganglioside profile case, CsA was found to delete GlcCer and reduce other neutral GSLs present Inhibition of MDR1mediated GlcCer translocation results in increased access to the cytosolic glucocerebrosidase [34] which is not defective in Gaucher LSD CsA treatment of a Fabry B-cell line (Fig 1B–F) also showed significant inhibition of accumulated Gb3, monitored by orcinol stain (Fig 1B) and VT1 ⁄ TLC overlay (Fig 1C) This indicates residual a-galactosidase activity in this cell line Metabolic labeling of neutral GSLs (including Gb3) within the Fabry cell line was prevented by CsA (Fig 1D), confirming MDR1 inhibition reduces de novo Gb3 synthesis Steady-state levels (Fig 1E) and metabolically labeled (Fig 1F) gangliosides in this Fabry cell line were unaffected by CsA GM3 is the A major ganglioside present but additional, more complex gangliosides were detected by metabolic labeling Because the Fabry mouse has no a-galactosidase activity and already accumulated Gb3 cannot therefore turnover, we designed a treatment protocol in which the effect of MDR1 inhibition by CsA on accumulation of Gb3 via de novo synthesis was assessed Tissue Gb3 expression The Gb3 expression profile for various tissues from wild-type and Fabry mice was first compared by VT1 TLC overlay (Fig 2) The Gb3 content was marked increased in the kidney, spleen and liver of Fabry mice A detectable increase was also observed in the heart B Fig Comparison of the Gb3 content of wild-type and Fabry mouse tissues GSLs were separated by TLC (C ⁄ M ⁄ W 65 : 25 : v ⁄ v ⁄ v) and visualized using orcinol spray for carbohydrate (A) or VT1 overlay (B) to detect Gb3 Lane 1, GSL standards, from the top: GlcCer, GalCer, LacCer, Gb3, Gb4, Gb5; lanes 2, 4, 6, 8, wild-type; lanes 3, 5, 7, 9, Fabry, lanes 2, heart; lanes 4, 5, spleen; lanes 6, kidney; lanes 8, liver 2066 FEBS Journal 273 (2006) 2064–2075 ª 2006 The Authors Journal compilation ª 2006 FEBS M Mattocks et al The most notable elevation was seen surprisingly, in the liver in which, under the conditions used, Gb3 was undetected in the wild-type This indicates Gb3 must undergo rapid turnover in the normal liver Alternatively, liver Gb3 may accumulate via increased serum Gb3 clearance rather than de novo synthesis Gb3 synthase is present in the liver, however [35,36], suggesting de novo synthesis in liver endothelial cell subsets and scavenger accumulation in Kupffer cells (see histology below) Renal Gb3 Renal Gb3 is the verotoxin receptor responsible for the development of hemolytic uremic syndrome (HUS) in man [14] HUS is a renal glomerular disease Gb3 is found in tubules and glomeruli in man [14] There is no adequate small animal model of VT1-induced HUS because Gb3 is not found in rodent renal glomeruli [13] Gb3 is present in rodent renal tubules and VT1 induces renal tubular necrosis [37] We considered that the increased renal Gb3 of the Fabry mouse might extend to the glomerulus to provide a model of the human disease In the cortex of wild-type kidney, subpopulations of renal tubules were VT1 stained but glomeruli were unreactive, consistent with our previous studies [13] However, although the VT1 staining of renal tubules is dramatically increased in the Fabry mouse (Fig 3A), compared with the sporadic VT1 staining seen in the wild-type animal [13], the glomeruli of Fabry mouse kidney remain completely unstained In Fabry kidney, virtually all cortical tubules were now stained This indicates that Gb3 is synthesized in all renal tubules in wild-type mice but is rapidly degraded in the majority VT1 renal tubular targeting in vivo (Fig 3B) was also significantly increased relative to wild-type mice [13], suggesting that Fabry mice should be hypersensitive to VT1 The deparaffinization necessary for immunostaining precludes identification of the tubule type stained Under the experimental conditions used, no in vivo staining of wild-type kidney tubules was seen (not shown) As with the VT1 cryosection staining, VT1 did not target the renal glomeruli of Fabry mice in vivo Serum Gb3 The low level of Gb3 in the Fabry mouse serum and the small volumes available precluded the use of TLC overlay to detect Gb3 A more sensitive VT1-based ELISA assay was used [38] This assay was linear < 60 ng standard Gb3 and was able to detect > ng Gb3 per lL serum sample Gb3 in the serum of wildtype mice was below the background of this assay MDR1 inhibition and GSL storage disease ERT and CsA treatment Effect on serum Gb3 Owing to the difficulty of drug administration in neonates and the availability of well-documented CsA dosage protocols for adult mice, it was decided that our initial studies on the feasibility of MDR1 inhibition as a potential treatment should be carried out in adult Fabry animals after ERT a-Galactosidase treatment will eliminate the serum Gb3 levels [9] and the effect of a maintenance dosing of CsA on the recovery of serum Gb3 levels after termination of ERT was determined ERT is an effective means of eliminating serum Gb3, and Gb3 remained subsequently undetectable in the serum of any animal until weeks post ERT At this time, the serum Gb3 has recovered for most control mice, whereas the level reached by the CsA-treated mice is reduced by  50% (Fig 4; P ¼ 0.028) The recovery of serum Gb3 post ERT was found to be, to some extent, variable and some mice within both the control and treated groups did not recover detectable serum Gb3 by the 9-week experiment termination For responding mice, CsA-treated Fabry mice had serum Gb3 levels of 3.31 ± 1.33 ngỈlL)1 compared with control Fabry serum levels of 8.21 ± 2.27 ngỈlL)1 as determined from a standard curve Serum Gb3 levels were monitored in all animals throughout the experimental period However, at the termination of the experiment a random selection of organs from control and CsA-treated mice were assigned for either GSL extraction or VT1 ⁄ immunohistological evaluation Effect of CsA on tissue Gb3 GSLs were extracted from kidney and liver of control and CsA-treated Fabry mice after weeks recovery post ERT, and from CsA-treated and untreated wildtype mice The Gb3 content was assessed via VT1 overlay CsA-dependent differences were seen only in the liver (Fig 5) The Gb3 content of the liver was increased in Fabry mice and this was reduced in CsAtreated, compared with control mice after recovery from ERT CsA treatment reduced the liver Gb3 content overall by  50% (P ¼ 0.013) Renal Gb3 content was much greater but significant changes after ERT and CsA treatment were not seen (Fig 5) GM2 ganglioside is the major ganglioside of mouse liver [39] and the only ganglioside we detected in the Fabry liver GSL extract GM2 levels were similar in Fabry and wild-type mouse liver Comparison of Gb3 and GM2 levels (Fig 5G) clearly show that although liver Gb3 FEBS Journal 273 (2006) 2064–2075 ª 2006 The Authors Journal compilation ª 2006 FEBS 2067 MDR1 inhibition and GSL storage disease M Mattocks et al A a b d c B Fig Comparison of VT1 staining of wild-type and Fabrys kidney tissue (A) VT1 staining of cryosections (a, b) Fabry, (c, d) wild-type kidney cortex Magnifications: (a) ·16, (b–d) ·40 Glomeruli are marked by arrows VT1 staining is brown The section is counter stained with hematoxylin (B) In situ staining of renal VT1 bound in vivo VT1 (50 lg per mouse) injected i.p and bound within the kidney was immunostained with anti-VT1(without counterstain) in fixed sections after paraffin removal The Fabry cortical section is shown VT1 in vivo renal tubular targeting is significantly increased in the Fabry mouse compared with the 5–10 VT1-labeled tubules which would be seen in an equivalent normal mouse kidney field [13] No VT1 containing glomeruli are seen Magnification: ·16 levels are reduced in the extracts of CsA-treated mice, the level of GM2 is unaffected Gb3 tissue histochemistry The localization of Gb3 within frozen sections of liver and selected tissues from Fabry and wild-type mice 2068 monitored by VT1 binding is shown in Fig VT1 Gb3 staining was not above background in the wildtype mouse liver (Fig 6A), but within the Fabry liver VT1 binding detected Gb3 in the stellate Kupffer cells, distributed throughout the section, and in cells lining the portal triad The levels detected in Fabry mouse liver were reduced in ERT Fabry animals maintained FEBS Journal 273 (2006) 2064–2075 ª 2006 The Authors Journal compilation ª 2006 FEBS M Mattocks et al MDR1 inhibition and GSL storage disease Fig Effect of CsA treatment on the serum Gb3 levels in Fabry mouse Serum Gb3 assessment at weeks post ERT Control Fabry mice (n), CsA-treated Fabry mice ()) Serum Gb3 levels for CsA-treated mice are  50% less than control, P ¼ 0.028 on CsA during recovery (Fig 6B) compared with ERT Fabry mice that recovered without CsA In ERT Fabry recovery control mice, subsets of endothelial cells in the central (Fig 6B c,d,ij) and portal (Fig 6B d) vein were Gb3 positive and many Kupffer cells expressed Gb3 (Figs 6Ba–d,ij) Some vessels within the portal triad were also stained (Fig 6B d) Extracellular matrix staining in the triad was seen In the livers of CsA-treated Fabry mice, VT1 staining of Kupffer cells was greatly reduced (Fig 6Be-h) Gb3 expression in central and portal vein endothelial cells was significantly reduced and many vessels negative for Gb3 were observed in CsA-treated mice Portal triad staining was largely unaffected by CsA treatment (Fig 6B k,l) In the heart, VT1 staining of a subpopulation of larger blood vessel endothelial cells was seen only in the Fabry mouse (Fig 7A, compared to Fig 7B) A patchwork staining which originates from a subset of fibrocytes between the cardiac muscle fibers and appears to ‘diffuse’ into the myofibrils, is also evident in the Fabry mice (Fig 7A) The VT1 binding in the Fabry mouse lung (Fig 7C) was increased in the bronchiolar epithelium Staining of bronchiolar epithelial cells in the lung was significantly elevated compared with the wild-type (Fig 7D) Although MDR1 is detected in the heart and lung [40], this staining was not consistently altered after CsA treatment There was virtually Wild-type liver Wild-type liver Fabry liver A sphingomyelin B GSLs C GlcCer LacCer Gb3 Gb4 Gb5 GM3 GM2 GM1 10 Wild-type kidney 11 12 13 14 10 Fabry kidney 1 Fabry liver sphingomyelin E D Fabry liver GSLs F 10 G Fig Comparison of Gb3 levels in wild-type and Fabry mouse liver and kidney: relative effect of CsA on Gb3 compared with other sphingolipids (A, B, C, F, G) Liver extracts, (D, E) kidney extracts (A, C, D) Wild-type, (B, E, F, G) Fabry mice extracts, as indicated (+) Marks extracts from CsA-treated Fabry mice Neutral GSLs (A, B, D, E) were separated in C ⁄ M ⁄ W 65 : 25 : v ⁄ v ⁄ v and neutral and acidic GSLs (C, F, G) were separated in C ⁄ M ⁄ 0.8% KCl aq 60 : 40 : v ⁄ v ⁄ v Gb3 detection by VT1 ⁄ TLC overlay (A, B, D, E) Lanes 1–3, 0.5, 1, lg Gb3 standard; lane 4, GM3 ganglioside standard; (B) lanes 5, 7, 9–11 CsA-treated Fabry mice; lanes 6, 8, 12–14 control Fabry mice; (E) lanes 5, 6, 10, CsA-treated Fabry mice; lanes 7–9, control Fabry mice Liver sphingomyelin and ganglioside detection, (C) (lanes 1,2) and (F) iodine vapour detects liver sphingomyelin (marked * C); (C) (lanes 3, 4) and (G) orcinol spray detects liver GSLs-resorcinol reactive GM2 ganglioside is arrowed (C) lanes and 3, GSL standards: from the top GlcCer, LacCer, Gb3,Gb4,Gb5 (Forssman), GM3, GM2,GM1; lanes and 4, lipid extract of wild-type liver; (F, G) lane 1, GSL standards; lanes and 4, lipid extracts of control Fabry liver; lanes 3, 5, lipid extracts of CsAtreated Fabry liver Gb3 is only detected in Fabry, as opposed to wild-type liver (compare A with B, and C with G) and the normal renal Gb3 doublet (D) is markedly enhanced in the Fabry mouse (E) Less Gb3 is detected in the liver of CsA-treated, compared with control Fabry mice (B, G) Although the level of Gb3 is reduced by CsA treatment, the levels of GM2 ganglioside (similar in wild-type, indicated by arrow, and Fabry mouse liver; compare C with G), and sphingomyelin (F) are unaffected The Gb3 detected in (B) was subject to densitometry and compared The CsA-treated Fabry liver Gb3 values were reduced by 45% (P ¼ 0.013) compared with controls FEBS Journal 273 (2006) 2064–2075 ª 2006 The Authors Journal compilation ª 2006 FEBS 2069 MDR1 inhibition and GSL storage disease M Mattocks et al A a b B a b c d e f g h i j k l Fig Verotoxin staining of frozen liver sections from Fabry mice treated ± CsA (A) Wild-type (a) compared with Fabry liver (b) VT1 staining in wild-type liver is undetectable Arrows in (b) indicate some of the VT1-stained (brown) Kupffer cells in the Fabry mouse liver (B) (a–d, I, j) Untreated, (e-h, k, l) CsA-treated Fabry mice (Liver sections from three individual mice in each category are shown.) Magnification: (a–c, e–g) ·40; (d, g, i–l) ·16 * ¼ central veins, p ¼ portal veins Inserts in (a) and (b) show Kupffer cell staining, and in (d) portal vein endothelial VT1 cell staining Most VT1 staining is lost after CsA treatment but portal triad staining was retained 2070 FEBS Journal 273 (2006) 2064–2075 ª 2006 The Authors Journal compilation ª 2006 FEBS M Mattocks et al MDR1 inhibition and GSL storage disease Fig Comparison of Verotoxin staining of other tissues (A, C, E) Fabry, (B, D, F) wildtype tissue, (insets) CsA-treated Fabry (A, B) Heart – endothelial staining in Fabry mouse; (C, D) lung – epithelial cell staining increased in Fabry mouse; (E, F) brain microvascular endothelial staining in Fabry mouse Magnification ·16 no VT1 staining of normal brain (Fig 7F) In Fabry brain, extensive staining of the microvasculature is evident (Fig 7E) The arachnoid membrane surrounding the brain is extensively stained in Fabry but not normal mouse brain Although MDR1 is highly expressed in the brain microvasculature [41], ERT is not effective to reduce the level of Gb3 in the brain [9] Discussion The differential sensitivity of ganglioside and neutral GSL synthesis to depletion of GlcCer via MDR1 inhibition [28] provides an attractive method for the selective reduction of neutral GSL synthesis in neutral GSL storage diseases Other substrate reduction approaches are less selective and hence have greater potential sideeffects Inhibitors of glucosyl ceramide synthase prevent the synthesis of both neutral GSLs and gangliosides Although the lack of GSLs can be tolerated in cultured cells [42], the glucosyl ceramide synthase knockout mouse is embryonic lethal [43] Imino sugars inhibit a-glucosidases as well as glucosyl transferases [22,23] The role of MDR1 in GSL synthesis, though established in vitro, has yet to be understood in vivo MDR1 knockout mice not show an overt phenotype, although skin fibroblasts from such mice are, as predicted, defective in neutral GSL synthesis [28] We showed that an alternative mechanism of Golgi membrane GlcCer translocation must exist in HeLa cells [28] because their neutral GSLs are unaffected by CsA Whereas the liver of MDR1 knockout mice show a GSL complement consistent with the translocase function of MDR1, the GSLs of some other tissues are complicated by the redundancy in this function (studies in progress) and the tissue differences in MDR1 expression Thus, an effect of MDR1 inhibition on GSL biosynthesis in vivo was by no means assured The possibility of using MDR1 inhibition as a new approach to neutral GSL storage diseases is supported by our finding that CsA completely reverses GSL accumulation in Gaucher lymphoblasts, in which there is FEBS Journal 273 (2006) 2064–2075 ª 2006 The Authors Journal compilation ª 2006 FEBS 2071 MDR1 inhibition and GSL storage disease M Mattocks et al an alternative cytosolic mechanism for breakdown [34] The significant effect in Fabry lymphoblasts to reduce Gb3 without effect on ganglioside synthesis supports this approach Prevention of Gb3 synthesis is the only feasible stratagem in the Fabry mouse and in those Fabry patients with no residual a-galactosidase activity In such cases, Gb3 already accumulated would not be reversed by MDR1 inhibition, or other mechanisms of substrate-reduction therapy A protocol using adult Fabry mice was designed to test the efficacy of MDR1 inhibition on de novo Gb3 synthesis, whereby animals were treated by ERT and the effect of CsA on ‘relapse’ of Gb3 accumulation monitored ERT primarily affects serum and liver Gb3 accumulation [9] and these tissues were therefore the primary focus of our study, although the location of Gb3 accumulated in other tissues was also investigated Our demonstration that CsA significantly reduces Fabry mouse serum and liver Gb3 levels, approaches proof of concept Total Gb3 extracted from the liver was reduced yet the level of GM2 ganglioside, the only ganglioside we detected in Fabry mouse liver, was not reduced by CsA treatment This is consistent with our cell culture [28] and Fabry lymphoblast studies in which MDR1 inhibition was found to prevent neutral but not acidic GSL synthesis Thus in vivo (at least within the liver), as well as in cell culture, GlcCer translocated to the Golgi lumen by MDR1 is a precursor for neutral GSL but not ganglioside synthesis This preferential effect on neutral GSL biosynthesis might be considered as a ‘signature’ for MDR1 involvement VT1 staining of Kupffer cells and endothelial cells within the central vein showed significantly less Gb3 accumulation after CsA treatment Because the phagocytic Kupffer cells are major reticulo-endothelial degradative sites, the Gb3 they contain could be serum ⁄ red blood cell-derived and the decrease seen after CsA result from the reduced serum Gb3 levels Kupffer cells are modified monocytes that share a common origin with endothelial cells However, we believe that this is unlikely to be the case because endothelial cell staining within the liver was also reduced and mice in which serum Gb3 was found to remain undetectable after ERT, were nevertheless found to have Gb3 in the hepatic extract and express Kupffer cell Gb3 Heart, lung, brain, kidney and spleen tissue show a clear increase in Gb3 staining in the Fabry, compared with normal mouse but this was not obviously affected by the current CsA protocol However, because these tissues are less sensitive than liver to ERT [9], the potential benefit of CsA in these tissues might accrue 2072 on prolonged treatment or treatment prior to GSL accumulation The increased in vivo VT1 renal targeting in the Fabry mouse suggests increased susceptibility to this toxin compared with wild-type, but the retained lack of glomerular binding indicates that the Fabry mouse will not serve as a model of HUS in man The increased Gb3 expression in virtually all the renal tubules of the Fabry mouse shows that the lack of Gb3 detection in most tubules of the wild-type mouse [13] is a result of rapid Gb3 turnover, rather than the lack of Gb3 synthesis A similar effect in man could be important in determining susceptibility to HUS following VTEC infection Our results indicate the feasibility of using inhibition of MDR1 as an approach to the treatment of Fabry disease Although the efficacy may not, as yet, be as dramatic as ERT, inhibition of MDR1 may prove most beneficial as an adjunct, rather than alternative to ERT It is clear that the dosage and treatment period in this model needs optimization and the effect of maintenance MDR1 inhibition from birth requires investigation In addition, more selective inhibitors of MDR1 than CsA are available CsA is, however, clinically used long-term and it might be expected that under such conditions, the effect on Fabry patient tissue Gb3 levels might be accumulative and more significant than the modest reductions we have seen following brief treatment of the Fabry mouse Other GSL storage diseases in which a similar approach might be beneficial would include Gaucher In this case, inhibition of GlcCer Golgi translocation should increase exposure to the cytosolic glucosidase (not deficient in Gaucher disease) to effect a reduction in GSL accumulation ERT is clinically effective in Fabry patients [4] but neurological symptoms are not addressed and treatment with the missing a-galactosidase is extremely costly, such that it is not universally available The search for alternative or complementary treatment strategies continues [18,19,44] Our studies suggest a new approach to the inhibition of substrate synthesis Future work with neonatal Fabry mice is required to establish a ‘proof of principle’ as to the efficacy of an MDR1 inhibition approach In summary, CsA treatment has been found to reduce the recovery of serum Gb3 levels in Fabry mice following a-galactosidase treatment In such mice, the expression of Gb3 within the liver is also reduced in comparison with Fabry mice allowed to recover from ERT without MDR1 inhibition These studies indicate that MDR1 inhibition represents a potential novel adjunct to the current treatment of neutral GSL storage diseases FEBS Journal 273 (2006) 2064–2075 ª 2006 The Authors Journal compilation ª 2006 FEBS M Mattocks et al Experimental procedures CsA treatment of LSD cultured cells EBV-transformed B-lymphoblastoid cell lines from Gaucher type and Fabry disease (kindly supplied by J Clarke, Hospital for Sick Children, Toronto, Canada) were cultured in RPMI +15% fetal bovine serum (FBS) ± lm CsA for days CsA induces a < 10% reduction in growth rate, compensated for in analyses Neutral GSLs from equal cell numbers were extracted and separated by TLC as described previously [28] The ganglioside fraction was prepared by anion exchange [28] Treatment of Fabry mice Twelve adult mice were treated i.p with a bolus injection of a-galactosidase (1.5 mgỈkg)1) Six mice were then injected twice a week i.p with CsA (30 lgỈg)1) and the remaining mice served as controls Similarly six wild-type mice were maintained on CsA and six animals were left untreated Serum Gb3 levels were monitored for nine weeks post ERT at which time some organs (wild-type and Fabry) were processed for Gb3 extraction, whereas others were processed for VT1 staining of cryosections Experimentation using the Fabry mouse is necessary to demonstrate the in vivo potential of MDR1 inhibition as an approach to treatment of Fabry disease in man and was carried out under ethical approval Mice were euthanized under conditions of minimized trauma Extraction of plasma Gb3 and quantitation by VT1 ELISA Determination of Gb3 levels in Fabry mouse plasma was performed effectively as described by Zeidner et al [38] Plasma samples ( 20–60 lL) were prepared weekly during the study and stored at )20 °C End-point plasma volumes ranged from 100 to 400 lL For lipid isolation, plasma samples were extracted overnight with mL of chloroform ⁄ methanol (2 : v ⁄ v) per 100 lL of plasma and then partitioned against ⁄ volume of water The lower phase was dried under a stream of nitrogen gas and then the residue was dissolved in chloroform The sample was applied to a silica gel 60 column ( 100 mg of silica per 100 lL plasma volume) Neutral lipids were removed by washing with column volumes of chloroform and then neutral glycosphingolipids were eluted with 10 column volumes of acetone ⁄ methanol (5 : v ⁄ v) The eluate was dried, dissolved in 10 times the original plasma volume of ethanol and stored at )20 °C Plasma extracts (50 lL) were added to duplicate ELISA plate wells (Nunc Polysorp DiaMed Mississauga, ON) Dilutions of standard human kidney Gb3 in ethanol were also plated in triplicate Serum Gb3 levels < ngỈmL)1 were MDR1 inhibition and GSL storage disease below the detection limit Gb3 standard was quantitated by sphingosine assay using the method of Naoi et al [45] The plates were placed at 37 °C overnight to evaporate the solvent All subsequent incubations were performed for h 37 °C and washes at room temperature Wells were blocked with 150 lL of 0.2% bovine serum albumin (BSA) in 50 mm Tris-buffered saline pH 8.0 (BSA-TBS) then washed twice with BSA-TBS Wells were subsequently incubated with 200 ng per well of VT1 in BSA-TBS, rabbit antiserum against the VT1 B subunit, diluted ⁄ 2000 in BSA-TBS, and finally goat anti-(rabbit HRP)-conjugate (Bio-Rad Laboratories, Hercules, CA) diluted ⁄ 2000 in BSA-TBS (all 50 lL per well) Verotoxin binding in the wells was visualized by incubation with 100 lL per well of 0.5 mgỈmL)1 ABTS in citrate-phosphate buffer, pH 4.0 Absorbance was measured at 405 nm after 30–40 of colour development at room temperature Serum Gb3 values were assessed for significance using a transformed two-sample Student’s t-test assuming equal variances Tissue Gb3 extraction Tissues were homogenized, extracted in 20 vol chloroform ⁄ methanol (2 : v ⁄ v) and filtered The extract was dried under N2 and saponified overnight in 0.1 n NaOH in MeOH at 37 °C [11] The glycolipid extract was neutralized, partitioned against water and was used for VT1 TLC overlay without further purification GSL extracted from 0.5 mg wet weight organ were applied per sample For ganglioside and Gb3 comparison in Fabry liver, the saponified extract was desalted on a SepPak cartridge after neutralization and total GSL separated by TLC Sphingomyelin was detected by iodine, gangliosides by resorcinol and total GSLs by orcinol spray In this case, lipids equivalent to mg liver were applied per sample VT1 TLC overlay of the GSL tissue extracts to detect Gb3 was performed as described [46] Some TLC overlays were subject to comparative densitometry using the image j 1.34 program Values were compared using an unpaired Student’s t-test VT1 tissue staining Five-micrometer frozen tissue sections were air-dried overnight at room temperature on the lab bench When dry, a PAP hydrophobic barrier pen was used to encircle sections Throughout all incubation steps, slides were kept in a humid chamber at room temperature Sections were blocked with endogenous peroxidase blocker (Universal Block, KPL Inc., Gaithersburg, MD) for 20 After extensive rinses with 1· NaCl ⁄ Pi solution, sections were blocked with 1% normal goat serum ⁄ NaCl ⁄ Pi (NGS–NaCl ⁄ Pi) for 20 Without washing, sections were then stained with VT1 (200 ngỈmL)1 in NGS–NaCl ⁄ Pi) for 30 After five vigorous rinses with NaCl ⁄ Pi, sections were incubated with rabbit anti-(VT1B FEBS Journal 273 (2006) 2064–2075 ª 2006 The Authors Journal compilation ª 2006 FEBS 2073 MDR1 inhibition and GSL storage disease M Mattocks et al 6869) (1 : 1000 in NGS–NaCl ⁄ Pi) for 30 min, washed and then incubated with HRP-conjugated goat anti-(rabbit IgG) (1 : 500 in NGS–NaCl ⁄ Pi) for 30 Following washing, sections were developed using DAB substrate for To stop the DAB reaction, sections were dipped in distilled water for Hematoxylin counterstain was applied for 30 s; excess 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endothelial staining in Fabry mouse; (C, D) lung – epithelial cell staining increased in Fabry mouse; (E, F) brain microvascular endothelial staining... Chiba Y, Sakuraba H, Kotani M, Kase R, Kobayashi K, Takeuchi M, Ogasawara S, Maruyama Y, Nakajima T, Takaoka Y et al (2002) Production in yeast of alpha-galactosidase A, a lysosomal enzyme applicable... inhibition of MDR1 as an approach to the treatment of Fabry disease Although the efficacy may not, as yet, be as dramatic as ERT, inhibition of MDR1 may prove most beneficial as an adjunct, rather than alternative