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 Bagovich
Trang 1diseases 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 Medin2,3and Clifford Lingwood1,4,5
1 Research Institute, The Hospital for Sick Children, Toronto, Canada
2 Ontario Cancer Institute, University Health Network, Toronto, Canada
3 Department of Medical Biophysics, University of Toronto, Canada
4 Department of Laboratory Medicine and Pathology, University of Toronto, Canada
5 Department of Biochemistry, University of Toronto, Canada
The lysosomal storage diseases (LSD) are genetic
defi-ciencies 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
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 2 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 1 (MDR1, P-glycoprotein) translocates glucosyl ceramide from the cytosolic to the luminal Golgi surface for neutral, but not acidic, gly-cosphingolipid (GSL) synthesis Here we show that the MDR1 inhibitor, cyclosporin A (CsA) can deplete Gaucher lymphoid cell lines of accumu-lated 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 1 (VT1)-staining renal tubules, and VT1 tubular targeting in vivo, is markedly increased in Fabry mice Adult Fabry mice were treated with a-galactosidase (enzyme-replacement therapy, ERT) to eliminate serum Gb3and lower Gb3levels in some tissues Serum Gb3 was monitored using a VT1 ELISA during a post-ERT recovery phase ± biweekly intra peritoneal CsA After 9 weeks, tissue Gb3 content and localization were determined using VT1⁄ TLC over-lay 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
Abbreviations
BSA, bovine serum albumin; CsA, cyclosporin A; ERT, enzyme replacement therapy; Gb 3 , globotriaosyl ceramide; GlcCer, glucosyl ceramide; GSL, glycosphingolipid; HUS, hemolytic uremic syndrome; LacCer, lactosyl ceramide; LSD, lysosomal storage disease; MDR1, multiple drug resistance protein 1 (P-glycoprotein); NGS, normal goat serum; VT1, verotoxin 1; TLC, thin layer chromatogram.
Trang 2accumulation results in the formation of lipid
inclu-sions and multilamellar structures which prevent
nor-mal 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-replace-ment therapy (ERT) has been developed, particularly
in the two neutral GSL storage diseases, Gaucher
(glucosyl ceramide accumulates) and Fabry
(globotria-osyl ceramide, Gb3, accumulates) [2–4]
a-Galactosi-dase 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],
ele-vated serum Gb3 levels (serum Gb3 is undetectable in
normal mice) can be deleted by ERT, but tissue Gb3is
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
replace-ment a-galactosidase is phosphomannosylated to
pro-mote such uptake [8] Within the Fabry mouse tissues,
liver Gb3 is most susceptible to a-galactosidase
ther-apy Although some lowering of spleen and heart Gb3
is seen, renal Gb3is 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
pathol-ogy is in the kidney [1], the major site of Gb3synthesis
in man [11], and in the heart, possibly due to the
association of Gb3synthesis with the microvasculature
GSL synthesis in man and mouse are distinct,
partic-ularly 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-decan-oylamino-3-morpholino-1-propanol (PDMP), or its
derivatives with improved selectivity [16], provide one
approach [17,18] Imino sugar-based glucosyl ceramide
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 resist-ance protein 1 (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] MDR1-mediated GlcCer translocation into the Golgi could be
a component of such resistance However, we found that MDR1-translocated GlcCer is used only for neut-ral GSL synthesis [28] because inhibition of MDR1 does not affect cellular ganglioside synthesis This pro-vides a degree of selectivity not available in the other approaches to substrate reduction therapy as a clinical management for Fabry disease In addition, the long-term clinical experience with drugs that modulate MDR1 in cancer, and, for cyclosporin A (CsA), immu-nosuppression, would provide significant advantage, in terms of defined toxicity and dosage Although MDR1 expression varies within tissues, expression in the kid-ney, 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 deter-mined the effect of CsA on Gb3synthesis in the Fabry mouse model Our studies have further delineated the abnormal Gb3synthesis 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
4 lm CsA for four days The GSL fractions were puri-fied and separated by thin layer chromatography (TLC) Figure 1A shows the accumulation of glucosyl ceramide (GlcCer) was prevented in CsA-treated Gau-cher B lymphoblasts Three cell lines were tested Glc-Cer accumulated in each, but in one cell line, lactosyl ceramide accumulated also (Fig 1A, lane 6) In each
Trang 3case, CsA was found to delete GlcCer and reduce
other neutral GSLs present Inhibition of
MDR1-mediated 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
major ganglioside present but additional, more com-plex 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 accumula-tion of Gb3via de novo synthesis was assessed
Tissue Gb3expression 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
GlcCer
LacCer
Gb 3
Gb 4
Gb 5
GM2
GM1
Fig 1 Effect of cyclosporin A (CsA) on cultured Gaucher and Fabry B-cell line glycosphingolipids (GSLs) The neutral GSL fraction (from
2 · 10 6 cells per lane) was separated by thin layer chromatogram (TLC) (C ⁄ M ⁄ W 65 : 25 : 4 v ⁄ v ⁄ v) The doublets corresponding to GlcCer and Gb3are shown by arrows (A) Gaucher lymphoblastoid cell lines, detected using orcinol spray Lane 1, GSL standards, GlcCer, GalCer, LacCer, Gb 3 , Gb 4 , Gb 5 (Forssman) as indicated Lanes 2, 4, 6, Neutral GSLs of untreated 5072, 5410, 5831 Gaucher cell lines Lanes 3, 5, 7 Neutral GSLs of CsA-treated 5072, 5410, 5831 cell lines (B–F) Fabry lymphoblastoid cell line Cells were grown with 14 C-serine 14 C-Radio-labeled GSLs were detected by phosphoimaging (B) Orcinol detection of total neutral GSL fraction, (C) VT1 overlay of panel B to detect Gb3 only, (D) 14 C-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 The14C-radiolabeled species below Gb 3 were not characterized (E) The ganglioside fraction from14C-labeled Fabry cells was separated by TLC (C ⁄ M ⁄ W 60 : 25 : 10 0.2 M CaCl2v ⁄ v ⁄ v) and detected using orcinol (GM3), or (F) phosphoimaging of the 14 C-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 Gb3and neutral GSL content without effect on the ganglioside profile.
Fig 2 Comparison of the Gb3content of wild-type and Fabry mouse tissues GSLs were separated by TLC (C ⁄ M ⁄ W 65 : 25 : 4 v ⁄ v ⁄ v) and visualized using orcinol spray for carbohydrate (A) or VT1 overlay (B) to detect Gb 3 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, 3 heart; lanes 4, 5, spleen; lanes 6, 7 kidney; lanes 8, 9 liver.
Trang 4The 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
Alternat-ively, liver Gb3 may accumulate via increased serum
Gb3 clearance rather than de novo synthesis Gb3
syn-thase 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 Gb3is 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 Gb3is not found in rodent renal glomeruli [13]
Gb3is 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
dis-ease 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
immunostain-ing 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 > 1 ng
Gb3 per lL serum sample Gb3 in the serum of
wild-type mice was below the background of this assay
ERT and CsA treatment Effect on serum Gb3 Owing to the difficulty of drug administration in neo-nates and the availability of well-documented CsA dos-age 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 Gb3levels [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 9 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
Gb3levels of 3.31 ± 1.33 ngÆlL)1compared with con-trol Fabry serum levels of 8.21 ± 2.27 ngÆlL)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⁄ immunohis-tological evaluation
Effect of CsA on tissue Gb3 GSLs were extracted from kidney and liver of control and CsA-treated Fabry mice after 9 weeks recovery post ERT, and from CsA-treated and untreated wild-type 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 CsA-treated, compared with control mice after recovery from ERT CsA treatment reduced the liver Gb3 con-tent 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 gan-glioside is the major gangan-glioside 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
Trang 5levels are reduced in the extracts of CsA-treated mice,
the level of GM2 is unaffected
Gb3tissue histochemistry
The localization of Gb3 within frozen sections of liver
and selected tissues from Fabry and wild-type mice
monitored by VT1 binding is shown in Fig 6 VT1
Gb3 staining was not above background in the wild-type mouse liver (Fig 6A), but within the Fabry liver VT1 binding detected Gb3in 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
a
A
B
b
d
c
Fig 3 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 hema-toxylin (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.
Trang 6on 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-trea-ted 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 lar-ger blood vessel endothelial cells was seen only in the Fabry mouse (Fig 7A, compared to Fig 7B) A patch-work staining which originates from a subset of fibro-cytes 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 consis-tently altered after CsA treatment There was virtually
Wild-type liver
1 2
G F
E D
3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10 11 12 13 14 1 2 3 4
5 4 3 2 1 5 4 3 2 1 10 9 8 7 6 5 4 3 2 1 9 8 7 6 5 4
3
2
1
GM1 GM2
GlcCer
LacCer
Gb 5
Gb 4
Gb 3
Fabry liver sphingomyelin GSLs Wild-type liver
Fabry liver GSLs Fabry liver sphingomyelin
Fabry kidney Wild-type kidney
Fig 5 Comparison of Gb 3 levels in wild-type and Fabry mouse liver and kidney: relative effect of CsA on Gb 3 compared with other sphingo-lipids (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 : 4 v ⁄ v ⁄ v and neutral and acidic GSLs (C, F, G) were separated in C ⁄ M ⁄ 0.8% KCl aq 60 : 40 : 8 v ⁄ v ⁄ v Gb 3 detection by VT1 ⁄ TLC overlay (A, B, D, E) Lanes 1–3, 0.5, 1, 2 lg
Gb3standard; 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 1 and 3, GSL standards: from the top GlcCer, LacCer, Gb3,Gb4,Gb5(Forssman), GM3, GM2,GM1; lanes 2 and 4, lipid extract of wild-type liver; (F, G) lane 1, GSL standards; lanes 2 and 4, lipid extracts of control Fabry liver; lanes 3, 5, lipid extracts of CsA-treated Fabry liver Gb 3 is only detected in Fabry, as opposed to wild-type liver (compare A with B, and C with G) and the normal renal Gb 3
doublet (D) is markedly enhanced in the Fabry mouse (E) Less Gb3is detected in the liver of CsA-treated, compared with control Fabry mice (B, G) Although the level of Gb3is 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 Gb 3 detected in (B) was subject to densitometry and com-pared The CsA-treated Fabry liver Gb3values were reduced by 45% (P ¼ 0.013) compared with controls.
Fig 4 Effect of CsA treatment on the serum Gb3levels in Fabry
mouse Serum Gb3 assessment at 9 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.
Trang 7a b c d
h g
f e
B
a
A
b
Fig 6 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.
Trang 8no VT1 staining of normal brain (Fig 7F) In Fabry
brain, extensive staining of the microvasculature is
evi-dent (Fig 7E) The arachnoid membrane surrounding
the brain is extensively stained in Fabry but not
nor-mal mouse brain Although MDR1 is highly expressed
in the brain microvasculature [41], ERT is not effective
to reduce the level of Gb3in the brain [9]
Discussion
The differential sensitivity of ganglioside and neutral
GSL synthesis to depletion of GlcCer via MDR1
inhi-bition [28] provides an attractive method for the
select-ive reduction of neutral GSL synthesis in neutral GSL
storage diseases Other substrate reduction approaches
are less selective and hence have greater potential
side-effects Inhibitors of glucosyl ceramide synthase
prevent the synthesis of both neutral GSLs and
gan-gliosides 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 estab-lished in vitro, has yet to be understood in vivo MDR1 knockout mice do not show an overt phenotype, although skin fibroblasts from such mice are, as pre-dicted, defective in neutral GSL synthesis [28] We showed that an alternative mechanism of Golgi mem-brane 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 func-tion of MDR1, the GSLs of some other tissues are complicated by the redundancy in this function (stud-ies 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 accu-mulation in Gaucher lymphoblasts, in which there is
Fig 7 Comparison of Verotoxin staining of
other tissues (A, C, E) Fabry, (B, D, F)
wild-type 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
micro-vascular endothelial staining in Fabry mouse.
Magnification ·16.
Trang 9an 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
activ-ity 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 Gb3accumulation monitored ERT primarily affects
serum and liver Gb3accumulation [9] and these tissues
were therefore the primary focus of our study,
although the location of Gb3 accumulated in other
tis-sues 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
precur-sor 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
phago-cytic Kupffer cells are major reticulo-endothelial
de-gradative 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
on prolonged treatment or treatment prior to GSL accumulation The increased in vivo VT1 renal target-ing in the Fabry mouse suggests increased susceptibil-ity 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 per-iod 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, clin-ically used long-term and it might be expected that under such conditions, the effect on Fabry patient tis-sue Gb3levels might be accumulative and more signifi-cant 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 treat-ment 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 Gb3levels 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 stor-age diseases
Trang 10Experimental procedures
CsA treatment of LSD cultured cells
EBV-transformed B-lymphoblastoid cell lines from Gaucher
type 1 and Fabry disease (kindly supplied by J Clarke,
Hospital for Sick Children, Toronto, Canada) were
cul-tured in RPMI +15% fetal bovine serum (FBS) ± 4 lm
CsA for 4 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
pre-pared by anion exchange [28]
Treatment of Fabry mice
Twelve adult mice were treated i.p with a bolus injection
of a-galactosidase (1.5 mgÆkg)1) Six mice were then
injec-ted twice a week i.p with CsA (30 lgÆg)1) and the
remain-ing mice served as controls Similarly six wild-type mice
were maintained on CsA and six animals were left
untreated Serum Gb3levels 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
Experimentat-ion 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
condi-tions of minimized trauma
Extraction of plasma Gb3and 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
dur-ing the study and stored at)20 C End-point plasma
vol-umes ranged from 100 to 400 lL For lipid isolation,
plasma samples were extracted overnight with 2 mL of
chloroform⁄ methanol (2 : 1 v ⁄ v) per 100 lL of plasma and
then partitioned against 1⁄ 5 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 4 column volumes of chloroform and then
neutral glycosphingolipids were eluted with 10 column
vol-umes of acetone⁄ methanol (5 : 1 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 Gb3levels < 2 ngÆmL)1were
below the detection limit Gb3standard was quantitated by sphingosine assay using the method of Naoi et al [45] The plates were placed at 37C overnight to evaporate the sol-vent All subsequent incubations were performed for 1 h
37C 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 1⁄ 2000 in BSA-TBS, and finally goat anti-(rabbit HRP)-conjugate (Bio-Rad Laboratories, Hercules, CA) diluted 1⁄ 2000 in BSA-TBS (all 50 lL per well) Verotoxin binding in the wells was visu-alized by incubation with 100 lL per well of 0.5 mgÆmL)1 ABTS in citrate-phosphate buffer, pH 4.0 Absorbance was measured at 405 nm after 30–40 min 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 Gb3extraction Tissues were homogenized, extracted in 20 vol chloro-form⁄ methanol (2 : 1 v ⁄ v) and filtered The extract was dried under N2and saponified overnight in 0.1 n NaOH in MeOH at 37C [11] The glycolipid extract was neutral-ized, 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 Gb3comparison in Fabry liver, the saponified extract was desalted on a SepPak cartridge after neutralization and total GSL separated by TLC Sphingo-myelin was detected by iodine, gangliosides by resorcinol and total GSLs by orcinol spray In this case, lipids equiv-alent to 2 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 over-night 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 min After extensive rinses with 1· NaCl ⁄ Pisolution, sections were blocked with 1% normal goat serum⁄ NaCl ⁄ Pi (NGS–NaCl⁄ Pi) for 20 min Without washing, sections were then stained with VT1 (200 ngÆmL)1
in NGS–NaCl⁄ Pi) for 30 min After five vigorous rinses with NaCl⁄ Pi, sections were incubated with rabbit anti-(VT1B