Tài liệu Báo cáo khoa học: 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 doc

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 Mattocks
1
, Maria Bagovich
1
, Maria De Rosa
1,4
, Steve Bond
2
, Beth Binnington
1
,
Vanessa I. Rasaiah
2
, Jeffrey Medin
2,3
and Clifford Lingwood
1,4,5
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:
(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
(Gb
3
), by preventing de novo synthesis. In the Fabry mouse model, Gb
3
is
increased in the heart, liver, spleen, brain and kidney. The lack of renal
glomerular Gb
3
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 Gb
3
and lower Gb
3
levels in
some tissues. Serum Gb
3
was monitored using a VT1 ELISA during a
post-ERT recovery phase ± biweekly intra peritoneal CsA. After 9 weeks,
tissue Gb
3
content and localization were determined using VT1 ⁄ TLC over-
lay and histochemistry. Serum Gb
3
recovered to lower levels after CsA
treatment. Gb
3
was undetected in wild-type liver, and the levels of Gb
3
(but not gangliosides) in Fabry mouse liver were significantly depleted by
CsA treatment. VT1 liver histochemistry showed Gb
3
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.
2064 FEBS Journal 273 (2006) 2064–2075 ª 2006 The Authors Journal compilation ª 2006 FEBS
accumulation 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, Gb
3
, accumulates) [2–4]. a-Galactosi-
dase administered to Fabry patients is able to reduce
serum levels of Gb
3
by 50% [5], liver Gb
3
and by
inference, kidney Gb
3
levels [5,6]. In the Fabry mouse
model, in which the a-galactosidase is abscent [7], ele-
vated serum Gb

3
levels (serum Gb
3
is undetectable in
normal mice) can be deleted by ERT, but tissue Gb
3
is
more refractory. This may be due, in part, to the direct
access of the enzyme to the serum substrate. To digest
accumulated Gb
3
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 Gb
3
is most susceptible to a-galactosidase ther-
apy. Although some lowering of spleen and heart Gb
3
is seen, renal Gb
3
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 pathol-
ogy is in the kidney [1], the major site of Gb
3

synthesis
in man [11], and in the heart, possibly due to the
association of Gb
3
synthesis with the microvasculature.
GSL synthesis in man and mouse are distinct, partic-
ularly in the kidney, where Gb
3
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 Gb
3
. 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 Gb
3
(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 Gb
3
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 Gb
3
synthesis in the Fabry
mouse model. Our studies have further delineated the
abnormal Gb
3
synthesis in this model and have shown
that the inhibition of MDR1 is a viable potential
approach to the reduction of Gb
3
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
M. Mattocks et al. MDR1 inhibition and GSL storage disease

FEBS Journal 273 (2006) 2064–2075 ª 2006 The Authors Journal compilation ª 2006 FEBS 2065
case, 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 Gb
3
, 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
Gb
3
) within the Fabry cell line was prevented by CsA
(Fig. 1D), confirming MDR1 inhibition reduces
de novo Gb
3
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 Gb
3
cannot therefore
turnover, we designed a treatment protocol in which
the effect of MDR1 inhibition by CsA on accumula-

tion of Gb
3
via de novo synthesis was assessed.
Tissue Gb
3
expression
The Gb
3
expression profile for various tissues from
wild-type and Fabry mice was first compared by VT1
TLC overlay (Fig. 2). The Gb
3
content was marked
increased in the kidney, spleen and liver of Fabry mice.
A detectable increase was also observed in the heart.
ABCDEF
GlcCer
GalCer
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 ⁄ W65:25:4v⁄ v ⁄ v). The doublets corresponding to GlcCer
and Gb
3
are 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 Gb
3
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. The
14
C-radiolabeled species below Gb
3
were not characterized. (E) The ganglioside fraction from
14
C-labeled Fabry cells
was separated by TLC (C ⁄ M ⁄ W60:25:100.2
M CaCl

2
v ⁄ 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 Gb
3
and neutral GSL content without effect on the ganglioside profile.
A
B
Fig. 2. Comparison of the Gb
3
content of wild-type and Fabry mouse tissues. GSLs were separated by TLC (C ⁄ M ⁄ W65:25:4v⁄ 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, Gb
3
,Gb
4
,Gb
5
; 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.
MDR1 inhibition and GSL storage disease M. Mattocks et al.
2066 FEBS Journal 273 (2006) 2064–2075 ª 2006 The Authors Journal compilation ª 2006 FEBS
The most notable elevation was seen surprisingly, in
the liver in which, under the conditions used, Gb
3
was

undetected in the wild-type. This indicates Gb
3
must
undergo rapid turnover in the normal liver. Alternat-
ively, liver Gb
3
may accumulate via increased serum
Gb
3
clearance rather than de novo synthesis. Gb
3
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 Gb
3
Renal Gb
3
is the verotoxin receptor responsible for the
development of hemolytic uremic syndrome (HUS) in
man [14]. HUS is a renal glomerular disease. Gb
3
is
found in tubules and glomeruli in man [14]. There is no
adequate small animal model of VT1-induced HUS
because Gb
3
is not found in rodent renal glomeruli [13].

Gb
3
is present in rodent renal tubules and VT1 induces
renal tubular necrosis [37]. We considered that the
increased renal Gb
3
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 Gb
3
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 Gb
3
The low level of Gb
3
in the Fabry mouse serum and
the small volumes available precluded the use of TLC
overlay to detect Gb
3
. A more sensitive VT1-based
ELISA assay was used [38]. This assay was linear
< 60 ng standard Gb
3
and was able to detect > 1 ng
Gb
3
per lL serum sample. Gb
3
in the serum of wild-
type mice was below the background of this assay.
ERT and CsA treatment
Effect on serum Gb
3
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 Gb
3

levels [9] and the effect of
a maintenance dosing of CsA on the recovery of serum
Gb
3
levels after termination of ERT was determined.
ERT is an effective means of eliminating serum Gb
3
,
and Gb
3
remained subsequently undetectable in the
serum of any animal until 9 weeks post ERT. At this
time, the serum Gb
3
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 Gb
3
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 Gb
3
by the 9-week experiment termination. For
responding mice, CsA-treated Fabry mice had serum
Gb
3
levels of 3.31 ± 1.33 ngÆlL
)1

compared with con-
trol Fabry serum levels of 8.21 ± 2.27 ngÆlL
)1
as
determined from a standard curve.
Serum Gb
3
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 Gb
3
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 Gb
3
content was assessed via VT1
overlay. CsA-dependent differences were seen only in
the liver (Fig. 5). The Gb
3
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 Gb
3
con-

tent overall by  50% (P ¼ 0.013). Renal Gb
3
content
was much greater but significant changes after ERT
and CsA treatment were not seen (Fig. 5). GM2 gan-
glioside 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 Gb
3
and GM2
levels (Fig. 5G) clearly show that although liver Gb
3
M. Mattocks et al. MDR1 inhibition and GSL storage disease
FEBS Journal 273 (2006) 2064–2075 ª 2006 The Authors Journal compilation ª 2006 FEBS 2067
levels are reduced in the extracts of CsA-treated mice,
the level of GM2 is unaffected.
Gb
3
tissue histochemistry
The localization of Gb
3
within frozen sections of liver
and selected tissues from Fabry and wild-type mice
monitored by VT1 binding is shown in Fig. 6. VT1
Gb
3
staining was not above background in the wild-
type mouse liver (Fig. 6A), but within the Fabry liver
VT1 binding detected Gb

3
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
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.
MDR1 inhibition and GSL storage disease M. Mattocks et al.
2068 FEBS Journal 273 (2006) 2064–2075 ª 2006 The Authors Journal compilation ª 2006 FEBS
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
Gb
3
positive and many Kupffer cells expressed Gb
3
(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). Gb
3
expression in central
and portal vein endothelial cells was significantly
reduced and many vessels negative for Gb
3
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
AB C
G
F
E

D
345678
910
12345678910
11 12 13 14
12 34
543215432110
9876543219
8
765432
1
GM1
GM2
GM3
GlcCer
LacCer
Gb
5
Gb
4
Gb
3
Fabry liver
Wild-type liver
sphingomyelin GSLs
Fabry liver GSLs
Fabry liver sphingomyelinFabry 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 ⁄ W65:25:4v⁄ 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
Gb
3
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 1 and 3, GSL standards: from the top GlcCer, LacCer, Gb
3
,Gb
4
,Gb
5
(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 Gb
3
is detected in the liver of CsA-treated, compared with control Fabry mice
(B, G). Although the level of Gb
3

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 Gb
3
detected in (B) was subject to densitometry and com-
pared. The CsA-treated Fabry liver Gb
3
values were reduced by 45% (P ¼ 0.013) compared with controls.
Fig. 4. Effect of CsA treatment on the serum Gb
3
levels in Fabry
mouse. Serum Gb
3
assessment at 9 weeks post ERT. Control
Fabry mice (n), CsA-treated Fabry mice ()). Serum Gb
3
levels for
CsA-treated mice are  50% less than control, P ¼ 0.028.
M. Mattocks et al. MDR1 inhibition and GSL storage disease
FEBS Journal 273 (2006) 2064–2075 ª 2006 The Authors Journal compilation ª 2006 FEBS 2069
a
b
c
d
h
g
f
e
i
j
kl

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.
MDR1 inhibition and GSL storage disease M. Mattocks et al.
2070 FEBS Journal 273 (2006) 2064–2075 ª 2006 The Authors Journal compilation ª 2006 FEBS
no 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 Gb
3
in 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.
M. Mattocks et al. MDR1 inhibition and GSL storage disease
FEBS Journal 273 (2006) 2064–2075 ª 2006 The Authors Journal compilation ª 2006 FEBS 2071
an alternative cytosolic mechanism for breakdown [34].
The significant effect in Fabry lymphoblasts to reduce
Gb
3
without effect on ganglioside synthesis supports
this approach. Prevention of Gb
3
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, Gb
3
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 Gb
3
synthesis, whereby animals
were treated by ERT and the effect of CsA on ‘relapse’
of Gb
3
accumulation monitored. ERT primarily affects
serum and liver Gb
3
accumulation [9] and these tissues
were therefore the primary focus of our study,

although the location of Gb
3
accumulated in other tis-
sues was also investigated.
Our demonstration that CsA significantly reduces
Fabry mouse serum and liver Gb
3
levels, approaches
proof of concept. Total Gb
3
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 Gb
3
accumulation after CsA treatment. Because the phago-
cytic Kupffer cells are major reticulo-endothelial de-
gradative sites, the Gb
3

they contain could be
serum ⁄ red blood cell-derived and the decrease seen
after CsA result from the reduced serum Gb
3
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 Gb
3
was found to
remain undetectable after ERT, were nevertheless
found to have Gb
3
in the hepatic extract and express
Kupffer cell Gb
3
.
Heart, lung, brain, kidney and spleen tissue show a
clear increase in Gb
3
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 Gb
3
expression in virtually all the
renal tubules of the Fabry mouse shows that the lack
of Gb
3
detection in most tubules of the wild-type
mouse [13] is a result of rapid Gb
3
turnover, rather
than the lack of Gb
3
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 Gb
3
levels 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 Gb
3
levels in Fabry mice
following a-galactosidase treatment. In such mice, the
expression of Gb
3
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.
MDR1 inhibition and GSL storage disease M. Mattocks et al.
2072 FEBS Journal 273 (2006) 2064–2075 ª 2006 The Authors Journal compilation ª 2006 FEBS
Experimental 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 Gb
3
levels were monitored for nine weeks

post ERT at which time some organs (wild-type and Fabry)
were processed for Gb
3
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 Gb
3
and quantitation
by VT1 ELISA
Determination of Gb
3
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 Gb
3
in ethanol were
also plated in triplicate. Serum Gb
3
levels < 2 ngÆmL
)1
were
below the detection limit. Gb
3
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 sol-
vent. All subsequent incubations were performed for 1 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 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 Gb
3
values were assessed for
significance using a transformed two-sample Student’s t-test
assuming equal variances.
Tissue Gb
3
extraction
Tissues were homogenized, extracted in 20 vol. chloro-
form ⁄ methanol (2 : 1 v ⁄ v) and filtered. The extract was
dried under N
2
and saponified overnight in 0.1 n NaOH in
MeOH at 37 °C [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 Gb
3
comparison 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
Gb
3

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 ⁄ P
i
solution, sections were blocked with 1% normal
goat serum ⁄ NaCl ⁄ P
i
(NGS–NaCl ⁄ P
i
) for 20 min. Without
washing, sections were then stained with VT1 (200 ngÆmL
)1
in NGS–NaCl ⁄ P
i
) for 30 min. After five vigorous rinses with
NaCl ⁄ P
i
, sections were incubated with rabbit anti-(VT1B
M. Mattocks et al. MDR1 inhibition and GSL storage disease
FEBS Journal 273 (2006) 2064–2075 ª 2006 The Authors Journal compilation ª 2006 FEBS 2073

6869) (1 : 1000 in NGS–NaCl ⁄ P
i
) for 30 min, washed and
then incubated with HRP-conjugated goat anti-(rabbit IgG)
(1 : 500 in NGS–NaCl ⁄ P
i
) for 30 min. Following washing,
sections were developed using DAB substrate for 5 min. To
stop the DAB reaction, sections were dipped in distilled
water for 4 min. Hematoxylin counterstain was applied for
30 s; excess staining was removed by immersing sections in
distilled water for 4 min and ‘blued’ by immersing in tap
water for 4 min. Sections were then dehydrated for 2 min.
in each of 70%, 95% and 100% ethanol, cleared in xylene
for 5 min and mounted in Permount.
For in vivo VT1 distribution, 50 lg VT1 was injected i.p.
Mice were killed after one hour and organs removed, fixed,
sectioned and deparaffinized sections stained with anti-VT1
as described previously [13] without counterstain.
Acknowledgements
This work was supported by CIHR grant #MT13073
(CAL) and NIH grant #HL70569 (JAM). We thank
Dr J. Phillips (Dept Pediatric Laboratory Medicine
HSC), for help in liver histology analysis.
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