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An accumulation of galactocerebroside in kidney from mouse globoid cell leukodystrophy (twitcher)
vol. 109, No. 3, 1982 December
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS Pages 634-636
15, 1982
AN ACCUMULATION OF GALACTOCEREBROSIDE IN KIDNEY FROM MOUSE GLOBOID CELL LEuK~DY~TROPHY (TWITCHER)
H. Ida, F. Umezawa, Department
Beceived
October
of Pediatrics, Medicine,
E. Kasai,
Y. Eto*
The Tokyo Jikei Minato-ku, Tokyo,
and K. Maekawa University Japan.
School
of
13, 1982
SUMMARY: The twitcher mouse is genetically determined mutant characterized by a deficiency of galactocerebroside beta-galactosidase. In this study, a significant accumulation of galactocerebroside was demonstrated in twitcher mouse kidney. The data suggest that mouse Krabbe's disease is not only involved in CNS, but also in visceral organs.
The twitcher
mouse has recently
human qloboid
cell
leukodystrophy
an autosomal
recessive
in peripheral
nerves
deficiency organs mice
of twitcher closely
resemble
presence
However,
that
found
a number
the apparent
lack
from the This
in kidney
was preserved
of human disease.
communication from
*To whom reprint
twitcher
requests
of globoid
well,
clearly
the
central
important
(4,
found
leukodystrophy
nervous
finding other
the in various in twitcher (Krabbe's
have been noticed system
and biochemically,
of galactocerebroside
myelin
in human GLD is than
demonstrates
be sent.
0006/291X/82/230634-05$01.00/C Copyright 0 1982 by Academic Press, Inc. All righrs of reproduction in any form reserved.
634
an accumulation
in
as contrasted
in globoid
5).
mice.
should
cell
by
demyelination
findings
characteristics
pathologically
An additional
system
was also
for
transmitted
Biochemically,
and biochemical
of different
is
by severe
cells.
in human globoid
disease;
of accumulation
nervous
The mouse disease
pathological
human and mouse Krabbe's
the mouse model
as a mouse model
beta-qalactosidase
These
those
2).
identified
It is characterized
(twi).
and the
mice.
(l-3).
between
cells
(1,
of galactocerebroside
disease)
with
gene
been
of galactocerebroside
BIOCHEMICAL
Vol. 109, No. 3, 1982 MATERIALS
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
AND METHODS
The colony of the twitcher mice was identified from phenotypically normal littermates of carrier to carrier matings provided from the Jackson Laboratory, Bar Harbor, Maine. The heterozygote mice were identified by the determination of galactocerebroside beta-galactosidase in mouse tail, as described by Kobayashi et al. (6). Matings between heterozygote mice produced the homozygous offspring in our laboratory. The animals were sacrificed on the 25th-day. The tissues from homozygotes were further confirmed by the determination of galactocerebroside beta-galactosidase. Galactocerebroside beta-galactosidase activity was assayed as described by Suzuki et al. (7), but at pH 4.0. The lipid analysis of mouse tissues was essentially carried out as described previously (8). The carbohydrate analvsis of glycosphingolipids by Gas chromatography was performed as a TMS derivatives as described by Sweely et al. (9). Protein determinations were carried out usinq the method of Lowry et al. (10). RESULTS Table liver
1 shows the
and kidney
mice was less the highest
of twitcher
activity
of cerebroside
chromatography
monohexoside
of kidney were
by the solvent
from
gl.ycolipids
layer
chromatogram.
lipid
compositions
Among these
twitcher
increased
mice.
The spots
times
Glucosylceramide,
ceramide
not
increased
normal,
lipids
to ceramide
v/v/v).
not
increased
normal
found
on the
ceramide
in littermate
digalactosyl
and thin-
of qlycosphingo-
Galactosyl
twitcher
Other
(ceramide-gal-gal)
analysis
mice.
developed
were within
dihexoside
the level
heterozygotes
coincident
(65:25:4,
were
in
1 shows
chromatogram
the quantitative
at the age of 25 days. were
contained
Fig.
on thin-layer
from twitcher
was 5-8
kidney
from
and neutral
2. shows
of kidney
in affected
tissues,
(ceramide-glc-gal-NAcgal) Table
in brain,
enzyme
beta-galactosidase.
such as ceramide
mice kidney
contents
of this
of chloroform-methanol-water
trihexoside
in twitcher
The activity
such as phospholipids
Other
beta-galactosidase
of glycosphingolipids
markedly
system
classes
cermide
mice.
10% of normals.
and homozygotes
limits.
of cerebroside
than
the thin-layer
lipid
activity
ceramide
content controls
and trihexosyl
kidney.
DISCUSSION The accumulation is
unique,
(4,
5).
since Non-neural
compositional
of galactocerebroside
in mouse kidney
human GLD does not accumulate tissues
changes
such as kidney
in GLD tissues
635
galactocerebroside
and liver
by both
from
do not
pathological
twitcher
mice
in kidney
show conspicuous and biochemical
studies
Vol. 109, No. 3, 1982 Table
1.
BIOCHEMICAL
Cerebroside-,+galactosidase of Twitcher mouse
AND BIOPHYSICAL activity
in
RESEARCH COMMUNICATIONS liver
brain,
and kidney
tissues.
cerebroside-s-galactosidase
s-galactosidase
Brain normal heterozygote affected
10.4 12.6 7.8 7.0 1.3 1.7
5.9
10.6 9.4
E
6.0 5.6 5.2 ::i
Liver normal heterozygote
5.0
affected
4.1 2.4 2.4
7:1 7.4 7.5 6.6
43.5 50.2 18.4 23.2 6.0 3.4
15.9 16.1 15.4 14.8 23.3 13.7
Kidney normal heterozygote affected
Activities
Suzuki
et al.
and found broside is
are
(4)
was similarly
Krabbe's though
this
to kidney
The unique and not in kidney
central
nervous
ceramide
is
system
is
is
of the
documented also
deficient
Adams
glycolipids
in
this
glycolipid et
al.
mutant
central
(11). mouse
probably minimal mice
of
dihexosyl
Lactosyl and 636
of this
ceramide
is
limited is normally
glycolipid
since
in the sheath,
normally
mice.
digalactosyl This
finding
beta-galactosidase increase
even
storage
in twitcher
ceramide.
a slight
system
the myelin
unusual,
there
Hense,
galactocerebroside
because
is
kidney.
nervous
demyelination
in
that
lysosomal
patients
qlucocere-
in twitcher
The accumulation
undergoes
predominant by
11).
of five
However,
in the
the
since
kidney
was concluded
of galactocerebroside
not apparent,
kidney
normal.
as an inborn
organs,
hour.
in the
and it
only
accepted
in other
and CNS (4,
than
kidney
to involve
accumulation apparent
per
of galactocerebroside
generally
in galactocerebroside compositions
30% higher
increase
is
mg protein
levels
in patient's
considered
disorder
present
also
is
per
galactocerebroside
high
abnormal
disease
disease.
The
examined
as n moles
them to be approximately
no specific
rich
expressed
of
lactosyl
was activity ceramide
BIOCHEMICAL
Vol. 109, No. 3, 1982
-m--me--A B
C
Fig.
is
found
is
not
1.
D
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
E
G
F
H
I
The thin layer chromatogram of saponified lipids of kidney of twitcher mice. The lipid was developed in the solvent system of chloroform-methanol-water (65:25:4, v/v/v) and visualized the spots spraying 50% sulfuric acid. The abbreviations used: CH; cholesterol, CMH; ceramide monohexoside, CDH,; ceramide lactoside, CDHb; ceramide digalactoside, CTH; ceramide trihexoside, SM; sphingomyelin. lane A, I; galactocerebroside, lane B, C; normal kidney lane D. E; heterozygous kidney, lane F, G; homozygous kidney, lane H; mixtures of galactocerebroside, ceramide lactoside and sulfatide.
in twitcher increased,
mouse brain
(data
because
digalactosyl
Ceramide
hexoside
not
shown).
ceramide
However,
in kidney
is a major
slycolipid
2.
compositions
Cer-gal
of
kidney
of
Cer-glc
CDH*
CTH**
Normal
13.8 14.7
10.5 12.0
25.2 20.3
10.5 16.0
Heterozygote
12.7 18.8
6.7 7.1
22.7 27.6
18.9 20.6
Homozygote
89.8 57.6
14.0 14.8
31.8 27.5
17.6 19.7
* CDH; values
cermide-gal-gal, are
expressed
**
twitcher
CTH;
as mg per
ceramide-gle-gal-NAcgal 100 gr
637
tissue.
glycolipid
in mouse
kidney.
Table
this
mice.
Vol. 109, No. 3, 1982
8lOCHEMlCAL
An accumulation
of galactocerebroside
new view
pathological
the
of the
identification
AND BIOPHYSICAL in twitcher
consideration
of affected
mice
RESEARCH COMMUNICATIONS
mouse kidney
in GLD and also
by analysis
provides
a
can be used for
of glycolipid.
REFERENCES: 1. Duchen L.W., Either Brain 103, 695-710.
E.M.,
Jacobs
2. Kobayashi T., Yamanaka T., Brain Res. 202, 479-483. 3. Suzuki
K. (1982)
Acta
J.M.,
Jacobs
Pediatr.
Scravilli
J.M.,
F. and Teixeira
Teixeira
Japonica,
in
F. and Suzuki
K. (1971)
6. Kobayashi 8-14.
T.,
Lipids
Nagara
5, H.,
9. Sweelv edited
Inherited Disease" pp. 747-769,
433-436. Suzuki
K. and Suzuki
7. Suzuki K. (1978) in "Methods in Enzymo1og.y" P. 456-458, Academic Press, New York. 8. Eto Y. and Suzuki
K. (1980)
press.
4. Suzuki K. and Suzuki Y. (1978) in " Metabolic Basis of ( J.B. Stanbury, Wyngaarden and P.S. Fredrickson, eds.) MC Graw Hill, New York. 5. Suzuki
F. (1980)
K. (1970)
J. Lipid
Res.
K. (1982) (V.
Biochem.
Ginsburg,
Med. 27,
eds.):vol.
11, 473-479.
C.C. and Vance D.E. (1967) In ' Lipid chromatograuhic analysis" by Marizetti, GW., vol. 1, P. 465, Marcel Dekker, New York,
10. Lowry O.H., Rosebrough Chem. -193, 265-275. 11. Adams E.P.
J.N.,
and Gray G.M.
Farr
(1968)
A.L.
and Randall
Chem. Phys.
638
Lipids
R.J. 1,
(1951) 147-155.
J. Biol.
50C
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS Pages 634-636
15, 1982
AN ACCUMULATION OF GALACTOCEREBROSIDE IN KIDNEY FROM MOUSE GLOBOID CELL LEuK~DY~TROPHY (TWITCHER)
H. Ida, F. Umezawa, Department
Beceived
October
of Pediatrics, Medicine,
E. Kasai,
Y. Eto*
The Tokyo Jikei Minato-ku, Tokyo,
and K. Maekawa University Japan.
School
of
13, 1982
SUMMARY: The twitcher mouse is genetically determined mutant characterized by a deficiency of galactocerebroside beta-galactosidase. In this study, a significant accumulation of galactocerebroside was demonstrated in twitcher mouse kidney. The data suggest that mouse Krabbe's disease is not only involved in CNS, but also in visceral organs.
The twitcher
mouse has recently
human qloboid
cell
leukodystrophy
an autosomal
recessive
in peripheral
nerves
deficiency organs mice
of twitcher closely
resemble
presence
However,
that
found
a number
the apparent
lack
from the This
in kidney
was preserved
of human disease.
communication from
*To whom reprint
twitcher
requests
of globoid
well,
clearly
the
central
important
(4,
found
leukodystrophy
nervous
finding other
the in various in twitcher (Krabbe's
have been noticed system
and biochemically,
of galactocerebroside
myelin
in human GLD is than
demonstrates
be sent.
0006/291X/82/230634-05$01.00/C Copyright 0 1982 by Academic Press, Inc. All righrs of reproduction in any form reserved.
634
an accumulation
in
as contrasted
in globoid
5).
mice.
should
cell
by
demyelination
findings
characteristics
pathologically
An additional
system
was also
for
transmitted
Biochemically,
and biochemical
of different
is
by severe
cells.
in human globoid
disease;
of accumulation
nervous
The mouse disease
pathological
human and mouse Krabbe's
the mouse model
as a mouse model
beta-qalactosidase
These
those
2).
identified
It is characterized
(twi).
and the
mice.
(l-3).
between
cells
(1,
of galactocerebroside
disease)
with
gene
been
of galactocerebroside
BIOCHEMICAL
Vol. 109, No. 3, 1982 MATERIALS
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
AND METHODS
The colony of the twitcher mice was identified from phenotypically normal littermates of carrier to carrier matings provided from the Jackson Laboratory, Bar Harbor, Maine. The heterozygote mice were identified by the determination of galactocerebroside beta-galactosidase in mouse tail, as described by Kobayashi et al. (6). Matings between heterozygote mice produced the homozygous offspring in our laboratory. The animals were sacrificed on the 25th-day. The tissues from homozygotes were further confirmed by the determination of galactocerebroside beta-galactosidase. Galactocerebroside beta-galactosidase activity was assayed as described by Suzuki et al. (7), but at pH 4.0. The lipid analysis of mouse tissues was essentially carried out as described previously (8). The carbohydrate analvsis of glycosphingolipids by Gas chromatography was performed as a TMS derivatives as described by Sweely et al. (9). Protein determinations were carried out usinq the method of Lowry et al. (10). RESULTS Table liver
1 shows the
and kidney
mice was less the highest
of twitcher
activity
of cerebroside
chromatography
monohexoside
of kidney were
by the solvent
from
gl.ycolipids
layer
chromatogram.
lipid
compositions
Among these
twitcher
increased
mice.
The spots
times
Glucosylceramide,
ceramide
not
increased
normal,
lipids
to ceramide
v/v/v).
not
increased
normal
found
on the
ceramide
in littermate
digalactosyl
and thin-
of qlycosphingo-
Galactosyl
twitcher
Other
(ceramide-gal-gal)
analysis
mice.
developed
were within
dihexoside
the level
heterozygotes
coincident
(65:25:4,
were
in
1 shows
chromatogram
the quantitative
at the age of 25 days. were
contained
Fig.
on thin-layer
from twitcher
was 5-8
kidney
from
and neutral
2. shows
of kidney
in affected
tissues,
(ceramide-glc-gal-NAcgal) Table
in brain,
enzyme
beta-galactosidase.
such as ceramide
mice kidney
contents
of this
of chloroform-methanol-water
trihexoside
in twitcher
The activity
such as phospholipids
Other
beta-galactosidase
of glycosphingolipids
markedly
system
classes
cermide
mice.
10% of normals.
and homozygotes
limits.
of cerebroside
than
the thin-layer
lipid
activity
ceramide
content controls
and trihexosyl
kidney.
DISCUSSION The accumulation is
unique,
(4,
5).
since Non-neural
compositional
of galactocerebroside
in mouse kidney
human GLD does not accumulate tissues
changes
such as kidney
in GLD tissues
635
galactocerebroside
and liver
by both
from
do not
pathological
twitcher
mice
in kidney
show conspicuous and biochemical
studies
Vol. 109, No. 3, 1982 Table
1.
BIOCHEMICAL
Cerebroside-,+galactosidase of Twitcher mouse
AND BIOPHYSICAL activity
in
RESEARCH COMMUNICATIONS liver
brain,
and kidney
tissues.
cerebroside-s-galactosidase
s-galactosidase
Brain normal heterozygote affected
10.4 12.6 7.8 7.0 1.3 1.7
5.9
10.6 9.4
E
6.0 5.6 5.2 ::i
Liver normal heterozygote
5.0
affected
4.1 2.4 2.4
7:1 7.4 7.5 6.6
43.5 50.2 18.4 23.2 6.0 3.4
15.9 16.1 15.4 14.8 23.3 13.7
Kidney normal heterozygote affected
Activities
Suzuki
et al.
and found broside is
are
(4)
was similarly
Krabbe's though
this
to kidney
The unique and not in kidney
central
nervous
ceramide
is
system
is
is
of the
documented also
deficient
Adams
glycolipids
in
this
glycolipid et
al.
mutant
central
(11). mouse
probably minimal mice
of
dihexosyl
Lactosyl and 636
of this
ceramide
is
limited is normally
glycolipid
since
in the sheath,
normally
mice.
digalactosyl This
finding
beta-galactosidase increase
even
storage
in twitcher
ceramide.
a slight
system
the myelin
unusual,
there
Hense,
galactocerebroside
because
is
kidney.
nervous
demyelination
in
that
lysosomal
patients
qlucocere-
in twitcher
The accumulation
undergoes
predominant by
11).
of five
However,
in the
the
since
kidney
was concluded
of galactocerebroside
not apparent,
kidney
normal.
as an inborn
organs,
hour.
in the
and it
only
accepted
in other
and CNS (4,
than
kidney
to involve
accumulation apparent
per
of galactocerebroside
generally
in galactocerebroside compositions
30% higher
increase
is
mg protein
levels
in patient's
considered
disorder
present
also
is
per
galactocerebroside
high
abnormal
disease
disease.
The
examined
as n moles
them to be approximately
no specific
rich
expressed
of
lactosyl
was activity ceramide
BIOCHEMICAL
Vol. 109, No. 3, 1982
-m--me--A B
C
Fig.
is
found
is
not
1.
D
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
E
G
F
H
I
The thin layer chromatogram of saponified lipids of kidney of twitcher mice. The lipid was developed in the solvent system of chloroform-methanol-water (65:25:4, v/v/v) and visualized the spots spraying 50% sulfuric acid. The abbreviations used: CH; cholesterol, CMH; ceramide monohexoside, CDH,; ceramide lactoside, CDHb; ceramide digalactoside, CTH; ceramide trihexoside, SM; sphingomyelin. lane A, I; galactocerebroside, lane B, C; normal kidney lane D. E; heterozygous kidney, lane F, G; homozygous kidney, lane H; mixtures of galactocerebroside, ceramide lactoside and sulfatide.
in twitcher increased,
mouse brain
(data
because
digalactosyl
Ceramide
hexoside
not
shown).
ceramide
However,
in kidney
is a major
slycolipid
2.
compositions
Cer-gal
of
kidney
of
Cer-glc
CDH*
CTH**
Normal
13.8 14.7
10.5 12.0
25.2 20.3
10.5 16.0
Heterozygote
12.7 18.8
6.7 7.1
22.7 27.6
18.9 20.6
Homozygote
89.8 57.6
14.0 14.8
31.8 27.5
17.6 19.7
* CDH; values
cermide-gal-gal, are
expressed
**
twitcher
CTH;
as mg per
ceramide-gle-gal-NAcgal 100 gr
637
tissue.
glycolipid
in mouse
kidney.
Table
this
mice.
Vol. 109, No. 3, 1982
8lOCHEMlCAL
An accumulation
of galactocerebroside
new view
pathological
the
of the
identification
AND BIOPHYSICAL in twitcher
consideration
of affected
mice
RESEARCH COMMUNICATIONS
mouse kidney
in GLD and also
by analysis
provides
a
can be used for
of glycolipid.
REFERENCES: 1. Duchen L.W., Either Brain 103, 695-710.
E.M.,
Jacobs
2. Kobayashi T., Yamanaka T., Brain Res. 202, 479-483. 3. Suzuki
K. (1982)
Acta
J.M.,
Jacobs
Pediatr.
Scravilli
J.M.,
F. and Teixeira
Teixeira
Japonica,
in
F. and Suzuki
K. (1971)
6. Kobayashi 8-14.
T.,
Lipids
Nagara
5, H.,
9. Sweelv edited
Inherited Disease" pp. 747-769,
433-436. Suzuki
K. and Suzuki
7. Suzuki K. (1978) in "Methods in Enzymo1og.y" P. 456-458, Academic Press, New York. 8. Eto Y. and Suzuki
K. (1980)
press.
4. Suzuki K. and Suzuki Y. (1978) in " Metabolic Basis of ( J.B. Stanbury, Wyngaarden and P.S. Fredrickson, eds.) MC Graw Hill, New York. 5. Suzuki
F. (1980)
K. (1970)
J. Lipid
Res.
K. (1982) (V.
Biochem.
Ginsburg,
Med. 27,
eds.):vol.
11, 473-479.
C.C. and Vance D.E. (1967) In ' Lipid chromatograuhic analysis" by Marizetti, GW., vol. 1, P. 465, Marcel Dekker, New York,
10. Lowry O.H., Rosebrough Chem. -193, 265-275. 11. Adams E.P.
J.N.,
and Gray G.M.
Farr
(1968)
A.L.
and Randall
Chem. Phys.
638
Lipids
R.J. 1,
(1951) 147-155.
J. Biol.
50C