is

associated

with

the

Dystrophin-Glycoprotein

Complex

at

astrocytic

endfeet

Megalencephalic

leukoencephalopathy

with

subcortical

cysts

-LRB-

MLC

-RRB-

is

a

progressive

cerebral

white

matter

disease

with

onset

in

childhood

caused

by

mutations

in

the

gene

is

a

protein

with

unknown

function

that

is

mainly

expressed

in

the

brain

in

astrocytic

endfeet

at

the

blood

brain

and

cerebrospinal

fluid

brain

barriers

It

shares

its

localization

at

astrocytic

endfeet

with

the

dystrophin-associated

glycoprotein

complex

-LRB-

DGC

-RRB-

The

objective

of

the

present

study

was

to

investigate

the

possible

association

of

with

the

DGC

To

test

this

hypothesis

-LRB-

co

-RRB-

localization

of

DGC-proteins

and

was

analyzed

by

immunohistochemical

stainings

in

gliotic

brain

tissue

from

a

patient

with

multiple

sclerosis

in

glioblastoma

tissue

and

in

brain

tissue

from

an

MLC

patient

In

control

tissue

a

direct

protein

interaction

was

tested

by

immunoprecipitation

Results

revealed

that

is

co-localized

with

DGC-proteins

in

gliotic

brain

tissue

We

demonstrated

that

both

and

a

member

of

the

DGC

were

redistributed

in

glioblastoma

cells

In

MLC

brain

tissue

we

showed

absence

of

and

altered

expression

of

several

DGC-proteins

We

demonstrated

a

direct

protein

interaction

between

and

From

these

results

we

conclude

that

is

associated

with

the

DGC

at

astrocytic

endfeet

Introduction

Megalencephalic

leukoencephalopathy

with

subcortical

cysts

-LRB-

MLC

MIM

-RRB-

is

a

childhood

leukoencephalopathy

with

autosomal

recessive

inheritance

-LSB-

-RSB-

Patients

develop

macrocephaly

during

the

first

year

of

life

followed

by

slow

motor

deterioration

with

cerebellar

ataxia

and

spasticity

-LSB-

-RSB-

Magnetic

resonance

imaging

-LRB-

MRI

-RRB-

shows

diffusely

abnormal

mildly

swollen

cerebral

white

matter

and

subcortical

cysts

in

anterior

temporal

and

frontoparietal

areas

-LSB-

-RSB-

Histopathologic

and

electron

microscopic

examination

of

brain

tissue

reveals

countless

intramyelinic

vacuoles

-LSB-

-RSB-

From

early

on

the

striking

similarity

in

MRI

features

of

MLC

and

congenital

muscular

dystrophy

-LRB-

CMD

-RRB-

with

merosin

deficiency

-LRB-

CMD

type

-RRB-

was

noted

-LSB-

-RSB-

Patients

with

also

have

diffusely

abnormal

mildly

swollen

cerebral

white

matter

and

sometimes

anterior

temporal

cysts

-LRB-

Fig.

-RRB-

-LSB-

-RSB-

Microscopic

examination

of

the

brain

of

a

patient

who

was

later

genetically

confirmed

to

have

-LRB-

B.

Echenne

personal

communication

-RRB-

also

revealed

myelin

vacuolation

-LSB-

-RSB-

is

caused

by

mutations

in

the

gene

encoding

the

chain

of

merosin

-LSB-

-RSB-

In

contrast

to

MLC

is

not

associated

with

muscle

disease

-LSB-

-RSB-

We

hypothesized

that

MLC

could

be

the

brain-only

variant

of

We

could

however

not

confirm

linkage

of

MLC

to

the

locus

and

instead

demonstrated

that

mutations

in

another

gene

cause

MLC

-LSB-

-RSB-

encodes

a

plasma-membrane

protein

of

unknown

function

Recently

the

was

found

to

be

located

in

astrocytic

endfeet

at

the

blood

brain

and

cerebrospinal

fluid

brain

barriers

-LSB-

-RSB-

Fig.

of

the

brain

of

a

patient

with

MLC

-LRB-

a

c

-RRB-

and

-LRB-

d

f

-RRB-

The

axial

images

-LRB-

a

b

d

e

-RRB-

reveal

extensive

signal

abnormalities

and

mild

swelling

of

the

cerebral

white

matter

in

both

MLC

-LRB-

a

b

-RRB-

and

-LRB-

d

e

-RRB-

The

corpus

callosum

and

internal

capsule

are

relatively

preserved

in

both

cases

-LRB-

a

d

-RRB-

The

sagittal

images

reveal

a

large

cyst

in

the

anterior

temporal

white

matter

and

smaller

cysts

in

the

frontal

subcortical

white

matter

in

the

MLC

patient

-LRB-

c

-RRB-

There

are

small

cysts

in

the

same

regions

in

the

patient

-LRB-

f

-RRB-

Both

MLC

and

patients

had

a

genetically

confirmed

diagnosis

Other

proteins

localized

at

astrocytic

endfeet

are

those

of

a

multi-subunit

complex

called

dystrophin-associated

glycoprotein

complex

-LRB-

DGC

-RRB-

-LSB-

-RSB-

The

DGC

is

expressed

in

various

tissues

and

has

been

characterized

best

in

skeletal

muscle

where

it

connects

the

cytoskeleton

of

muscle

fibers

to

the

extracellular

matrix

-LSB-

-RSB-

Mutations

in

different

DGC

components

lead

to

various

muscular

dystrophies

In

a

subset

of

muscular

dystrophies

the

congenital

muscular

dystrophies

-LRB-

CMDs

-RRB-

the

muscular

phenotype

is

often

combined

with

brain

involvement

including

white

matter

abnormalities

most

notably

in

-LSB-

-RSB-

The

brain

DGC

is

involved

in

the

adhesion

of

perivascular

glia

to

the

extracellular

matrix

-LSB-

-RSB-

At

the

astrocytic

endfeet

the

DGC

consists

of

four

main

components

-LSB-

-RSB-

-LRB-

-RRB-

α

and

β-dystroglycan

which

form

the

backbone

of

the

DGC

by

forming

a

transmembrane

link

between

the

actin

cytoskeleton

and

the

extracellular

matrix

α-Dystroglycan

is

a

heavily

glycosylated

peripheral

membrane

protein

that

binds

to

extracellular

ligands

such

as

merosin

and

agrin

β-Dystroglycan

is

a

transmembrane

protein

that

anchors

the

extracellular

α-dystroglycan

to

the

plasma

membrane

and

interacts

intracellularly

with

dystrophin

-LRB-

and/or

utrophin

which

can

replace

dystrophin

-RRB-

and

syntrophin

-LRB-

-RRB-

Dystrophin

and/or

utrophin

are

linker

molecules

that

can

form

a

bridge

between

actin

and

β-dystroglycan

-LRB-

-RRB-

α-Dystrobrevin

which

is

linked

to

dystrophin

and/or

utrophin

binds

to

syntrophin

-LRB-

-RRB-

Syntrophin

which

binds

to

the

water

channel

and

the

potassium

channel

is

responsible

for

the

polarized

expression

of

these

proteins

in

astrocytic

endfeet

The

striking

similarities

in

brain

MRI

features

and

pathology

between

MLC

and

together

with

the

specific

localization

of

both

and

merosin

at

astrocytic

endfeet

brought

us

to

the

hypothesis

that

there

may

be

an

association

between

and

DGC

in

the

brain

In

the

present

paper

we

report

on

our

studies

to

test

this

hypothesis

We

investigated

the

co-localization

of

and

DGC-proteins

in

gliotic

white

matter

because

staining

is

more

intense

in

gliotic

brain

tissue

than

in

normal

tissue

-LSB-

-RSB-

Previous

studies

in

humans

and

mice

have

shown

that

if

members

of

the

DGC

are

mutated

or

missing

the

complex

may

be

disrupted

which

affects

the

localization

of

other

protein

members

-LSB-

-RSB-

We

therefore

also

tested

our

hypothesis

by

studying

the

localization

of

and

several

DGC-proteins

in

glioblastoma

and

MLC

brain

tissue

Besides

we

assessed

a

direct

association

between

and

DGC-members

by

co-immunoprecipitation

experiments

Materials

and

methods

Immunohistochemical

staining

of

human

brain

tissue

Human

control

brain

tissue

specimens

both

frozen

and

paraffin-embedded

consisting

of

neocortex

and

white

matter

were

obtained

at

autopsy

from

patients

without

neurological

disease

and

neuropathologic

abnormalities

Additionally

frozen

brain

tissue

was

obtained

from

a

multiple

sclerosis

patient

to

study

gliotic

white

matter

in

which

has

a

normal

localization

but

expression

is

increased

-LSB-

-RSB-

During

life

all

patients

or

their

next

of

kin

had

given

consent

for

autopsy

and

the

use

of

brain

tissue

for

research

purposes

Human

glioblastoma

multiforme

tissue

-LRB-

frozen

-RRB-

and

brain

biopsy

tissue

from

MLC

patients

-LRB-

both

frozen

and

paraffin-embedded

-RRB-

were

obtained

for

diagnostic

purposes

and

used

for

research

purposes

with

informed

consent

of

patients

and

families

Light-microscopy

of

glioblastoma

tissue

was

used

to

select

areas

that

display

characteristic

glioblastoma

features

including

necrosis

giant

cells

and

endothelial

proliferation

for

further

studies

Brain

biopsies

were

obtained

in

the

following

MLC

patients

-LRB-

decreased

expression

-LSB-

-RSB-

frozen

tissue

-RRB-

-LRB-

homozygous

for

the

c.

C

-LRB-

-RRB-

mutation

paraffin-embedded

tissue

-RRB-

and

-LSB-

compound-heterozygous

for

the

A

and

c.

A

mutations

leading

to

splice-defects

and

truncation

of

the

protein

-LRB-

and

-RRB-

paraffin-embedded

tissue

-RSB-

Immunohistochemistry

was

performed

as

described

previously

-LSB-

-RSB-

with

antibodies

against

the

following

proteins

-LRB-

polyclonal

-LSB-

-RSB-

-RRB-

merosin

-LRB-

monoclonal

-LSB-

-RSB-

-RRB-

-LRB-

polyclonal

Chemicon

Huissen

The

Netherlands

-RRB-

-LRB-

polyclonal

USBiological

Swampscott

Massachusetts

-RRB-

β-dystroglycan

-LRB-

monoclonal

Santa

Cruz

Heidelberg

Germany

-RRB-

α-dystroglycan

-LRB-

monoclonal

UpstateCellSignaling

solutions

-RRB-

agrin

-LRB-

monoclonal

-LSB-

-RSB-

-RRB-

glial

fibrillary

acidic

protein

-LRB-

GFAP

polyclonal

DAKO

Heverlee

Belgium

-RRB-

and

NeuN

-LRB-

monoclonal

Chemicon

-RRB-

In

all

slides

except

the

slides

used

for

double

stainings

the

nuclei

are

counterstained

with

Haematoxylin

Double

immunofluorescence

staining

was

performed

as

described

-LSB-

-RSB-

with

agrin

β-dystroglycan

-LRB-

monoclonal

-RRB-

or

syntrophin

-LRB-

monoclonal

Affinity

BioReagents

Raamdonksveer

The

Netherlands

-RRB-

versus

In

negative

controls

the

primary

antibodies

were

omitted

Double-stainings

were

performed

as

described

previously

-LSB-

-RSB-

In

frozen

tissue

double-stainings

were

done

with

agrin

-LRB-

red

-RRB-

antibodies

versus

-LRB-

blue

polyclonal

Lab

Vision

NeoMarkers

Fremont

CA

USA

-RRB-

antibodies

and

in

paraffin

tissue

with

agrin

or

α-dystroglycan

antibodies

versus

NeuN

or

GFAP

For

the

agrin

-LRB-

brown

-RRB-

NeuN

-LRB-

blue

-RRB-

double-stainings

agrin

was

visualized

with

biotinylated

secondary

antibodies

-LRB-

DAKO

-RRB-

and

NeuN

with

poly-HRP

-LRB-

Immunologic

Duiven

The

Netherlands

-RRB-

For

the

agrin

-LRB-

red

-RRB-

GFAP

-LRB-

blue

-RRB-

double-stainings

agrin

was

detected

with

poly-HRP

-LRB-

Immunologic

-RRB-

and

GFAP

with

biotinylated

secondary

antibodies

-LRB-

DAKO

-RRB-

In

negative

controls

the

primary

antibodies

were

omitted

Immuno-precipitation

and

western

blotting

Preparation

of

brain

tissue

and

immunoprecipitation

experiments

were

performed

as

previously

described

-LSB-

-RSB-

without

antibodies

as

a

negative

control

with

μg

-LRB-

home

made

-RRB-

with

the

nonspecific

CHOP

-LRB-

Santa

Cruz

Heidelberg

Germany

-RRB-

as

an

extra

negative

control

and

with

-LRB-

USBiological

Swampscott

Massachusetts

-RRB-

antibodies

Western

blots

were

incubated

with

the

antibodies

overnight

at

°C

and

complexes

were

visualized

using

ECL

Western

Blot

detection

reagents

-LRB-

Amersham

Buckinghamshire

United

Kingdom

-RRB-

Results

Co-localization

of

and

DGC-members

To

test

whether

co-localizes

with

DGC-proteins

at

astrocytic

endfeet

we

performed

double-immunofluorescent

staining

experiments

in

gliotic

brain

tissue

with

the

transmembrane

protein

-LRB-

Fig.

d

g

green

-RRB-

versus

the

extracellular

matrix

protein

agrin

-LRB-

Fig.

red

-RRB-

the

intracellular

protein

syntrophin

-LRB-

Fig.

red

-RRB-

and

the

transmembrane

protein

β-dystroglycan

-LRB-

Fig.

red

-RRB-

There

was

partial

overlap

-LRB-

yellow

-RRB-

between

all

three

DGC-proteins

-LRB-

red

-RRB-

and

-LRB-

green

-RRB-

The

co-localization

-LRB-

Fig.

yellow

-RRB-

of

-LRB-

Fig.

green

-RRB-

and

β-dystroglycan

-LRB-

Fig.

red

-RRB-

was

most

striking

If

agrin

β-dystroglycan

or

syntrophin

was

omitted

fluorescent

labeling

was

only

visible

for

the

appropriate

isotype

thereby

excluding

cross-reactivity

-LRB-

not

shown

-RRB-

On

omission

of

the

primary

antibody

no

staining

was

observed

-LRB-

not

shown

-RRB-

Fig.

of

and

DGC-proteins

a

i

Double

immuno-fluorescence

labelling

of

-LRB-

a

d

g

green

-RRB-

agrin

-LRB-

b

red

-RRB-

syntrophin

-LRB-

e

red

-RRB-

and

β-dystroglycan

-LRB-

h

red

-RRB-

in

gliotic

human

brain

tissue

There

is

partial

overlap

-LRB-

c

f

i

yellow

-RRB-

between

and

these

DGC-proteins

The

overlap

-LRB-

i

yellow

-RRB-

between

the

transmembrane

proteins

-LRB-

green

-RRB-

and

β-dystroglycan

-LRB-

red

-RRB-

is

most

striking

In

all

panels

the

ocular

magnification

and

μm

scale

bars

are

indicated

in

the

lower

right

hand

corner

and

are

redistributed

in

glioblastoma

cells

Staining

normal

human

brain

tissue

with

and

antibodies

confirmed

their

specific

expression

around

blood

vessels

consistent

with

the

previously

described

perivascular

localization

-LRB-

Fig.

c

-RRB-

-LSB-

-RSB-

In

glioblastoma

tissue

both

and

had

altered

expression

patterns

The

proteins

were

no

longer

confined

to

astrocytic

endfeet

but

were

redistributed

over

the

entire

glioblastoma

cell

-LRB-

Fig.

d

-RRB-

Anti-agrin

staining

showed

expression

of

the

protein

in

the

extracellular

matrix

around

blood

vessels

of

all

sizes

in

normal

brain

tissue

-LRB-

Fig.

-RRB-

whereas

it

was

exclusively

expressed

around

larger

blood

vessels

in

glioblastoma

tissue

-LRB-

Fig.

-RRB-

Double

staining

of

agrin

-LRB-

red

-RRB-

with

-LRB-

blue

-RRB-

an

endothelial

marker

in

glioblastoma

tissue

confirmed

agrin

and

staining

around

larger

blood

vessels

-LRB-

Fig.

-RRB-

and

absence

of

agrin

around

small

vessels

-LRB-

Fig.

boxed

area

-RRB-

This

is

in

line

with

previously

published

results

-LSB-

-RSB-

On

omission

of

the

primary

antibody

no

staining

was

observed

-LRB-

not

shown

-RRB-

Fig.

of

in

glioblastoma

tissue

Immunohistochemical

staining

of

-LRB-

-RRB-

and

agrin

in

control

brain

-LRB-

a

c

e

-RRB-

and

tumor

tissue

-LRB-

b

d

f

g

-RRB-

a

has

a

perivascular

localization

in

normal

tissue

b

In

glioblastoma

tissue

the

restriction

of

to

perivascular

areas

is

lost

and

the

protein

has

an

intracellular

location

in

glioblastoma

cells

is

expressed

in

perivascular

astroglial

endfeet

in

control

brain

is

localized

in

glioblastoma

cells

e

f

Agrin

is

expressed

in

the

extracellular

matrix

around

blood

vessels

of

all

sizes

in

normal

tissue

-LRB-

e

-RRB-

and

only

around

large

vessels

in

glioblastoma

tissue

-LRB-

f

-RRB-

g

Double-staining

of

agrin

-LRB-

red

-RRB-

-LRB-

blue

-RRB-

in

glioblastoma

tissue

shows

agrin

and

-LRB-

endothelial

marker

-RRB-

staining

around

larger

blood

vessels

The

boxed

area

in

panel

g

shows

that

agrin

staining

is

absent

around

small

vessels

In

all

panels

the

ocular

magnification

and

μm

scale

bars

are

indicated

in

the

lower

right

corner

Absence

of

and

altered

expression

of

DGC-proteins

in

MLC

brain

tissue

staining

revealed

perivascular

expression

of

the

protein

in

control

brain

tissue

-LRB-

Fig.

-RRB-

-LSB-

-RSB-

and

absence

of

staining

in

MLC

tissue

-LRB-

Fig.

-RRB-

The

distribution

of

merosin

β-dystroglycan

and

was

similar

in

control

-LRB-

Fig.

e

g

-RRB-

and

MLC

tissue

-LRB-

Fig.

f

h

-RRB-

In

control

tissue

showed

a

perivascular

localization

in

configuration

suggesting

presence

in

distal

atroglial

processes

-LRB-

Fig.

-RRB-

while

in

MLC

patient

tissue

additionally

showed

a

diffuse

cytoplasmic

staining

of

astrocytes

-LRB-

Fig.

-RRB-

In

control

tissue

agrin

-LRB-

Fig.

-RRB-

and

α

dystroglycan

-LRB-

Fig.

-RRB-

were

seen

in

the

perivascular

extracellular

matrix

whereas

these

proteins

were

observed

both

in

the

perivascular

extracellular

matrix

and

within

cells

in

MLC

tissue

-LRB-

Fig.

n

-RRB-

Fig.

of

proteins

associated

with

the

DGC

in

MLC

patients

In

control

tissue

the

perivascular

staining

of

is

visible

-LRB-

a

-RRB-

but

no

staining

is

observed

in

MLC

brain

tissue

-LRB-

b

-RRB-

Normal

perivascular

basal

lamina

staining

with

merosin

is

seen

in

control

-LRB-

c

-RRB-

and

MLC

-LRB-

d

-RRB-

brain

tissue

β-dystroglycan

has

a

normal

perivascular

distribution

in

both

control

-LRB-

e

-RRB-

and

MLC

-LRB-

f

-RRB-

brain

tissue

-LRB-

-RRB-

has

a

normal

perivascular

distribution

in

both

control

-LRB-

g

-RRB-

and

MLC

-LRB-

h

-RRB-

brain

tissue

is

present

in

distal

astrocytic

processes

around

blood

vessels

in

control

tissue

-LRB-

i

-RRB-

In

MLC

tissue

-LRB-

j

-RRB-

is

expressed

both

in

distal

astrocytic

processes

around

blood

vessels

and

in

cell

bodies

of

astrocytes

-LRB-

boxed

area

-RRB-

Agrin

is

expressed

in

the

extracellular

matrix

around

blood

vessels

in

control

tissue

-LRB-

k

-RRB-

In

MLC

tissue

-LRB-

l

-RRB-

agrin

is

expressed

in

both

the

extracellular

matrix

around

blood

vessels

-LRB-

boxed

area

-RRB-

and

in

cells

as

indicated

by

arrows

Immunostaining

for

α

dystroglycan

shows

a

normal

perivascular

distribution

in

control

tissue

-LRB-

m

-RRB-

and

expression

of

α

dystroglycan

around

blood

vessels

-LRB-

boxed

area

-RRB-

and

in

cells

in

MLC

tissue

-LRB-

arrows

in

n

-RRB-

o

Double-staining

of

NeuN

-LRB-

blue

-RRB-

agrin

-LRB-

brown

-RRB-

in

MLC

tissue

shows

cytoplasmic

agrin

staining

in

NeuN

positive

cells

-LRB-

arrows

-RRB-

p

Double-staining

of

Agrin

-LRB-

red

-RRB-

GFAP

-LRB-

blue

-RRB-

in

MLC

brain

tissue

The

picture

on

the

left

shows

an

astrocyte

that

is

only

GFAP

positive

-LRB-

arrow

-RRB-

while

the

picture

on

the

right

shows

an

astrocyte

that

is

both

GFAP

and

agrin

positive

-LRB-

arrow

brown

staining

in

cell

body

-RRB-

In

all

panels

the

ocular

magnification

μm

scale

bars

and

patient

numbers

are

indicated

in

the

lower

right

corner

The

cells

expressing

these

latter

two

DGC-proteins

had

the

morphological

characteristics

of

neurons

Double

stainings

of

agrin

and

α-dystroglycan

with

NeuN

a

neuronal

marker

in

MLC

brain

tissue

confirmed

that

both

agrin

-LRB-

brown

-RRB-

-LRB-

Fig.

-RRB-

and

α-dystroglycan

-LRB-

not

shown

-RRB-

co-localized

with

NeuN

-LRB-

blue

-RRB-

Although

most

redistribution

appeared

to

be

in

neurons

double

stainings

of

agrin

-LRB-

red

-RRB-

and

GFAP

-LRB-

blue

-RRB-

showed

that

also

some

astrocytes

displayed

positive

cell

body

staining

for

agrin

-LRB-

Fig.

-RRB-

On

omission

of

the

primary

antibody

no

staining

was

observed

-LRB-

not

shown

-RRB-

Direct

protein

interaction

between

and

To

verify

interaction

of

DGC-proteins

with

immunoprecipitation

experiments

were

performed

using

human

control

brain

lysates

Figure

shows

that

after

immunoprecipitation

with

both

and

antibodies

and

probing

the

precipitate

for

the

presence

of

a

band

at

kDA

was

detected

the

size

expected

for

This

band

was

not

present

in

a

pull-down

without

antibodies

or

with

an

unrelated

antibody

-LRB-

anti-CHOP

-RRB-

Of

concern

in

this

experiment

was

the

use

of

cross-linking

reagents

since

this

might

cause

nonspecific

interactions

When

cross-linking

reagents

were

left

out

we

failed

to

detect

interactions

which

is

in

agreement

with

previous

studies

on

interaction

between

DGC-members

-LSB-

-RSB-

Fig.

-LRB-

IP

-RRB-

of

human

brain

lysates

Antibodies

used

for

IP

are

indicated

above

the

figure

The

dash

indicates

pull-down

without

antibodies

anti-CHOP

was

used

as

a

nonspecific

antibody

The

resulting

Western

blot

was

probed

with

antibodies

against

The

arrowhead

shows

the

position

of

at

kDa

and

the

asterisk

indicates

a

nonspecific

band

that

is

pulled

down

with

all

the

antibodies

used

Discussion

The

DGC

is

important

in

linking

cytoskeletal

proteins

to

the

extracellular

basal

lamina

It

is

present

in

several

tissues

including

muscle

heart

nerve

and

brain

The

composition

of

this

complex

shows

some

differences

for

different

tissues

Structural

defects

in

a

number

of

DGC-members

cause

muscular

dystrophies

Best

known

is

Duchenne

muscular

dystrophy

related

to

mutations

in

dystrophin

Another

muscular

dystrophy

is

the

CMD

in

which

the

MRI

features

are

strikingly

similar

to

those

of

MLC

is

caused

by

mutations

in

the

chain

of

merosin

-LSB-

-RSB-

A

specific

group

of

CMD

variants

is

known

as

dystroglycanopathies

and

includes

Walker

Warburg

syndrome

Fukuyama

type

of

CMD

muscle

eye

brain

disease

CMD

type

and

type

They

are

characterized

by

hypoglycosylation

of

α-dystroglycan

-LSB-

-RSB-

and

most

of

the

dystroglycanopathies

lead

to

both

muscle

disease

and

brain

involvement

including

cerebral

white

matter

abnormalities

We

hypothesized

that

could

be

associated

with

the

DGC

in

the

brain

To

strengthen

our

hypothesis

we

showed

co-localization

between

and

DGC-proteins

around

blood

vessels

by

immunofluorescent

stainings

A

common

feature

of

the

DGC

is

that

mutations

affecting

one

of

its

components

often

lead

to

destabilization

of

the

complex

with

reduced

or

altered

expression

of

other

DGC-members

For

instance

in

muscle

and

brain

tissue

of

patients

with

a

dystroglycanopathy

immunostaining

is

reduced

for

α-dystroglycan

as

well

as

merosin

and

agrin

-LSB-

-RSB-

In

patients

with

Duchenne

muscular

dystrophy

and

Fukuyama

type

of

CMD

decreased

expression

of

multiple

DGC-proteins

has

been

found

in

muscle

and

brain

-LSB-

-RSB-

In

brain

tissue

of

syntrophin

knock

out

mice

altered

expression

of

has

been

reported

-LSB-

-RSB-

In

glioblastoma

tissue

abnormal

expression

of

and

syntrophin

over

the

entire

surface

of

glioblastoma

cells

and

absence

of

α-dystroglycan

from

glial

structures

have

been

reported

-LSB-

-RSB-

Agrin

was

only

present

around

larger

vessels

but

absent

around

small

vessels

in

glioblastoma

tissue

-LSB-

-RSB-

We

decided

to

use

these

DGC

destabilization

phenomena

to

test

our

hypothesis

Because

of

the

co-localization

of

and

DGC-members

at

perivascular

regions

and

the

disassembly

of

the

DGC

in

glioblastoma

tissue

we

were

interested

in

whether

is

redistributed

in

this

type

of

brain

tumor

We

therefore

tested

the

expression

pattern

of

by

immunohistochemical

staining

in

glioblastoma

tissue

We

confirmed

previous

findings

for

agrin

and

-LSB-

-RSB-

and

demonstrated

that

like

is

redistributed

in

glioblastoma

cells

We

were

also

interested

whether

mutations

would

lead

to

destabilization

of

the

complex

with

reduced

or

altered

expression

of

DGC-members

in

MLC

brain

We

demonstrated

the

absence

of

and

altered

expression

of

agrin

and

α-dystroglycan

in

brain

tissue

of

MLC

patients

whereas

merosin

β-dystroglycan

and

retained

their

normal

perivascular

localization

Agrin

and

α-dystroglycan

were

redistributed

in

both

neurons

and

astrocytes

although

most

positive

staining

cells

appeared

to

be

neurons

All

the

above

results

provide

circumstantial

evidence

for

an

association

between

and

the

DGC

To

show

a

direct

protein

protein

interaction

we

performed

co-immunoprecipitation

experiments

that

revealed

an

association

between

and

All

together

the

above

findings

provide

strong

evidence

for

an

association

between

and

the

DGC

So

far

almost

all

defects

in

DGC-proteins

have

been

associated

with

a

muscular

dystrophy

which

is

absent

in

MLC

This

difference

can

be

ascribed

to

a

difference

in

DGC

composition

for

muscle

as

compared

to

brain

Whereas

merosin

is

expressed

both

in

muscle

and

brain

is

not

expressed

in

muscle

-LRB-

http://biomed.ngic.re.kr/cgi-bin/cards/carddisp?MLC1&search = KIAA0027

-RRB-

Both

in

MLC

and

the

water

content

of

the

affected

white

matter

is

abnormally

high

due

to

intramyelinic

vacuole

formation

-LSB-

-RSB-

The

DGC

is

crucial

for

anchoring

of

water

and

potassium

channels

at

the

perivascular

endfeet

-LSB-

-RSB-

Destabilization

of

the

DGC

may

lead

to

changes

in

glial

polarity

disturbances

of

the

blood

brain

barrier

and

alterations

in

ion

and

water

homeostasis

of

the

brain

-LSB-

-RSB-

and

result

in

an

increased

myelin

water

content

Our

findings

may

therefore

have

implications

for

the

pathophysiology

of

white

matter

abnormalities

observed

in

MLC

and

the

dystroglycanopathies

B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O B O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O