Expression patterns of MLC1 protein in the central and peripheral
Oscar Teijido,aRicardo Casaroli-Marano,bTatjana Kharkovets,cFernando Aguado,b
Antonio Zorzano,aManuel Palacín,aEduardo Soriano,bAlbert Martínez,b,⁎and Raúl Estéveza,⁎
aDepartment of Biochemistry and Molecular Biology, Faculty of Biology, University of Barcelona and Institute for Research in Biomedicine,
Barcelona Science Park, Josep Samitier 1-5. Barcelona, E-08028, Spain
bDepartment of Cell Biology, Faculty of Biology, University of Barcelona and Institute for Research in Biomedicine, Barcelona Science Park,
Josep Samitier 1-5. Barcelona, E-08028, Spain
cZentrum für Molekulare Neurobiologie (ZMNH), Universität Hamburg, Falkenried 94, D-20246 Hamburg, Germany
Received 12 December 2006; revised 12 January 2007; accepted 28 January 2007
Available online 23 February 2007
Mutations in MLC1 cause megalencephalic leukoencephalopathy with
subcortical cysts (MLC), a disorder characterized clinically by
macrocephaly, deterioration of motor functions, epilepsy and mental
decline. Recent studies have detected MLC1 mRNA and protein in
astroglial processes. In addition, our group previously reported MLC1
expression in some neurons in the adult mouse brain. Here we
performed an exhaustive study of the expression pattern of MLC1 in
the developing mouse brain by means of optic and electron microscopy.
In the central nervous system, MLC1 was detected mainly in axonal
tractsearlyin development. Inaddition,MLC1wasalsoobservedin the
peripheral nervous system and in several sensory epithelia, as retina or
saccula maculae. Post-embedding immunogold experiments indicated
that MLC1 is localized in astrocyte–astrocyte junctions, but not in the
dystrophin–glycoprotein complex. In neurons, MLC1 is located at the
plasma membrane and vesicular structures. Our data provide a mouse
MLC1expressionmapthat couldbe usefulto understandthephenotype
of MLC patients, and suggested that MLC disease is caused by an
astrocytic and a neuronal dysfunction.
© 2007 Elsevier Inc. All rights reserved.
Keywords: Myelin; Leukodystrophy; MLC; Neuron; Astrocyte; Develop-
Megalencephalic leukoencephalopathy with subcortical cysts
[MLC (MIM 604004)] is an autosomal recessive neurological disorder
inchildren characterizedclinically bymacrocephaly,mostly duringthe
first year of life, deterioration in motor functions, cerebellar ataxia and
mental decline (Goutieres et al., 1996; Singhal et al., 1996; van der
Knaap et al., 1995a,b). Magnetic resonance imaging (MRI) together
with clinical symptoms is diagnostic and shows swollen brain with
diffusely abnormal cerebral white matter and subcortical cysts in the
anterior-temporal and frontoparietal region (van der Knaap et al.,
patient shows that swelling is caused by the presence of numerous
vacuoles between the outer lamellae of myelin sheets, but not in the
inner or medial ones. This finding would explain the relatively mild
clinical course of MLC despite its dramatic histopathological features
(van der Knaap et al., 1996).
The first gene responsible for MLC was localized in the
chromosome 22qtel (Topcu et al., 2000) and identified as MLC1
(Leegwater et al., 2001). MLC1 codes for a membrane protein of
unknown function with low homology to potassium channels. In situ
hybridization studies (ISH) have reported MLC1 mRNA in adult
mouse brain astrocytes, ependymal cells and Bergmann glia in
cerebellum(Schmittetal.,2003).Our previousstudiesusing ISHand
immunohistochemistry demonstrated that MLC1 was broadly
expressed in the adult mouse brain, including brain barriers such as
ependyma, pia matter and astrocyte endfeet membranes adjacent to
blood vessels localized in astrocyte–astrocyte membrane contacts
(Teijido et al., 2004). In addition, MLC1 expression was detected in
mouse neurons, mainly with an axonal localization in several brain
areas, such as hippocampal mossy fibers and cortical pyramidal cells
MLC1 expression in distal astroglial processes, although it did not
Neurobiology of Disease 26 (2007) 532–545
☆This study was supported in part from networks G03/054 and C03/08 to
MP, the Comissionat per a Universitats I Recerca to MP, FIS PI04/1680 to
RE, grant #302005 from Fundació La Caixa to MP and RE, by PI04/2433 to
AM and by BFU 2004-01154 to FA. RE is a researcher from the Programa
Ramón y Cajal of the Spanish Ministry of Science and Technology.
⁎Corresponding authors. A. Martínez is to be contacted at fax: +34 93
4034717. R. Estévez, Present address: Unitat de Fisiologia, IDIBELL-
Universitat de Barcelona, Feixa Llarga S/N, E-08907 L'Hospitalet, Spain.
Fax: +34 93 4039781.
E-mail addresses: email@example.com (A. Martínez), firstname.lastname@example.org
Available online on ScienceDirect (www.sciencedirect.com).
0969-9961/$ - see front matter © 2007 Elsevier Inc. All rights reserved.
address whether this protein was also expressed in human neurons
(Boor et al., 2005).
Here we examined MLC1 localization during mouse develop-
ment using immunohistochemistry with optical and electron
microscopy experiments. Our results provide a mouse MLC1
expression map that could be useful in understanding the phenotype
of MLC patients, revealed that MLC1 is also expressed in the PNS
and in several sensory epithelia and refined MLC1 subcellular
Materials and methods
The antibodies against the N-terminal region of mouse MLC1
have been characterized extensively (Teijido et al., 2004). Other
antibodies used were mouse anti-Neurofilament-200 (NF-H)
(1:1000) (Sigma St. Louis, MO), mouse anti-Myelin Basic
Protein (MBP) (1:50) (Chemicon International Inc., Temecula,
CA, USA), FITC-labeled α-Bungarotoxin (α-BTX) (1:200)
(Amersham, Arlington Heights, IL), mouse anti-Thy 1.1 (1:10)
(Chemicon), mouse anti-Vimentin (1:50) (Chemicon), mouse anti-
Glial Fibrillary Acidic Protein (GFAP) (Chemicon) (1:500), anti-
calretinin (Swant) (1:100) and anti NF68 (Chemicon) (1:100).
Tissue membrane isolation and Western Blot analyses
Embryonic E13, E16, E18, postnatal P1, P3, P7, P21 and
adult stage mouse brains were dissected out and separated into
forebrain–midbrain and hindbrain sections. These experiments
were carried out in accordance with the European Community
Council Directive for the care and use of laboratory animals.
Fig. 1. Analysis of MLC1 expression during development. (A) Western blot analysis of MLC1 expression in forebrain/midbrain and hindbrain at distinct developmental
of entire mouse at E13 showing MLC1 expression in the peripheral nervous system. Immunoreactive axons were distributed in the medulla (med), dorsal root ganglia
4V, 4th ventricle; CB, primordium of cerebellum; FB, forebrain; HB, hindbrain; MB, midbrain; SC, spinal cord. Scale bars (B) 500 μm; (C) 200 μm; (D) 200 μm.
533O. Teijido et al. / Neurobiology of Disease 26 (2007) 532–545
Tissues were homogenized in eight volumes of buffer containing
25 mM HEPES, 250 mM sucrose, 4 mM EDTA, 1 mM PMSF
and 1× protease inhibitor cocktail (Roche Diagnostics, Germany).
The homogenate was centrifuged at 5000×g at 4 °C for 10 min
followed by an ultracentrifugation at 150,000×g for 90 min at
4 °C. Rat optic, sciatic nerves and retina were removed and
homogenized in 0.1 M PBS, 0.1% Triton X-100. Western blot
analyses were performed as described (Teijido et al., 2004).
Immunohistochemistry in whole E13 and CNS
For studies on prenatal mouse brains, mothers were
anesthetized and embryos were removed. Whole E13 and
released heads of E16 and E18 were embedded in a fixative
solution containing 4% paraformaldehyde (PFA) in 0.1 M PBS
for 48 h at 4 °C. Postnatal and adult mice were anaesthetized and
perfused with 4% PFA in 0.1 M PBS. The brains were dissected
out and post-fixed in the same fixative solution for 48 h at 4 °C.
Embryo and postnatal samples were cryoprotected with 30%
sucrose in PBS 0.1 M. E13 embryos were immersed in a solution
containing 0.1 M PBS, 8% gelatin, 15% sucrose, while the other
stages were immersed in O.C.T. solution (TissueTek). Samples
were frozen with dry ice-cooled isopenthane and were stored at
−80 °C. E13 was cut in a cryostat (14 μm thick) in the saggital
plane while the other stages were cut along the transversal plane
(40 μm thick).
The sections were rinsed in 0.1 M PBS and treated with
endogenous peroxidase inhibitor buffer containing 2% H2O2and
10% methanol. Three washes with buffer containing 0.1 M PBS
Triton X-100 0.1% (for embryos), 0.1 M PBS Triton X-100 0.3%
(for P0, P5 and P15) and 0.1 M PBS Triton X-100 0.5% (for adult
mouse) were performed to permeabilize the membranes. To
suppress non-specific binding, the sections were incubated in
0.2% gelatine, 10% normal goat serum and 0.1 M PBS 0.1, 0.3 and
0.5% Triton X-100 respectively, as indicated above, for 60 min at
room temperature (RT). Thereafter, the slices were incubated
overnight at 4 °C using rabbit anti-MLC1 polyclonal antibodies
(1:100). After three PBS-Triton X-100 rinses, an incubation with a
Expression of MLC1 at embryonic stages
Granule cell layer
External plexiform layer
Mitral cell layer
Internal plexiform layer
Lateral olfactory tract
Habenulo peduncular tract
Medial habenular nucleus
Periaqueductal gray area
Optical nucleus layer
Commissure of superior colliculus
External germinative layer
Purkinje cell layer
Pontine reticular nucleus
Ventral cochlear nucleus
Dorsal cochlear nucleus
Gigantocellular reticular nucleus
Spinal trigeminal nucleus
Spinal trigeminal tract
Medial longitudinal fasciculus
Medullary reticular formation
Very low (+/−), low (+), moderate (++), high (+++), and very high (++++) levels of MLC1 expression were shown. No detectable expression was also indicated (−).
534O. Teijido et al. / Neurobiology of Disease 26 (2007) 532–545
secondary anti-rabbit biotinylated antibody was performed (1:200)
(Vector Laboratories, Inc., Burlingame, CA, USA) for 60 min at RT,
and then with a streptavidin–horseradish peroxidase complex
(Amersham) for 120 min at RT. Sections were developed with
0.03% diaminobenzidine and 0.003% H2O2, mounted onto slides,
dehydrated and coverslipped with DPX mounting media. Incuba-
tion with non-immunized IgGs and omission of primary antibodies
were used as negative controls.
Immunohistochemistry in retina, inner ear and PNS
Mouse spinal cords and rat nerves, retina and rat femoral
muscles were dissected out, cryoprotected, frozen and sliced in a
cryostat (14 μm thick). Slices were processed for immunohis-
tochemistry and DAB staining as described above. To process
slices for immunofluorescence, they were rinsed in 0.1 M PBS,
permeabilized in 0.1 M PBS 0.5% Triton X-100 and blocked in
0.1 μM PBS, 0.2% gelatine, 10% normal goat serum and 0.5%
Triton X-100 for 60 min at RT. Slices were incubated overnight at
4 °C with rabbit anti-MLC1 polyclonal antibodies (1:25) in
combination with other antibodies (see above). Slices were then
rinsed with 0.1 M PBS, 0.5% Triton X-100, incubated with
secondary fluorescent antibodies (1:250) (goat anti-mouse and goat
anti-rabbit Alexa fluor 488 and 568; Molecular Probes, Inc.,
Eugene, OR, USA), rinsed in 0.1 M PBS and finally mounted with
Inner ears were dissected from the temporal bone and fixed
at 4 °C for 1.5 h in 4% PFA in PBS. From P8 onward, bones
were decalcified with 10% EDTA in PBS over 48 h and post-
fixed with 4% PFA for 15 min. For immunohistological staining,
8-μm cryosections were prepared after incubation in 30%
sucrose in PBS and then embedded in Tissue Freezing Medium
(Leica). Sections were blocked with 3% NGS, 2% BSA and
0.5% NP-40 in PBS. Dilutions of primary and secondary
fluorescence-labeled antibodies were applied in PBS with 3%
normal goat serum and 0.1% NP-40. TOTO®-3 iodide was used
for staining nuclei.
Naval Medical Research Institute (NMRI) adult mice (n=2)
were perfused with 4% PFA and 0.1% glutaraldehyde in 0.12 M
phosphate buffer. Brains were removed and small samples of
hippocampus, cortex and cerebellum were dissected, cryopro-
tected gradually in sacarose and cryofixed by immersion in liquid
propane. Freeze substitution was performed at −90 °C during 3
Fig. 2. Distribution of MLC1 in the olfactory bulb along development. (A–D) Horizontal sections of E18 (A), P5 (B) and adult (C–D) olfactory bulbs showing
the pattern of expression of anti-MLC1 antibody. The highest labeling was observed in the inner plexiform layer (IPL) and in the glomerular layer (GlL)
throughout development. D is a high magnification of boxed area in C. MLC1 showed strong protein expression in the glomeruli, the IPL and axons crossing the
granule cell layer (GL). Abbreviations: EPL, external plexiform layer; GL, granule cell layer; GlL, glomerular layer; IPL, internal plexiform layer; ML, mitral
cell layer; SEL, ependyma/subependymal layer. Scale bars (A, B, C, D) 200 μm.
535O. Teijido et al. / Neurobiology of Disease 26 (2007) 532–545
days in an “Automatic Freeze Substitution System” (AFS, Leica),
using methanol containing 0.5% uranyl acetate as substitution
medium. Infiltration was carried out in Lowicryl HM20 at
−50 °C and then polymerized with UV lamps. Ultrathin sections
were collected and processed for a post-embedding MLC1
immunostaining using a rabbit anti-N-terminal MLC1 antiserum
(1:10) and 15 nm colloidal gold-coated secondary antibodies
(BBI; 1:75). In control experiments, the primary MLC1 antibody
was omitted. No immunogold labeling occurred under these
conditions. Rat and optic sciatic nerve were processed in a similar
MLC1 in mouse nervous system development
As a first step to study the regional expression of MLC1 during
development, we analyzed brain MLC1 protein levels at a range of
developmental stages. Mouse brains from E13, E16, E18, P1, P3,
P7, P21 and adult stages were taken out and dissected into
forebrain/midbrain and hindbrain regions. Membrane homogenates
were extracted and MLC1 protein was detected by Western blot
using an antibody against an N-terminal region of the protein. As
already described (Teijido et al., 2004), bands of ∼35 and 70 kDa
(only the ∼35 kDa band is shown in Fig. 1A) corresponding to
MLC1 were observed, with increased expression levels throughout
development (Fig. 1A). Although MLC1 signal was very low at
E13 developmental stage, it was clearly visible with higher
In agreement with its detection by Western blot, whole E13 was
immunostained (Fig. 1B) and MLC1 was detected in the CNS (Fig.
1C), mostly in the Rhombencephalon, and the PNS (Fig. 1D). In
the latter (Fig. 1D), MLC1 was highly expressed in Dorsal Root
Ganglia (DRGs) processes and in Trigeminal Ganglia, and in
nerves innervating tongue, stomach, genital primordium, lung and
heart (Fig. 1B).
An MLC1 expression map in embryonic and postnatal stages
is provided in Tables 1 and 2, respectively. In the following
figures, we indicated MLC1 distribution in the developing CNS
mouse brain following the antero-posterior axis.
In the olfactory bulb, expression levels increased along
development (Fig. 2). At prenatal stages, staining was observed
Fig. 3. Expression of MLC1 in the anterior forebrain along development. (A–B) MLC1 staining of the anterior forebrain at E18 (A) and P5 (B) stages
showed immunopositive fibers in the cortical subplate/white matter (arrowheads), the striatum (STR), medial septum (SP), and lateral optic tract (LOT). (C)
At adult stages, staining was also observed in fibers of the corpus callossum and in neurons of distinct areas such as the amygdaloid complex (AM)
(arrows). (D–F) Electron microscopy localization of MLC1 in the neocortex. Post-embedding immunostaining showed MLC1 protein in pre-synaptic (D–E)
and post-synaptic (D and F) elements. Abbreviations: AM, amygdala; AT, axon terminal; cc, corpus callosum; DB, diagonal band; LOT, lateral olfactory
tract; LV, lateral ventricle; NC, neocortex; Pir, piriform cortex; S, dendritic spine; SP, septum; STR, striatum; vhc, ventral hippocampal commissure. Scale
bars (A–C) 500 μm; (D–F) 0.25 μm.
536O. Teijido et al. / Neurobiology of Disease 26 (2007) 532–545
in the inner plexiform layer (IPL) and in the glomerular layer,
while the remaining layers, such as the external plexiform layer
(EPL), showed very low or null staining (Fig. 2A). At postnatal
stages, signal was remarkably intense in the glomeruli and the
IPL, and diffuse staining was observed in the EPL (Fig. 2B). In
addition, stained axonal fibers crossing granular cell layer
appeared at P5 (Fig. 2B) and were strongly stained at adult stage
(Figs. 2C and D). Finally, strong labeling was observed in the
glomerular layer and in fibers of the inner plexiform layer at adult
stage (Figs. 2C–D).
In the basal forebrain, stained axonal tracts were observed in
the striatum, medial septum and diagonal band (DB) along
development (Figs. 3A, B, C) and, from P10, highly immunos-
tained cell bodies were observed in the amygdaloid complex (Fig.
3C). Regarding the main anterior commissural projections, while
MLC1 staining was observed in the ventral hippocampal
commissure in all developmental stages (Figs. 3A and B), the
corpus callossum showed stained fibers only in the mature brain
(Fig. 3C). Finally, the lateral optic tract was highly immunos-
tained by MLC1 antibodies in both embryonic and postnatal
stages (Figs. 3A–B).
In the embryonic cerebral cortex, MLC1 protein was detected
only in the reciprocal thalamocortical and corticothalamic fibers
invading the subplate (Fig. 3A, arrowheads). From P0 onwards, in
addition to these fibers located in the white matter, staining was
predominantly found in II–III and V–VI layers (Figs. 3B and C).
Throughout development, adult cerebral cortex showed the
greatest MLC1 expression and its pattern was similar in all the
cortical regions. Thus, we detected high levels in the frontal
association, somatosensory, lateral orbital and motor cortical areas
and lower levels in the piriform, entorhinal, insular and cingular
areas (Fig. 3C). In cortical areas with high MLC1 expression,
such as the somatosensory cortex, staining was predominantly
observed in dendrites and axons, but not cell bodies of cortical
neurons, in the distinct layers and in astroglial cells in layer I
(data not shown). To characterize the subcellular localization of
the MLC1 protein, post-embedding immunostaining was per-
formed in the neocortex. Immunoreaction was observed mainly in
plasma membrane and intracellular membranous structures in
dendrites (not shown), axons and the pre-synaptic and post-
synaptic elements, i.e. axon terminals and dendritic spines,
respectively (Figs. 3D–F).
Fig. 4. Distribution of MLC1 in the hippocampus throughout development. (A) At E18, stained fiber bundles were detected in the white matter (WM),
entorhinohippocampal termination zone (arrowheads) and the fimbria (f). (B) At P5, strongest expression was observed in the stratum oriens (so), stratum
lacunosum moleculare (slm) and the CA3 stratum radiatum (sr). (C) At adult stages, stained axons were detected in alveus, CA1 stratum lacunosum moleculare
(slm),CA3 stratumradiatum(sr),inner molecularlayerandhilus (H). (D) Enlargementof the region markedby the boxesin C showinglabeledaxons inthe inner
molecularlayer andin the hilarregion. Abbreviations: DG, dentategyrus; f, fimbria;iml, innermolecularlayer; H, hilus;mml, medial molecular layer;oml, outer
molecular layer; sg, stratum granulosum; sl, stratum lucidum; slm, stratum lacunosum moleculare; sm, stratum moleculare; so, stratum oriens; sp, stratum
pyramidale; sr, stratum radiatum; WM, white matter. Scale bars (A–C) 150 μm; (D) 50 μm.
537O. Teijido et al. / Neurobiology of Disease 26 (2007) 532–545
Expression of MLC1 at postnatal stages
Granule cell layer
External plexiform layer
Mitral cell layer
Internal plexiform layer
Lateral olfactory tract
IVand VI layers
Periaqueductal gray area
Optical nucleus layer
Commissure of superior colliculus
Purkinje cell layer
Internal granular layer
Pontine reticular nucleus
Ventral cochlear nucleus
Dorsal cochlear nucleus
Gigantocellular reticular nucleus
Spinal trigeminal nucleus
Spinal trigeminal tract
Ventral spinocerebellar tract
Medullary reticular formation
Tegmental pontine nuclei
Mesencephalic tract of n5
Sensory root of n5
Principal sens 5 nucleus
Motor 5 nucleus
Dorsal motor vagus
Medial/outer molecular layer
Inner molecular layer
Habenulo peduncular tract
Medial habenular nucleus
Lateral geniculate nuclei
Anteroventral thalamic nucleus
Superior thalamic radiation
Very low (+/−), low (+), moderate (++), high (+++), and very high (++++) levels of MLC1 expression were shown. No detectable expression was also
538O. Teijido et al. / Neurobiology of Disease 26 (2007) 532–545
Regarding the hippocampal formation, and similar to
observations in the other brain regions, MLC1 expression
increased along development. Thus, at embryonic stages, the
protein was detected only in fiber bundles located in the white
matter, entorhinohippocampal termination zone and fimbria (Fig.
4A). At early postnatal stages (P5), MLC1 was detected in the
stratum oriens, stratum lacunosum moleculare, stratum radiatum
of CA3 and in the dentate molecular layer (Fig. 4B). Finally, as
described elsewhere (Teijido et al., 2004), adult hippocampus
showed high MLC1 expression in the stratum lucidum and
539O. Teijido et al. / Neurobiology of Disease 26 (2007) 532–545
stratum oriens, and, to a lesser extent in the CA1 stratum
lacunosum moleculare, CA3 stratum radiatum and hilus (Figs.
4C–D). In the dentate gyrus, stained fibers were also observed in
the inner tier of the molecular layer, corresponding to the
commissural/associative termination layer (Figs. 4C–D). No
staining was observed in the principal cell layers, i.e. the pyra-
midal and the granule cell layers.
In the diencephalon, strong expression was detected in the
habenulo peduncular, mamillothalamic and optic tracts as well as
in the cerebral peduncle at embryonic and postnatal stages
(Tables 1 and 2). In addition, at postnatal stages, staining was
also observed in fibers crossing distinct regions, such as the
thalamus and hypothalamus, and in cell bodies of distinct
neuronal and glial populations in many nuclei (Table 2).
In the mesencephalon, only a few stained fibers in the dorsal
raphe were observed at embryonic stages (Table 1). However,
MLC1 expression progressively appeared in other tracts and
nuclei. Thus, at adult stages, moderate staining was detected in
fibers surrounding the periaqueductal gray area, and in both the
inferior and superior colliculi (Table 2).
Regarding the hindbrain, distinct axonal tracts were observed
in brainstem at all stages of the developing brain. Thus, the most
intense staining was in the spinal trigeminal tract (sp5), facial
nerve (FN) and medial longitudinal fasciculus (MLF) at
embryonic (Fig. 5A) and postnatal stages (Figs. 5B and E)
and some fibers appeared in the pontine, vestibular, hypoglossal
and ventral cochlear nuclei in developing and mature brain
(Tables 1 and 2). In addition, cell bodies in the both the
mesencephalic trigeminal tract nucleus (Me), corresponding to
trigeminal propioceptors (Figs. 5A–C) and the pontine inter-
mediate reticular zone (IRZ) (Figs. 5A, B and D) were strongly
Finally, the cerebellum and the projection tracts that connect
this region to others were labeled from perinatal stages. The
external germinative and the molecular layers of the cerebellum
in perinatal (Figs. 5A–B) and postnatal stages (Figs. 5E–G),
respectively, were MLC1-positive. In the latter, staining corre-
sponded to the apical processes of Bergmann glia (Fig. 5G), as
described (Schmitt et al., 2003; Teijido et al., 2004). In addition,
olivocerebellar fibers (OCF), the cerebellar commissure (CeC)
and fibers in cerebellar white matter were strongly stained (Figs.
5A, B, E–G). In a recent study, we demonstrated MLC1
expression in astrocytic processes at electron microscope level by
pre-embedding immunostaining techniques (Teijido et al., 2004).
In the present study, in order to establish the precise distribution
of the protein in these processes, we performed post-embedding
immunolocalization with gold-coated antibodies. By this techni-
que, staining was located exclusively in the protoplasmic and
perivascular astrocyte–astrocyte junctions, but not in the
perivascular astrocyte–endothelial cell junction (Figs. 5H–I).
MLC1 in the PNS
In the adult spinal cord (Figs. 6A–C), grey matter showed
diffuse MLC1-immunostaining, including motoneurons in the
ventral horn which sent several well-defined stained axons along
the white matter, reaching the motor nuclei of brainstem. High
immunostaining was also detected in fibers from sensory nerves
reaching the dorsal horn (Fig. 6A). As detected in the CNS
(Boor et al., 2005; Schmitt et al., 2003; Teijido et al., 2004),
MLC1 was also present in astrocytes surrounding blood vessels
in the white matter of spinal cord (Fig. 6C).
To follow the motor distribution of MLC1, we first
analyzed whether MLC1 was expressed in extracts from rat
sciatic nerve by Western blot. Two bands presumably
corresponding to MLC1 were detected, because they showed
the same molecular weight as MLC1 expressed in HeLa cells
(Fig. 6D). Longitudinal sections from human (Fig. 6E), rat
(Fig. 6F) and mouse nerves (data not shown) revealed marked
MLC1 immunostaining in several myelinic tracts, as well as in
astrocytes surrounding the nerve. To study MLC1 localization,
double staining immunocytochemical experiments with different
antibodies were performed. MLC1 combined with the axonal
neurofilament-H (NF-H) presented clear co-localization (Fig.
6F). In contrast, double immunolabeling with myelin-basic
protein (MBP) showed that MLC1 was not expressed in myelin
sheets (Fig. 6G). Electron microscopy post-embedding immu-
nostaining studies showed that MLC1 was located inside the
axon, associated with vesicles or membranous structures (Fig.
6H). These data indicated that MLC1 is localized in axonal
tracts but not in myelin.
Double staining experiments were also performed at neuro-
muscular junctions. MLC1 was localized in axons and in a pre-
synaptic localization as showed by double immunostaining with
α-bungarotoxin (Fig. 6I).
MLC1 in sensory organs
The observation that MLC1 was distributed through sensory
pathways in the brain suggested that it would be also located in
sensory organs. Thus, specific bands corresponding to MLC1
protein were detected in rat retina and optic nerve homogenates,
which presented the same size as control mouse MLC1 in HeLa
transfected cells (Fig. 7A).
Immunohistochemistry studies indicated that in the retina,
mainly the ganglionar cells were MLC1-immunoreactive (Fig. 7B),
Fig. 5. Expression of MLC1 in the hindbrain. (A–E) Micrographs showing MLC1 staining at E16 (A), P0 (B–D) and P5 (E) stages. Stronger staining was
observed in the inner cerebellar peduncle (ICP), spinocerebellar tract (sp5), cerebellar commissure (CeC), fibers around periaqueductal gray (PAG) area and
facial nerve (FN). In addition, stained neurons were observed in the mesencephalic nuclei (Me) and in the intermediate reticular zone (IRZ), as shown at
higher magnifications of boxed areas in C and D, respectively. (F–G) MLC1 expression in the adult cerebellum. Protein was found in the molecular layer
and in the fibers of the white matter. G is an enlargement of boxed area in F. (H–I) At electron microscopy level, post-embedding staining showed
immunoreactivity in the astrocyte–astrocyte junctions of protoplasmic (H) and perivascular (I) astroglial processes. Abbreviations: AC, astrocyte; BV, blood
vessel; CB, cerebellum; CeC, cerebellar commissure; DTg, dorsal tegmental nucleus; EC, endothelial cell; FN, facial nerve; GL, granular layer; IC, inferior
colliculus; ICP, inferior cerebellar peduncle; IO, inferior olive; IRZ, intermediate reticular zone; Me, mesencephalic trigeminal nucleus; ML, molecular
layer; MLF, medial longitudinal fasciculus; OCF, olivo-cerebellar fibers; P, pons; PAG, periaqueductal gray; PCL, Purkinje cell layer; SC, superior
colliculus; SO, superior olive; sp5, spinal trigeminal tract; Ve, vestibular nuclei; WM, white matter. Scale bars (A, B) 500 μm; (C, D) 20 μm; (E) 500 μm;
(F) 200 μm; (G) 50 μm; (H, I) 0.25 μm.
540O. Teijido et al. / Neurobiology of Disease 26 (2007) 532–545
as shown by the co-localization of MLC1 with anti-thy1.1 (Fig.
7C). However, the astrocytic Müller cells did not express MLC1,
as assessed using the marker anti-vimentin (Fig. 7D). Ganglionar
cells are retinal neurons that send their axons along the optic nerve.
Therefore, an abundant MLC1 immunostaining was detected in
axonal tracts in the optic nerve, as shown by the co-localization
with NF-H (Fig. 7E). In addition, in the optic nerve, MLC1 was
strongly expressed in numerous astrocytes surrounding the axons
Fig. 6. MLC1 in the peripheral nervous system. (A) MLC1 was localized in the adult mouse spinal cord. White matter presented a conspicuous distribution of
MLC1 in axonal tracts while grey matter showed a diffuse labeling. High immunostaining was detected also in fibers from sensory nerves reaching the dorsal
horn. (B) Detail of MLC1 immunostaining in motoneurons. (C) In a longitudinal section of rat spinal cord, MLC1 was also detected in astrocytes surrounding
blood vessels in white matter of spinal cord. (D) Using anti-MLC1 antibodies, MLC1 protein was detected in extracts from rat sciatic nerve by Western blot.
Extracts from transiently transfected HeLa cells with mouse MLC1 (mMLC1T) were used as controls. Molecular weight markers indicate 37.3 and 54.3 kDa. (E)
MLC1 was detected in several axonal tracts in human nerve. Arrows indicate staining in astrocytes surrounding endothelial cells close to the nerve. (F) Double
immunolabeling experiments in rat sciatic nerve, combining MLC1 (in red) with neurofilament-H (NF-H, in green) showed nearly complete co-localization
(MERGE, in yellow). (G) Double immunostaining of MLC1 (in red) and myelin-basic protein (MBP, in green) demonstrated that MLC1 was not expressed in
myelin sheets. (H) Using electron microscopy post-embedding immunostaining, MLC1 was specifically detected in axonal tracts, associated with membranous
structures, but not in myelin. (I) Double staining at neuromuscular junctions showed MLC1 (in red) labeling the neuronal processes, but not the pre-synaptic
motor plate, labeled with α-bungarotoxin (BTX, in green). Abbreviations: DH, dorsal horn; EC, endothelial cell; GM, grey matter; M, myelin; VH, ventral horn;
WM, white matter. Scale bars (A) 200 μm; (B) 50 μm; (C) 200 μm; (E) 200 μm; (F, G, I) 10 μm; (H) 1 μm.
541O. Teijido et al. / Neurobiology of Disease 26 (2007) 532–545
and blood vessels, as demonstrated by co-localization with glial
fibrillary acidic protein (GFAP) (Fig. 7F). Post-embedding
immunostaining did not show MLC1 expression in myelin, but
expression was located in the axon, in association with membranes
(Fig. 7G), as observed in the sciatic nerve (Fig. 6H).
We also studied whether MLC1 was expressed in the inner ear
(Fig. 8). MLC1 showed post-synaptic localization on the inner
hair cells (IHC) and in afferent fibers of the IHC in the organ of
Corti (Fig. 8A), as evidenced by co-localization with calretinin
(Dechesne et al., 1994). MLC1 was strongly expressed in the
spiral ganglion (Fig. 8B) in non-myelinated and myelinated parts
of the auditory nerve, where it overlapped partially with NF68
(Fig. 8C). In the spiral ganglion, only very few neuronal cell
bodies were MLC1-positive. In contrast, we detected expression
in the vestibular ganglion in more neuronal cell bodies (Fig. 8D).
Expression of MLC1 in the fibers of both ganglia was positive.
MLC1 was also observed in the sensory epithelia of the
vestibular organ (saccula maculae), where it was expressed
mainly in calretinin-positive fibers (Fig. 8E).
To gain an insight into the pathophysiology of MLC, which is
caused by mutations in MLC1, here we analyzed its expression in
the CNS mouse brain during development and in the PNS in adult
Our results confirmed the initial hypothesis that MLC1 was
expressed in neurons and astrocytes. The supporting evidence was
as follows: (1) ISH detected MLC1 expression in neurons (Teijido
et al., 2004); (2) an antibody against MLC1 detected expression in
neurons in adult mouse, the developing mouse and humans
(Teijido et al., 2004 and this work); and (3) the pattern of
Fig. 7. MLC1intheretinaandopticnerve.(A)MLC1expressioninrateye,asassessedbyWesternblot,showedabundantMLC1expressionintheopticnerveand
retina. Molecular weight markers indicate 37.5 and 52.8 kDa. (B) Localization of MLC1 in the retina. MLC1 was expressed mainly in the ganglional cell layer
(GCL). (C) Specific labeling of MLC1 in ganglionar cells (in red) was demonstrated by double immunostaining with thy1.1 (in green), showing a clear co-
localization (MERGE, in yellow). (D) MLC1 (in red) was not detected in glia-like Müller cells, labeled with vimentin (in green). (E) Several optic nerve axonal
processes coming from ganglionar cells were MLC1-immunoreactive (in red), as shown with co-localization (MERGE, in yellow) with NF-H (in green). (F)
MLC1 was also co-expressed with glial fibrillary acidic protein (GFAP, in green), indicating again that MLC1 was also expressed in astrocytes (MERGE, in
yellow), mostly around blood vessels. Note that antibody signal was stronger in astrocytic processes than in neuronal processes. (G) At the electron microscope
level, post-embedding immunostaining showed MLC1 expression in axonal tracts of the optic nerve, associated with membranes, but not in myelin.
Scale bars (B–F) 10 μm; (G) 1 μm.
542O. Teijido et al. / Neurobiology of Disease 26 (2007) 532–545
expression in neurons was consistent during development. We
propose that the discrepancies between the results reported by other
research groups (Boor et al., 2005; Schmitt et al., 2003) can be
explained by the sensitivity of detection. MLC1 is strongly
enriched in mouse astrocytes compared to neurons, and this may
explain the lack of detection of MLC1 mRNA and protein in
neurons by the other research laboratories (Boor et al., 2005;
Schmitt et al., 2003). Clearly, a knockout mouse would be the best
control to address the specificity of our antibodies; however, the
abovementioned controls strongly supported our data.
At structural level, our results revealed that MLC1 was
present in protoplasmatic and perivascular astrocyte–astrocyte
junctions, but not in endothelial–astrocyte junctions (Teijido et
al., 2004). Around blood vessels, most gold particles were
Fig. 8. MLC1 in the inner ear. (A) Confocal images in the organ of Corti showed that MLC1 was expressed (in red) post-synaptically to inner hair cells (IHC), as
evidenced by the lack of colocalization with calretinin. Co-localization between both markers was found in the afferent fibers of the IHC cells. (B) MLC1
expression in the spiral ganglia showed strong expression in fibers, but only in a few neuronal cell bodies. (C) In the spiral ganglia, expression overlapped
partially (in yellow), with NF68, an axonal marker. (D) MLC1 was also expressed in the vestibular ganglion, including many neuronal bodies. (E) MLC1
expression in the sensory epithelia of the vestibular organ stained mainly fibers that were calretinin-positive. Abbreviations: IHC, inner hair cells; Scale bars (A)
100 μm; (B, D) 25 μm; (C, E) 10 μm.
543 O. Teijido et al. / Neurobiology of Disease 26 (2007) 532–545
accumulated where endfeet membranes about on other endfeet
membranes. We believe that the latter result was correct due to
the higher resolution of the post-embedding technique using gold-
coated antibodies compared with the pre-embedding method. In
the pre-embedding method the DAB precipitate can diffuse in the
surrounding cells, making more difficult the interpretation of the
results. Then, we can conclude that in mouse, MLC1 is not a
component of the dystrophin–glycoprotein complex (DGC),
because it is not located in the perivascular membrane (Amiry-
Moghaddam et al., 2004). In agreement with this result, we failed
to detect a biochemical and functional interaction between MLC1
and some proteins of the DGC complex, as the potassium channel
Kir4.1 (Amiry-Moghaddam et al., 2003) or the water channel
AQP4 (Amiry-Moghaddam et al., 2004) (Teijido, Pusch, Estévez
In MLC, megalencephaly is the first symptom which appears
within the first year of life (van der Knaap et al., 1995a). MR images
show subcortical cysts in the temporal lobes and also in the
frontoparietal subcortical area. Histopathological examination of a
brain biopsy from an MLC patient shows that swelling is caused by
the presence of numerous vacuoles between the outer lamellae of
myelin sheets (van der Knaap et al., 1996). Interestingly, vacuoles
have been also detected in several knockout mice models. For
the outside of myelin sheaths (Neusch et al., 2001), whereas Cx32/
Cx47 dKO have vacuoles mainly in the periaxonal space
(Menichella et al., 2006). It has been speculated that vacuoles are
caused by accumulation of ions, as potassium, and water that are
unable to redistribute in these mutants mice (Menichella et al.,
2006). Here we show that MLC1 is located in astrocyte–astrocyte
junctions, where it could work in astrocyte communication.
Considering the phenotype of MLC vacuolization in the outer
lamellae of myelin, MLC1 could also participate in the process of
potassium buffering (Kofuji and Newman, 2004) within the
astrocyte network in intimate contact with blood vessels.
Epilepsy is frequently present in MLC, being easily controlled
with antiepileptic drugs (Yalcinkaya et al., 2003). Our results also
indicated that MLC1 was associated mainly with membranes in
neurons. More precisely, colloidal gold particles were mostly
associated with vesicle structures, along axons. Considering the
observation of MLC1 expression in synaptic elements, i.e. axon
terminals and dendritic spines, MLC1 may be involved in synaptic
transmission. Lack of function of MLC1 could modify neuronal
function by altering neuronal transmission. Alternatively, epilepsy
could also be due to an astrocyte dysfunction (Tian et al., 2005).
As many other white matter disorders (Aicardi, 1993; Kaye,
2001; Schiffmann and Boespflug-Tanguy, 2001; Schiffmann and
van der Knaap, 2004; Singhal, 2005), MLC is characterized by a
decline in cognitive function and motor dysfunction. The most
dramatic disorders in these patients are motor impairments that
progressively increase (Singhal et al., 1996; van der Knaap et al.,
1995a). Most patients lose the ability to walk and reach wheelchair-
dependency after childhood, probably due to cerebellar ataxia and
minor pyramidal tract dysfunction (Koussa et al., 2005; Pascual-
Castroviejo et al., 2005; Saijo et al., 2003; Santos-Moreno and
Campos-Castello, 2002; Singhal etal., 1996; Topcu et al., 1998;van
der Knaap et al., 1995a). In accordance with the cerebellar ataxia
present in most MLC patients, MLC1 was highly expressed in the
cerebellum and its extrinsic connections early in the development.
Thus, MLC1 was expressed in the axonal tracts that connect
cerebellum, i.e. theolivocerebellar fibers(OCF), thespinocerebellar
tracts, the cerebellar commissure (CeC) and the inferior cerebellar
1 and 2). In addition, in adult stages, MLC1 was expressed in
cerebellar astrocytes, mainly in Bergmann glia.
In addition, our expression data indicated expression of MLC1 in
nor sensory abnormalities at neurological examination. Thus, conduc-
tion of all types of information, as visual and somatosensory evoked
potentials, is initially normal, later slowed and may become negative,
probably due to a CNS white matter disturbance (van der Knaap et al.,
1995a). Due to the fact that MLC1 was detected in sensory epithelia, a
peripheral contribution of this protein to the alterations described in
latest stages of MLC disease, apart to the disturbances caused by the
white matter pathology, may also be important. Alternatively, the
peripheral function of MLC1 may be compensated by other genes.
Some patients do not show linkage to the MLC1 locus (Blattner et al.,
2003; Patrono et al., 2003), indicating that other genes could play
similar tasks like MLC1.
In conclusion, this study provides an expression map for
MLC1 that will be useful to study MLC pathophysiology.
Moreover, our results further our knowledge about the precise
localization of MLC1 in neurons and astrocytes. We speculate
that some human phenotypes may be caused by an astrocytic
(Gorospe and Maletkovic, 2006) defect whereas others may be
caused by a neuronal defect.
We thank Marjo van der Knaap for reviewing the manuscript.
We thank Ole Peter Ottersen for initial EM experiments. We thank
Thomas J Jentsch for providing support to Tatjana Kharkovets. The
editorial assistance of Tanya Yates is acknowledged.
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