Developmental Brain Research 143 (2003) 33–46
D evelopmental changes in the Ca
aspartate–glutamate carrier aralar1 in brain and prominent expression
in the spinal cord
Milagros Ramos , Araceli del Arco
Juan Ramon Martınez-Morales , Keiko Kobayashi , Tomotsugu Yasuda , Elena Bogonez ,
Paola Bovolenta , Takeyori Saheki , Jorgina Satrustegui
, Beatriz Pardo , Alberto Martınez-Serrano ,
Departamento de Biologıa Molecular, Centro de Biologıa Molecular Severo Ochoa, Universidad Autonoma de Madrid, 28049 Madrid, Spain
bFacultad de Ciencias del Medio Ambiente, Universidad de Castilla La Mancha, Toledo, Spain
Departamento de Neurobiologıa del Desarollo, Instituto Cajal, Consejo Superior de Investigaciones Cientıficas, Madrid, Spain
dDepartment of Biochemistry, Faculty of Medicine, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8520, Japan
Accepted 18 March 2003
Aralar1 and citrin are two isoforms of the mitochondrial carrier of aspartate–glutamate (AGC), a calcium regulated carrier, which is
important in the malate–aspartate NADH shuttle. The expression and cell distribution of aralar1 and citrin in brain cells has been studied
during development in vitro and in vivo. Aralar1 is the only isoform expressed in neurons and its levels undergo a marked increase during
in vitro maturation, which is higher than the increase in mitochondrial DNA in the same time window. The enrichment in aralar1 per
mitochondria during neuronal maturation is associated with a prominent rise in the function of the malate–aspartate NADH shuttle.
Paradoxically, during in vivo development of rat or mouse brain there is very little postnatal increase in total aralar1 levels per
mitochondria. This is explained by the fact that astrocytes develop postnatally, have aralar1 levels much lower than neurons, and their
increase masks that of aralar1. Aralar1 mRNA and protein are widely expressed throughout neuron-rich areas in adult mouse CNS with
clear enrichments in sets of neuronal nuclei in the brainstem and, particularly, in the ventral horn of the spinal cord. These aralar1-rich
neurons represent a subset of the cytochrome oxidase-rich neurons in the same areas. The presence of aralar1 could reflect a tonic activity
of these neurons, which is met by the combination of high malate–aspartate NADH shuttle and respiratory chain activities.
2003 Elsevier Science B.V. All rights reserved.
Theme: Development and regeneration
Topic: Genesis of neurons and glia
Keywords: Mitochondria; Aspartate–glutamate carrier; Calcium dependent mitochondrial carrier; Brain development; Neuron maturation; Malate–aspartate
1 . Introduction
mitochondrial carriers (CaMCs) , and are activated by
calcium on the external face of the inner mitochondrial
The sequences of the two isoforms of AGC are highly
homologous, with 71% identity in the N-terminal half that
harbours the EF-hand domains and 84% within the C-
terminal half that encompasses the mitochondrial carrier
homology sequence . Both are nuclear-coded proteins,
with genes in human chromosome 7 [citrin [15,32] and 2
(aralar1, [7,30])]. The two isoforms have different tissue
distributions in adult animals, aralar1 being expressed
Aralar1 and citrin are two isoforms of the aspartate/
glutamate carrier (AGC) [15,23,39,40]. The AGC catalyses
an important reaction of the malate–aspartate NADH
shuttle that functions to deliver cytosolic redox equivalents
to mitochondria. The two isoforms of the AGC are the first
known members of the subfamily of calcium binding
*Corresponding author. Tel.: 134-91-3974872; fax: 134-91-3974799.
E-mail address: email@example.com (J. Satrustegui).
0165-3806/03/$ – see front matter
2003 Elsevier Science B.V. All rights reserved.
M. Ramos et al. / Developmental Brain Research 143 (2003) 33–46
predominantly in brain and skeletal muscle while citrin is
preferentially expressed in liver. However, both isoforms
are expressed in heart and many other adult tissues [1,41].
The adult distribution of aralar1 and citrin results from
specific developmental patterns. In muscle, both isoforms
are present in the dermomyotome at early embryonic
stages (E11 in mouse) and aralar1 becomes the major
isoform by E18 . In liver, there is an apparent isoform
switch around birth with a decrease in aralar1 levels and a
marked increase in those of citrin [10,41]. This switch
affected different cell types. Aralar1 expression decreased
in the haematopoietic cells from the embryonic liver while
citrin expression increased in fully differentiated hepat-
ocytes. As a result of this switch, citrin and aralar1 become
the major AGC isoforms in adult liver and haematopoietic
system, respectively. This is highly relevant, since a
genetic deficiency in citrin causes type 2 citrullinaemia
, a human disease that affects liver function but not the
haematopoietic or other systems.
Aralar1 is thought to be the main AGC isoform in brain.
The presence of an extramitochondrial site for calcium
regulation of the AGC allows the regulation by Ca
the malate–aspartate NADH shuttle, providing a novel
mechanism to transduce Ca
into the mitochondria in the form of reducing equivalent
supply, without the need of mitochondrial Ca
The crucial role of calcium in the CNS suggests that this
mechanism may be an important one. As a starting point in
understanding the role of brain AGC isoforms, we were
interested in the developmental changes in citrin and
aralar1 expression in the CNS. Both isoforms are present
in the early embryonic (E11) murine brain, but aralar1
becomes the major isoform toward the end of embryo-
genesis (E18) and it is the only one observed in mito-
chondrial fractions isolated from adult rat brain .
Beyond this, it is still unclear whether these isoforms are
expressed by neurons, glial cells or neural stem/progenitor
cells. Clarifying this point has been the first aim of this
Since the AGC is located in mitochondria its function
depends not only on the isoform levels prevalent in a
particular cell type but also on the number and differentia-
tion stage of mitochondria. Postnatal brain development
involves changes in the number and differentiation stage of
mitochondria, the extent of which is still unresolved
[6,12,31]. Therefore, we have also asked whether AGC
levels vary during neuronal maturation in vitro and during
postnatal development in vivo and have assessed whether
these changes simply follow those of mitochondrial con-
tent or increase beyond mitochondrial numbers.
A third aim of this work was to analyse whether aralar1
is distributed evenly in the postnatal and adult brain or
enriched in certain CNS areas. We have addressed this
question by studying the expression of the two AGC
isoforms in adult mouse brain by in situ hybridisation, by
Western blot studies in dissected brain areas, and through
immunocytochemistry in CNS sections. Since aralar1 is
localised to mitochondria, we have compared its distribu-
tion with that of cytochrome oxidase, the terminal enzyme
in the electron transport chain .
Our results indicate that aralar1, the only AGC isoform
of the adult brain, is localised preferentially in neurons,
where it becomes highly enriched during differentiation
both in vitro and in vivo. Surprisingly, this enrichment was
not homogeneous throughout the CNS, but was particu-
larly evident in neurons with a high cytochrome oxidase
content, suggesting that the main function of aralar1 in
neurons is to fuel energy-consuming functions.
2 . Methods
2 .1. Preparation of mitochondrial fractions
Mitochondrial fractions (P2) were obtained as indicated
byVilla et al. . Rat and mouse brains or neuronal, glial
and stem cells, were homogenised (1:10 w/v) in buffer A
(0.25 M sucrose, 20 mM Hepes pH 7.4, 10 mM KCl, 1.5
mM MgCl , 1 mM EDTA, 1 mM EGTA, 1 mM dithio-
threitol supplemented with a mixture of protease inhibitors,
1 mM phenylmethylsulfonylfluoride, 5 mM iodoacetamide,
and 0.1 mg/ml bacitracin). Nuclei and cell debris were first
removed by centrifugation (7003g, 10 min) and mito-
chondria then spun down (14,5003g, 15 min).
signals arising in the cytosol
2 .2. Neuronal cell culture
Cortical neuronal cultures were prepared from 18-day-
old Wistar rat embryos as previously described [28,29].
Cerebral cortices were enzymatically dissociated in phos-
phate-buffered saline (PBS) containing 1% bovine serum
albumin, 0.4 mg/ml papain, and 6 mM glucose. Disso-
ciated cells were collected by centrifugation (8003g, 5
min) and resuspended in medium supplemented with 20%
horse serum for 3 h. Then the medium was replaced,
reducing serum concentration to 5%. The medium was
partially replaced every second day with serum free
defined medium. This medium was based in a serum-free
defined medium formulation  with the modifications
described by Ruiz et al. . Cells were plated at a density
of 13 10 cells/cm on poly-L-lysine and laminin-coated
2 .3. Glial cell culture
Mixed glial (astroglial and oligodendroglial) cell cul-
tures were established as described by McCarthy and de
Vellis . Briefly, cerebral cortex from 1- to 2-day-old rat
pups were dissected and mechanically dissociated in HBSS
medium without Caand Mg
KH PO , 138 mM NaCl, 0.3 mM Na HPO , glucose 5.6
mM). Dissociated cells were collected by centrifugation
(5.33 mM KCl, 0.44 mM
M. Ramos et al. / Developmental Brain Research 143 (2003) 33–4635
(8003g, 5 min) and seeded at a density of one brain per
10-cm diameter plates precoated with poly-L-lysine. The
medium used in these cultures was DMEM–F12 (1:1)
glutamine and antibiotics. The medium was changed after
the first 4 h and then three times a week. The cultures were
maintained for a period of 10 days.
clone W82002 (ATCC) . This probe hybridises to a 2-
and a 4-kb fragment of mouse genomic DNA digested with
EcoRI, corresponding to exons 14 and 15, respectively.
The rat Aralar1 probe used was generated by RT-PCR
using 2 mg total RNA obtained from adult rat brain as
template. The oligonucleotides used, RAT1-AW (59-
plified a 650-bp fragment. This fragment hybridises to a
2.6-kb EcoRI rat genomic DNA fragment corresponding to
exons 16, 17 and 18. The DNA probe for the mito-
chondrial gene 12S rRNA was obtained as described by
Ostronoff et al. . DNA probes were labelled by nick
translation at 15 8C for 1 h using 50 mCi of (a P)dCTP
(3000 Ci/mmol). Hybridisation was carried out at 42 8C
for 18 h. After hybridisation, membranes were washed
according to the following protocol: (1) 63SSC at room
temperature for 10 min (where SSC is 0.15 M NaCl and
0.015 M sodium citrate); (2) 23SSC containing 0.1% SDS
at 65 8C for 10 min; and (3) 0.13SSC containing 0.1%
SDS at 65 8C for 10 min. Membranes were exposed to
X-ray films and analysed by laser densitometric scanning.
2 .4. Neural stem cell cultures
The cerebral cortex (and underlying neuroepithelium,
ventricular zone) from 16- and 18-day-old embryos was
removed from mice and rats, respectively. Cells were
enzymatically dissociated in phosphate-buffered saline
(PBS) containing 1% bovine serum albumin, 0.4 mg/ml
papain, and 6 mM glucose. Dissociated cells were col-
lected by centrifugation (8003g, 5 min) and plated at
1310 cells/ml into 25-cm
pretreatment. The culture medium was composed of
DMEM–F12 (1:1), N2 supplements (Invitrogen) and 1%
bovine serum albumin. Human recombinant (hr-)FGF-2
and hr-EGF (Research and Development Systems) were
each used at 20 ng/ml . After 7 days in vitro, spheres
grown in flasks were removed, spun down (8003g, 5 min)
and resuspended in 2 ml of medium. The spheres were
mechanically dissociated into single cells by trituration
with a fire polished Pasteur pipettes. This procedure was
repeated every 6–8 days until the desired number of cells
were reached. Cells were used at passage 6 where cultures
are highly pure in neurosphere forming cells.
flasks with no substrate
2 .7. MTT reduction assays
To study the MTT (3(4,5-dimethylthiazol-2-yl)2,5-
diphenyltetrazolim bromide) reduction capacity of cerebral
cortex neurons during in vitro culture, we used a modi-
fication of the assay developed by Hansen et al. . After
4–15 days in vitro (DIV), the culture medium was
replaced by 0.5 ml MEM and 125 ml of a 5 mg/ml stock
solution of MTT and incubated in the dark for 30 min at
37 8C to allow for formazan formation. Cells were then
lysed in SDS (20%, w/v), in 50% aqueous N,N-dimethyl
formamide, pH 4.7), and after incubation for 18 h at 37 8C,
the amount of formazan was estimated from the absor-
bance at 570 nm, with the lysis solution as blank.
To study MTT reduction by mitochondria, neuronal or
astrocyte cultures (10cells/cm ) were switched to a
glucose-free medium (HCSS: 5.4 mM KCl, 0.12 M NaCl,
0.8 mM MgCl , 1 mM CaCl
7.4). About 4 h later, cultures were washed in Ca
HCSS and preincubated for 30 min in the same medium
containing 20 mM digitonin. This digitonin concentration
was sufficient to make Ca
without loss of cytosolic components . Then, MTT (1
mg/ml) was added alone or together with either 1 mM
glutamate, 5 mM malate and 10 mM lactate, or 15 mM
glycerol-3-phosphate and 10 mM lactate, and incubated at
37 8C in the dark for 30 min. Cell lysis and determination
of formazan formation were performed as indicated above.
2 .5. Western blot analysis
Mitochondrial fractions (P2) were subjected to electro-
phoresis on a 8% polyacrylamide gel in the presence of
SDS and transferred to nitrocellulose membranes (Sleicher
and Schuell). Antibodies against aralar1 , citrin 
and b-F ATPase  were used as first antibodies with a
goat anti-rabbit IgG-peroxidase conjugated as a secondary
antibody and processed with the luminiscence technique
ECL (Amersham Pharmacia).
and 25 mM HEPES, pH
2 .6. Southern blot analysis
Total cellular DNA was extracted from cultured cells,
and rat and mice brain tissue after digestion with RNAse
and proteinase K, using standard protocols. DNA samples
were digested with EcoRI. The DNA fragments were
resolved on 0.8% agarose gels and transferred to nylon
membranes (Hybond-N, Amersham Pharmacia Biotech).
probes. Two types of DNA probes were used in this study,
those derived from Aralar1 as a nuclear-encoded gene,
and those derived from the gene for 12S rRNA as a
mitochondrial gene . The mouse Aralar1 probe used
was a 381-bp PstI fragment obtained from the mouse EST
available to mitochondria
2 .8. In situ hybridisation
The 381- and 557-bp fragments of aralar1 and citrin
cDNA described by del Arco et al. , were transcribed
M. Ramos et al. / Developmental Brain Research 143 (2003) 33–46
to generate digoxigenin-labelled anti-sense and sense
cRNA probes. In situ hybridisations were performed on
free-floating 50-mm-thick vibratome sections from em-
bryonic spinal cord (E14.5) or adult mouse brains and
spinal cord, following standard protocols. Briefly, animals
were anaesthetised, perfused with 4% paraformaldehyde in
phosphate buffer (PB) and the isolated brain was postfixed
overnight in the same fixative. Embryos were fixed by
immersion. After washes in PBS the brain or the spinal
cords was included in an agarose gel and sliced on a
vibratome (Leica). Sections were collected in PBS and
processed in free-floating. After permeabilisation with
Proteinase K (10 mg/ml in PB-saline containing 0.1%
Tween) for 3–5 min at room temperature, sections were
post-fixed in 4% paraformaldehyde in PB, pre-hybridised
for 1 h at 65 8C in 50% formamide and incubated with
probes for 16 h at 65 8C. Post-hybridisation washes were
performed at the same temperature and in the same buffer.
The staining patterns described below were obtained only
with antisense riboprobes and not with control sense
min at 37 8C in a humidified chamber and some were also
incubated at 37 8C for 5–20 min in Tris–hydrochloride 10
mM pH 7.4 containing 10 mg/ml proteinase K (Roche).
Sections were then briefly rinsed with PBS (4 8C) to stop
the reaction, and processed for immunohistochemistry.
None of these treatments modified aralar1 or COI immuno-
3 . Results
3 .1. AGC isoforms in different brain cell types
To determine whether the two AGC isoforms are
differentially expressed in neurons, glial and neural stem
cells, we investigated the levels of aralar1 and citrin in
primary cultures of cortical neurons and glial cells ob-
tained from rat embryos at E18, and in neural stem cells
obtained from both mouse and rat embryos at E16 and
Mitochondrial fractions from the above cultures were
processed in parallel with mitochondrial fractions from
HEK293 cells with known citrin-to-aralar1 ratios ; this
was necessary to correct for the different sensitivity of
anti-aralar1 and anti-citrin antibodies. The levels of the
b-subunit of the b-F ATPase were used to control for the
amount of mitochondrial protein loaded (Fig. 1A). Calcula-
tion of aralar1 and citrin content standardised to that of
b-F ATPase indicated that neurons had no citrin and a
aralar1/b-F ATPase ratio of about 3, whereas glial cells
showed citrin/b-F ATPase and aralar1/b-F ATPase ratios
of 2.7 and 0.8, respectively (Fig. 1A). Mouse and rat
neural stem cells displayed an AGC content somehow
similar to those of neurons with lower levels of aralar1
expression and no detectable citrin (Fig. 1B). In conclu-
sion, aralar1 seems to be the only isoform present in both
neurons and neural stem cells, while citrin is prevalent in
2 .9. Immunocytochemistry
The animals were anaesthetised with sodium pentobarbi-
tal, and perfused through the cardiac ventricle, first with 50
ml of 0.9% NaCl followed by 250 ml of fixative solution
containing 4% paraformaldehyde in 0.1 M phosphate
buffer, pH 7.4, at room temperature. Brain and spinal cord
were removed, postfixed at 4 8C for 24 h, and cryop-
rotected by immersion in 30% sucrose. Free-floating
cryostat 40-mm-thick sections were first quenched with 3%
hydrogen peroxide in 10% methanol for 20 min in
potassium-phosphate-buffered saline (KPBS) to remove
endogenous peroxidase activity. Following this treatment,
the sections were preincubated for 2–3 h in KPBS
containing 5% horse serum and 0.25% triton X-100, to
block unspecific binding of antibodies and then incubated
overnight with anti-aralar1 antibody (dilution 1:100) or
anti-cytochrome oxidase subunit I (COI) (2.5 mg/ml;
Molecular Probes) in 1% horse serum and 0.25% Triton
X-100 in KPBS. Secondary biotinylated antibody (goat-
anti-rabbit or horse-anti-mouse; Vector 1:150) was then
incubated for 1–2 h, followed by a 1-h reaction with
avidin–biotin–peroxidase complexes (regular ABC kit
Vectastain, Vector). Sections were developed using 0.05%
3,3-diaminobenzidine (Sigma) as a chromogen in the
presence of 0.03% H O in KPBS for 1–2 min. Sections
were mounted onto polylisine-coated slides, dehydrated,
delipidated, and mounted in DPX (BDH). Controls in-
cluded omission of the primary serum or its substitution by
non-immune rabbit serum. No specific staining was visible
on such preparations.
To unmask epitopes, before undergoing the conventional
immunohistochemical analysis for aralar1 and COI de-
scribed above, some sections were prewarmed during 10
3 .2. Aralar1 levels strongly increase during in vitro
Neurons undergo maturation during brain development
and during in vitro culture [5,6,18] with changes that may
include modifications in the number and differentiation
state of the mitochondria. We therefore asked whether the
content of mitochondrial DNA and that of AGC isoforms
varied in neurons during in vitro culture. Under our culture
conditions mitochondrial maturation increased steadily and
reached a maximum between 9 and 11 days in vitro, as
judged by the increase in the capacity to reduce MTT (Fig.
2A), an assay sensitive to mitochondrial inhibitors [2,17].
Since neuronal survival drops beyond 15 days in culture,
we compared AGC levels and mitochondrial DNA content
between three and 10 DIV, the time when neuronal
differentiation is maximal . Fig. 2 shows that the levels
M. Ramos et al. / Developmental Brain Research 143 (2003) 33–4637
Fig. 1. AGC isoforms in primary neuronal (1–3 DIV) and astrocyte cultures, and neural stem cells. (A) Mitochondrial fractions (25 mg protein/lane) from
glial cells (GLIA), and cortical neurons (neurons) derived from embryonic rat brain and HEK293 cells (HEK) were probed by Western blotting with
antibodies against aralar1 (1:5000) (a) or citrin (1:10,000) (c) and with antibodies against bF1-ATPase. Histograms (b,d) represent the quantification of the
intensities of the bands with respect to that of bF1-ATPase, and with respect to HEK293 cells that have a citrin/aralar1 ratio of 2.4  and are
means6S.E.M. of two to five experiments. (B) Mitochondrial fractions (25 mg protein/lane) from rat or mouse neural stem cells (NSC), and HEK293 cells
(HEK), were probed by Western blotting as described above (a,c). The histogram (b) corresponding to means6S.E.M. of two experiments, represents the
quantification of the results in (a). As shown in (c), citrin levels were not detectable in either rat or mouse neural stem cells.
of aralar1 in mitochondrial fractions increased markedly
with the time of culture (Fig. 2B,C), while those of citrin
remained undetectable (Fig. 2D). During the same period
of time, mitochondrial DNA, measured as the ratio of
mitochondrial DNA/total DNA, showed a remarkable 3.3-
fold increase (Fig. 2E,F), reflecting mitochondrial prolifer-
In conclusion, aralar1 content is strikingly high in
differentiated neurons because both the mitochondrial
content (Fig. 2E,F) and the level of aralar1 expression per
mitochondria (aralar1/b-F ATPase ratio increases 2.7-
fold) increase during in vitro maturation.
3 .3. The rise in aralar1 levels during in vitro neuronal
differentiation correlates with an increase in the activity
of the malate–aspartate NADH shuttle
The exchange of aspartate against glutamate catalyzed
by the AGC is the only irreversible reaction in the malate–
aspartate NADH shuttle and, as such, is a site for its
control . An increased expression of the AGC should
up-regulate the activity of the shuttle leading to an increase
in NADH production and MTT reduction in mitochondria
. In order to test this possibility, MTT reduction was
measured in digitonin (20 mM) permeabilized neurons
M. Ramos et al. / Developmental Brain Research 143 (2003) 33–46
Fig. 2. MTT reduction, AGC isoforms and mitochondrial DNA content during in vitro differentiation in primary neuronal cultures. (A) MTT reduction
capacity of cerebral cortex neurons during in vitro culture. The results are means6S.E.M. of three to four experiments. (B,D). Mitochondrial fractions (25
mg protein/lane) derived from neurons of three to 10 DIV and from HEK293 cells were probed by Western blotting for aralar1 and citrin levels as
described in the legend to Fig. 1. (C) Quantification of the results in (B) corresponding to means6S.E.M. of five experiments. (E) Total cellular DNA (15
mg DNA/lane) were probed by Southern blotting with a probe for mitochondrial ribosomal RNA 12S and with a probe for nuclear DNA (aralar1). (F)
Quantification of the results in (E) expressed as means6S.E.M. of three experiments. The differences between three and 10 DIV were significant,
*P,0.05; **P,0.005 (paired, t- test).
incubated with no additional substrates (none) or either 1
mM glutamate, 5 mM malate, 10 mM lactate (GML), or 5
mM glycerol-3-phosphate, 10 mM lactate (G3P), or 5 mM
succinate (Succ). The first two conditions, GML and G3P,
provide substrates for the malate–aspartate or alfa-
glycerophosphate shuttle, respectively, while the third fuels
The glycerophosphate shuttle is very active in brain
. Accordingly, we observed that the G3P shuttle was
much more active than that of malate–aspartate in imma-
ture neurons at 3DIV (Fig. 3A) as measured by the levels
of MTT reduction. In fact, it is as active in immature
neurons as in glial cells (results not shown). As expected
by the increase in mitochondrial content, MTT reduction
was higher in neurons at 10 DIV in all the conditions
tested (Fig. 3A,B). However, proportionally, the largest
increase observed with neuronal differentiation was that
associated to the malate–aspartate shuttle (Fig. 3B, GML),
suggesting that aralar1 expression and malate–aspartate
shuttle function are important events in this process.
M. Ramos et al. / Developmental Brain Research 143 (2003) 33–4639
aralar1 along postnatal development up to P7 in both rats
and mice but this ratio levelled off and even decreased
slightly beyond this stage in both species [Fig. 4A(b),
The stabilization of the aralar1-to bF1-ATPase ratio
beyond P7 is mainly due to the increase in bF1-ATPase
during this period [Fig. 4A(a), B(a)]. This increase is
consistent with the data of Schonfeld and Bohnensack
, who found that mitochondrial content in rat brain
increased steadily (from 5% to about 8% of total protein)
between P0 and 2 months. To study whether the increase
in mitochondrial protein content is associated with mito-
chondrial proliferation, we determined the variations in
mitochondrial DNA during postnatal development. Fig.
4A(d,e), B(d,e) shows that the ratio of mitochondrial DNA
to nuclear DNA increases only slightly until P7 but it is
more evident at P17 and beyond. Thus, our results indicate
that the increase in mitochondrial DNA is a relatively late
3 .5. Expression of AGC isoforms in adult CNS
We have previously shown that Aralar1 and to a lower
extent Citrin are expressed in the CNS during embryonic
development with similar expression patterns . We
have now shown that citrin is clearly expressed in cultured
glial cells (Fig. 1). We were therefore surprised by the
observation that citrin was not detectable in postnatal
brains. Even more so, considering that there are between
10 and 50 times more glial cells than neurons in the central
nervous system of vertebrates . To verify our findings
and to get more information on the precise distribution of
aralar1, we hybridised vibratome sections of adult mouse
brain with probes for citrin and aralar1 mRNAs. No signal
above background level was ever observed with the probe
for citrin (data not shown), confirming that this AGC
isoform is completely down regulated in the brain after
birth. In contrast, aralar1 mRNA was widely distributed in
many neuron-rich areas, as determined by comparing the
pattern of expression obtained in the presence of anti-sense
probe (Fig. 5), with those obtained with the sense control
or omitting the probe from the hybridisation mixture (inset
in Fig. 5I). Staining was in general associated to large cell
body neurons scattered throughout the brain. Particularly
evident were the mitral cell and glomerular layers of the
olfactory bulb (Fig. 5A–C); neurons of the layer 2 and 5
of the cortex (Fig. 5D–F) and the pyramidal layer and the
dentate gyrus of the hippocampus (Fig. 5D,G,H). In the
cerebellum, aralar1 was strongly expressed in the Purkinje
cells and in the deep nuclei (Fig. 5I–K). The brainstem
was the brain region with the major accumulation of
aralar1-positive neurons, including the pontine (Fig. 5L,M)
and inferior olivary (not shown) nuclei, that project to the
cerebellum, and the nuclei of the cranial nerves, including
the facial and the vestibular ones (Fig. 5L,N,O).
Fig. 3. MTT reduction by mitochondria in primary neuronal cultures
during in vitro differentiation. (A) Digitonin (20 mM)-permeabilized
neuronal cultures at three or 10 DIV were tested for reduction of MTT in
the presence of either 1 mM glutamate, 5 mM malate, 10 mM lactate
(GML), 15 mM glycerol-3-phosphate and 10 mM lactate (G3P), 2 mM
succinate (Succ) or in the absence of additions (none). The results
represent means6S.E.M. of two experiments performed in triplicate. The
presence of substrates resulted in a higher MTT reduction where indicated
(*P,0.05; **P,0.005, paired t-test). (B) The -fold increase in MTT
reduction in each condition during neuronal differentiation is shown.
Differences with respect to no substrates are indicated (**P,0.005,
3 .4. Postnatal development of AGC isoforms in mouse
and rat brain in vivo
The results described above indicated that the levels of
aralar1 expression and the activity of the malate–aspartate
shuttle increased during neuronal differentiation in vitro.
Therefore, we next asked whether similar changes occurred
in vivo, during brain postnatal development. To this end
we analysed the level of aralar1 and citrin expression by
Western blotting of crude mitochondrial fractions obtained
from whole forebrain of mouse and rat animals at different
developmental stages (E18, P0, P7, P17, P90). The levels
of aralar1 expression in mitochondrial fractions slightly
increased throughout postnatal development in both murine
species [Fig. 4A,(a)B(a)], whereas those of citrin remained
undetectable at all stages [Fig. 4A(c)B(c)]. The levels of
bF1-ATPase that were used as control for the amount of
mitochondrial protein also increased during this time
showed that mitochondria become slightly enriched in
M. Ramos et al. / Developmental Brain Research 143 (2003) 33–46
Fig. 4. AGC isoforms and mitochondrial DNA content during rat and mouse brain development. A (a,b,c) B (a,b,c). Mitochondrial fractions (25 mg
prot/lane) from total brain obtained from E18, P0, P7, P17 and 3-month-old rat (A) and mice (B) were probed for aralar 1(a), citrin (c) as described in the
legend to Fig. 1, and aralar1 contents normalised to b-F ATPase are shown in b. The results are means6S.E.M. of four experiments. A(d,e) B(d,e). Total
rat (A) or mouse (B) brain DNA obtained at the stages indicated above (15 mg DNA/lane) were analysed by Southern blotting with a probe for
mitochondrial ribosomal RNA 12S and with a probe for nuclear DNA (aralar1). Panels (e) show the quantification of the results in (d) expressed as
means6S.E.M. of three experiments. In panels A(b,e) and B(e) the statistical significance of the difference between E18 and the different ages was:
*P,0.05 (paired t-test).
Immunohistochemistry with anti-aralar1 antibodies con-
firmed that the protein was widely distributed throughout
the brain, and many brainstem nuclei were also enriched in
aralar1 protein (Fig. 6), particularly the facial (Fig. 6A–
C), and superior olivary nuclei (Fig. 6D–F) while neuron
bodies were weakly stained in the pontine nucleus (Fig.
6G–I). However, immunostaining of the hippocampal
pyramidal CA1–CA2 layers, or the dentate gyrus was
much weaker (results not shown). Similarly, no particular
staining was detected in other regions with ongoing cell
production such as the subventricular zone (results not
shown). Throughout the mouse brain the distribution
pattern of aralar1 appeared to be quite similar to that of
cytochrome oxidase, a marker of mitochondrial respiratory
chain with high expression in grey matter [9,14]. In
contrast to what happens in the brain, where both AGC
isoforms are present in early embryos, we found that only
aralar1, but not citrin, is expressed during embryonic
development in the spinal cord and localised to the
developing motor neurons (Fig. 7A). Strong motor neuron
expression was also observed in the adult spinal cord
where aralar1 mRNA is abundantly localised also in
neurons of the intermediate and dorsal grey matter (Fig.
7B,C). This strong expression of aralar1 in the spinal cord
M. Ramos et al. / Developmental Brain Research 143 (2003) 33–46 41
Fig. 5. Distribution of Aralar1 transcripts in the adult mouse brain.Vibratome sections from adult mouse brain were hybridised with digoxigenin-labelled
probes specific for the aralar1 gene. Images represent sagital (A–C, E, F, I–O) or frontal (D, G, H) views of the olfactory bulb (A–C); dorsal half of the
prosencephalon at mid-diencephalic levels (D); neocortex (E, F); hippocampal formation (G, H); cerebellum (I–K) and brainstem (L–O). Higher power
images show the strong localisation of aralar1 in different neuronal cell types, including the mitral (B) and periglomerular cells (C) of the olfactory bulb;
neurons of layer 5 of the cortex (F); pyramidal cells of the hippocampus (H), Purkinje cells (J) and deep nuclei (K) of the cerebellum; pontine (M), facial
(N) and medial vestibular nuclei (O) of the brainstem. Note that myelinated axonal tracts in corpus callosum, the internal capsule (arrowheads in D) or the
white matter of the cerebellum, show artifactual staining due to unspecific retention of the colour reaction product as shown in the inset in I. Abbreviations:
(cx) cortex; (dcn) deep cerebellar nuclei; (dg) dentate gyrus; (ep) ependymal layer; (fn) facial nucleus (gl) glomerular layer; (ha) habenula; (hp)
hippocampus; (mc), mitral cells; (ml) mitral cell layer; (mVe) medial vestibular nucleus; (PC) Purkinje cells; (pcl) Purkinje cell layer; (pgc) periglomerular
cells; (pn) pontine nuclei; (rsgc) retro-splenial granular cortex; (vmt) ventral posterior nuclei of the thalamus. Scale bar: A, D, I, L, 625 mm; E, K, M, N,
125 mm; B, C, F, H, J, O, 30 mm.
M. Ramos et al. / Developmental Brain Research 143 (2003) 33–46
Fig. 6. Distribution of aralar1 protein in the adult mouse brain. Cryostat sections from adult mouse brain were immunostained with antibodies specific to
the aralar1 protein. Images represent coronal sections of three nuclei of the adult mouse brainstem with noticeable aralar1 staining pattern. Facial nucleus
(A–C), superior olivary complex (D–F) and pontine nuclei (G–I). B,E,H are higher power images of the bracketed area in A, D and G, respectively. C, F
and I are higher power views of the bracketed area in B, E and H, respectively. Scale bar: A (D and G) 800 mm, B (E and H) 100 mm, C (F and I) 25 mm.
was confirmed by immunohistochemical studies of aralar1
protein in mouse (Fig. 7D,E) and rat sections (Fig. 7F,G).
Staining was observed not only in the cell bodies but also
along the neurites of the motor neurons as they entered in
the ventral roots (Fig. 7L,M). Aralar1-rich neurons in the
spinal cord are localised in regions with high COI levels
both in mouse (compare Fig. 7D,E, and H,I) and rat
(compare Fig. 7F,G and J,K). Similar large cell body
neurons are labelled with both antibodies (Fig. 7E,G and
I,K). The cellular localisation of aralar1 immunostaining
was uniform throughout the cytosol with occasional punc-
Only a subset of the COI positive neuronal bodies are
highly labelled with anti-aralar1 antibodies. For example,
while the number and distribution of COI and aralar 1
positive neurons is very similar in the ventral spinal cord
(Fig. 7P,Q), the dorsal spinal cord contains many COI
positive neurons but few aralar1-labelled neurons (Fig.
M. Ramos et al. / Developmental Brain Research 143 (2003) 33–46 43
Fig. 7. Distribution of Aralar1 transcripts and protein in the mouse and rat spinal cord. Vibratome (A–C) or cryostat (D–Q) sections from embryonic
E14.5 (A) or adult mouse spinal cord (B–E, H, I) or adult rat spinal cord (F, G, J–Q) were hybridised with digoxigenin-labelled probes specific for the
aralar1 gene (A–C), immunostained with antibodies specific to the aralar1 protein (D, F, H, J, L–N, P) or immunostained with antibodies specific to the
cytochrome c oxidase protein (E, I, G, K, O, Q). Images present cross-sectional (A, B, D–Q) or longitudinal (C) views of the spinal cord (hemicords in B,
D–G, L; dorsal horn in N, O; ventral horn in M, P, Q). Images in H–K are higher power views of the bracketed area in D–G, respectively. Panel M is a
higher power view of the bracketed area in L. Note the intense staining in the motor neurons in the embryonic (blown up in the inset in (A) and adult spinal
cord. Aralar1 protein is localised in the cell bodies (arrow in M) and along the axons (arrowheads in M). No aralar1-positive cells were detected in the
white matter. Abbreviations: (a) anterior; (d) dorsal; (drg) dorsal root ganglia; (mn) motor neurons; (v) ventral. Scale bar: A, 100 mm; B,C, 50 mm; D,E 100
mm; F,G, 250 mm; H–K, 25 mm; L, 150 mm; M, 50 mm; N–Q, 100 mm.
M. Ramos et al. / Developmental Brain Research 143 (2003) 33–46
7N,O). Treatments to unmask epitopes and increase im-
munolabelling (prewarming, protease addition, ), did
not increase aralar1 staining any further.
Thus, the spinal cord appeared as a CNS area par-
ticularly enriched in Aralar 1 expression. The enrichment
of aralar1 in the spinal cord was further verified by
assessing the ratio of aralar1/bF1-ATPase content of the
spinal cord as compared to other CNS regions. Western
blot analysis indeed revealed that the ratios (ng aralar1/
arbitrary bF1-ATPase units) were 4.7660.171 in cere-
bellum and 8.061.38 in spinal cord [n53, P50.029 (one
tailed, paired, student t-test)].
place in neurons. Neuronal mitochondria also undergo
differentiation during this period, and this explains the
small but consistent increase in aralar1 levels in brain
mitochondria from E18 to P7 in both rat and mice [Fig.
4A(b), B(b)]. The second phase (from P7 to 2 months)
corresponds to the final maturation of synapses in the
cortex . This second phase coincides with the largest
increase in mitochondrial content [Fig. 4A(e), B(e)] and
corresponds to the proliferation of glial cells that have
fewer mitochondria than neurons [27,36]. Moreover, these
glial mitochondria are poor in aralar1, and therefore, the
multiplication of glial cells would mask any specific
proliferation or differentiation of neuronal mitochondria in
this time window. This probably accounts for the postnatal
plateau or even decrease in aralar1 levels in adult brain
mitochondria (Fig. 4).
The impact of mitochondrial proliferation on neuronal
function in vitro may be also estimated from the changes
in the MTT reduction assay during maturation. Since this
assay is carried out in digitonin-treated cells, it estimates
exclusively the mitochondrial capacity to reduce MTT.
Notably, neuronal maturation resulted in an increase in
MTT reduction with all three added substrates (GML, G3P,
Succ), and even with endogenous substrates (none) (Fig.
3A,B), which clearly reflects the increase in mitochondrial
content. On the other hand, the increase in MTT reduction
in the presence of the substrates of the malate–aspartate
shuttle (GML) clearly supersedes that obtained with all
others, underlying the functional importance of mitochon-
drial differentiation and enrichment of aralar1 in mito-
chondria during neuronal maturation.
The malate–aspartate NADH shuttle is most important
in mature neurons, since glucose metabolism is mainly
aerobic, and the shuttle is required to meet NAD demands
from glycolysis . On the other hand, glial cells are far
less dependent on the transport of reducing equivalents to
mitochondria, since in different glial cell types, such as
retinal Muller cells, glucose is transformed into pyruvate
which is reduced to lactate and exported from the cells
. Accordingly, LaNoue et al.  have found that
malate–aspartate shuttle activity in Muller cells is very
small. Glutamate utilisation by mitochondria from as-
trocytes and Muller cells is largely dependent on glutamate
dehydrogenase, which is highly enriched in these cells
[25,38]. This probably explains why aralar1 is preferen-
tially enriched in neurons rather than glial cells in vivo.
As a member of the malate–aspartate shuttle, a reason-
able premise is that the metabolic requirement for aralar1
should be uniform, and therefore, the protein should be
evenly distributed among all neuronal mitochondria. We
were therefore surprised to find that aralar1 is enriched in a
subset of CNS neurons in brainstem areas and in the spinal
cord.We have not identified neurochemically which are the
neurotransmitters in these aralar1-enriched neurons from
the spinal cord, and, therefore, any comment on the
function of aralar1 in these cells is highly speculative. The
4 . Discussion
The results from this study show that aralar1 is the only
AGC expressed in murine CNS neurons where it under-
goes a very marked enrichment during maturation in vitro.
Cultured neural stem cells behave similarly to neurons
showing, albeit at lower level, aralar1 but not citrin
expression (Fig. 1B). Finally, mixed glial cell cultures
have the lowest content in aralar1 per mitochondria but
they do express citrin.
Aralar1 enrichment during neuronal maturation in vitro
may be accounted for by two mechanisms: an increase in
the number of mitochondria per cell (mitochondrial prolif-
eration), as reflected in the increase of the mitochondrial
DNA/nuclear DNA ratio during maturation (Fig. 2F) and
the increase in aralar1 levels per mitochondria (Fig. 2B,C),
or mitochondrial differentiation. The process of mitochon-
drial proliferation in vitro shown in this study fits with the
findings of Cordeau-Lossouarn et al. , who compared
neuronal mitochondria differentiation in vitro and in vivo.
Using synaptophysin and neuron specific enolase as
markers, these authors found that in vitro differentiation
between DIV1 and DIV20, for brain neuronal cultures
obtained from 15-day-old rat embryos, is equivalent to in
vivo differentiation between foetal day 15 and postnatal
day 10. During this time, mitochondrial content increased
2-3-fold in vitro from plating to DIV 13, which matches
with the 3.3-fold increase in mitochondrial/nuclear DNA
found in this study.
Paradoxically, the developmental pattern of aralar1
levels in brain mitochondria during postnatal development
fails to show any conspicuous enrichment in this protein,
either in rat or mouse brain [Fig. 4A(b), B(b)]. The most
likely explanation of this paradox is the following. In brain
cortex, the kinetics of accumulation of mitochondrial mass
can be correlated with the successive steps of brain
maturation . There is a first phase of accumulation from
E15 to P7, spanning over a period where neurons migrate,
and sprouting of neurites and formation of transient
connections occur. In this phase mitochondrial prolifer-
ation is small, as judged by the changes in mit DNA/
nuclear DNA ratios [Fig. 4A(e), B(e)], and it possibly takes
M. Ramos et al. / Developmental Brain Research 143 (2003) 33–4645
cell body of many cholinergic motoneurons is located
close to the same ventral root areas of the spinal cord,
which are enriched in aralar1. It has been reported that
specific motoneuron functions may require malate–aspar-
tate shuttle activity . The acetyl-CoA moiety of acetyl-
choline is formed in mitochondria and then exported to the
cytosol via the tricarboxylate carrier. While defects in this
carrier would be expected to impair acetylcholine syn-
thesis, under certain conditions inhibition of malate–aspar-
tate shuttle activity (with transaminase inhibitors) also
results in decreased acetylcholine synthesis. For example,
Cheeseman and Clark  found that glucose incorporation
into acetylcholine was blocked by transaminase inhibitors,
suggesting that inhibition of the shuttle activity leads to
decreased carbon flux through glycolysis and the citric acid
cycle, and decreased intramitochondrial citrate, the molec-
ular exporter of acetyl-CoA from mitochondria. However,
it is unlikely that aralar1 overexpression reflects a higher
demand of the malate aspartate shuttle for acetylcholine
synthesis, since the brain cholinergic neurons were not
stained with anti-aralar1 antibodies (results not shown).
On the other hand, the uniform-distribution concept of
mitochondrial, housekeeping proteins is probably grossly
incorrect, since Wong-Riley and colleagues have shown
that cytochrome oxidase, the terminal enzyme in the
respiratory chain, has a most uneven distribution in CNS
neurons [9,21,36]. The enrichment in cytochrome oxidase
in sets of neurons and subsets of neuronal segments is
thought to be required to meet energy demands imposed by
repolarization of postsynaptic membranes depolarised by
glutamate or other excitatory neurotransmitters. Neuronal
bodies or neuronal segments receiving inhibitory inputs
have lower energy demands and levels of cytochrome
oxidase since repolarization subsequent to hyperpolarisa-
tion is largely passive . The presence of aralar1 in a
subset of cytochrome oxidase-rich neurons in the brain and
spinal cord could thus reflect a higher energy demand of
these neurons, which is met by the combination of high
malate–aspartate NADH shuttle and respiratory chain
activities. These neurons are likely to be tonically active,
maintaining a greater capacity to fuel high rates of
spontaneous or synaptically evoked activity.
The presence of an extramitochondrial site for calcium
binding in aralar1 may allow the regulation by Ca
malate–aspartate NADH shuttle, by global and/or rela-
tively small Casignals, without the need of mito-
chondrial calcium uptake . Whether this regulatory
mechanism is involved in the tonic and/or stimulated
activity of the spinal cord neurons rich in aralar1 remains
to be established.
Ciencia y Tecnologıa, Comunidad Autonoma de Madrid,
Fondo de Investigaciones Sanitarias del Ministerio de
Sanidad y Consumo, by Health Sciences Research Grants
(H11-Genome-002) from the Ministry of Health and
Welfare in Japan, by Grants-in-Aid for Scientific Research
(B-12470518) from the Japan Society for the Promotion of
Science and by an institutional grant from the Fundacion
Ramon Areces to the Centro de Biologıa Molecular
‘Severo Ochoa’. We thank Prof. J.M. Cuezva for the
generous gift of anti-b-F ATPase antibodies, and Prof.
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