Brain Struct Funct
DOI 10.1007/s00429-013-0691-7
ORIGINAL ARTICLE
Localisation of N-acetylaspartate in oligodendrocytes/myelin
Kaja Nordengen • Christoph Heuser •
Johanne Egge Rinholm • Reuben Matalon
Vidar Gundersen
•
Received: 14 August 2013 / Accepted: 14 December 2013
Ó Springer-Verlag Berlin Heidelberg 2013
Abstract The role of N-acetylaspartate in the brain is
unclear. Here we used specific antibodies against N-acetylaspartate and immunocytochemistry of carbodiimidefixed adult rodent brain to show that, besides staining of
neuronal cell bodies in the grey matter, N-acetylaspartate
labelling was present in oligodendrocytes/myelin in white
matter tracts. Immunoelectron microscopy of the rat hippocampus showed that N-acetylaspartate was concentrated
in the myelin. Also neuronal cell bodies and axons contained significant amounts of N-acetylaspartate, while
synaptic elements and astrocytes were low in N-acetylaspartate. Mitochondria in axons and neuronal cell bodies
contained higher levels of N-acetylaspartate compared to
the cytosol, compatible with synthesis of N-acetylaspartate
in mitochondria. In aspartoacylase knockout mice, in
which catabolism of N-acetylaspartate is blocked, the levels of N-acetylaspartate were largely increased in oligodendrocytes/myelin. In these mice, the highest myelin
concentration of N-acetylaspartate was found in the cerebellum, a region showing overt dysmyelination. In organotypic cortical slice cultures there was no evidence for Nacetylaspartate-induced myelin toxicity, supporting the
notion that myelin damage is induced by the lack of Nacetylaspartate for lipid production. Our findings also
implicate that N-acetylaspartate signals on magnetic resonance spectroscopy reflect not only vital neurons but also
vital oligodendrocytes/myelin.
Keywords Cerebellum Immunogold leukodystrophy Canavan disease Glutamate Aspartate
Electronic supplementary material The online version of this
article (doi:10.1007/s00429-013-0691-7) contains supplementary
material, which is available to authorized users.
Introduction
K. Nordengen C. Heuser J. E. Rinholm V. Gundersen (&)
Department of Anatomy, Institute of Basic Medical Sciences,
University of Oslo, PO Box 1105, 0317 Oslo, Norway
e-mail: vidar.gundersen@medisin.uio.no
C. Heuser
Institute of Molecular Medicine and Experimental Immunology,
Rheinische Friedrich Wilhelms University, Bonn, Germany
J. E. Rinholm
Janelia Farm Research Campus, Howard Hughes Medical
Institute, Ashburn, USA
R. Matalon
Department of Pediatrics, University of Texas Medical Branch,
Galveston, USA
V. Gundersen
Department of Neurology, Oslo University Hospital,
Rikshospitalet, Oslo, Norway
N-acetylaspartate (NAA) is exclusively localised in the
brain, where it is one of the most abundant small molecular
weight compounds (Tallan et al. 1956; Reichelt and Fonnum 1969). Despite that NAA was identified almost
50 years ago, its function in the brain is still obscure. NAA
is synthesised in neurons from aspartate and acetyl-coenzyme A by the enzyme aspartate N-acetyltransferase (AspNAT) (Ariyannur et al. 2010; Truckenmiller et al. 1985).
From neurons NAA is thought to be released to the
extracellular space and taken up by oligodendrocytes. In
the latter cells NAA is converted back to aspartate and
acetate by aspartoacylase (ASPA), which seems to be
predominantly present in oligodendrocytes (Kaul et al.
1991; Chakraborty et al. 2001; Klugmann et al. 2003;
Madhavarao et al. 2004; Wang et al. 2007; Mersmann et al.
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Brain Struct Funct
2011; Moffett et al. 2011). Acetate produced in this reaction can generate lipids for maintaining myelination
(Chakraborty et al. 2001), although this is debated (Baslow
2003). Suggestions that deficient catabolism of NAA could
lead to dysmyelination come from studies of Canavan
disease, an early-onset leukodystrophy caused by mutations in the gene for ASPA (Surendran et al. 2003; Kaul
et al. 1993), in which there are defects in the myelination of
large white matter tracts (Canavan 1931; Mahloudji et al.
1970).
Previous immunocytochemical studies in rodents have
reported that NAA is located predominantly in neurons
throughout the brain (Simmons et al. 1991; Moffett et al.
1991, 1993; Moffett and Namboodiri 1995, 2006),
although a light microscopic report suggesting the presence
of NAA in oligodendrocytes also has been presented
(Moffett and Namboodiri 2006). However, precise information about the cellular and subcellular localisation of
NAA in the intact brain has been hampered by the lack of
studies of its ultrastructural localisation. For instance, it is
not known in which part of the neuron NAA is most concentrated, or if NAA is located in other types of brain cell,
such as astrocytes and oligodendrocytes. Such information
is essential for the understanding of the role of NAA in the
brain.
To determine the ultrastructural distribution of NAA
within and between different types of brain cell we used
immunocytochemistry (including postembedding immunogold electron microscopy) with specific NAA antibodies,
in combination with brain cell type markers, to label brain
tissue from normal rats and wild type and ASPA knockout
mice (Matalon et al. 2000; Surendran et al. 2003).
fixation the animals were deeply anesthetised by intraperitoneal injections with pentobarbital (50 mg/ml).
Using a peristaltic pump adult Wistar rats (4–5 weeks;
Scanbur, Sollentuna, Sweden) were perfused through the
left cardiac ventricle with 5 % EDC and 1 mM Nhydroxysuccinimide (NHS) in 10 mM HEPES buffer (pH
7.4) (2 rats) or with 4 % EDC ? 1 mM NHS ? 1 %
DMSO in 0.9 % NaCl (1 rat) (for NAA immunoperoxidase and immunogold cytochemistry). Aspartoacylase
(ASPA) knockout (KO) mice (strain 129 Sv/Ev) and wild
type (WT) littermates were bred from heterozygous parents (Matalon et al. 2000). Mouse genotypes were
determined by PCR. As described previously a prominent
phenotypic feature of the ASPA KO mice was ataxia [for
details see (Matalon et al. 2000)]. At 3–4 weeks of age
wild type and ASPA KO mice were perfused through the
left cardiac ventricle with one of the following mixtures
of fixatives: (A) 5 % EDC and 1 mM NHS in 10 mM
HEPES buffer (pH 7.4) (NAA immunoperoxidase and
NAA and MPB immunogold cytochemistry) and
(B) 2.5 % glutaraldehyde (GA) and 1 % formaldehyde
(FA, made freshly from paraformaldehyde) in 0.1 M
sodium phosphate buffer (PB, pH 7.4) (morphologic
electron microscopic analysis).
To make organotypical cortical slice cultures we used
wild type mice (ICR) at postnatal day 8 (P8) (from Harlan,
France). After culturing the slices were fixed with 4 % FA
for 1 h in room temperature.
For Western blotting, Wistar rats, ASPA KO and WT
mice were decapitated, the hippocampus and the whole
brain, respectively, quickly dissected out on ice and frozen
fresh in liquid N2.
Antibodies
Materials and methods
Animals
All animals were treated in strict accordance with the
guidelines of the Norwegian Committees on Animal
Experimentation (Norwegian Animal Welfare Act and
European Communities Council Directive of 24 November
1986-86/609/EDC) and the Institutional Animal Care and
Use Committee at The University of Texas Medical
Branch. Animals were kept at 19–22 °C and about 50 %
relative humidity on a 12-h light/dark cycle with food and
water available ad libitum.
For NAA immunocytochemistry, we used the hydrophilic fixative 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) with and without addition of dimethyl
sulfoxide (DMSO), which might increase penetration of
EDC in the tissue. We always prepared fresh EDC when
performing the perfusion fixations. Prior to perfusion
123
Rabbit polyclonal anti-NAA antibodies were raised against
NAA bound to thyroglobulin by EDC. These NAA antibodies have previously been tested and they were found to
be specific (Moffett et al. 1991). We purified the NAA
antiserum by adsorption on series of agarose columns
bearing Sepharose–EDC–thyroglobulin. In addition, we
performed extensive specificity testing of the NAA antibodies against 16 small molecular compounds endogenous
to the brain. Test specimens (Ottersen and Storm-Mathisen
1984) were made by spotting brain protein–EDC–small
molecular compound conjugates onto cellulose nitrate and
acetate filters (Millipore filters). The test filters were processed with the NAA antibodies (along with the tissue
sections) according to a three-layer peroxidase–antiperoxidase method. The tested compounds were as follows:
glutamine, N-acetylaspartylglutamate (NAAG), GABA,
taurine, N-acetylglutamate, succinate, L-aspartate, L-lactate,
L-glutamate, pyruvate, NAA, a-ketoglutarate, citrate,
Brain Struct Funct
b-hydroxybutyrate, oxaloacetate. To avoid cross-reactivities, the NAA antibodies were used with addition of soluble
complexes of EDC-treated N-acetylaspartylglutamate plus
N-acetylglutamate (1 mM of each). As an additional
specificity control we added complexes of EDC-treated
NAA (1 mM) to the NAA antibodies before applying them
on the sections. Rabbit polyclonal anti-ASPA antibodies
were
raised
against
amino
acids
192–204
(DQMRKMIKHALD) (rabbit number 6284, Biosynthesis,
Texas, US). Mouse monoclonal myelin basic protein
(MBP) was raised against a peptide containing amino acids
84–89 (MAB387; Millipore). Rat monoclonal antibody
against myelin basic protein (MBP) was raised against a
peptide containing amino acids 82–87 (MAB386; Millipore) (Einstein et al. 2009). Chicken polyclonal antibodies
against neurofilament 200 kDa (NF200) were from Abcam
(ab4680) (Sparrow et al. 2012; Wirt et al. 2010) and rabbit
polyclonal oligodendrocyte transcription factor 2 (Olig2)
antibodies were from Millipore (ab9610) (Zeis et al. 2008).
Secondary antibodies comprised the following: biotinylated donkey anti-rabbit (GE Healthcare UK, RPN 1004V),
goat F(ab0 )2 anti-rabbit coupled to 10 nm gold particles
(British Biocell International, 15731), goat anti-mouse
coupled to 15 nm gold particles (British Biocell International, 15752), Alexa 633 goat anti-rat (Invitrogen,
A21094), Alexa 488 goat anti-chicken (Invitrogen,
A11039), Alexa 568 goat anti-rabbit (Invitrogen, A11036),
mouse anti-rabbit peroxidase (Sigma-Aldrich, A1949). The
tertiary immune reagent was streptavidin–biotinylated
horseradish peroxidise complex (GE Healthcare UK, RPN
1051V).
Light microscopy
For light microscopic studies parasagittal sections of the
fixed brains (see above) were cryoprotected in 30 %
sucrose before 20-lm-thick sections were cut on a freezing
microtome. The sections were subjected to immunocytochemistry according to a three-layer immunoperoxidase
method as described before (Gundersen et al. 1998). After
incubation with primary NAA antibodies (dilution
1:5,000), biotinylated secondary antibodies (dilution 1:100)
and with streptavidin–biotinylated horseradish peroxidise
(dilution 1:100), the epitope–antibody binding was visualised with hydrogen peroxide/diaminobenzidine (DAB).
Then the sections were mounted using gelatine, and dried
over night.
High-resolution digital images of the DAB stained tissue
were obtained using an automated Olympus BX52 microscope equipped with a 209 objective (Olympus Uplan
Apo, NA 0.70), a motorised stage (LEP MAC5000, LUDL
Electronic Products Ltd, Hawthorne, NY, USA), an Optronics MicroFire digital camera (Optronics Goleta, CA,
USA), and Neurolucida 7.0 Virtual Slice software (MBF
Bioscience, Inc, Williston, VT, USA).
Electron microscopy
Disregarding the electron microscopic preparation protocol
all ultrathin sections were contrasted with uranyl acetate
(1 %) and lead citrate (0.3 %) and viewed in a FEI Tecnai
12 electron microscope with a Veleta digital camera and
iTEM software (Olympus).
Morphologic electron microscopic visualisation
For morphological analysis the mice tissue fixed with
2.5 % GA and 1 % FA were treated with 1 % osmium
tetroxide in PB before embedding in Durcupan ACM
(Sigma-Aldrich). Ultrathin sections were cut (about
100-nm thick, gold colour) on an Ultramicrotome (Zeiss),
placed on 300 mesh nickel grids, contrasted and viewed in
the electron microscope.
Preembedding DAB immunocytochemistry
40-lm-thick parasagittal sections of the rat brains fixed
with 5 % EDC were cut on a vibratome. The sections were
processed with the NAA antibodies (dilution 1:5,000)
according to the immunoperoxidase DAB detection
described above. To preserve the ultrastructural morphology, the sections were processed without detergent. After
termination of the peroxidase reaction, the stained sections
were dehydrated and embedded in Durcupan ACM (SigmaAldrich), before ultrathin sections were cut (Ultramicrotome, Zeiss) on 300 mesh nickel grids, contrasted and
viewed in the electron microscope.
Postembedding immunogold cytochemistry
Tissue specimens (about 1 mm3) of the corpus callosum,
the striatum, the cerebellum, the hippocampal CA1/dentate
gyrus from ASPA KO and WT mice, as well as specimens
of rat CA1 hippocampus/dentate gyrus, were gradually
saturated in 10, 20 and 30 % glycerol to cryoprotect the
tissue. The specimens were embedded in Lowicryl HM20
according to a freeze-embedding protocol (Bergersen et al.
2008). Ultrathin sections of each brain region were cut on
an Ultramicrotome (Zeiss) (about 100 nm, gold colour),
and placed on 300 mesh nickel grids. The ultrathin sections
were then treated with the antibodies according to an immunogold protocol (Bergersen et al. 2008). To make the
epitopes optimally accessible for immunogold postembedding, the sections were etched with 2 % hydrogen
peroxide in 20 min. To reduce background labelling, the
sections were incubated in glycine/borohydride before they
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Brain Struct Funct
were placed in a solution with 2 % human serum albumin
(HSA). In the next step, the sections were incubated over
night with the primary antibodies. Rabbit anti-NAA antibodies and the mouse anti-MBP antibody were used at
dilutions of 1:300 and 1:50. The NAA antibodies were
either used alone in single labelling experiments or together with the MBP antibody in double labelling experiments. Then the sections were treated with secondary
antibodies (goat anti-rabbit IgG and goat anti-mouse IgG,
dilution 1:20).
Electron micrographs (926,500 and 943,000 primary
magnification) were randomly taken from the brain areas
under investigation. The plasma membranes of the below
mentioned profiles and the centre of each gold particle
were registered using Image J (http://rsb.info.nih.gov/ij/).
The density of gold particles (average number of gold
particles/lm2) was calculated using an ImageJ plugin and a
custom Python program (available from http://www.neuro.
ki.se/broman/maxl/software.html). We outlined the plasma
membranes of myelin in white matter tracts (in the corpus
callosum, the striatum, the cerebellum and the alveus of
hippocampus), as well as the plasma membrane of the
central myelinated axons. Only myelin sheaths with clearly
visible myelin lamellae surrounding a central axon were
included in the quantitative analysis. At places the tissue
used for NAA quantitation (fixed in 5 % EDC/4 %
EDC ? DMSO) membranes appeared somewhat blurred.
Care was taken to include in the analysis only stretches of
myelin showing distinct lamellae. The myelin included in
the quantitative NAA analyses had an apparently normal
morphology and contained more than five concentrically
arranged myelin lamellas surrounding a central axon. In
addition, we outlined the plasma membranes of pyramidal
cell bodies in the CA1 hippocampus, dentate granular cell
bodies, perivascular astrocytes, MBP positive oligodendrocyte cell bodies in the corpus callosum, myelin, axons
and nerve terminals making asymmetric synapses and the
corresponding spines in the stratum radiatum of CA1 hippocampus, nerve terminals making symmetric synapses, as
well as mossy fibre terminals and postsynaptic dendritic
thorns in the dentate hilus. We recorded NAA immunogold
densities over mitochondria-free cytosol and mitochondria
in axons, granule and CA1 pyramidal cell bodies, dendritic
shafts, terminals making asymmetric synapses, as well as
in oligodendrocyte cell bodies. In the other profiles only
cytosol quantifications were performed. A total of over
10,000 profiles in 3 rats and 16 mice have been quantified
for this study.
Hippocampal pyramidal cells and dentate granular cells
were identified by their characteristic localisation in the
pyramidal cell and granular cell layer. Perivascular astrocytes were identified by their localisation at the blood–
brain barrier, contacting the basal membrane of endothelial
123
cells (Mathiisen et al. 2010). Oligodendrocyte cell bodies
were identified by the localisation in the white matter and
the presence of MBP labelling. Excitatory nerve terminals
were identified by forming asymmetric synapses with
dendritic spines, while inhibitory nerve terminals were
identified by forming symmetric synapses with dendritic
shafts or granular cell somata. Mossy fibre terminals were
identified by their large size, making multiple synaptic
contacts with dendritic thorns.
Background NAA labelling was calculated in sections
exposed to NAA antibodies that were pre-neutralised with
soluble NAA–EDC complexes. Background NAA labelling
was calculated for all tissue profiles included in the quantitative analysis, these background values were subtracted
from the averages of the immunogold labelling produced
by the NAA antibodies. In the MBP immunogold quantifications labelling over mitochondria was used as background labelling.
Organotypic cortical slice cultures
Wild type mice (ICR) at postnatal day 8 (P8) (from Harlan,
France) were decapitated and 300 lm coronal slices from
the cerebral cortex were cut on a vibratome in a slicing
solution consisting of Earl’s balanced salt solution (EBSS)
(Invitrogen, 24010043) with 25 mM HEPES added, pH
7.3. Organotypic cortical slices were cultured on polytetrafluoroethylene filter membranes (0.45 lm pore size;
Millipore) lying on 30 mm culture plate inserts (0.4 lm
pore size; Millicell; Millipore) in culture dishes with wells
containing 1 ml of culture medium according to (De and
Yu 2006). All slices were cultured in 50 % Minimal
Essential Medium (MEM) (Invitrogen, 41090028), 23 %
EBSS, 25 % horse serum (Invitrogen, 26050070), penicillin (25 U/ml), streptomycin (25 lg/ml), 1.125 % nystatin
(12.5 U/ml) and 5 mM Tris base the first 3 days. Some
slice cultures were exposed to 1 mM NAA, which started
on the third day in vitro (DIV3), and the medium was then
replaced every third day. The cultures were kept at 37 °C
in a humidified atmosphere with 5 % CO2. After 2 weeks,
the slices were fixed with 4 % formaldehyde (1 h, room
temperature).
The organotypic cortical slice cultures were processed
according to an immunofluorescence protocol (Ormel et al.
2012a, b), with some modifications. In brief, the slices
were preincubated in 0.05 % Triton and 10 % goat serum
in 0.01 M PBS, before they were incubated over night
(room temperature) with primary antibodies (rat anti-MBP,
dilution 1:300; chicken-anti NF200, dilution 1:10,000;
rabbit anti-Olig2, dilution 1:1,000). The slices were then
treated with secondary antibodies (Alexa 633 goat anti-rat,
dilution 1:200; Alexa 488 goat anti-chicken, dilution 1:200;
Alexa 568 goat anti-rabbit, dilution 1:200) for 4 h. Pictures
Brain Struct Funct
of the labelled slices were taken on a Zeiss LSM 510 Meta
confocal microscope using a 209 objective (PLAN APOCHROMAT, 920, NA 0.8). The 488, 568 and 633 fluorophores were obtained sequentially, with a pinhole
ranging from 1.1 to 1.4 Airy units, and with a scan speed of
1.6 ls/pixel. They were excited sequentially with a laser
wavelength of 488, 561 and 633, and the emitted light was
obtained by a band pass filter of 505–550, a band pass filter
of 575–615 and a meta filter of 636–754 and recoloured
red, blue and green, respectively. Images of organotypic
cultures of the myelin within the grey matter of the cortex
were taken, covering layers I–VI. For each cultured slice,
three confocal z-stacks covering the thickness of the slice
were taken. The myelination was quantified by measuring
the intensity of MBP labelling relative to the axon labelling
(NF200) and oligodendrocyte labelling (Olig2), averaging
the fluorescence intensity in the three stack images showing the strongest labelling intensity.
control in each immunogold experiment we incubated
ultrathin tissue sections with NAA antibodies that were
pre-neutralised with soluble NAA–ECD complexes. This
reduced, or the almost abolished NAA labelling in all tissue profiles under investigation (by 50–90 % in neuronal
and oligodendrocyte/myelin profiles, respectively, see
Supplementary Tables 1, 2 and 3). The resulting NAA
densities after neutralising the NAA antibodies were
regarded as background NAA labelling and were analysed
in each tissue profile included in the study. All of the NAA
immunogold density values given below are the densities
of gold particles produced by the NAA antibodies minus
the densities of gold particles produced by the NAA antibodies preabsorbed by NAA. Thus, the specificity controls
show that the NAA antibodies are specific and recognise
EDC-fixed NAA.
Statistical analysis
All previous studies on NAA localisation in the brain are
based on immunoperoxidase light microscopy. Here our
intention was to study the localisation of NAA at the
subcellular level using the electron microscopic immunogold method. We tested tissue fixed in EDC with and
without the addition of DMSO (to facilitate penetration of
EDC in the tissue, see ‘‘Materials and methods’’). With the
addition of 1 % DMSO membranes were somewhat more
blurred compared to membranes fixed without DMSO, but
they could be reliably identified in the electron microscope.
There was no clear difference in the NAA immunogold
labelling pattern with and without DMSO (Fig. 2).
We undertook a detailed immunogold study of the distribution of NAA between several types of subcellular
profiles in the adult rat hippocampus. In light of the previous reports of NAA in neurons, we were surprised to find
immunogold labelling of NAA in the myelin, which was
stronger than that in the cytosol of any neuronal profiles
included in the study, such as nerve terminals and axons,
dendritic spines, dendritic shafts and neuronal cell bodies
(Fig. 2a–d). Indeed, immunogold quantifications (Fig. 2f,
g) showed that the density of NAA immunogold particles
was higher in the myelin compared to in the cytosol of their
central axons in the white matter alveus (for a discussion of
the effects of addition of DMSO in the EDC fixative on the
NAA labelling in the myelin versus axons, see ‘‘Discussion’’). Scattered myelin in the neuropil of the CA1 hippocampus contained similar densities of NAA as that in the
white matter alveus (not shown). The NAA immunogold
signal in the myelin was also higher than in the cytosol of
pyramidal and granule cell bodies, excitatory and inhibitory types of nerve terminal, dendritic spines and shafts,
and perivascular astrocytes in the hippocampal grey matter
neuropil in the CA1 region and the dentate gyrus.
The quantitative results were statistically evaluated by an
unpaired t test (two tails, Graph Pad), an unpaired Mann–
Whitney test (two tails, Graph Pad) and a non-parametric
one-way ANOVA test (Kruskal–Wallis multiple comparison test, GraphPad).
Results
The specificity of the NAA antibodies
We tested the antibodies against a battery of 16 small
molecular compounds known to exist in the brain at high
concentrations (Fig. 1; see dot blot explanation). As an
intrinsic specificity control for each immunoperoxidase
experiment, dot blots (cf Ottersen and Storm-Mathisen
1984) were incubated with the NAA antibodies along with
parasagittal brain tissue sections. Only the spot containing
NAA was stained and the tissue showed an overall neuronal labelling pattern similar to that observed previously
(Fig. 1a) (e.g., Moffett et al. 1991). After adding soluble
complexes of NAA–EDC to the NAA antibodies the NAA
labelling of tissue sections and dot blots were abolished
(Fig. 1b). In addition, when we probed the antibodies on
Western blots of hippocampal homogenates there were no
labelled bands (Fig. 1c), neither on non-fixed nor on EDCfixed blots, showing that the NAA antibodies do not crossreact with brain proteins. As a control of the negative
Western blots, dot blots were incubated with the NAA
antibodies along with the Western blots, showing staining
of the NAA spot, which was abolished after preabsorbing
the NAA antibodies with NAA. As an intrinsic specificity
Electron microscopic immunogold localisation of NAA
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Brain Struct Funct
Fig. 1 Specificity testing of the NAA antibodies. The antibodies
were tested against a battery of 16 small molecular compounds known
to exist in the brain at high concentrations (see dot blot explanation).
a When dot blots were incubated with the NAA antibodies along with
parasagittal brain tissue sections, only the spot containing NAA was
stained and the tissue showed overall a neuronal labelling pattern in
the grey matter. b Pre-incubating the NAA antibodies with soluble
complexes of NAA–EDC caused the NAA labelling of tissue sections
and dot blots to disappear. c Western blots of hippocampal
homogenates treated with the NAA antibodies. Note that there were
no labelled bands neither on non-fixed nor on EDC-fixed blots, while
dot blots run along with the Westerns showed staining of the NAA
spot (NAA), which was abolished after preabsorbing the NAA
antibodies with NAA (preabs). Scale bars in a and b 2,000 lm
Interestingly, the density of NAA immunogold particles
was higher in the mitochondria than in the cytosol of axons, granule and pyramidal cell bodies, but not in synaptic
elements and astrocytes. The NAA signal was stronger in
the cytosol of the myelin than in the mitochondria of these
neuronal profiles, except for in the mitochondria of axons.
Taking the NAA immunogold values produced by the
NAA antibodies after preabsorption with NAA–EDC
complexes as a measure of background values, the profiles
containing background levels of NAA varied somewhat
between tissues fixed with and without DMSO. In the
presence of DMSO the NAA labelling in the cytosol of
CA1 dendritic spines and CA3 thorny excrescences, and in
astrocytes, as well as in the mitochondria of dendritic
shafts was at background level. In the absence of DMSO
background levels of NAA were observed in the cytosol of
CA1 pyramidal cell bodies, excitatory and inhibitory terminals and dendritic spines, as well as in the mitochondria
of excitatory terminals and dendritic shafts. With DMSO,
the NAA immunogold density in the cytosol of granule cell
bodies was significantly higher than in the cytosol of other
neuronal compartments, except for in pyramidal cell bodies, while without DMSO the cytosol of axons contained
significantly higher levels of NAA compared to the cytosol
of the other neuronal profiles (Fig. 2f, g). The NAA signal
was stronger in the cytosol of the myelin than in the
mitochondria of the neuronal profiles, except for in the
mitochondria of axons fixed in the presence of DMSO
(Fig. 2f, g). To clarify if the myelin labelling was a phenomenon that could be observed only with the postembedding immunogold method (Fig. 2d1) we performed
electron microscopic preembedding immunocytochemistry. Here only myelin which has been cut open at the
section surface will be available for staining with the NAA
antibodies, which will diffuse along the myelin sheaths and
react with EDC-bound NAA. Indeed, also the preembedding immunoperoxidase method produced labelling of
NAA in the myelin (Fig. 2e; compare d1 with e1).
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Brain Struct Funct
Fig. 2 NAA is localised in the myelin. a–d Electron micrographs of
the CA1 hippocampus fixed with EDC (without DMSO) showing
immunogold labelling for NAA (gold particles, indicated by arrows)
in a pyramidal cell body (pc) (a), a nerve terminal (t) and a dendritic
spine (s) forming an asymmetric synapse, and a surrounding astrocyte
(astro) (b), dendritic shaft (c) and the myelin (my) and its central axon
(ax). Note the strong immunogold labelling of the myelin (d1, high
magnification of the area indicated by rectangle in d). e Electron
microscopic immunoperoxidase labelling. Note that precipitation
products of NAA have similar localisation in the myelin as the
immunogold particles (e1, high magnification of the area indicated by
rectangle in e). Scale bars a–e 200 nm, 50 nm (d1, e1). f–g
Quantitative analysis of the density of NAA gold particles (mean
number of gold particles per lm2 ± SD) in a rat fixed with EDC in
the presence of DMSO (f) and in two rats fixed with EDC without
DMSO (g). * The value in the myelin of the alveus (my) (n = 95/72)
is significantly different from the cytosol (open columns) and
mitochondrial (grey columns) values in axons (ax) (n = 95/72),
CA1 pyramidal cells (pc) (n = 18/30), excitatory terminals (ex t)
(n = 37/63), dendritic spines (sp) (n = 37/63) and dendritic shafts
(den sh) (n = 14/41) and perivascular astrocytes (astrocyte) (n = 26/
46) in the CA1 stratum radiatum, as well as in inhibitory terminals
(inh term) (n = 20/57), mossy fibre terminals (mft) (n = 11/48), the
postsynaptic mossy fibre dendritic thorns (mf th) (n = 15/114) and
granular cell bodies (gc) (n = 19/34) in the dentate gyrus (p \ 0.01,
non-parametric one-way ANOVA, Kruskal–Wallis multiple comparison test, GraphPad). **The value in gc-c (in f) and ax-c (in g) is
significantly different from the values in all the other profiles, except
from pc (p \ 0.05, non-parametric one-way ANOVA, Kruskal–
Wallis multiple comparison test, GraphPad). ***The values in the
mitochondria were significantly different from the values in the
cytosol (p \ 0.01, non-parametric one-way ANOVA, Kruskal–Wallis
multiple comparison test, GraphPad). The values presented are the
densities of gold particles produced by the NAA antibodies minus the
densities of gold particles produced by the NAA antibodies preabsorbed by NAA complexes. DThe NAA density values in CA1
dendritic excitatory terminals, spines, inhibitory terminals, mossy
fibre dendritic thorns and astrocytes were at background labelling
levels (see Supplementary Fig. 1)
Light microscopic localisation of NAA
the electron microscope, the NAA immunoperoxidase
staining in the white matter most likely represents
labelled myelin/oligodendrocytes. In addition, as
described previously we observed NAA staining in various types of neuronal cell bodies throughout the brain
(Fig. 1). For example, in the hippocampus CA1 pyramidal cell bodies and granule cell bodies were moderately stained, while CA3 pyramidal cell bodies and
scattered neurons, in, e.g., the dentate hilus, were
strongly stained (Fig. 3e). Also some populations of
neuronal cell bodies in, e.g., the cerebral cortex showed
rather strong NAA staining (Fig. 3e).
Based on the findings at the electron microscopic level
we re-examined the light microscopic NAA staining
pattern. Indeed, when we investigated the immunoperoxidase staining produced by the NAA antibodies at high
magnification in the white matter we observed labelling
of fibre-like structures, as well as of cell body-like
structures (Fig. 3). This labelling pattern was evident in
several white matter regions, such as the alveus of the
hippocampus, the corpus callosum, as well as in the
cerebellum and the striatum (Fig. 3a–d). As evidenced in
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Brain Struct Funct
Fig. 3 Light microscopic staining pattern of NAA. In white matter
tracts in the hippocampal alveus (a), the corpus callosum (b), the
striatum (c) and the cerebellum (d) the NAA staining is present in
distinct dotted (open arrowheads) and cell body-like (closed arrowheads) structures. e In the grey matter of the hippocampus and the
cerebral cortex the NAA staining is located in neuronal cell bodies
and proximal dendrites. This is most prominent in CA3 pyramidal
cells, scattered neurons in the hippocampal neuropil, e.g., in the
dentate hilus, and some cortical neurons, while CA1 pyramidal cells
and granule cells are less intensely stained. c cerebral cortex, cc
corpus callosum, a alveus, o str. oriens, pcl pyramidal cell layer, r str.
radiatum, lm str. lacunosum molecular, ml dentate molecular layers,
gcl granule cell layer. Scale bars 10 lm (a–d) and 1,000 lm (e)
Increased NAA labelling of myelin in ASPA knockout
mice
against ASPA confirmed that the ASPA protein was absent
from the ASPA knockout mice brains (Fig. 4a). We reasoned that since in these mice the catabolism of NAA is
blocked in oligodendrocytes/myelin, this should lead to an
increase in NAA in the myelin, which in turn should be
detected by our immunogold labelling method. In contrast,
if the NAA staining over myelin represents ‘‘unspecific’’
sticking of antibodies to protein-rich areas, we would not
expect an increase in the staining in ASPA knockout
myelin.
Immunogold electron microscopy
Based on the somewhat surprising findings of strong NAA
labelling in the myelin described above we wanted to test
the NAA antibodies in a positive control system. To this
end we used brains from ASPA knockout mice. Western
blots of whole brain homogenates stained with antibodies
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Brain Struct Funct
Fig. 4 ASPA knockout brains contain apparently normal myelin.
a Western blots of whole brain homogenates demonstrating the lack
of ASPA in ASPA knockout mice. In wild type (WT) brains ASPA is
present (36 kDa band, arrowhead), while it is absent in the ASPA
knockout brains (arrowhead). b–d Myelin with normal appearance is
present in the ASPA knockout. Electron micrographs of myelin show
examples from the cerebellum (b), the striatum (c) and the alveus of
the hippocampus (d). The tissue was prepared for good preservation
of the ultrastructure (fixed with high glutaraldehyde, treated with
osmium tetroxide and embedding in epoxy resin). Scale bars 100 nm.
e Myelin with normal appearance in the ASPA knockout has similar
levels of myelin basic protein (MBP) as wild type myelin. Quantification of MBP immunogold labelling (mean number of gold
particles/lm2 ± SEM) in undistorted myelin from tissue fixed for
preservation of immunogold NAA sensitivity (fixed with EDC,
embedding in Lowicryl). Values in WT were not significantly
different from values in ASPA knockout mice (p [ 0.05, unpaired
t test, two tails, GraphPad). The quantitative analysis is based on 4
ASPA knockout mice (75 myelinated axons in total) and 4 wild type
mice (110 myelinated axons in total)
Patho-anatomically brains of ASPA knockout mice
show dysmyelination (Surendran et al. 2003; Namboodiri
et al. 2006; Matalon et al. 2000; Mattan et al. 2010). Hence,
when comparing NAA levels in the myelin in ASPA
knockout brains with those in wild type brains it is essential
that myelin with a normal appearance is included. First, we
therefore determined if myelin with normal morphology
was present in the ASPA knockout brain. For this, we used
tissue prepared for optimal morphologic visualisation of
lipid-bounded structures (tissue fixed with a high glutaraldehyde concentration, postfixed with osmium tetroxide,
and embedded in epoxy resin). By electron microscopy we
found that the white matter in several brain regions contained myelin with intact morphology (Fig. 4b–d),
although parts of the myelin was disrupted (see below). As
in the rat tissue, for electron microscopic immunogold
labelling, we continued using tissue prepared for optimal
NAA immunogold sensitivity (tissue fixed with EDC
without glutaraldehyde, treated with uranyl acetate and
embedded in Lowicryl). This tissue shows somewhat suboptimal morphology, e.g., with some blurring of lipid
membranes, compared to tissue prepared for optimal
morphology. To ensure that the myelin we picked out on
morphological grounds (see ‘‘Materials and methods’’) was
apparently normal we determined the myelin level of
myelin basic protein (MBP). Immunogold quantifications
showed that in the myelin with a normal appearance the
MPB levels were similar in ASPA knockout and wild type
tissues (Fig. 4e).
In confidence that we could reliably identify apparently
normal myelin we immunogold labelled ultrathin sections
from wild type and ASPA knockout brains with the NAA
antibodies. Similar to rats, mouse brains contained rather
high levels of NAA in the myelin, as well as in oligodendrocyte cell bodies (identified by labelling for MBP),
although axons and neuronal cell bodies were labelled to
some extent (Fig. 5). The density of NAA gold particles in
the myelin was much higher in ASPA knockout brains
compared to wild type brains in all regions investigated
(Figs. 5a–d, 6). The increase in the NAA labelling of
myelin in ASPA knockout mice versus wild type mice was
strongest in the cerebellum and the striatum, and with less
prominent changes in the corpus callosum and the alveus of
hippocampus (Fig. 6a). Quantification of the NAA signal
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Brain Struct Funct
Fig. 5 NAA in the myelin increases in ASPA knockout mice. a–
d Electron micrographs showing myelin (my) and axonal (ax)
labelling for NAA (gold particles, some indicated by red arrowheads)
in wild type and ASPA knockout mice in the white matter of the
cerebellum (a), striatum (b), hippocampus (c) and corpus callosum
(d). Axons are outlined by a grey pseudo-coloured line. e, f Electron
micrographs of NAA labelling (small gold particle, some indicated by
red arrowheads) in granule cell bodies (gc) (e) and myelin basic
protein positive oligodendrocyte cell bodies (oligod, large gold
particles, some indicated by arrows) (f) in wild type and ASPA
knockout mice, respectively. Insets (e1, e2, f1 and f2) show NAA gold
particles (some indicated by red arrowheads) at higher magnification.
Large gold particles (arrows) in f1 and f2 signal MBP. n nucleus,
m mitochondrion, *plasma membrane. Scale bars a–d 200 nm, e,
f 400 nm; insets (e1, e2, f1 and f2) 200 nm
in the myelin gave ratios of gold particle densities between
wild type and ASPA knockout mice of 9.3, 4.5, 3.3 and 3.0
in the cerebellum, the striatum, the corpus callosum and the
alveus of hippocampus, respectively. Interestingly, in oligodendrocyte cell bodies the NAA gold particle ratio
(ASPA knock out mice:wild types) was 7.5. In contrast, the
ratios in neuronal profiles (i.e., axons in all regions and
granule cell bodies in the hippocampus) were much lower
and approximately 1–2.3. Furthermore, the density of NAA
gold particles was analysed in pairs of surrounding myelin
and the central axon. In both ASPA knockout mice and
wild type mice the densities of gold particles signalling
NAA were significantly higher in the myelin compared to
axons for all brain regions under investigation, except for
in the wild type cerebellum (Fig. 6b). Since ASPA catalyses the conversion of NAA to aspartate and acetyl, we
used the immunogold method to quantify aspartate labelling in the myelin and compared these values to those for
glutamate. Somewhat surprising, in the myelin of ASPA
knockout brains we found that only the glutamate, but not
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Brain Struct Funct
Fig. 6 Quantification of NAA labelling in wild type and ASPA
knockout mice. a, b Bar charts showing the density of NAA
immunogold particles (mean number of gold particles/lm2 ± SEM in
4 WT and 4 ASPA knockout mice) in the myelin (m) and axons (ax)
in the white matter of the cerebellum (cb), striatum (str), alveus of
hippocampus (hc) and corpus callosum (cc), as well as in myelin basic
protein positive oligodendrocyte cell bodies (oligod) and dentate
granule cell bodies (gc). The values presented are the densities of gold
particles produced by the NAA antibodies minus the densities of gold
particles produced by the NAA antibodies preabsorbed by NAA.
Included in the analyses were in average 67 myelinated axons, 20
oligodendrocytes and 20 granular cells per mouse. Asterisks in
a indicate that values in ASPA knockout mice are significantly
different from those in wild type mice (p \ 0.05, unpaired t test, two
tails, GraphPad). Asterisks in b indicate that values in the myelin are
significantly different from those in axons (p \ 0.05, unpaired t test,
two tails, GraphPad)
the aspartate level, was significantly reduced (see Supplementary Fig. 1 and Supplementary Fig. 1 Discussion).
Also mouse neuronal profiles contained a higher density
of NAA immunogold particles in mitochondria than in the
cytosol. We quantified the density of NAA immunogold
particles in the cytosol and mitochondria of dentate granule
cell bodies, hippopcampal axons and corpus callosum oligodendrocyte cell bodies in four wild type mice. In dentate
granule cell bodies the cytosol versus mitochondrial values
[average number of NAA gold particles/lm2 ± SD in n
profiles) were 4.2 ± 1.2 (n = 80) versus 21.9 ± 38.5
(n = 91) (p \ 0.05, unpaired t test (two tails, Graph Pad)],
and in hippocampal axons the cytosol versus mitochondrial
values were 6.1 ± 10.6 (n = 382) versus 22.9 ± 30.1
(n = 80) [p \ 0.05, unpaired Mann–Whitney test (two tails,
Graph Pad)], respectively. However in oligodendrocyte cell
bodies there was no significant difference between the NAA
values in the cytosol and mitochondria (20.3 ± 13.8
(n = 61) versus 25.1 ± 20.1 (n = 73) [p [ 0.05, unpaired
Mann–Whitney test (two tails, Graph Pad)]. In the mouse
hippocampus the mitochondrial NAA values in axons were
similar to the NAA values in the myelin (16.7 ± (n = 382)
versus 22.9 ± 30.1 (n = 80) [p [ 0.05, unpaired Mann–
Whitney test (two tails, Graph Pad)].
In short, the electron microscopic immunogold data of
mice tissue show that deletion of ASPA results in a large
increase of NAA immunoreactivity in the myelin as compared to in neuronal profiles.
Light microscopic immunoperoxidase
Also in the mice, light microscopic immunoperoxidase
microscopy showed NAA staining in the white matter, both
in wild type and ASPA knockout brains. In line with the
electron microscopic data, white matter tracts in ASPA
knockout brains contained considerably stronger staining
compared to wild type brains (Fig. 7a–d vs. e–h). There
was no clear change in the neuropil NAA staining pattern
in the ASPA knockout mice (Fig. 7). After pre-neutralising
the NAA antibodies with NAA, staining of tissue sections
was abolished (Fig. 7i–l), confirming that the NAA staining of the myelin is specific (see Fig. 1).
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Brain Struct Funct
Fig. 7 Lack of ASPA leads to increased NAA levels in white matter
structures. Light micrographs of immunoperoxidase NAA-stained
sections of wild type (WT) (a–d) and ASPA knockout (ASPA KO)
(e–h) brain tissue. In all brain regions studied, the white matter (wm)
displayed a clear increase in the NAA labelling in ASPA KO
compared to WT tissue. The most prominent increase of NAA
staining from WT to ASPA KO was found in the cerebellum (a vs e).
Note that the grey matter (gc, cerebellar granule cell layer; neup,
striatal and hippocampal neuropil) did not show any increase in NAA
staining. The NAA staining is present in distinct elongated/dotted
(open arrowheads) and cell body-like (closed arrowheads) structures.
i–l The NAA labelling was effectively inhibited by preabsorbing the
NAA antibodies with soluble NAA–carbodiimide complexes, demonstrating the specificity of the NAA labelling. Dashed lines outline
the borders between white and grey matter. alv the alveus of
hippocampus. Scale bars 10 lm
Myelin alterations in the ASPA knock out mouse
myelin (Woelcke histochemical stain), as well as with a
histological stain (toluidine blue) (see Supplementary
Methods). At the light microscopic level there were clear
signs of overall reduction in myelination in white matter
tracts, as well of spongy white matter degeneration, while
the grey matter neurons did not show any obvious
pathology (Supplementary Fig. 2). Vacuolation in the
white matter was most prominent in the cerebellum compared to other brain areas, such as the striatum, the corpus
callosum and the alveus of hippocampus (Supplementary
Fig. 2).
Patho-anatomically dysmyelination is a prominent feature
in brains of ASPA knockout mice (Namboodiri et al. 2006;
Matalon et al. 2000; Mattan et al. 2010). To characterise
the histopathologic changes in the ASPA knockout brain in
more detail we undertook an electron microscopic investigation of the tissue prepared for optimal morphologic
visualisation of lipid-bounded structures (Fig. 8). In the
white matter we found that, besides examples of myelin
with normal appearance (Figs. 4, 8), there were large
vacuoles of loosely arranged myelin without contact with
central axons. This was most prominent in the cerebellum
(Fig. 8a), but it was observed also in the other white matter
areas included in the study, as for example in the striatum
(Fig. 8b). Between these large myelin vacuoles there were
abnormal stretches of disrupted myelin with and without
signs of a central axon (Fig. 8c, d). In contrast to the white
matter, the grey matter neuropil showed apparently normal
ultrastructure (Fig. 8e).
To obtain an overview of the white matter dysmyelination in the ASPA knock out brains, they were stained for
123
NAA is not toxic to myelin
The reason why ASPA deficiency causes disruption of
myelin is not settled. One possibility is that a high concentration of NAA in the myelin could be toxic. To test this
we exposed organotypic cortical slice cultures to high
external concentrations of NAA (1 mM). The effect of
NAA on myelinated axons was assessed in the confocal
microscope by analysing the intensity of fluorescent
staining for myelin basic protein, olig2 and neurofilament
Brain Struct Funct
Fig. 8 White matter in ASPA knockout mice contains normal and
distorted myelin. a, b Electron micrographs showing myelin-lined
vacuoles (my) in the cerebellum (a, a1) and the striatum (b, b1).
Apparently normal myelinated axons are present next to the myelin
vacuolation (a2, b2). Note the ‘‘naked’’ axon in a2. In addition to the
myelin vacuoles, disorganised myelin (my) with and without a central
axon (ax) (c and d, corpus callosum) was present in all regions. (e,
hippocampus) The ultrastructure of neuropil profiles, including
synapses between nerve terminal (ter) and dendritic spines (spine)
and astrocytic processes (astro), appears morphologically normal.
Scale bars a, b 1,000 nm; a1, a2, b1, b2, c, d and e 200 nm
200. These signals mark the integrity of the myelin, oligodendrocyte cell bodies and axons, respectively. We
could not find any evidence for NAA toxicity; there was no
change in oligodendrocyte (myelin and cell body) or axon
staining intensities after NAA exposure (Fig. 9).
Discussion
We report here for the first time that NAA is localised in
the myelin in the adult brain. Our immunogold data indicate that NAA is more concentrated in the myelin
123
Brain Struct Funct
Fig. 9 High extracellular NAA concentration does not damage
oligodendrocyte cell bodies, myelin or axons in organotypic cortical
slice cultures. The slices were cultured in control solution (n = 10
slices) and in solution containing 1 mM NAA (n = 9 slices). The
slice cultures were immunolabelled for Olig2 (oligodendrocyte cell
body marker) (a), NF 200 (axon marker) (b) and MBP (myelin
marker) (c). Scale bar 50 lm. The bar charts indicate normalized
mean fluorescence intensity ± SEM in slice cultures incubated at
control conditions (control) and a high NAA concentration (NAA).
Values in control were not significantly different from values in NAA
treated slice cultures (p [ 0.05, unpaired t test, two tails, GraphPad)
compared to neuronal dendrites, nerve terminals and some
types of cell bodies, although we did not quantify the NAA
levels in the neuronal cell bodies that showed the strongest
staining at the light microscopic level (e.g., CA3 hippocampal, dentate hilar and some cortical neurons). In several
white matter tracts we found that NAA was higher in the
myelin than in the cytosol of the central axon. We used the
hydrophilic carbodiimide EDC as a fixation agent, which
will not penetrate equally into brain tissue. To compensate
for this, one of the rats included in the present quantifications was fixed with the addition of the lipophilic agent
DMSO, enhancing penetration of EDC through membranes. However, in our hands the addition of DMSO did
not alter the NAA labelling pattern between the myelin and
axons to any considerable extent. The slight difference in
neuronal profile labelling observed in the rats fixed with
and without DMSO could rather be random, as the NAA
signal in some of these profiles was very low and showed
strong variation, than due to DMSO. Since DMSO will
destroy the ultrastructure of the brain tissue, we used rather
low concentrations (1 %). We noticed that even this low
DMSO concentration produced somewhat more blurring of
membranes than was observed without DMSO. Thus, the
use of a higher DMSO concentration, as that (5 %) used in
Moffett et al. (1993) and Moffett and Namboodiri (1995),
would probably have led to more intense labelling of the
myelin and even stronger of the axon, but not permitted
electron microscopic immunogold microscopy. Based on
these considerations, it could be that our labelling ratios of
NAA in the myelin and axons are overestimations of the
true value. Indeed, when using a high concentration of
DMSO to enhance EDC fixation in the brain there was an
increased NAA staining of, e.g., fibre tracts (Moffett et al.
1993; Moffett and Namboodiri 1995).
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Brain Struct Funct
NAA is present in the myelin
We believe that the NAA antibodies are specific and detect
carbodiimide-fixed NAA in the brain tissue, and in particular in the myelin, for the following reasons: (1) In
control dot blots only the NAA spot was stained. (2) Both
the immunoperoxidase and the immunogold NAA signals
were strongly reduced by pre-neutralisation of the NAA
antibodies with NAA; this was particularly evident for the
myelin. (3) There was no labelling of Western blots of
whole brain homogenates, suggesting that the NAA antibodies do not cross-react with brain proteins, including
those present in the myelin. (4) At the electron microscopic
level, the NAA antibodies produced myelin labelling both
with preembedding immunoperoxidase and postembedding
immunogold labelling. (5) In ASPA knockout mice the
NAA labelling was significantly increased in myelin with
normal morphology, as well as in oligodendrocyte cell
body cytoplasm. This strongly suggests that the NAA
signal was not due to unspecific binding of the antibodies
to the myelin. (6) The NAA antibodies produced a similar
neuronal staining pattern as described previously.
Our findings are in agreement with a previous report of
high levels of NAA in mature oligodendrocyte cultures
in vitro (Bhakoo and Pearce 2000). Also previous immunocytochemical studies have detected staining for NAA in
the white matter (Simmons et al. 1991; Moffett and
Namboodiri 2006), although most focus has been given to
neuronal staining (Moffett and Namboodiri 2006; Moffett
et al. 1991, 1993). Immunoperoxidase microscopy does not
have the sufficient resolution to reliably discriminate
between axons and myelin, or to reveal the identity of the
labelled cell bodies in the white matter. Our immunogold
labellings strongly suggest that, besides localisation in
axons, NAA is present at rather high concentrations in the
myelin and oligodendrocyte cell bodies in different white
matter tracts in the brain.
The role of NAA in the myelin
The question is what the mechanism behind the presence of
NAA in oligodendrocytes/myelin is. The NAA synthesising enzyme Asp-NAT seems to be located in neuronal cell
bodies (Ariyannur et al. 2010). In agreement with this, we
found that this part of the neuron harbours the highest
concentration of NAA, along with axons. In comparison,
the NAA levels were low in presynaptic terminals, postsynaptic dendritic spines and shafts. From the light
microscopic study of Ariyannur et al. (2010) it is difficult
to infer about Asp-NAT in axons, but our results indicate
that NAA could also be produced in axons. Interestingly,
we found that the concentration of NAA in neuronal cell
bodies and axons was higher in mitochondria compared to
in the cytosol. This suggests that the synthetic pathway of
NAA involves formation in mitochondria (see Patel and
Clark 1979; Madhavarao et al. 2003; Arun et al. 2009),
which also is in agreement with the presence of Asp-NAT
in the mitochondria of neuroblastoma cells (Ariyannur
et al. 2010). Also in oligodendrocyte cell bodies NAA was
present in mitochondria, but at similar levels as in the
cytosol. In accordance with the lack of Asp-Nat in oligodendrocytes, this indicates a flux of NAA across mitochondrial membranes without de novo synthesis in this cell
type. Whether such mitochondrial transport of NAA also
suggests catabolism by ASPA in mitochondria is not
known.
In the grey matter the presence of NAA in the myelin
(myelin in the neuropil of the CA1 hippocampus contained
similar NAA levels as in white matter tracts) is probably
brought about by transport from neuronal cell bodies to
oligodendrocytes/myelin via the extracellular fluid, which
contains rather high extracellular NAA concentrations
(about 20 lM; Gotoh et al. 1997). In the white matter, in
which there are no neuronal cell bodies, NAA is probably
synthesised in axons (see above). Although NAA can be
transported by the sodium dicarboxylate transporter 3
(NaDC-3) in oligodendrocyte cell lines (Long et al. 2013),
the molecular identity of the transporter carrying NAA
across plasma membranes in different type of mature brain
cells is unknown. Thus, little is known about how and at
which cellular/subcellular sites NAA is transported. In this
respect, it is interesting that substrates for myelin production are able to cross the myelin membranes and the oligodendrocyte plasma membrane, as was recently shown for
lactate (Rinholm et al. 2011). The reason why lactate is
transported across the myelin is not settled, but it may be to
supply the myelin with substrates for lipid production
(Rinholm et al. 2011), or it may be to support the central
axon with a substrate for energy production (Lee et al.
2012; Rinholm and Bergersen 2012). Analogous with this,
it may be that the NAA produced in the axon is carried first
over the axonal plasma membrane into the myelin. Here
NAA could support the myelin with acetate for lipid production, or NAA could be transported through the entire
myelin and released to the extraaxonal space. Then NAA
could be taken up by nearby oligodendrocyte cell bodies/
proximal processes and used to synthesise lipids, which in
turn could be transferred to the myelin (Fig. 10). Favouring
local myelin synthesis of lipids from NAA is that ASPA
activity (Wang et al. 2007), as well as components of lipid
synthesising machinery (Chakraborty and Ledeen 2003; for
review see Ledeen 1992) is found in purified myelin. In
short, our data is in agreement with the view that there is
axon-to-myelin transfer NAA that acts as a donor of acetate
for myelin lipid production (Burri et al. 1991; Mehta and
Namboodiri 1995; Chakraborty et al. 2001).
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Brain Struct Funct
enhanced NAA concentrations in oligodendrocyte myelin,
and not in neurons (cf. the present results). Moreover, in a
patient with hypoacetylaspartia, shown to have a mutation
in the gene for Asp-NAT (Wiame et al. 2010), NAA
signals on MRS were absent, while magnetic resonance
imaging of the patient did not show any clear sign of
pathology in the grey matter (Martin et al. 2001; Boltshauser et al. 2004). In support of our idea that oligodendrocytes/myelin significantly contribute to the NAA
MRS signal are also results of MRS studies in the healthy
human brain. Several studies have shown high NAA
signals in white matter tracts compared to areas of grey
matter (Tedeschi et al. 1995; Soher et al. 1996; Schuff
et al. 2001).
A note on Canavan disease pathology
Fig. 10 Schematic representation of the proposed flux of NAA from
axons to the myelin/oligodendrocytes. Lipids can be produced in the
myelin and/or in the oligodendrocyte cell body. According to this
scheme the NAA labelling observed in the myelin could represent
NAA that is directly degraded as a lipid precursor in the myelin, or
NAA on its way through the myelin to the oligodendrocyte cell body
Our data suggest that NAA is not a selective marker of
neurons. NAA can also indicate oligodendrocytes/myelin.
This should have implications for the interpretation of
NAA signals on brain MRS, which are taken to be a
selective marker for healthy neurons (Arnold et al. 2001).
This is mostly based on the assumption that NAA is
synthesised by and contained in neurons and that the
maintenance of constant NAA levels in the brain indicates
neuronal health and integrity (Tsai and Coyle 1995). We
suggest that oligodendrocytes/myelin significantly contribute to the NAA MRS signals in the white matter.
Hence, in the white matter these signals may mainly
represent intact myelin and viable oligodendrocytes,
rather than viable axons. This is in agreement with some
lines of evidence that suggest that the NAA signal on
MRS does not solely stem from neurons. Time-lapse
studies of multiple sclerosis (MS) and mitochondrial
encephalopathy with lactic acidosis and stroke-like episodes (MELAS) (De Stefano et al. 1995; Kamada et al.
2001) have shown reversible changes of NAA MRS signals. The pathology underlying these reversible changes
are associated with demyelination followed by remyelination, i.e., reversible damage of the myelin and preservation of quite normal grey matter, at least in the early
stages of the disease. Moreover, it has been shown that
focal MS plaques, as revealed by MR imaging, contain
reduced MRS signals of NAA (Narayana et al. 1998),
further strengthening the link between NAA levels and
myelin viability. Likewise, in Canavan disease, a hyperacetylaspartia, there is a large increase in the MRS signal
of NAA in the brain (Janson et al. 2006), which reflects
123
The pathogenic mechanisms underlying development of
Canavan disease are not settled. It is known that there is a
large build-up of NAA in Canavan disease (Matalon et al.
2000; Janson et al. 2006; Traka et al. 2008; Mersmann
et al. 2011). Here we have analysed NAA levels in ASPA
knockout mice, which is a model for Canavan disease
(Namboodiri et al. 2006; Matalon et al. 2000; Mattan et al.
2010). In these mice we show that the major increase in
NAA is localised in oligodendrocytes, including the myelin. It has been proposed that the dysmyelination observed
in Canavan disease involves lack of acetate formed from
NAA through the ASPA reaction (Madhavarao et al. 2005;
Namboodiri et al. 2006; Wang et al. 2009). This probably
compromises oligodendrocyte maturation and myelination,
in turn leading to the white matter degeneration observed in
Canavan disease (Mattan et al. 2010).
Interestingly, when organotypic cortical slice cultures
were exposed to high NAA concentrations (1 mM) we did
not observe any toxic effects on the myelin, further supporting the notion that it is the lack of acetate and not high
NAA concentrations per se that causes dysmyelination in
Canavan disease. As the extracellular NAA concentration
in the brain is about 0.4–0.9 mM in Canavan disease
(Wevers et al. 1995; Burlina et al. 1999), the external NAA
concentration put on the slice cultures should be sufficient
to induce myelin damage. Interestingly, NAA does not
evoke NMDA glutamate receptor responses in oligodendrocytes in the cerebellar white matter (Kolodziejczyk
et al. 2009). Activation of such NMDA receptors is one
mechanism by which myelin can be damaged (Karadottir
et al. 2005).
In the ASPA knockout we observed that the myelin
morphology varied from normal to clearly distorted within
individual white matter regions. In addition, some white
matter areas seemed to be more prone to myelin damage
than others, with the cerebellum and the striatum showing
Brain Struct Funct
the most prominent changes (cf. Fig. 8 and Supplementary
Fig. 2; see also Traka et al. 2008). Interestingly, the NAA
increase in ASPA knockout myelin was strongest in the
cerebellum and the striatum (cf. Fig. 6a), suggesting that
some regions, such as the cerebellum and the striatum, are
more dependent on NAA-derived acetate for myelin production, while in other regions acetate could to a greater
extent be derived from other sources, such as lactate. In
this respect, it was recently shown that lactate could be
taken up into the myelin and support cortical myelination
(Rinholm et al. 2011). The fact that the white matter
region most prone to damage harboured the largest
increase in NAA supports a link between the degree of
white matter vacuolation/dysmyelination and the myelin
levels of NAA. The fact that these changes were large in
the cerebellum correlates with the behavioural phenotype
of the ASPA knockout mice and patients, inasmuch as
they suffer from a severe ataxia. Moreover, our morphological findings in the ASPA knockout mice are in
agreement with previous reports of Canavan disease,
which described that the pathology was marked in the
white matter, especially in the cerebellum, versus in the
grey matter, and that many cortical white matter tracts
seemed to be preserved (Canavan 1931; Adachi et al.
1972). In this respect, it is noteworthy to mention that in a
transgenic mouse model of Canavan disease there was a
marked vacuolation also in the grey matter, which, in line
with our results, was mostly due to myelin sheath
destruction (Traka et al. 2008).
Conclusion
Here we show that besides certain neuronal cell bodies,
oligodendrocytes/myelin contains high concentrations of
NAA. We propose that NAA is not solely a marker of
neurons but also of oligodendrocytes/myelin. In the Canavan disease mouse model the increase in NAA is prominent in myelin and oligodendrocytes, and cerebellum
shows the highest myelin increase. This is the brain region
with the most pronounced myelin pathology in the Canavan mice. As NAA is toxic neither to myelin nor to axons,
our data fit with the notion that myelin damage caused by
deficient NAA catabolism is related to lack of acetate for
myelin production.
Acknowledgments We thank Anna-Bjørg Bore for technical
assistance, especially with the Woelcke staining. We thank Sylvia
Szucs for help with handling the Canavan mice and the ASPA antibodies. The NAA antibodies were a kind gift from John R. Moffett
and Joseph H. Neale (Georgetown University, Washington, US). This
work was supported by grants from the Research Council of Norway
(grant number 170441/V40).
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