Neuroscience Letters 254 (1998) 17–20

Pattern of apolipoprotein D immunoreactivity in human brain

Ana Navarro, Jorge Tolivia*, Aurora Astudillo, Eva del Valle

Departamento de Morfologı́a y Biologı´a Celular, Facultad de Biologı́a y Medicina,
Universidad de Oviedo, Julián Claverı́a s/n, Oviedo 33006, Spain

Received 17 June 1998; received in revised form 24 July 1998; accepted 3 August 1998

Abstract

Presence of intracytoplasmatic apolipoprotein D (apo D), a lipophilic ligand transporter, was investigated in normal human
brains between 20 and 55 years, using an anti-human apolipoprotein D antibody and extravidin-biotin-enhanced immunohis-
tochemistry. Apo D immunoreactivity was found in neuroglial cells of white matter in all sampled brain regions studied but also in
pial cells and perivascular cells. Immunoreactive neurons do not present a uniform pattern throughout the gray matter. The pons
and the brainstem show a high immunoreactivity for apo D in several nuclei (olivary, arciforme, cuneado, raphe). In the
cerebellum the immunoreactivity appears in some neurons of the Purkinje layer. Finally in the cerebral cortex apo D positive
neurons were not observed. These results suggest that apo D role may vary depending of cellular synthesis or location.  1998
Elsevier Science Ireland Ltd. All rights reserved

Keywords: Apolipoprotein D; Immunocytochemistry; Human; Nervous system

Human apolipoprotein D (apo D) is a glycoprotein com-
ponent of the plasma lipoprotein particles with its maximum
concentration being localized in high density lipoprotein
(HDL) and with trace amounts in other lipoproteins. It
was first isolated and partially characterized by McConathy
and Alaupovic [8] and subsequently by others authors
[5,21]. The human apo D cDNA was cloned and sequenced
and the analysis of the deduced aminoacidic sequence
revealed that apo D shows no sequence similarities to
other known apolipoproteins and extensive homology to
lipocalin family of proteins [6]. Most members of this
family bind and transport small hydrophobic molecules.
Recent reports have shown that apo D can bind and trans-
port a variety of molecules including cholesterol, heme-
related compounds, odorant substances and some steroid
hormones [9,12,22]. Thus, it has been suggested that apo
D may be a protein ‘multigand-multifunction’ [16].
Apo D is unique among apolipoproteins, being expressed
throughout the body and at high levels in organs other than
the liver and intestine [13–16]. It has been reported that apo
D could be used to a prognostic marker of some types of
* Corresponding author. Fax: +34 985 103618 or +34 985 232225.
cancer [1,4] and other pathologies [18–20]. Recent data
have raised the possibility that apo D expression is a marker
of cell differentiation and growth arrest [7].
The finding of apo D synthesis in brain [13–16] and in
regenerating and remyelinating peripheral nerve [2,17] has
suggested that this protein play a significant role in lipid
transport and neuronal regeneration in CNS [10,18]. In ani-
mal brains apo D production, by astrocytes, oligodendro-
cites and neurons, has been shown by in situ hybridization
[13,14,16] or in vitro [11] studies. In the present study the
expression of apolipoprotein D in sampled human brain was
investigated to provide further information regarding the
response of different types of neurons to apo D expression.
Eighteen human brains belonging to males 20–55 years
old were used for this study. Specimens were obtained
within 6–12 h postmortem from subjects who had no
known symptoms of nervous system disease before death;
the cause of death in each case was not neurological. The
specimens were fixed for 2 weeks in 10% buffered formalin,
sampled, dissected out and embedded in paraffin. Samples
were taken of cortex (frontal, parietal and temporal), peri-
ventricular white matter, lenticular nucleus, cerebellum and
brainstem.
Immunohistochemical assays were performed on 10 mm

0304-3940/98/$19.00  1998 Elsevier Science Ireland Ltd. All rights reserved

PII S0304- 3940(98) 00639- 9
18 A. Navarro et al. / Neuroscience Letters 254 (1998) 17–20

Fig. 1. Immunostained sections, for apo D, of different brain areas. (A) White matter of central nervous system, the glial cells appear clearly
marked for apo D (arrows) (35 years). (B) Cerebellar cortex immunoreactive (arrows) and non-immunoreactive (arrowheads) Purkinje cells can
be observed. In the white matter a great number of immunoreactive glial cells (arrows) appear (35 years). (C) Dentate nucleus of cerebellum,
neurons with high immunoreactivity (arrows) (36 years). (D) Purkinje layer, group of Purkinje neurons in which only one immunoreactive cell is
present (arrows) (35 years). (E) Parietal cortex, the immunoreactivity is only present in the glial cells (arrows), neurons are not positives for apo D
(arrowheads) (24 years). (F) Nucleus cunneatus, an intense immunoreactivity can be observed in all neurons (54 years). Scale bars, (A,C,D) 28
mm; (B,F) 14 mm; (E) 17.5 mm.
 A. Navarro et al. / Neuroscience Letters 254 (1998) 17–20
in thickness tissue sections that were treated with 0.1%
Triton X-100 in H2O for 10 min. Endogenous peroxidase
and non-specific binding were blocked by sequential incu-
bation of the sections in 3% hydrogen peroxide solution and
in normal serum. Incubation with a specific antibody against
apo D (1:2000) dilution was carried out overnight at 4°C
(provided by Dr. C. López-Otı́n, Departamento de Bio-
quı́mica y Biologı́a Molecular, Universidad de Oviedo).
Purification protocol of polyclonal antiserum against apo
D was described in previous papers [4,7]. The immunoreac-
tivity was detected using the Extravidin-biotin-peroxidase
kit (Sigma Extra-3). Peroxidase activity was demonstrated
by incubation with 0.05% DAB in 50 mM Tris buffer pH
7.5, containing 0.05% H2O2. Finally the sections were coun-
terstained with formaldehyde-thionine, dehydrated, cleared
in eucalyptol and mounted with Eukitt.
For control purposes, representative sections were pro-
cessed in the same way with a non-immune serum or with
specifically absorbed sera instead of the primary antibody.
Under these conditions no specific immunostaining was
observed. Sections were photographed in a Nikon Labophot
microscope on panchromatic film (Agfapan, APX25). To
diminish the contrast coloration, in black and white photo-
graphy, the sections were photographed with a blue filter.
In sampled human brain, high apo D immunoreactivity
was found in the neuroglial cell of the white matter (Fig.
1A,B). The soma and the processes of these cells show an
intense deposit of apo D. By contrast, only a few neuroglial
cells of the grey matter appear stained for apo D (Fig.
1E).These observations are in correlation with the studies
achieved in other species [3,10,13,17]. Neurons apo D posi-
tive were only found in some of the different brain regions
studied in the present work. The neuroglial deposits of apo
D show a granular aspect, while a more diffuse deposit was
observed in the apo D positive neurons. Unlike the gray
matter no changes were observed in white matter tracts of
different brain areas, where astrocytes and oligodendrocytes
appear strongly labeled.
The immunohistochemical staining of several human
brain areas showed that neurons do not accumulate apo D
uniformly. In the samples containing the pons and the brain-
stem, apo D immunoreactivity was detected in many of the
neuronal nuclei: olivary, arciforme, cuneado, raphe, etc.
(Fig. 1C,F). However a few isolated apolipoprotein D-
immunoreactive neurons were observed in nearest nuclei,
amid a vast majority of unlabeled neurons.
In the cerebellum, strongly immunoreactive neurons to
the apo D were scattered throughout the Purkinje layer
(Fig. 1B,D). In the molecular and granular layer most of
the cells positive for apo D were identified as neuroglia
while only an occasional displaced Purkinje cell apo D
positive neuron was observed. Dentate nucleus, within the
deeper white matter of the cerebellum, presented a high apo
D immunoreactivity in most of their neurons (Fig. 1C).
In the frontal, parietal and temporal cortex neurons apo D
positive were not observed, and only a few and scattered
19
neuroglial cells (Fig. 1E) show positive immunoreactivity
for apo D. Some apo D positive neurons were present in the
basal nuclei, where the higher immunoreactivity appears in
myelinic tracts.
The functional role of apo D in CNS is not yet known. In
brains of several species, apo D is produced by neuroglia
and neurons [3,15,16]. Smith et al. [16] have suggested that
apo D may transport cholesterol or its derivatives across the
blood–brain barrier maintaining appropriate cholesterol
levels in compartments not directly in contact with the
blood. Patel et al. [11] have found that apo D is constitu-
tively secreted by cultured astrocytes and that the secreted
protein is associated with cholesterol and other lipids. A
potential role for apo D in neural regeneration has been
suggested by the observation that its levels increase mark-
edly in the regenerating nerve. This increase appears to be
due to an induction of apo D gene expression in endoneurial
fibroblast [2,17]. Recent works of neuronal degeneration
have implicated the increased in neuronal apo D immunor-
eactivity with a potential role in neural regeneration [10,18].
High levels of apo D immunoreactivity in rat hippocampus
following excitotoxic injury with kainic acid have been
reported in neurons that probably may be ultimately des-
tined to cell death. Authors found that apo D is increased in
some pyramidal neurons but not in glial cells or any vascu-
lar compartments in rats that suggest a neuronal synthesis
[10]. A similar increase of apo D levels has been found in
Niemann-Pick disease type C mouse brains [18]. These
findings could explain the lack of neuronal apo D immunor-
eactivity in normal human cortex, hippocampus and cere-
bellum. However it is insufficient to explain why other
nuclei present a strong labeling to apo D in all neurons
where we could not suppose a neuronal loss. While a true
high affinity physiological ligand of apo D has not been
found, it is possible that apo D has multiple physiological
ligands and functions and that both vary according to the
site of synthesis or localization.
The present study highlights the features of apo D immu-
noreactivity in different neuronal populations throughout
CNS in humans and provide more data to elucidate its func-
tional role. Clearly, additional works need to be done in
humans to observe the role of apo D in development
aging and pathology of the CNS.
This research was supported by a FISS (96/1546) Grant.
We thank Dr. C. López-Otı́n for providing the antibody for
apo D and for helpful discussion.
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