Protective effect of BDNF against beta-amyloid induced neurotoxicity in vitro
and in vivo in rats
S. Arancibia⁎, M. Silhol, F. Moulière, J. Meffre, I. Höllinger, T. Maurice, L. Tapia-Arancibia
Univ Montpellier 2, Montpellier, F-34095, France
Inserm, U710, Montpellier, F-34095, France
EPHE, Paris, F-75007, France
a b s t r a c ta r t i c l ei n f o
Received 16 November 2007
Revised 31 March 2008
Accepted 15 May 2008
Available online 15 July 2008
Dentate gyrus hilus
We examined the potential protective effect of BDNF against β-amyloid-induced neurotoxicity in vitro and in
vivo in rats. In neuronal cultures, BDNF had specific and dose–response protective effects on neuronal toxicity
induced by Aβ1–42and Aβ25–35. It completely reversed the toxic action induced by Aβ1–42and partially that
induced by Aβ25–35. These effects involved TrkB receptor activation since they were inhibited by K252a.
Catalytic BDNF receptors (TrkB.FL) were localized in vitro in cortical neurons (mRNA and protein). In in vivo
experiments, Aβ25–35 was administered into the indusium griseum or the third ventricle and several
parameters were measured 7 days later to evaluate potential Aβ25–35/BDNF interactions, i.e. local
measurement of BDNF release, number of hippocampal hilar cells expressing SRIH mRNA and assessment
of the corpus callosum damage (morphological examination, pyknotic nuclei counting and axon labeling
with anti-MBP antibody). We conclude that BDNF possesses neuroprotective properties against toxic effects
of Aβ peptides.
© 2008 Published by Elsevier Inc.
Alzheimer's disease (AD) is a progressive neurodegenerative
disorder characterized by mild cognitive impairments at onset and
deficits in multiple cortical functions in later stages. In the dementia
stages, numerous senile plaques and neurofibrillary tangles accom-
panied by neuronal loss are observed. The senile plaques are
essentially composed of amyloid β-peptide (Aβ), a 40–42 amino acid
peptide fragment of the β-amyloid precursor (APP) (Glenner and
Wong,1984), but also of Aβ25–35oligomers (Gruden et al., 2007; Kubo
et al., 2002). Aβ accumulation can result in oxidative stress,
inflammation, and neurotoxicity, all of which can initiate the
pathogenic cascade, ultimately leading to apoptosis and deterioration
of the neurotransmission systems (Yankner, 1996).
Recent findings have suggested that a decrease in brain-derived
neurotrophic factor (BDNF) levels could be associated to the
pathogenesis of AD. BDNF is an endogenous protein from the
neurotrophin family involved in the structural and functional
plasticity of the brain (McAllister et al., 1999; Poo, 2001). It protects
neurons against different kinds of brain insult (Lindvall et al., 1994;
Tapia-Arancibia et al., 2004) and also plays important roles in the
neural development and maintenance of central and peripheral
neurons (Lewin and Barde, 1996; Thoenen, 1995). In AD patients, it
has been observed that the precursor form of BDNF and mature BDNF
(Peng et al., 2005; Michalski and Fahnestock, 2003) or its mRNA
(Holsinger et al., 2000; Phillips et al., 1991) are decreased in the
parietal cortex and hippocampus even in pre-clinical stages of AD.
BDNF serum concentrations also vary over the course of the disease
and are correlated with the severity of dementia (Laske et al., 2007).
Strikingly, Murer et al. (1999) demonstrated in AD brains that neurons
containing neurofibrillary tangles, a hallmark of the disease, do not
contain BDNF-immunoreactive material whereas most intensely
BDNF-labeled neurons were devoid of tangles. Taken together, these
findings support a role of BDNF in the etiology of AD and suggest a
potential neuroprotective action of BDNF in AD treatment.
We examined the presence of BDNF receptors and the impact of
BDNF administration on the toxic effects of Aβ peptides (Aβ25–35and
Aβ1–42) in primary cultures of cortical neurons. In parallel, we inves-
tigated whether our in vitro results could be in keeping with some in
vivo Aβ/BDNF interactions supporting eventual protective effects in
areas related to cognitive functions, i.e. gyrus dentate and corpus
callosum. Adult rats were thus injected with aggregated Aβ25–35
Neurobiology of Disease 31 (2008) 316–326
⁎ Corresponding author.INSERM Unité 710, Université de Montpellier 2, Place Eugène
Bataillon, cc 105, 34095 MONTPELLIER Cedex 5, France.^Fax: (+33/0) 4 67 14 33 86.
E-mail address: sandor.arancibia@univ^-montp2.fr (S. Arancibia).
Available online on ScienceDirect (www.sciencedirect.com).
0969-9961/$ – see front matter © 2008 Published by Elsevier Inc.
Contents lists available at ScienceDirect
Neurobiology of Disease
journal homepage: www.elsevier.com/locate/ynbdi
griseum (IG)orin the3rd ventricle(icv).Theformerisa medialcortical
region near the corpus callosum and classically felt to be a displaced
hippocampal anlage (Wyss and Sripanidkulchai, 1983). Following the
number of somatostatin (SRIH) neurons in the dentate gyrus hilus and
Materials and methods
Adult pregnant females or male Sprague–Dawley rats (Depré, St
Doulchard,France)(230–250g)were housedforat least 1week before
the experiments and kept under constant temperature (21 ± 1°C) and
lighting (light on from 07:00 am to 07:00 pm) regimens. Food pellets
and water were freely available throughout the experiment. Proce-
dures involving animals and their care were conducted in conformity
with French laws on laboratory animals that are in compliance with
international laws and policies (EC Council Directive 86/609, OJ L
358,1,24 November 1987). The Animal Welfare Committee at the
University of Montpellier II approved all protocols and particular
efforts were made to minimize the number of animals used and
potential pain and distress.
(Weil am Rhein, Germany) or NeoMPS (Strasbourg, France). Neurobasal
media, B-27 supplement and fetal bovine serumwere from GIBCO Invi-
trogen (Cergy Pontoise France). BDNF was a generous gift from
Regeneron Pharmaceutical (USA) and NGF was from Genentech, Inc.
(USA). All other chemicals, unless specifically mentioned, were pur-
chased from Sigma-Aldrich (St Quentin Fallavier, France).
Antibodies against BDNF (sc-546, lot E0704), TrkB.FL (sc-12, lot
J111) and TrkB (TK−) (sc-119, lot I1004 epitope mapping at the C-
terminus recommended for detection of truncated receptors) were
from Santa Cruz Biotechnology (Santa Cruz, CA). MBP mouse
monoclonal antibody against myelin basic protein was from Boehrin-
ger, Mannheim, Germany. The Alexa Fluor 488 secondary antibodies
(TrkB, BDNF and MBP detection) were from Molecular Probes (Leiden,
Cerebral cortical cultures greatly enriched in neurons were
prepared from embryonic day 17 rat fetuses obtained from Sprague–
Dawley rats, as previously described (Tapia-Arancibia and Astier,
1989) with minor modifications. Cells plated at 2.5 × 105cells/cm2
were cultured in poly-D-lysine coated 24-well plates and maintained
in Neurobasal medium supplemented with B-27 components (Invi-
trogen, Life Technologies, Cergy Pontoise, France) that contains a great
number of trophic and protective antioxidant compounds and
prevents the proliferation of glial cells (Brewer et al., 1993). The
potential proliferation of remaining non-neuronal cells was inhibited
by treatment with 10 μM cytosine arabinoside for 48 h between days 3
and 5 after plating. Cultures were grown for 6 days before treatments,
which were performed between 6–8 days in vitro (DIV).
RNA extraction and cDNA synthesis
Total RNA was extracted from cortical cultures using the High Pure
RNA Isolation Kit (Roche Diagnostique, Meylan, France) according to
the manufacturer's instructions. RNA concentration and purity were
evaluated by spectrometry on the basis of optical density (OD) mea-
surements at 260 and 280 nm. cDNA synthesis was performed as
already described (Silhol et al., 2007).
Quantitative real-time PCR
Real-time PCR was performed using a LightCycler rapid thermal
cycler system (Roche Diagnostics, Mannheim, Germany) according to
the manufacturer's instructions. The PCR reactions were performed in
a final volume of 20 μl with 1 × LC-DNA Master SYBR Green I mix, 3
mM MgCl2, 0.5 μM of each primer and 1/5 diluted RT mixture for trkB.
FL and trkB.T1, and 1/10 diluted RT mixture for cyclophilin (and water
as negative control) was added as PCR template. The amplification
conditionswerethe sameas already described(Silhol et al., 2007). The
amounts of target genes were normalized against cDNA of the
housekeeping gene cyclophilin in the corresponding samples. Primers
were developed using the Primer 3 software package (Rozen and
Skaletsky, 2000). Primer sequences specific to the genes examined
and predicted product sizes are shown in Table 1.
Cultures were grown in the B27-supplemented Neurobasal serum-
free medium. Since we observed that, in the presence of B27, amyloid
peptides were not toxic for neurons up to high concentrations the B27
supplement was removed from the cultures during exposure to Aβ
peptides between 6 and 8 DIV. Cells were washed for 1 h with
Neurobasal medium and then incubated for 48 h in the presence or
absence of Aβ peptides with or without BDNF. Control cells were
incubated in Neurobasal medium alone.
Measurement of in vitro cell viability
Neuronal survival was determined at 8 DIV by trypan blue
exclusion (Pike et al., 1993), which detects dead cells, or using the 3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay (Roche), which detects active mitochondrial dehydrogenases
of living cells to reduce MTT to a water-insoluble blue formazan
product (Ivins et al., 1999). Since initial assays showed that the two
methods gave comparable results, we continued our studies with the
MTT method. In brief, cultures grown in 24-well plates were rinsed
with 1 ml Locke's solution, then supplemented with MTT, and
incubated for 2 h in a 5% CO2incubator at 37°C. The reaction product,
solubilized in dimethylsulfoxide, was measured with an ELISA plate
reader at 570 nm. The optical density of dimethyl sulfoxide was used
as background and subtracted. MTT assays can quantify the survival
promoting the effects of neurotrophins (Manthorpe et al., 1986); the
viability of cortical neurons (Tong et al., 2004) or neuroblastoma cells
(Olivieri et al., 2003) after Aβ treatment and cerebellar granule neuron
apoptosis (Skaper et al., 1998). In each experiment, cell viability was
determined from four wells for each condition and normalized to
parallel controls with each well being treated as a single observation.
Data were obtained from at least four separate cultures and expressed
asmeans± S.E.M.Statisticalcomparisonwasdeterminedbyan ANOVA
test with Student's t test as the post hoc test.
On the day of the experiment, cells were fixed in 4% PFA in saline
phosphate buffer (PBS). The antibodies used included rabbit IgG
polyclonal antibodies against BDNF (1/50), TrkB.FL (recognizing the C-
Primers and expected sizes of PCR products with each primer pair
GeneForward primerReverse primerSize (bp)
S. Arancibia et al. / Neurobiology of Disease 31 (2008) 316–326
terminal cytoplasmic domain of the receptor) (1/100) and TrkB.T1 (1/
100). Cells were first incubated for 4 to 6 h in blocking buffer (PBS
containing 0.1% Triton X-100 and 2% BSA) and then incubated
overnight at 4°C in a humid chamber with primary BDNF and TrkB
antibodies in the same buffer. After rinsing in PBS, they were
incubated for 2 h at room temperature in a humid chamber with
secondary antibody diluted in the blocking buffer: an anti-rabbit IgG
conjugated with Alexa Fluor 488 (1/1000). After careful rinsing, cells
were mounted in FluorSave reagent (VWR International, Strasbourg,
France) and observed under a DMR fluorescent microscope (Leica,
Adjacent sections (10 μm) obtained from brains of control and
treated rats were incubated overnight at room temperature in a
humid chamber with the primary antibody anti MBP (1/500) in PBS
containing 0.4% Triton X-100 and 3% goat normal serum. After rinsing
in PBS, they were incubated for 2 h at room temperature in a humid
chamber with the secondary antibody, an anti-mouse IgG conjugated
with Alexa Fluor 488 (1/1000) (Molecular Probes, Leiden, Nether-
lands) diluted in PBS containing 0.1% Triton X-100 and 1.5% goat
normal serum. After careful rinsing, slices were mounted in FluorSave
reagent (VWR international, Strasbourg, France) and observed under a
DMR fluorescent microscope (Leica, Rueil-Malmaison, France).
In the two cases (cells and slices), controls of the immunostaining
specificity consisted of (1) omitting the primary antibody and
applying the secondary antibody alone and (2) exciting each
fluorochrome by inappropriate wavelengths. This allowed us to
confirm that the secondary antibody used did not induce artifactual
fluorescent labeling and that there was no overlap in the emission
spectra of the two fluorochromes.
After a 7 day laboratory acclimation period, rats were anesthetized
with sodium pentobarbital (40 mg/kg) and a push-pull device (Phymep,
Paris) consisting of a stainless steel cannula (0.5 mm outer diameter)
fitted with a stylette was stereotaxically implanted into the IG according
to Paxinos and Watson's (1997) coordinates (AP: 3.3 mm, L: 0.0 mm and
DV: 2.8 mm). After surgery, the animals were caged separately, handled
every day for 1 week and, after recovery of their preoperative body
weight, subjected to push-pull perfusion or injections. Briefly, as
previously reported (Arancibia, 1987; 2007), push-pull perfusion was
performed on unanesthetized animals. Artificial cerebrospinal fluid was
infused at a flow rate of 15 μl/min and samples (225 μl) were collected
every 15 min for 2 h following intracerebral (ic) administration of Aβ25–35
peptide or Aβ25–35peptide + BDNF. Perfusates were centrifuged and
evaporated in a Speed-Vac concentrator (Savant Instruments, Hicksville,
NY) and immediately stored at −20°C until BDNF ELISA immunoassay. As
control groups, we used rats injected with either scramble Aβ25–35
peptide or saline (sham-operated). As no differences were observed
between these controls, they could be used indiscriminately.
Fig. 1. Presence of BDNF and its receptors in primary cultures of rat cerebral cortical
neurons. (A) Changes in trkB.FL and trkB.T1 mRNA expression as a function of days in
vitro of cortical cultures determined by real-time PCR. A peak of trkB.FL mRNA levels
was observed at 8 DIV, whereas trkB.T1 levels increased later, from 8 DIV. (B)
Immunofluorescence detection of BDNF, TrkB.FL and TrkB.T1 proteins in cortical
neurons at 8 DIV. At day 8, cells were fixed and immunostained with specific antibodies
against BDNF, TrkB.FL and TrkB.T1 as indicated in Materials and methods. BDNF and
TrkB.FL, the catalytic receptor, were strongly represented but the TrkB.T1 signal was
weakly labeled. Scale bar=50 μm.
Fig. 2. Dose-dependent neurotoxicity of Aβ1–42and Aβ25–35peptides in rat cortical
neurons. Increasing concentrations of Aβ1–42(A) and Aβ25–35(B) were added to cells in
neurobasal medium on day 6 of culture and the toxicity was estimated at day 8 by the
MTT assay. The experimental data were from four different cultures with n=4 dishes/
culture at each concentration. Values represent mean±SEM. ⁎⁎pb0.01, ⁎⁎⁎pb0.001
compared with non-treated groups.
S. Arancibia et al. / Neurobiology of Disease 31 (2008) 316–326
All push-pull experiments were performed in conscious rats at the
same time of the day to avoid variations due to possible circadian
BDNF release. Samples were collected for 2 h and at the end of the
experiments animals were killed and their brains removed and fixed
for histological examination or in situ hybridization. Rats with an
unsuitable cannula location, or presenting a glial cells reaction around
the scar, were excluded from the study.
BDNF release was measured with a conventional two site enzyme-
immunoassay system (ELISA) assay, as previously reported (Arancibia et
al., 2007), according to the manufacturer's protocol (Promega, Charbon-
nières, France). Briefly, each evaporated sample was reconstituted with
100 μl H20 and used to determine BDNFrelease. The assaysensitivity was
15 pg/ml and the cross reactivity with other related neurotrophic factors
was less than 3%. The BDNF concentration was expressed as picograms
per fraction. The intra- and inter-assay coefficients of variation were 3%
and 8%, respectively. Non-detectable values were considered, by conven-
tion, as being equal to the assay detection limit.
Toxicity in vivo studies
Aggregates of Aβ25–35or scrambled peptide were prepared by
incubating the peptides at 3 mg/ml concentration in sterilewater for 4
days at 37°C. Formation of aggregates was confirmed visually by
phase-contrast microscope inspection (200X). Subsequently, 9 nmol/3
μl of aggregated Aβ25–35was administered to unanesthetized rats
through the push segment of the push-pull cannula (1 μl/min). Sham-
operated rats received the same volume of vehicle. Histological
observations were performed 7 days after Aβ25–35administration.
When the neuroprotective effect of BDNF was examined, it was
administered (6 μg/6 μl) twice: 1 day before and 1 h before Aβ25–35
administration and the studies were always performed 7 days later. At
the 7th day, animals were killed by decapitation. A scheme showing
the experimental design is illustrated in Fig. 6.
After in vivo experiments, rats were killed and their brains quickly
frozen in liquid nitrogen. The frozen brains were mounted on a
cryostat (Leica, Rueil-Malmaison, France) and serially cut into 10 μm
coronal sections. The mounted slides were stained with Harris'
hematoxylin solution for 3 min, dehydrated in alcohol and mounted
in Entellan (Merck, Darmstadt, Germany). This method is used to
assess the cytoarchitecture as well as degenerative changesin neurons
in areas of neurodegeneration according well established criteria
(Farber 1982; Garcia et al., 1995; Giovannelli et al., 1998; Stepanichev
et al., 2004). Briefly, undamaged neurons were recognized as cells
with round-shaped blue nuclei and clear perinuclear cytoplasm.
Damaged neurons appeared as cells with altered nuclei (pyknosis,
karyorrhexis and karyolysis) and cytoplasm with loss of hematoxylin
Brains dissected from another independent group of animals were
devoted to immunological assay with MBP antibody as above
In situ hybridization of SRIH
Somatostatin in situ hybridization was performed on frozen brains
as previously described (Arancibia et al., 2001) with digoxigenin-
labeled oligonucleotide probe. Briefly, a 45-mer oligonucleotide
antisense probe for somatostatin-14 (5′ to 3′ sequence: CCAGAA-
GAAAGTTCTTGCAGCCAGCTTTGCGTTCCCGGGGTGT) (Genosys, Cam-
bridge, UK) was end-labeled with digoxigenin-11-dideoxyuridine-5-
triphosphate. Negative controls were carried out by omitting the
labeled probe from the hybridization buffer, or by incubating the
sections with a 45-mer oligonucleotide sense probe based on the
Fig. 3. Neuroprotective effect of BDNF on rat cortical neurons exposed to Aβ1–42and
Aβ25–35for 2 days. 20 μM Aβ1–42(A) and 20 μM Aβ25–35(B) were added to cells in the
presence of increasing concentrations of BDNF in neurobasal medium on day 6 of
culture and toxicity was estimated at day 8 by the MTT assay. The experimental data
were from three different cultures with n=4 dishes/culture at each BDNF concentration.
Values represent mean±SEM. ⁎pb0.05, ⁎⁎pb0.01 and ⁎⁎⁎pb0.001 compared with
groups without BDNF.
Fig. 4. Effect of NGF on rat cortical neurons exposed to Aβ1–42and Aβ25–35for 2 days. 20
μM Aβ1–42and 20 μM Aβ25–35were added to cells in the presence of 50 ng/ml NGF in
neurobasal medium on day 6 of culture and toxicity was estimated at day 8 by the MTT
assay. The experimental data were from two different cultures with n=4 dishes/culture
for each experimental condition. Values represent mean±SEM. ⁎pb0.05 compared with
groups without NGF. NGF alone, only at this high concentration (50 ng/ml), increased
cell survival by 10% and weakly protected cells, only against Aβ1–42-induced toxicity.
S. Arancibia et al. / Neurobiology of Disease 31 (2008) 316–326
Cellular counting and analysis
DIG-labeled cells and hematoxylin-stained cells (n = 5 rats/group;
8–10 sections per animal) were quantified using a Sony CCD XC-77
video camera with high resolution (570(H)3485 (V) TV lines) coupled
to a Macintosh computer (Power PC G3) and NIH imageJ software
(1.37v, W. Rasband, NIH, Bethesda, USA) (Arancibia et al., 2001;
Givalois et al., 2004). For each animal, the cell number or the DIG-
labeled cells were counted from 8 serial, non-adjacent 10 μm-thick
coronal sections from the dorsal through the antero-posterior extent
of the hippocampus between bregma −2.8 to −4.3 according the atlas
of Paxinos and Watson. The count of stained sections was done by
three different scientists unaware of the experimental conditions and
independently from each other. Hilar neurons were considered
according classical criteria (Lowenstein et al., 1992). Data were
expressed as means ± S.E.M. Statistical comparisons of values were
performed using a one-way analysis of variance (ANOVA; Statview
4.5) followed by a Fisher's PLSD test, as previously reported (Arancibia
et al., 2001).
In vitro data
Primary cultures of cortical neurons
We first investigated the developmental expression of mRNAs
encoding TrkB.FL and TrkB.T1 receptors in cortical neurons in primary
cultures by real-time PCR in three independent experiments and each
with n = 4 wells (Fig. 1A). The results are presented as the ratio
between trkB.FL or trkB.T1 and cyclophilin mRNAs and all of these
parameters were determined in the same sample obtained from the
same RT. We could thus compare the relative amounts of each mRNA
at the same stage. We observed thattrkB.FL mRNA levelsprogressively
increased from 3 DIV to reach maximal levels at 8 DIV. In contrast,
mRNA coding for the TrkB.T1 receptor was weakly expressed up to 8
DIV but its expression sharply increased at 14 DIV. Then, we used
immunocytochemical staining to examine the presence of BDNF, TrkB.
FL and TrkB.T1 proteins at 8 DIV since the toxicity and pharmacolo-
gicalexperimentswereperformedat thisstage.Fig.1Bshows that,at 8
DIV, neurons were strongly immunostained for BDNF and TrkB.FL but
weakly stained for TrkB.T1.
Effect of increasing concentrations of Aβ1–42and Aβ25–35peptides on cell
As expected, 48 h exposure to Aβ1–42or to Aβ25–35induced a toxic
dose-dependent effect on cortical neurons (Figs. 2A and B) with a
maximal effect of around 40% and 60% at 20 μM, respectively. The
scrambled peptides failed to affect cell survival compared with
vehicle-treated control groups: 102 ± 4% at 20 μM Aβ1–42or 100 ±
4% at 20 μM Aβ25–35 vs. 98 ± 3% control group. From these
experiments, it was concluded that Aβ25–35 was more toxic than
Aβ1–42on in vitro cortical neurons.
Effect of increasing BDNF concentrations on Aβ1–42and Aβ25–35-treated
We examined the ability of BDNF to protect neurons against beta
amyloid peptide-induced toxicity. BDNF was able to significantly
protect cortical neurons from both 20 μM Aβ1–42and Aβ25–35-induced
toxicity. This BDNF protection was dose-dependent and significant
from 10 ng/ml and maximal at 50 ng/ml for Aβ25–35-induced toxicity.
Nevertheless, protection against Aβ25–35-induced toxicity (Fig. 3B)
was not complete (around 80%) whereas at 50 ng/ml BDNF completely
reversed Aβ1–42-induced toxicity (Fig. 3A). At 100 ng/ml, BDNF even
surpassed the control group, probably by protecting cells from natural
death. We also examined the effect of NGF (nerve growth factor) on
Aβ1–42- and Aβ25–35-induced toxicity. In spite of the strong concen-
tration used (50 ng/ml), this neurotrophin presented a very weak
protective effect on 20 μM Aβ1–42-induced toxicity and no effect on 20
μM Aβ25–35-induced toxicity (Fig. 4).
Trk involvement in BDNF protective activity
To investigate the potential involvement of Trk receptors in the
protective effects of BDNF described here, we treated cells with K252a,
a Trk receptor inhibitor (Koizumi et al., 1988; Tanaka et al., 1997), 5
min before BDNF addition. K252a (100 nM) completely blocked the
protective effects of BDNF on Aβ1–42- and Aβ25–35-induced toxicity
(Figs. 5A, B). K252a added alone significantly (ap b 0.001) decreased
cell survival compared to the control group (40–50%), probably by
blocking the protective effect of BDNF endogenously released by
In vivo data
Since our in vitro studies demonstrated that Aβ25–35was more
toxic than Aβ1–42, and that we previously showed that Aβ25–35retains
the ability to self aggregate and mediate toxicity invivo (Maurice et al.,
1996; Meunier et al., 2006), we injected animals with this fragment
either in the IG or in the 3rd ventricule.
Fig. 5. Trk involvement in BDNF protection against toxicity induced by Aβ1–42and
BDNF and 20 μM Aβ1–42(A) and Aβ25–35(B) peptide addition to rat cortical neurons.
Substances were added to cells in neurobasal medium on day 6 of culture and toxicity
was estimatedat day8bythe MTTassay.The experimental datawerefrom twodifferent
cultures with n=4 dishes/culture for each experimental condition. Values represent
mean±SEM. ⁎⁎pb0.01 compared with the grouptreated with 50 ng/ml BDNFand 20 μM
Aβ1–42but without K252a, ⁎pb0.05 compared with the group treated with 50 ng/ml
BDNF and 20 μM Aβ25–35without K252a.apb0.01 compared to the control group.
S. Arancibia et al. / Neurobiology of Disease 31 (2008) 316–326
Effect of Aβ25–35 on in vivo BDNF release measured by push-pull
perfusion of IG
Seven days after chirurgical cannula implantation, BDNF release
from IG was estimated using push-pull perfusion. Under control
conditions, BDNF release exhibited basal values ranging between 7 ± 4
pg/15 min, with peaks of 40 ± 5 pg/15 min throughout 120 min of
perfusion. At the end of this procedure, animals were injected with
Aβ25–35(group 1) or BDNF (group 2) and allowed to rest for 7 days
before the next perfusion. Animals of group 2 received, 24 h after the
1st BDNF injection, a 2nd administration of BDNF + Aβ25–35, and a new
push-pull perfusion on the same animal was performed on day 14.
Animalsof group 1 (Aβ25–35alonewithout BDNF) werealso perfused7
days after the first perfusion. A scheme of the experimental design is
shown in Fig. 6A. In group 1, Aβ25–35administered rats, the values of
BDNF release estimated in the perfusate by ELISA assay did not yield
differences as compared to values obtained for control rats (repre-
sentative experiments are shown in Fig. 6B). In contrast, animals of
group 2 yielded BDNF release values that were significantly higher
than the control values (first perfusion of the same animal, 7 days
previously). These higher values concerned the basal release (281 ±
26%), peaks (205 ± 11%) or total release (247 ± 22%). Quantitative data
are shown in Fig. 6C.
Secondarily, we used the icv route of Aβ administrationtocompare
the results of BDNF release obtained through IG administration with
those obtained fromothermedial regions (3rdventricle) close toareas
examined in this study. We succeeded in showing, and for the first
time, detectable basal BDNF levels in the third ventricle in rats, which,
similarly to BDNF release arising from IG, was not affected by Aβ
treatment. BDNF release was 11. 7 ± 2.2 pg/15 min (Aβ25–35group) vs.
11.9 ± 1.8 pg/15 min (control group) (n = 5).
Number of dentate gyrus hilar cells expressing SRIH mRNA in histological
sections from hippocampus
A significant (p b 0.02) decrease in the number of cells expressing
SRIH mRNA was observed in the dorsal hilar region of Aβ-treated
animals (Fig. 7B) as compared to sham-operated animals (Fig. 7A)
[135 ± 13 cells (Aβ25–35group) vs. 170 ± 7 cells (control group)]. In
contrast, when both BDNF and Aβ25–35were co-administered, the
harmful effect of Aβ25–35alone was completely reversed (Fig. 7C) and
even surpassed the control values (219 ± 8 cells), as shown in the
Fig. 6. Invivo BDNFrelease from IG inpush-pull cannulated, unanesthetized, free-moving rats 7 days after stereotaxic surgery. (A) Experimental invivo design. All animals underwent
surgery to implant the ic or icv push-pull cannula on day0. The first group underwent a first push-pull perfusion at the 7th day, at the end of which the Aβ25–35or scramble peptide or
saline solution was administered (group 1). When BDNF release was analyzed, a 2nd push-pull perfusion was performed 7 days later, on day 14. Then the animals were decapitated
and their brainwas removed and frozen for morphological studies. Otherwise, the animals were killed 7 days after Aβ25–35administration. In the second paradigm (group 2), animals
underwent a first push-pull perfusion at the 7th day, at the end of which BDNF was injected. The next day the animals received a second dose of BDNF followed by Aβ25–35
administration. On day 14, a 2nd push-pull perfusion was performed or, if BDNF release was not determined, the animals were decapitated and their brain was removed and frozen
for morphological studies. (B) Individual representations. Three different experimental conditions are illustrated: sham-control rat (♦), Aβ25–35injected rat (■) and Aβ25–35+BDNF
(▲) injected rats. Note that peak-like secretion was observed in the three experimental conditions. (C) Statistical representation including all animals studied. BDNF secretion was
calculated as total, peaks and basal amount of BDNF. Note that administration of Aβ25–35alone had no effect on BDNF release, but administration of Aβ25–35+BDNF significantly
modified the three parameters.
S. Arancibia et al. / Neurobiology of Disease 31 (2008) 316–326
statistical representation (Fig. 7D) (n = 5 rats/group). The analysis of
hematoxylin-stained sections adjacent to those processed for in situ
hybridization experiments allowed us to express data as percentage of
somatostatin cells vs. total cells found in the dorsal hilus: 32.5 ± 2.4%
(control group), 23.8 ± 3.2% (Aβ25–35group) and 47.5 ± 3.7% (Aβ25–35+
BDNF group). Examination of cingular and parietal cortex revealed
that Aβ treatment had no toxic effect on the number of SRIH neurons
but that BDNF administration induced a significant positive effect on
parietal SRIH neurons (Fig. 7D).
Effect of Aβ25–35administration on histological integrity of corpus
callosum and on immunostaining labeling
after Aβ25–35 administration or when co-administered with BDNF
according to the protocol specified in Fig. 6. The axonal histological
lesion was characterized by a white matter rarefaction and a clear loss
at −1.8 AP bregma, the lesion was laterally extended up to 1.9 mm
whereas at bregma −3.3, the lesion attained laterally 3.7 mm. This
injury pattern was observed throughout the corpus callosum as
demonstrated in our serial study in slices included between bregma
hematoxylin staining method. Histological observations and counting
of damaged cells (Table 2) showed that BDNF co-treatment signifi-
cantly prevented the Aβ25–35harmful effects.
Fig. 9 shows the corpus callosum morphology at higher magnifica-
tion in controls, Aβ25–35, and Aβ25–35 + BDNF-treated animals. In
control animals, MBP immunostaining of corpus callosum appeared
uniform and the staining showed the typical railroad structure. When
animals received Aβ25–35administration the MBP pattern revealed a
clear lesion in the corpus callosum consisting in myelin fragmentation
which was appreciably attenuated in BDNF-treated animals. Myelin
damage appeared as a toxic sign whose improvement by BDNF
treatment, although evident, was less striking than other histological
features observed here. This fact is probably in keeping with the
latency required to rescue axons.
Our findings show that BDNF has neuronal protective effects
against Aβ peptide toxicity in vivo and in vitro. The mechanisms by
which amyloid peptides are neurotoxic are not yet understood and
attempts to find protective molecules are exciting and promising. As
BDNF is able to protect neurons against cellular damage (Knüsel et al.,
1992; Lindvall et al., 1994), we hypothesized that it would be pro-
tective against Aβ aggression. Given that in this paradigm cortical
neurons express TrkB receptors (mRNA and protein) and the signal-
transducing BDNF-specific receptor (Tapia-Arancibia et al., personal
observations), we assessed the protective effect of BDNF against
toxicity induced by Aβ peptides on neuronal survival in vitro.
Consistent with previous observations (Geci et al., 2007; Pike et al.,
1995; Yao et al., 2005), our data showed a dose-dependent toxic effect
of amyloid peptides in cortical neurons. Our comparative in vitro
study, performed under similar experimental conditions, indicated
that Aβ25–35was more toxic for cellular survival than Aβ1–42.
Interestingly, we found that BDNF had a protective dose–response
differences. Actually, this protection was more pronounced on toxicity
Fig. 7. Representative histological sections of rat hippocampus from differently treated animals analyzed by in situ hybridization to detect cells expressing SRIH mRNA in the dentate
gyrus hilus. Controlhistological section corresponding to a sham-operated rat(A), toanAβ25–35injectedrat(B)and toan Aβ25–35+BDNF injectedrat(C). (D) Histogram illustratingthe
previous conditions using the number of cells expressing SRIH mRNA as the quantification index. In addition to the hilus, two other regions were also analyzed and represented, i.e.
the cingular cortex and the parietal cortex.
Counting of pyknotic nuclei in the corpus callosum
pb0.01 vs. control.
S. Arancibia et al. / Neurobiology of Disease 31 (2008) 316–326
induced by Aβ1–42than that induced by Aβ25–35since it completely
the toxicity induced by Aβ25–35. BDNF-mediated protection involved TrkB
receptor activation since the effect was completely inhibited by K252a, a
potent tyrosine kinase inhibitor (Koizumi et al., 1988). NGF, another
neurotrophin of the same family, had a very weak effect in rescuing cells
from amyloid peptide-induced death, which indicated a specific action of
BDNF at similar concentrations.
Fig. 8. Representative micrographs of histological features of the corpus callosum stereologically ranging between bregma +0.2 and −3.8, in an Aβ25–35injected rat and an
Aβ25–35+BDNF injected rat. Note that in these hematoxylin-stained slices, the corpus callosum exhibits a clear loss of tissue integrity and cellular damage in the Aβ25–35
injected rat. Scale bar=25 μm.
S. Arancibia et al. / Neurobiology of Disease 31 (2008) 316–326
In view of these results, we used the experimental paradigm of
intracerebral injection of Aβ25–35peptide in the in vivo studies. The
amyloid β-derived peptide Aβ25–35contains hydrophobic transmem-
brane residues 25–35 (GSNKGAIIGLM) of the Aβ protein and
aggregates as insoluble fibrils (Yankner et al., 1990) that retain the
toxic effect of larger Aβ peptides (Pike et al., 1993). We have already
reported that the Aβ25–35peptide retains the ability to self-aggregate
and induce in vivo toxicity (Maurice et al.,1996; Meunier et al., 2006).
In addition, it has been described that soluble D-ser26Aβ1–40, possibly
produced during aging, is released from plaques and converted by
proteolysis to toxic D-ser26Aβ25–35which enhances excitotoxicity in
AD (Kubo et al., 2002). As a route of Aβ25–35 administration, we
essentially chose an in situ injection into the IG, a single median
region, thus avoiding the bilateral injection protocol used by others
(Stepanichev et al., 2004). The IG, although it has been reported to be
refractory to some kind of neuronal aggressions, surrounds some
critical regions engaged in the AD onset, i.e. cingular cortex, corpus
callosum and hippocampus, and represents a phylogenic old olfacto-
recipient outpost of the hippocampus (Adamek et al., 1984).
After Aβ25–35 administration, we observed no modification in
BDNF release from IG measured in the push-pull perfusate, which
probably indicates that there are no changes in endogenous BDNF
content at this Aβ25–35concentration. In contrast, previous exogenous
administration of BDNF strongly increased BDNF release according to
invitro data showing that BDNF increases BDNFrelease (Canossa et al.,
1997). The expression of trkB.FL mRNA in cingular cortex surrounding
the IG region as well as in the hilar region of the dentate gyrus
(Arancibia et al. personal observations; Merlio et al., 1992; Yan et al.,
1997) further supports the physiological significance of our findings
concerning BDNF release (IG) and BDNF protective action (hilus and
We also examined the number of hippocampal hilar cells
expressing SRIH mRNA, which represents a valuable parameter to
investigate the toxic effect of Aβ25–35(Aguado-Llera et al., 2007). This
particularclass of interneurons was chosen becauseof their sensitivity
to excitotoxicity and vulnerability to a variety of insults and
neurological diseases including Alzheimer disease (Chan-Palay,
1987; Lowenstein et al., 1992; Tallen, 2007; Ylinen et al., 1991). It
has been hypothesized that vulnerability of hilar SRIH interneurons,
which is different from that of interneurons from the rest of the
hippocampus, is due to their lack of Ca2+binding proteins (Tallent,
2007). The striatal-enriched protein tyrosine phosphatase (STEP), a
key regulator of ERK/MAPK signaling (Choi et al., 2007), would be
involved in this excitotoxic event. The significant reduction in the
number of SRIH hilar cells after the Aβ administration reported here is
in keeping with recent invitro data (Geci et al., 2007). Although we did
not inject Aβ directly into the hippocampus, Aβ might exert their
effects in distant targets of the application site as it has been reported
(Sigurdsson et al., 1997; Stepanichev et al., 2000), inducing for
example a neural disconnection between some neuronal structures
(Ahmed et al., 1995; Kunzle, 2004). Disconnection of DG from the
entorhinal cortex, its major input, is observed in early stages of AD
(Ohm, 2007). The hilar interneurons (also innervated by fibers arising
from entorhinal cortex) which control the DG granule cell activity
(Amaral et al., 2007) would be affected in this process. The reduction
in SRIH interneurons reported here was completely prevented by
previous BDNF administration, which could have easily diffused (Yan
et al.,1994; Anderson et al.,1995) from the IG or transported from the
3rd ventricle to the dentate gyrus (Mufson et al., 1996). Interestingly,
SRIH has also been described as a neurotrophic factor (Schwartz et al.,
1998; Blake et al., 2004) presumably involved in the AD etiology
(Dournaud et al.,1994; Vecsei and Klivenyi, 1995), notably in its early
onset (Ramos et al., 2006). Moreover, it has been reported that
somatostatin is involved in the catabolism of Aβ through neprilysin
activation (Saito et al., 2005), a rate-limiting enzyme for Aβ
degradation (Hama and Saido, 2005). BDNF-induced SRIH increase
could result in neprilysin-mediated Aβ degradation thus contributing
to the neuroprotective effect.
Corpus callosum damage was other of the parameters examined in
our in vivo experiments after Aβ25–35 administration alone or
combined with BDNF administration. Its atrophy or rarefaction has
been shown to be a reliable and sensitive in vivo marker of cortical
neuronal loss associated with cognitive impairment in AD (Hampel et
al., 2002; Teipel et al., 1998, 2003) and correlated with dementia
Fig. 9. Representative histological micrographs of brain rats subjected to different experimental treatments. (A) Corpus callosum morphology under control conditions (left);
degeneration afterAβ25–35administration (middle); protection byBDNF co-administrated with Aβ25–35(right) (60X magnification). PanelsB and C showareasindicated in the inserts
in (A) from histological sections stained with antibody anti-MBP (myelin basic protein) at 200X and 400X magnifications, respectively. Arrows show corpus callosum damage after
Aβ25–35administration. Right micrographs show protection by previous BDNF administration. Scale bars=150 μm (A); 250 μm (B) and 500 μm (C).
S. Arancibia et al. / Neurobiology of Disease 31 (2008) 316–326
severity in these patients (Wiltshire et al., 2005; Yamauchi et al.,
2000). Our data showed that Aβ administration causes nuclei cell
pyknosis and corpus callosum disruption, indicating fragmentation of
the axonal cytoarchitecture. Previous treatment with BDNF notably
attenuated these Aβ25–35-induced damage. Our data using anti-MBP
antibody are in keeping with our histological results.
The neuronal injuries reported here could be explained by an
oxidative damage of Aβ25–35(Stepanichev et al., 2004) considered as a
fundamental pathogenic mechanism of Alzheimer's disease (Perry et
al., 2004). BDNF could act as an antioxidative factor since it is known
that it increased the level of activity of some antioxidant enzymes
(Mattson et al.,1995). Aβ/BDNF interaction could also be explained by
Aβ interference with signaling pathways used by BDNF to exert its
protective effects, i.e. on BDNF-induced Arc (activity-regulated
cytoskeleton-associated gene) protein expression (Echeverria et al.,
2007; Wang et al., 2006), CREB phosphorylation (Tong et al., 2004) or
its nuclear translocation (Arvanitis et al., 2007). Arc synthesis controls
local actin synthesis, synaptic plasticityand cognitive functions (Wang
et al., 2006). Whatever the case, β-amyloid peptides may engender a
dysfunctional encoding state in neurons, leading to neurodegenera-
tion (Tong et al., 2004), and BDNF signaling might be compromised
early in the course of AD (Murer et al., 1999).
Overall, both the in vitro and in vivo results presented here
validated our hypothesis that exogenous administration of BDNF
exerts neuroprotective actions against toxic effects of Aβ peptides in
regions related to cognitive functions providing new insight for future
The authors thank M. Edmond Savary for technical help and
bibliographical layout. We also thank Dr. E. Aliaga for valuable advice
and Montpellier RIO Imaging for the technical facilities in micro-
scopical imagery. This work was supported by grants from the Region
Languedoc-Roussillon, Montpellier, France and ECOS-Sud action (No.
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