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)
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