Current Alzheimer Research, 2011, 8, 000-000 1
1567-2050/11 $58.00+.00 © 2011 Bentham Science Publishers Ltd.
Loss of Medial Septum Cholinergic Neurons in THY-Tau22 Mouse Model:
What Links with tau Pathology?
Karim Belarbi1,2,3,#, Sylvie Burnouf1,2,3,#, Francisco-Jose Fernandez-Gomez1,2,3,
Jeremy Desmercières1,2,3, Laetitia Troquier1,2,3, Jonathan Brouillette1,2,3, Leslie Tsambou1,2,3,
Marie-Eve Grosjean1,2,3, Raphaëlle Caillierez1,2,3, Dominique Demeyer1,2,3, Malika Hamdane1,2,3,
Katharina Schindowski1,2,3, David Blum1,2,3 and Luc Buée1,2,3,*
1Université Lille Nord de France, Lille, France; 2Inserm U837, Jean-Pierre Aubert Research Centre, Lille France;
3UDSL, IMPRT, Lille France
Abstract: Alzheimer’s disease (AD) is a neurodegenerative disorder histologically defined by the cerebral accumulation
of amyloid deposits and neurofibrillary tangles composed of hyperphosphorylated tau proteins. Loss of basal forebrain
cholinergic neurons is another hallmark of the disease thought to contribute to the cognitive dysfunctions. To this date, the
mechanisms underlying cholinergic neurons degeneration remain uncertain. The present study aimed to investigate the re-
lationship between neurofibrillary degeneration and cholinergic defects in AD using THY-Tau22 transgenic mouse model
exhibiting a major hippocampal AD-like tau pathology and hyperphosphorylated tau species in the septohippocampal
pathway. Here, we report that at a time THY-Tau22 mice display strong reference memory alterations, the retrograde
transport of fluorogold through the septohippocampal pathway is altered. This impairment is associated with a significant
reduction in the number of choline acetyltransferase (ChAT)-immunopositive cholinergic neurons in the medial septum.
Analysis of nerve growth factor (NGF) levels supports an accumulation of the mature neurotrophin in the hippocampus of
THY-Tau22 mice, consistent with a decrease of its uptake or retrograde transport by cholinergic terminals. Finally, our
data strongly support that tau pathology could be instrumental in the cholinergic neuronal loss observed in AD.
Keywords: Alzheimer’s disease, Cholinergic neurons, Nerve growth factor, Tau, Transgenic model.
tive disease leading to memory loss, cognitive impairments
and dementia. AD is histologically defined by the pathologi-
cal accumulation of two brain lesions: amyloid deposits and
neurofibrillary tangles. Amyloid deposits are extracellular
lesions in which the major component is the ?-amyloid pep-
tide A? [1, 2], formed following the sequential cleavage of
its precursor, the amyloid precursor protein. Neurofibrillary
tangles result from the neuronal abnormal accumulation of
hyperphosphorylated tau protein isoforms [3-6]. Tau proteins
normally play an important role in the polymerization of
microtubules and axonal transport [7, 8]. In AD, neurofibril-
lary pathology follows a stereotyped and sequential pathway,
appearing first in the entorhinal cortex and the hippocampal
formation and then reaching isocortical areas [9, 10]. Several
studies outline the relationship between neurofibrillary tan-
gles spreading and cognitive deficits [11, 12], further sup-
porting that tau pathology plays a key role in the pathologi-
cal events cascade leading to AD symptoms.
Alzheimer disease (AD) is a progressive neurodegenera-
cholinergic system defect is another hallmark of AD that is
thought to participate to the cascade leading to memory im-
Although it may not be the initial pathological event,
*Address correspondence to this author at the Inserm U837, “Alzheimer &
Tauopathies”, Faculté de Médecine – Pôle Recherche, 59045, Lille Cedex,
France; Tel: +33320298866; Fax: +33320538562;
pairment in the disease. Loss of basal forebrain cholinergic
neurons is constantly found in postmortem brains of subjects
with endstage AD [13-16], resulting notably in a loss of cho-
line acetyltransferase (ChAT) immunoreactivity. Basal fore-
brain cholinergic neurons provide the major cholinergic in-
nervation to the cortex and the hippocampus and play a role
in memory behavior . This function is supported by a
cross-talk, since basal forebrain cholinergic neurons are sup-
plied with neurotrophins such as nerve growth factor (NGF)
via retrograde axonal transport from their fields of innerva-
tion . So far, cholinesterase inhibitors remain the most
common form of drug therapy prescribed for symptomatic
relief of cognitive dysfunction in AD [19-21].
gic neurons degeneration during the progression of AD re-
main unknown. Experimental studies exploring cholinergic
loss etiology in AD have been mainly realized on models
mimicking the amyloid pathology side of the disease. Re-
sults tend to highlight a possible role of the peptide A? that
has been shown to induce neuronal death through the
P75NTR receptor expressed by cholinergic neurons .
However, in vivo studies reveal some discrepancies, basal
forebrain cholinergic neurons number being decreased in
some [23, 24] but unchanged in other animal models of amy-
loid pathology [25, 26]. These discrepancies, together with
recent reports showing that in AD cholinergic system can be
affected by tau pathology early in the course of the disease
Molecular and cellular mechanisms underlying choliner-
2 Current Alzheimer Research, 2011, Vol. 8, No. 6 Belarbi et al.
[27, 28], suggest that cholinergic defects observed in AD
may rather be caused by tau pathology.
between tau pathology and cholinergic defects in AD, capi-
talizing on our recently developed THY-Tau22 transgenic
mouse model that exhibits progressive development of AD-
like neurofibrillary degeneration together with cognitive
The present study aims to investigate the relationship
MATERIAL AND METHODS
following the approval of the local Animal Resources Com-
mittee, standards for the care and use of laboratory animals
and with French and European Community rules. 12-13-
month-old heterozygous THY-Tau22 male mice (THY-
Tau22; C57Bl6/J background) and wild-type control litter-
mates (WT) were used in this study. Genotyping was per-
formed using PCR on DNA isolated from tail biopsy. Mice
were housed in standard cages (n=4/5 per cage) with ad libi-
tum access to rodent chow and water and maintained in a 14h
light/10h dark cycle. 13 THY-Tau22 and 13 WT mice were
used for Y-maze task and subsequently for ChAT im-
munofluorescence (n=5/group), NGF assay and quantitative
PCR (n=8/group) analyses. Additional mice were used for
western-blot analysis (n=5-7) and fluorogold injection study
(n=7-8). Mice used for histological analyses were anesthe-
tized and transcardially perfused sequentially with 0.9%
NaCl and 4% paraformaldehyde (PAF) in phosphate buff-
ered saline (PBS) (0.1 mol/L PBS, pH 7.4). Brains were re-
moved, post-fixed 24h in 4% PAF and cryoprotected in 20%
sucrose in PBS. Series of free-floating coronal 40?m sec-
tions were obtained on a freezing microtome (Leica) and
collected in PBS-azide 0.02%. Coronal sections of the me-
dial septum were collected separately, on the basis of two
anteroposterior anatomical landmarks, as described else-
where . Mice used for NGF assay, quantitative PCR and
western-blot analysis were sacrificed by cervical dislocation.
Brains were removed and hippocampi of each hemisphere
were dissected out separately at 4°C using a coronal acrylic
slicer (Delta Microscopies, France) and stored at -80°C until
use. The same experimenter dissected all the samples for
All experiments were performed in compliance with, and
ous spatial novelty preference Y-maze test [33, 34]. The
maze was made of clear wood. The three arms (22cm long,
6.4cm wide and 15cm deep) were randomly designated: start
arm, in which the mouse started to explore (always open),
novel arm, which was blocked during the first phase, but
open during the second phase, and other arm (always open).
The maze was placed in a separate room with dim illumina-
tion (6 lux). The floor of the maze was covered with saw-
dust, which was mixed after each individual trial to eliminate
olfactory stimuli. Visual cues were placed around the maze.
During the first phase (exposure phase), the mouse was
placed in the start arm and was allowed to explore the start
arm and the other arm for 5 minutes (beginning from the
time the mouse first left the start arm), the novel arm being
Mice were tested in a hippocampal-dependent spontane-
blocked by an opaque door. The mouse was then removed
from the maze and returned to its home cage for 2 minutes.
During the second phase (test phase), the door was removed
and the mouse was placed back in the maze in the same start-
ing arm. The mouse was then allowed to explore all three
arms of the maze for 1 minute (from the time the mouse first
left the start arm). The amount of time the mouse spent in
each arm of the maze was recorded during both the exposure
and the test phases using EthovisionXT (Noldus). The per-
centage of time spent in each arm was calculated. These
animals were then sacrificed to allow immunohistochemical
detection of ChAT and NGF determinations (see below).
trogen) was injected into the dentate gyrus/hilus of THY-
Tau22 and WT mice. One injection of 0.25?l of 1% fluoro-
gold in PBS was made in each hemisphere at the following
coordinates AP -2.3 ML ± 1.6 DV -1.65. After one week,
mice were sacrificed for histological analyses as described
Fluorogold (hydroxystilbamidine methanesulfonate, Invi-
buffer (100mM Pipes, 500mM NaCl, 2mM EDTA, 0.1%
NaN3, 0.2% Triton X-100, 2% bovine serum albumin, pH 7)
supplemented with protease inhibitors (Complete Mini,
Roche) and centrifuged at 16000 g for 30 minutes at 4°C.
The supernatant was frozen at -80°C for later analysis. En-
zyme-linked immunosorbant assay (ELISA) was performed
for NGF using Emax ImmunoAssay kit (Promega). This
assay does not distinguish between mature neurotrophin
NGF and its precursor pro-NGF. All antibodies and reagents
were provided in the kit and used according manufacturer’s
instructions. Briefly, all assays were performed at room tem-
perature unless otherwise specified. Samples were run in
triplicate. Samples were diluted with block and sample
buffer (BSB) as needed to bring concentrations within the
range of the standard curve, and 100μl were placed in desig-
nated wells of a 96-well plate previously coated with primary
NGF antibody in carbonate buffer (25mM Na2CO3 and
25mM NaHCO3, pH 9.7, incubated at 4°C) and then blocked
with BSB. Samples and standards were incubated, rinsed 5x
with Tris-buffered saline with detergent (TBST; 20mM Tris-
HCl, 150mM NaCl, and 0.05% Tween 20, pH 7.6) and 100μl
of the second primary antibody dilution were placed in each
well. After incubation, wells were rinsed 5 times with TBST,
and the horseradish peroxidase-conjugated secondary anti-
body placed in each well and incubated. Wells were rinsed 5
times with TBST and the tetramethylbenzidine substrate
placed in each well. After incubation, the reaction was
stopped by the addition of 1N HCl, and the absorbance read
at 450nm on a plate reader (Multiskan Ascent, Thermo Lab-
Tissue was homogenized on ice in cold homogenization
Western Blot Analysis
containing 0.5% (w/v) CHAPS (Amersham Biosciences),
protease inhibitors (Complete Mini, Roche) and phosphatase
inhibitors (125nM okadaic acid and 1mM sodium orthova-
Tissue was homogenized in 300μl RIPA buffer (Pierce)
Loss of Medial Septum Cholinergic Neurons Current Alzheimer Research, 2011, Vol. 8, No. 6 3
nadate) using a glass/Teflon Potter homogenizer (70
strokes), sonicated and let under agitation for 1h at 4°C.
Lysates were centrifuged at 12000g for 20 minutes at 4°C.
The supernatant was removed and kept. The remaining pellet
was homogenized in 100μl of the same buffer, sonicated, let
under agitation and centrifuged again. Supernatants were
pooled and proteins were quantified using the BCA system
(Pierce). For Western Blot analysis, samples were prepared
in reducing conditions (NuPage sample buffer with sample
reducing agent, Invitrogen) and heated at 100°C for 10 min-
utes. Then, 15μg of proteins were separated on a 4-12% Nu-
PAGE Novex gel (Invitrogen) and transferred to a nitrocellu-
lose membrane. Membranes were saturated in 5% non-fat
dry milk in TNT and incubated with appropriate antibodies
allowing detection of pro-NGF (Santa Cruz sc-549; 1/1000)
and glyceraldehyde 3-phosphate dehydrogenase (GAPDH)
(Santa Cruz sc-25778; 1/10000). Signals were visualized
using chemiluminescence kits (ECL, Amersham Bio-
sciences). Results are expressed normalized to GAPDH.
RNA Extraction and Quantitative PCR Analysis
Mini Kit (Qiagen), was reverse transcribed using random
hexamer primers and High capacity cDNA RT kit (Applied
Biosystems). RNA levels were determined by quantitative
PCR on ABI Prism 7900 Real time PCR system (Applied
Biosystems) using SYBR Green QPCR master mix (Applied
Biosystems). Primers used
AGCTTTCTATACTGGCCGCA-3’ (NGF forward), 5’-
TGTACGGTTCTGCCTGTACG-3’ (NGF reverse), 5’-
AGCATACAGGTCCTGGCATC-3’ (cyclophilin forward),
verse). Results are expressed normalized to cyclophilin
RNA, isolated from tissue using RNeasy Lipid Tissue
(Eurogentec) were: 5’-
serum for 2 hours and incubated with primary antibody
raised against ChAT (Chemicon AB143; dilution 1:1000) for
48h at 4°C. Incubation with the secondary antibody from
Molecular Probes (Invitrogen) was done at room temperature
for 2 hours. All washing steps (3x10 minutes) and antibody
dilution were done using PBS. Sections were finally
mounted in Vectashield mounting medium (Vector Labora-
Sections of interest were blocked in 1% normal donkey
Fluorogold-Positive Cells and ChAT-Positive Cells Quanti-
(Imager Z1, Zeiss). The quantifications of fluorogold-labeled
cells and ChAT-immunoreactive neurons were realized on
sections of similar rostrocaudal levels selected without
knowledge of the genotype. All quantifications were per-
formed blindly using ImageJ software by at least two ob-
servers (S.B. and F.J.F.-G.). The medial septum space en-
compassed the triangular area that contains 95% or more of
the labeled neurons dorsal to a theoretical line at the level of
the anterior commissure [32, 35, 36]. Results are expressed
Images were acquired using Apotome imaging system
as density of labeled cells in the medial septum space of
THY-Tau22 mice and WT littermate controls.
mean. Between-group comparisons were performed by Stu-
dent’s unpaired t test. Y-maze analysis was performed using
a one-tailed t-test to evaluate the score as compared to
chance. Statistical significance was set at p<0.05.
Data are presented as means ± standard error of the
THY-Tau22 Mouse Model
mutated at sites G272V and P301S under a neuronal Thy 1.2
promoter . In contrast to many other tau transgenic mod-
els, THY-Tau22 mice do not show any signs of motor defi-
cits or changes in motor activity at any age investigated,
therefore allowing behavioral testing without interference
due to motor abnormality . Here, we evaluated short-
term spatial memory in 12-13-month-old animals using a
hippocampal-dependent spontaneous spatial novelty prefer-
ence Y-maze task (n=13/group). During the exposure phase,
both genotypes similarly explored the maze Fig. (1A),
spending a similar amount of time in each of the arms Fig.
(1B-C). During the test phase Fig. (1D-E), animals from the
WT group spent a significantly greater proportion of time in
the novel arm than would be expected from chance
(t(12)=7.763; p<0.0001) whereas THY-Tau22 mice did not
(t(12)=1.102; p=0.2920). Analysis of the proportion of time
spent in the novel arm by each group indicates that WT mice
showed a significantly greater preference for the novel arm
than THY-Tau22 mice p=0.0088; Fig. (1D vs. E). Therefore
THY-Tau22 mice exhibit an impairment in hippocampal-
dependent short-term spatial memory .
THY-Tau22 transgenic mice express human 4-repeats tau
Tau Pathology and Septohippocampal Pathway
memory impairment is associated with a major hippocampal
AD-like tau pathology characterized by the presence of hy-
per- and abnormally phosphorylated tau species and with the
presence of hyper-phosphorylated tau proteins in the fimbria-
fornix fibers [29-31]. We aimed to ascertain if this tau pa-
thology distribution had any functional consequence on the
septohippocampal pathway. Thus, we evaluated septohippo-
campal retrograde transport using hippocampal injection of
the retrograde tracer fluorogold and confirmed that THY-
Tau22 exhibited a significant reduction in the number of
fluorogold-positive neurons within the medial septum as
compared to littermate controls (-27.2±4.5%; p=0.016; n=7-
8/group) Fig. (2) and ).
As we previously extensively described, such spatial
Tau Pathology, Medial Septum Cholinergic Neurons and
pendent upon the integrity of the septohippocampal system
[37, 38] and this dependency is likely due to cholinergic neu-
rons reliance on neurotrophic factor NGF synthesized in the
hippocampus [39-42]. We therefore hypothesized that medial
septum cholinergic neurons could be affected and that NGF
Several studies support that cholinergic neurons are de-
4 Current Alzheimer Research, 2011, Vol. 8, No. 6 Belarbi et al.
Fig. (1). Y-maze evaluation of hippocampal-dependent memory.
During the exposure phase, both genotypes (n=13/group) similarly
explored the maze (A), spending a similar amount of time in each
of the arms (B-C). During the test phase (D-E), animals from the
WT group spent a significantly greater proportion of time in the
novel arm than would be expected from chance (t(12)=7.763;
p<0.0001) whereas THY-Tau22 mice did not (t(12)=1.102;
p=0.2920). Analysis of the proportion of time spent in the novel
arm by each group indicates that WT mice showed a significantly
greater preference for the novel arm than THY-Tau22 mice
(p=0.0088; bar N in D vs. E). S: start arm; O: other arm; N: novel
arm. White bars: WT littermates, Black bars: THY-Tau22 mice.
Fig. (2). Septohippocampal retrograde transport in THY-Tau22
mice. Septohippocampal neurons were retrogradely labeled by hip-
pocampal injection of the retrograde tracer fluorogold. THY-Tau22
mice exhibited a significant reduction of medial septum labeled
neurons density as compared to littermate controls (-27.2±4.5%;
p=0.016). Scale bar = 200?m. N=7-8/group. White bars: WT lit-
termates, Black bars: THY-Tau22 mice.
availability could be disrupted in THY-Tau22 mice. Interest-
ingly, we observed that the reduction in the number of
fluorogold-labeled neurons was associated with a significant
decrease in the number of ChAT-positive neurons in the me-
dial septum of THY-Tau22 as compared to littermate con-
trols (-28.7±10.3%; p=0.029) Fig. (3). Concerning NGF
status, no difference could be found in either NGF mRNA or
NGF precursor pro-NGF hippocampal levels Fig. (4B and
C). However, we showed by ELISA (that does not distin-
guish between mature NGF and its precursor pro-NGF) a
significant increase in NGF (both mature and precursor pro-
teins) hippocampal level in THY-Tau22 mice reaching
53.3±13.6% (p=0.0013) of littermate control level Fig. (4A).
Together, these results support that degeneration of choliner-
gic neurons of the medial septum is accompanied with hip-
pocampal accumulation of mature NGF protein consistent
with a decrease of its uptake or retrograde transport by sep-
tohippocampal cholinergic neurons.
Fig. (3). ChAT immunoreactive cholinergic neurons in the medial
septum of THY-Tau22 mice. ChAT immunofluorescent staining
was performed on cerebral coronal sections of THY-Tau22 and
wild-type mice. THY-Tau22 mice exhibited a marked decrease of
ChAT immunoreactive neurons density within the medial septum as
compared to littermate controls (-28.7±10.3%; p=0.029). Scale bar
= 200?m. N=5/group. White bars: WT littermates, Black bars:
Fig. (4). Hippocampal NGF levels in THY-Tau22 mice. Hippo-
campal levels of NGF (A) measured by ELISA (that does not dis-
tinguish between mature NGF and its precursor pro-NGF) were
increased in THY-Tau22 mice as compared to littermate controls
(+53.3±13.6%; p=0.0013). Hippocampal levels of NGF mRNA (B)
and pro-NGF protein (C) both showed no significant differences
between THY-Tau22 mice and littermate controls. N=5-8/group.
White bars: WT littermates, Black bars: THY-Tau22 mice.
between neurofibrillary degeneration and basal forebrain
cholinergic loss in AD. During the progression of AD, neu-
rofibrillary degeneration has been described to appear first in
the entorhinal cortex and in the hippocampus, and then to
spread to isocortical areas [9, 10]. Interestingly, more recent
studies re-evaluating the cytoskeletal changes in the basal
forebain reported that hyperphosphorylated tau proteins were
also detected in basal forebrain cholinergic neurons early in
the course of the disease [27, 28]. Whereas these reports
This study aimed to further investigate the relationship
Loss of Medial Septum Cholinergic Neurons Current Alzheimer Research, 2011, Vol. 8, No. 6 5
strongly support the idea that tau pathology may be involved
in the cholinergic neurons degeneration observed in AD,
very few experimental studies addressing this hypothesis
have been realized so far. Interestingly, using the THY-
Tau22 transgenic model that fairly reproduces AD-like pa-
thology in the hippocampus , we previously reported the
presence of hyperphosphorylated tau proteins in the fimbria-
fornix fibers of 12-month-old transgenic mice as well as in a
few neurons of the basal forebrain . These observations
supported that THY-Tau22 line was a valuable model to
study the relationship between tau pathology and cholinergic
defects in AD.
septohippocampal tau hyperphosphorylation  is associ-
ated with septohippocampal retrograde transport defects and
loss of ChAT immunoreactive neurons within the medial
septum. Previous Morris Water Maze studies  and pre-
sent Y-maze task indicate that THY-Tau22 mice exhibit hip-
pocampal-dependent memory impairments. Whether these
alterations are mostly related to the major hippocampal pa-
thology observed in our model or to the cholinergic degen-
eration remains to be determined. However, basal forebrain
cholinergic neurons are the major source of cholinergic in-
nervation for the cortex and the hippocampus. Cholinergic
neurotransmission is known to regulate synaptic plasticity
and to play a role in memory behavior [17, 43, 44]. In line,
cholinergic depletion decreases adult neurogenesis  and
increases the vulnerability of hippocampal neurons to insult
thereby contributing to the appearance of hippocampal-
dependent spatial memory deficits [46, 47]. It is thus reason-
able to propose that the cholinergic neuronal loss observed in
the THY-Tau22 model may emphasize the hippocampal dys-
function primarily caused by tau pathology and contributes
significantly to the observed cognitive impairments.
The present study supports that the previously reported
fect involving abnormal tau species within the septohippo-
campal tract, to hippocampal pathology impacting on cho-
linergic terminals or to cholinergic tau pathology remains to
be determined. If a contribution of tau in cholinergic neurons
cannot be ruled out to explain cholinergic loss in THY-
Tau22 mice, only sparse medial septum neurons exhibiting
somatodendritic tau hyperphosphorylation were observed
supporting that other mechanisms could be involved .
One would suggest that defect of axonal transport within the
septohippocampal pathway has a role. Indeed, the function
of cholinergic neurons on the maintenance of memory func-
tion is supported by a cross talk with the hippocampus, since
hippocampal neurons produce the neurotrophin NGF that
plays an important role in the survival and phenotype main-
tenance of cholinergic neurons through its retrograde signal-
ing [39, 48]. In the THY-Tau22 mouse model, we observed
an increase of NGF hippocampal protein levels measured by
ELISA without change in NGF mRNA and pro-NGF levels.
These data would be consistent with a decrease in the mature
NGF uptake by septohippocampal cholinergic neurons rather
than with a defect in hippocampal NGF production. Even if
we cannot firmly conclude that NGF accumulation is a cause
or a consequence of the cholinergic neuronal loss, the pres-
ence of hyper-phosphorylated tau species in the septohippo-
campal pathway supports that before degenerating choliner-
gic neurons may present retrograde transport alterations that
Whether cholinergic loss is ascribed to a trafficking de-
could likely explain these observations. Further experiments
aiming to evaluate the time–course of retrograde NGF trans-
port in the THY-Tau22 model are however needed to bring
new information concerning the mechanisms by which tau
pathology leads to cholinergic degeneration in our model.
porting that tau pathology is instrumental in the cholinergic
defects observed in AD. Basal forebrain cholinergic neurons
play a major role in memory function and display early dys-
function in AD, making them a prime target for AD early-
stage therapeutic development. Whereas cholinesterase in-
hibitors treatments show a limited efficacy, one can predict
that preventing the cholinergic neurons degeneration would
lead to significant improvement of AD treatment. Besides
the fact that our results provide new insight into AD cho-
linergic defects etiology study, the present report confers to
THY-Tau22 model a major interest in the development and
evaluation of novel drugs therapies aiming to counteract cho-
linergic degeneration in AD.
To the best of our knowledge, this is the first study sup-
This work was supported by Inserm, CNRS, IMPRT,
University Lille 2, Lille County Hospital (CHR-Lille), Ré-
gion Nord/Pas-de-Calais, FEDER, ADERMA, DN2M and
grants from ANR-08-MNPS-002/AMYTOXTAU, ANR-
JC07-184902/ADONTAGE, Deutsche Forschungsgemeins-
chaft (DFG – ZI 1143/1-1), France Alzheimer, Fédération
pour la Recherche sur le Cerveau and LECMA and from the
European Community: MEMOSAD (FP7 contract 200611).
K.B., S.B., J.D. and L.T. are recipients of a PhD scholarship
co-sponsored by Région Nord/Pas-de-Calais. F.J.F.-G. is a
postdoctoral fellow from the Consejería de Educación of
JCCM. J.B. is a postdoctoral fellow from Canadian Institutes
of Health Research (CIHR). We also thank all members of
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