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Impaired Adult Neurogenesis in the Dentate Gyrus of a
Triple Transgenic Mouse Model of Alzheimer’s Disease
Jose
´J. Rodrı
´guez
1
*, Victoria C. Jones
1
, Masashi Tabuchi
1
, Stuart M. Allan
1
, Elysse M. Knight
1
, Frank M.
LaFerla
2
, Salvatore Oddo
2
, Alexei Verkhratsky
1,3
1Faculty of Life Sciences, The University of Manchester, Manchester, United Kingdom, 2Department of Neurobiology and Behaviour, University of California Irvine, Irvine,
California, United States of America, 3Institute of Experimental Medicine, ASCR, Prague, Czech Republic
Abstract
It has become generally accepted that new neurones are added and integrated mainly in two areas of the mammalian CNS, the
subventricular zone and the subgranular zone (SGZ) of the dentate gyrus (DG) of the hippocampus, which is of central
importance in learning and memory. The newly generated cells display neuronal morphology, are able to generate action
potentials and receive functional synaptic inputs, i.e. their properties are similar to those found in mature neurones. Alzheimer’s
disease (AD) is the primary and widespread cause of dementia and is an age-related, progressive and irreversible
neurodegenerative disease that deteriorates cognitive functions. Here, we have used male and female triple transgenic mice
(3xTg-AD) harbouring three mutant genes (b-amyloid precursor protein, presenilin-1 and tau) and their respective non-
transgenic (non-Tg) controls at 2, 3, 4, 6, 9 and 12 months of age to establish the link between AD and neurogenesis. Using
immunohistochemistry we determined the area density of proliferating cells within the SGZ of the DG, measured by the
presence of phosphorylated Histone H3 (HH3), and their possible co-localisation with GFAP to exclude a glial phenotype. Less
than 1% of the HH3 labeled cells co-localised with GFAP. Both non-Tg and 3xTg-AD showed an age-dependent decrease in
neurogenesis. However, male 3xTg-AD mice demonstrated a further reduction in the production of new neurones from
9 months of age (73% decrease) and a complete depletion at 12 months, when compared to controls. In addition, female 3xTg-
AD mice showed an earlier but equivalent decrease in neurogenesis at 4 months (reduction of 63%) with an almost inexistent
rate at 12 months (88% decrease) compared to controls. This reduction in neurogenesis was directly associated with the
presence of b-amyloid plaques and an increase in the number of b-amyloid containing neurones in the hippocampus; which in
the case of 3xgTg females was directly correlated. These results suggest that 3xTg-AD mice have an impaired ability to generate
new neurones in the DG of the hippocampus, the severity of which increases with age and might be directly associated with the
known cognitive impairment observed from 6 months of age onwards . The earlier reduction of neurogenesis in females, from
4 months, is in agreement with the higher prevalence of AD in women than in men. Thus it is conceivable to speculate that a
recovery in neurogenesis rates in AD could help to rescue cognitive impairment.
Citation: Rodrı
´guez JJ, Jones VC, Tabuchi M, Allan SM, Knight EM, et al. (2008) Impaired Adult Neurogenesis in the Dentate Gyrus of a Triple Transgenic Mouse
Model of Alzheimer’s Disease. PLoS ONE 3(8): e2935. doi:10.1371/journal.pone.0002935
Editor: Katrina Gwinn, Baylor College of Medicine, United States of America
Received March 24, 2008; Accepted July 21, 2008; Published August 13, 2008
Copyright: ß2008 Rodrı
´guez Arellano et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which
permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Alzheimer’s Research Trust Programme Grant (ART/PG2004A/1) to JJR and AV.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: Jose.Rodriguez-arellano@manchester.ac.uk
Introduction
The classical view that all neurones are generated (via neurogen-
esis) during prenatal development and early postnatal life has been
challenged by the seminal study of Altman and Das (1965) [1] and
now it is generally accepted that neurogenesis does also occur in
adulthood mainly in two areas of the mammalian CNS [1–5]. These
areas, which are involved in both plasticity and stability of the brain,
are the anterior part of the subventricular zone (SVZ) along the
lateral ventricles, which is also an important a site of gliogenesis [6,7]
and the subgranular zone (SGZ) of the dentate gyrus (DG) of the
hippocampus [4,5]. In both areas neurogenesis progress as a complex
multi-step process which starts with the proliferation of precursors
residing in the SVZ or in the SGZ. For the hippocampus, it has been
estimated that several thousand new cells are generated daily [5].
However, within several days after their birth at least fifty percent of
the newborn cells die [5]. The cells surviving this initial period of cell
death differentiate mainly into granule neurones and endure for
several months. These newly generated neurones receive synaptic
inputs, extend axons along the mossy fibres tract and exhibit
electrophysiological properties similar to those of mature dentate
granule cells [8,9]. In addition these new cells express a full
complement of membrane receptors [10]. From a functional point
of view, hippocampal neurogenesis plays an important role in
memory processes. Decline in neurogenesis within SGZ has been
involved in cognitive impairments linked with ageing and neurode-
generative disorders, and was suggested to play a role in Alzheimer
disease (AD) [5,11].
AD is a progressive neurodegenerative disease which is the
primary cause of dementia in the elderly and is characterized by
damage of the brain regions associated with learning and memory,
such as the hippocampus [12,13]. Decline in neurogenic capacity
could participate in AD-associated cognitive impairments and
contribute to early AD symptoms such as the inability to acquire
and store new information [14–16]. Incidentally, the use of
endogenous neuronal precursors to replace lost and/or damaged
cells has been proposed as a potential therapeutic approach to
treat AD [17,18]. Experimental studies of neurogenesis in various
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AD animal models, however, resulted in contradictory findings
[19–26]. For example, animals carrying presenilin or some APP
mutant genes demonstrated an impaired neurogenesis in the DG
[20–22,24,27,28]. Conversely, recent studies, performed on the
APP
Sw, Ind
(Swiss/Indian mutation) PDGF-APP mutant and on
FAD post-mortem human material [18,29], reported an increase
of neurogenesis. It has to be noted, though, that the study on
human FAD cases analysed only immature newly generated
neurones without providing definitive probes of further develop-
ment and/or progress into mature cells.
In the present study we sought to determine the rate and possible
changes in hippocampal neurogenesis in the recently developed
triple-transgenic AD (3xTg-AD) mouse model, that harbours the
mutant genes for amyloid precursor protein (APP
Swe
), for presenilin
1PS1
M146V
and for tau
P301L
[30,31]. These animals are recognised
as relevant AD model since they show temporal- and region-specific
Aband tau pathology, which closely resembles that seen in the
human AD brain [30,31]. As well as progressively developing
plaques and tangles the 3xTg-AD mice also show clear functional
and cognitive impairments including LTP, spatial memory and long
term memory deficits; which are manifest in an age-related manner
importantly preceding the appearance of histological markers
[30,31]. Cognitive deficits in the 3xTg-AD model correlate with
the accumulation of intraneuronal Ab[30–34]. Subsequently, we
decided to investigate a gender difference in the number of
proliferating cells, because it is well establish that AD affects women
more than men [35,36]. Finally we also aimed to correlate the rate of
neurogenesis with the presence of intracellular b-amyloid.
Materials and Methods
Mice
All animal procedures were performed according to the Animal
Scientific Procedures Act of 1986 under the license from the
United Kingdom Home Office.
The generation of the 3xTg-AD mice was done as previously
described [30,31,37]. Briefly, human amyloid precursor protein
with the Swedish mutation (APP
Swe
) and human tau with the
P301L (tau
P301L
) mutation were microinjected into single-cell
embryos from homozygous presenilin 1PS1
M146V
knockin mice.
The background of the PS1 knockin mouse is a hybrid 129/
C57BL6. The Non-Tg mice used were from the same strain and
genetic background as the PS1 knockin mice, but they harbor the
endogenous wild-type mouse PS1. All 3xTg-AD and Non-Tg mice
were obtained from crossing homozygous breeders. Male and
female mice were independently group housed and kept on daily
12 h light-dark cycles dark schedule. All mice were given ad
libitum access to food and water.
Fixation and tissue processing
Male and female 3xTg-AD and their respective non-transgenic
(non-Tg) controls were anaesthetized with an intraperitoneal
injection of sodium pentobarbital at different time points (2, 3, 4,
6, 9 and 12 months of age; n = 3–7). The brains were fixed by
perfusion through the aortic arch with 25 ml of 3.8% acrolein
(TAAB, UK) in a solution of 2% paraformaldehyde and 0.1 M
phosphate buffer (PB) pH 7.4, followed by 75 ml of 2%
paraformaldehyde. Brains were removed from the cranium and
cut into 4–5 mm coronal slabs of tissue containing the entire
rostrocaudal extent of the hippocampus. This tissue was then post-
fixed for 30 minutes in 2% paraformaldehyde and sectioned at
40–50 mm on a vibrating microtome (VT1000, Leica, Milton
Keynes, UK). To remove excess reactive aldehyde groups, sections
were treated with 1% sodium borohydride in 0.1 M PB for
30 minutes. The tissue sections were then freeze-thawed to
optimize the penetration of immunoreagents. For this procedure,
sections were incubated in cryoprotectant solution containing 25%
sucrose and 3.5% glycerol in 0.05 M PB at pH 7.4 and
subsequently rapidly immersed in chlorodifluoromethane followed
by liquid nitrogen and then thawed at room temperature in PB.
Sections were then rinsed in 0.1 M PB followed by 0.1 M Tris-
buffered saline (TBS), pH 7.6.
Antibodies
A polyclonal affinity-purified rabbit antiserum raised against
phosphorylated Histone 3 (Upstate, USA; #06-570) and a
monoclonal mouse antiserum generated against GFAP from pig
spinal cord (Sigma-Aldrich Company Ltd., UK; #G3893) were used
for the determination of proliferating cells and glia. Specificity of
these antisera was confirmed by immunoblot and western blot [38].
For identification of intracellular beta amyloid (Ab)depositsweused
a monoclonal mouse antiserum that reacts with abnormally
processed isoforms, as well as precursor forms of Ab,recognizing
an epitope within amino acids 3–8 (EFRHDS; anti-Ab 6E10 [SIG-
39320]. Signet Laboratories, Dedham, MA).The immunolabelling
pattern we obtained with this antibody is equivalent to that obtained
previously in different brain regions [30,31].
To assess for non-specific background labelling or cross reactivity
between antibodies derived from different host species, a series of
control experiments were performed. Omission of primary and/or
secondary antibodies from the incubation solutions resulted in a total
absence of target labelling. These primary antibodies are therefore
regarded as specific to their designated targets.
Immunohistochemistry
To optimize detection of all HH3 and GFAP cells and
containing profiles we used the highly sensitive avidin–biotin
peroxidase complex (ABC) method [39]; and to minimize
methodological variability, sections through the dorsal hippocam-
pus containing both hemispheres of all animals were processed at
the same time using precisely the same experimental conditions.
For this procedure, the vibratome sections were first incubated for
30 minutes in 0.5% bovine serum albumin in TBS to minimize
non-specific labelling. The tissue sections were then incubated for
48 hours at 4uC in 0.1% bovine serum albumin in TBS
containing: (1) rabbit polyclonal antiserum for HH3 (1:1,000)
and (2) mouse monoclonal antiserum for GFAP (1:60,000).
Subsequently, the HH3 and GFAP antibodies were detected in
a sequential manner on the same sections. For HH3 labelling,
sections were washed and placed in (1) 1:200 dilutions of
biotinylated donkey anti-rabbit IgG (Jackson Immunoresearch,
Stratech Scientific Ltd., Soham, UK) and (2) 1:200 dilutions of
biotin-avidin complex from the Elite kit (Vector Laboratories Ltd.,
Peterborough, UK). All antisera dilutions were prepared in TBS,
and the incubations were carried out at room temperature. The
peroxidase reaction product was visualized by incubation in a
solution containing 0.022% of 3,39diaminobenzidine (DAB,
Aldrich, Gillingham, UK) and 0.003% H
2
O
2
in TBS for
6 minutes. For GFAP labelling, sections were then rinsed again
in TBS and incubated (1) 1:200 dilution of biotynilated horse anti-
mouse IgG (1:200; Vector Laboratories Ltd., Peterborough, UK)
and (2) placed in a 1:200 dilution of biotin-avidin complex from
the Elite kit (Vector Laboratories Ltd., Peterborough, UK). The
GFAP peroxidase product was then visualized in a solution
prepared from the Novared or SGZ kits (Vector Laboratories Ltd.,
Peterborough, UK) for 3–4 minutes. This allowed us to see the
GFAP labelling in red and/or blue respectively; allowing us to
differentiate it from the HH3 labelled cells (brown).
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The same immunoperoxidase approach, but for single labelling,
was used for the detection of intracellular Ab. Briefly, adjacent
sections were incubated for 48 hours at 4uCin0.1%bovineserum
albumin in TBS containing mouse monoclonal antiserum for Ab
(1:2000). Subsequently, sections were then washed and placed in (1)
1:200 horse anti-mouse IgG (Vector Laboratories Ltd., Peterbor-
ough, UK) and (2) 1:200 dilution of biotin-avidin complex from the
Elite kit (Vector Laboratories Ltd., Peterborough, UK). The
peroxidase reaction product was visualized by incubation in a
solution containing 0.022% of 3,39diaminobenzidine (DAB,
Aldrich, Gillingham, UK) and 0.003% H
2
O
2
in TBS for 6 minutes.
HH3 Area density and Abcell number
To determine the area density (Sv, number/mm
2
) of HH3-
immunoreactive neurons, the labelled cells were counted on both
hemispheres in six non-consecutive coronal Vibratome sections,
separate by at least 80 mm, taken through representative sections
of both the dorsal (3) and ventral (3) DG of the hippocampus at
Figure 1. Photomicrographs showing phosphorilated Histone H3 (HH3, a proliferating mitotic marker) within the dentate gyrus of
Non Tg mice. A–B: Single labelling of HH3 positive cells (arrows) in the dentate gyrus of 2 (A) and 12 months (B) Non Tg mice. C–D: Dual labeling
of HH3 positive cells (arrows) and glial cells (GFAP, blue) in the dentate gyrus of 2 (C) and 12 months (D) Non Tg mice. E–F: Bar graphs showing the
area density of HH3 positive cells within the dorsal dentate gyrus (all layers included) of Non-Tg males (E) and females (F) mice. GCL: Granular Cell
Layer, ML: Molecular Layer. ** =p,0.01 compared to 2 months; *** =p,0.001 compared to 2 months;
NN
=p,0.01 compared to 3 months;
## =p,0.01 compared to 2 and 3 months; ### =p,0.001 compared to 3 months; e=p,0.05 compared to 4 months; ee =p,0.01 compared
to 4 months; ‘=p,0.05 compared to 4 and 6 months; +=p,0.05 compared to 2, 3 and 6 months.
doi:10.1371/journal.pone.0002935.g001
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levels 1.22 mm/2.46 mm and 2.54 mm/3.80 mm posterior
anterior to bregma, respectively, according to the mouse brain
atlas of Paxinos and Watson (1986) [40]. The number of HH3
positive cells and the area measurements of the complete dentate
gyrus and its different layers (granule cell layer –GCL-, molecular
layer –ML- and hilus) were determined blindly.
The number of Abcontaining neurones was examined in the CA1
region of the hippocampus CA1, since this field shows the earliest
and strongest accumulation of Abintracellular deposits. This
quantification was carried out on six non-consecutive hippocampal
sections of the same animals used for the proliferation analysis.
Statistical analysis
An analysis of variance (ANOVA) was used to examine differences
in the mean area density of labelled HH3 cells between the 3xTg-AD
and non-Tg animals and sexes, followed by unpaired t-test
comparisons at the different time points. Spearman correlation
was used to correlate the mean area density of HH3 positive cells
with the mean number of Abcontaining neurons (implemented
through GraphPad Prism 4.0, GraphPad Software, Inc.).
Results
In the dorsal hippocampus and more specifically within the
dentate gyrus of both non-Tg and 3xTg-AD mice a fair number of
newly generated cells could be visualized, as indicated by HH3
immunoreactivity (HH3-IR; Figs.1A, 2A). These newly formed
cells showed the distinctive characteristics of proliferating cells;
they were mainly localized in the inferior part of the granule cell
layer (GCL) and demonstrated typical morphology such as
irregular shape and small size; sometimes they appeared close
together and/or formed clusters (Fig. 1A–B).
Effects of ageing on neurogenesis in non-Tg animals
Quantitative analysis of the rate of cell proliferation showed a
reduction in the number of HH3-IR cells with age. This reduction
was apparent within the dentate gyrus of non-Tg males and
females mice (F
5,20
= 5.643, p = 0.0021 and F
5,16
= 44.8,
p,0.0001, respectively; Fig 1E–F) and in the GCL where we
observed the highest number of proliferating cells (F
5,20
= 5.362,
p = 0.0028 and F
5,16
= 17.54, p,0.0001, respectively; Fig 3A–B).
Decrease in proliferation rate with age was quite significant: at
6 months of age in both sexes the proliferation rate was reduced
by more than 60% when compared to the 2 months old animals
(males 2.6460.23 vs. 6.6161.74; females 1.4860.39 vs.
7.4660.45, Fig. 1E–H). Decrease in the number of HH3 cells in
dentate gyrus and the subgranular zone of the GCL appears
earlier in females (3–4 month) than in males as confirmed by the t-
test analysis (Figs. 1E–F; Fig. 3).
Figure 2. Brightfield micrographs showing HH3 labelled cells within the dentate gyrus of 3xTg-AD e. A–B: Dual labeling of HH3 positive
cells (arrows) and glial cells (GFAP, red) in the dentate gyrus of 2 (A) and 12 months (B) 3xTg-AD mice. C–D: Bar graphs showing the area density of
HH3 positive cells within the dorsal dentate gyrus (all layers included) of 3xTg-AD males (C) and females (D) mice. GCL: Granular Cell Layer, ML:
Molecular Layer. ** =p,0.01 compared to 3 months; *** =p,0.001 compared to 3 months;
N
=p,0.05 compared to 2 months
NN
=p,0.001
compared to 2 months; ## =p,0.01 compared to 2 and 3 months; e=p,0.05 compared to 4 months; ‘=p,0.05 compared to 2 and 4 months.
doi:10.1371/journal.pone.0002935.g002
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Dual labelling showed that the majority of HH3-IR cells did not
possess the astroglia marker GFAP; in fact, less then 1% of HH3-
IR cells co-expressed GFAP in the dentate gyrus, including the
GCL (data not shown).
Impairment of neurogenesis in 3xTg-mice
Similarly to the controls, the rate of neurogenesis in 3xTg-AD
mice was decreased with age (Fig. 2, 3) in both males and females
throughout the dentate gyrus (F
5,20
=5.437, p=0.0026 and
F
5,18
=12.39, p,0.0001, respectively) and in the GCL
(F
5,20
= 8.524, p = 0.0002 and F
5,18
=11.81, p,0.0001, respectively;
Figs 2C–D). Decrease in proliferation rate in 3xTg-AD animals was
about 60–90% more pronounced compared to control animals: at
6 months compared to the 2 months 3xTg-AD mice (males
2.2360.5 vs. 5.7161.47; females 0.47+0.23 vs 5.0961) Fig. 2 C–
D). Furthermore, at 12 months of age males and females showed
very little capacity of forming new cells within the GCL (Fig. 2 C–D;
Fig. 3); whilst in both males and females non-Tg animals we could
still observe approximately a 20–35% of the number of HH3-IR
observed at young ages (2–3 months ; Fig 3).
Neurogenesis depression in 3xTg-mice is gender
dependent
When we analysed the number of HH3-IR cells within the GCL
of the 3xTg-AD mice and compared it with the cell proliferation
rate of the non-Tg it became evident that at young ages (2 and
3 months) the levels were very similar whilst at older ages,
especially 9 and 12 months the neurogenesis levels have decreased
over 70% in both groups (Figs. 1 E–F, 2 C–D, 3). The
quantification and consequent statistical analysis showed that the
age associated reduction in HH3-IR cells in 3xTg-AD males
compared to non-Tg animals start to be significant at 9 months
(73%; showing a trend to significant difference) and is completely
disappeared at 12 months of age (Fig. 3A; age effect, F
5,1
= 53.57,
p,0.0001; group effect F
5,1
= 1.73, p = 0.1744; age x group effect
F
5,1
= 3.75, p = 0.5353; p,0.05 at 12 months). In contrast in
females 3xTg-AD, compared to non-Tg animals, HH3-IR cells
were already significantly reduced at 4 months of age (63%) being
maximal (88% reduction) at 12 months (Fig. 3B; age effect,
F
5,1
= 68.08, p,0.0001; group effect F
5,1
= 10.31, p,0.0001; age
x group effect F
5,1
= 2.31, p = 0.4961; p,0.05 at 4 and 6 months;
p,0.001 at 9 and 12 months). This impairment in neurogenesis
rate within the 3xTg-AD mice is mainly due to changes at dorsal
more than ventral hippocampal levels (Fig. 3), which is consistent
with their specific preferential roles in learning and memory and
affective behaviour respectively [41].
Relationship between intraneuronal b-amyloid
accumulation and neurogenesis
The 3xTg-AD mice had also an age-dependent increase in the
number of hippocampal neurones accumulating b-amyloid
(Fig. 4A–D). Within the hippocampus neurones containing b-
amyloid could be found as early as 2 months of age, and this
number was higher in females compared to males (Fig. 4C–D).
Quantitative analysis showed that in both males and females
3xTg-Ad mice there was an age-dependent increase of b-amyloid
containing neurones that was maximal and very significant at
9 months of age when compared to young animals (Fig. 4 C–D;
F
4,10
= 4.231, p = 0.0293 and F
4,10
= 4.948, p = 0.0184, respec-
tively). The fully formed b-amyloid plaques within the neuropil,
however, were observed much later, at 9 and 12 months (Fig. 4B).
Increase in the number of b-amyloid containing hippocampal
neurones seem to match with the reduction of GCL proliferating
cells in both males and females (Fig. 4E–F). However, only females
showed a significant indirect correlation between the number of
HH3-IR cells with the number of b-amyloid positive cells
Figure 3. Bar graphs showing the mean area density HH3 labelled cells within the GCL of the dentate gyrus of both 3xTg-AD and
control nonTg-AD mice. A–B: Males (A) and females (B) dorsal GCL. C–D: Males (C) and females (D) dorsal GCL. Asterisks indicate a significant
difference in the means.
doi:10.1371/journal.pone.0002935.g003
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(R
2
= 0.7018; Fig 4F). This finding showing a higher prevalence of
b-amyloid positive cells in 3xTg-AD females is also in agreement
with the recently reported 20% increase in plaque load observed in
APP23 transgenic mice females when compared to males [42].
Furthermore,The presence of high number of b-amyloid contain-
ing neurones and occurrence of plaques at 12 months were
occasionally concomitant with the first appearance of phosphori-
lated Tau (Fig. 4G).
Figure 4. Photomicrographs showing the presence of b-amyloid within the pyramidal neurones of CA1 as well as the presence of a
plaque in 12 months 3xTg-AD mice (B) compared to a nonTg control animal (A). C–D: Bar graphs showing the number of cells containing
b-amyloidin the hippocampal CA1 of males (C) and females (D) 3xTg-AD mice. E–F: Linear correlations between the mean number of cells containing
b-amyloid in the hippocampal CA1 and the mean area density of HH3 positive cells in the GCL of the dentate gyrus of males (E) and females (F) 3xTg-
AD mice. In Gwe can see the accumulation phosphorilated Tau within the CA1 of a 3xTg-AD mice. Or: CA1 Stratum Oriens, Py: CA1 stratum
Pyramidale, Rad: CA1 Stratum Radiatum, LMol: CA1 Stratum Lacunosum Moleculare. *=p,0.05 compared to 2 months; ** =p,0.01 compared to
2 months; ‘=p,0.05 compared to 3 months.
doi:10.1371/journal.pone.0002935.g004
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Discussion
In the present study we have used single and dual labelling
immunohistochemistry to demonstrate changes in cell prolifera-
tion and neurogenesis within the hippocampal dentate gyrus of an
AD animal model. The experiments were performed on a recently
developed 3xTg-AD mouse model, which is recognised as an
extremely relevant, since these transgenic animals show temporal-
and region-specific Aband tau pathology, which closely resembles
that seen in the human AD brain [30,31]. These pathological
hallmarks are concomitant with clear functional and cognitive
impairments including LTP, spatial memory and long term
memory deficits, which are manifest in an age-related manner
[30–34]. We found that only a very small proportion of HH3-IR
proliferating cells in either 3xTg-AD or Non-Tg mice express
GFAP (,1%), suggesting that those HH3-IR cells are likely of a
neuronal lineage and thus are an indicator of neurogenesis.
Our main findings are that 3xTg-AD mice have a decreased GCL
neurogenesis, which is also is gender dependent. As happens in
normal rodents (including our control non-Tg animals) the decrease
in the dentate gyrus GCL neurogenesis develops with age [5,14]
However, we found that this effect is much exacerbated in 3xTg-AD,
being at least 60% stronger than in normal animals. These results are
in agreement with findings in other transgenic models of AD in
which transgenic mice having mutant forms of APP or presenilin-1
demonstrated impaired neurogenesis [20–22,24,27,28]. Since none
of models used previously fully reproduces the features of familial
and/or sporadic AD, our findings made in the 3xTg-AD mice
become of major relevance and importance.
We also demonstrated that the impairment of neurogenesis in the
dentate gyrus of the hippocampus is closely associated with AD
pathogenesis. Indeed hippocampus is affected early in AD; impaired
memory related to hippocampal damage may be associated with
deregulations of neurogenesis [5,11,29,43]. Two previous studies,
however, are in contradiction with our findings; one performed on
the APP
Sw, Ind
(Swiss/Indian mutation) mutant and one on FAD
post-mortem human material, reporting an increase of neurogenesis
[18,29]. These discrepancies could be explained by methodological
and preservation differences, including the post-mortem fixation
delay which could contribute to antigenmasking and in consequence
a misevaluation of the proliferation rates [44]. In addition, in these
studies the Doublecortin (Dcx) labelling, which marks both young
and immature neurons was used. This could affect the data
interpretation because more than 50% of the newborn cells die
[5]. Another discrepancy may reside in the fact that even if they use
5-Bromo-29-Deoxyuridine (BrdU) as an accurate index to label
proliferating cells, very few of them survives to 4 weeks [28].
Furthermore, these methods suffer from uncertainty since they may
not detect a distinct proliferative state but instead mark repaired
DNA in post-mitotic neurons and/or an abortive cell cycle [45,46].
In this study we also investigated the gender difference of the rate
of neurogenesis in 3xTg-AD mice. We found that neurogenesis in
female 3xTg-AD mice is affected earlier; already at 4 months of age
we found significant depression of neurogenesis in female AD
animals. This difference is in line with the recently reported sexual
dimorphism observed in cognitive performance such as the Morris
water-maze in which female 3xTg-AD mice also perform worse than
males [37]. In addition, it correlates to the well known fact, that AD
affects women earlier and with more severity than men (according to
some findings the incidence of the disease is double in females
[35,36]. Importantly such a gender-based predisposition toward
females is specific of AD and not found in other dementias [36].
Several lines of evidence suggest that this prevalence is directly
related with the circulating levels of estrogens [35,36,47]. As a result
females have higher levels of cell proliferation, but not cell survival
when compared with males cell proliferation rate in turn depends on
the endocrine status [48]. Only proestrus females, with high levels of
estradiol, show higher levels of cell proliferation; which seems to be
mediated through its effect on estrogen receptors [34,48,49]. Be it all
as it may, all these results are in agreement with our findings of a
greater degree of Abpathology in female versus male 3xTg-AD
animals from 4 months of age; this is also in agreement with recent
results observed in anothere AD transgenic mouse model (APP23) as
well as in line with clinical evidence of higher prevalence of AD in
females [35,36,42,50,51]. However, future studies are needed to
confirm this estrogen active role on cell proliferation and
neurogenesis whilst lately it has been shown controversial evidence
in some mouse strains, such as C57BL/6, in which female
hippocampal cell proliferation is not influenced by estrous cycle or
ovariectomy [52].
Generation of new neurones is an important feature of the adult
brain; in hippocampus the newborn neuronal cells, governed by
multiple factors, undergo complex stages of morphological and
functional maturation and integrate into existing neural circuitry
[53]. The hippocampal neurogenesis can be directly involved in
variety of cognitive processes; decreased neurogenesis negatively
affects certain learning and memory processes such as spatial
memory [51,53]. In contrast, increased load on the cognitive
processes (e.g. enriched environment) and physical exercises
positively affect neurogenesis [54]. On the other hand the role of
impaired neurogenesis in cognitive deficits in neurodegenerative
diseases is much less characterized. However, our observations
showing an impairment in neurogenesis correlate with the recent
evidence of age dependent impairment in spatial and long-term
memory tasks observed in the 3xTg-AD animals [32–34]; suggesting
a critical implication of neurogenesis in cognitive deficits
Thus, we consider that our data are specifically important as we
directly addressed the contradicting issue of the neurogenesis status
in the AD. By using longitudinal study on newly developed
transgenic model of the disease we demonstrated clear inhibition
of neurogenesis in diseased animals; this inhibition is specifically
pronounced in females, which correlates with clinical observations.
Therefore we conclude that inhibited neurogenesis can play an
active role in development of cognitive deficits and progression of
the AD.
Acknowledgments
The authors would like to thank Mr. Harun Noristani and Markel
Olabarria for their help editing this manuscript.
Author Contributions
Conceived and designed the experiments: JJRA. Performed the experi-
ments: JJRA MT EMK. Analyzed the data: JJRA VCJ EMK. Contributed
reagents/materials/analysis tools: SMA FML SO AV. Wrote the paper:
JJRA AV.
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