Neuroscience 129 (2004) 583592

ALTERED MITOGEN-ACTIVATED PROTEIN KINASE SIGNALING, TAU

HYPERPHOSPHORYLATION AND MILD SPATIAL LEARNING
DYSFUNCTION IN TRANSGENIC RATS EXPRESSING THE -AMYLOID
PEPTIDE INTRACELLULARLY IN HIPPOCAMPAL AND
CORTICAL NEURONS
V. ECHEVERRIA,a A. DUCATENZEILER,a E. DOWD,a,d
J. JNNE,e S. M. GRANT,a M. SZYF,a F. WANDOSELL,f
J. AVILA,f H. GRIMM,g S. B. DUNNETT,d T. HARTMANN,g
L. ALHONENe AND A. C. CUELLOa,b,c*

with the initial steps in the tau-phosphorylation cascade,

alterations in ERK2 signaling and impairment of higher CNS
functions in male rats. 2004 IBRO. Published by Elsevier
Ltd. All rights reserved.

a
Departments of Pharmacology and Therapeutics, McGill University,
3655 Promenade Sir-William-Osler, Montreal, Quebec, Canada H3G
1Y6

Key words: -amyloid, transgenic rat, ERK/MAPK, tau phosphorylation, Morris water maze.

b
Anatomy and Cell Biology, McGill University, Montreal, Canada H3G
1Y6

In genetically inherited cases of Alzheimers disease (AD),

there is increased production of the -amyloid fragment
(A) of the amyloid precursor protein (APP) and aggregated fibrils of this peptide form the core of the neuritic
plaques which are widespread across the neocortex and
hippocampus of all AD patients (for reviews see Walsh et
al., 2002). Thus, strong evidence favors a central role for
A in the AD disease process. The most established hypothesis of the pathophysiology of AD, the so-called amyloid hypothesis, states that A plaques or A oligopolymers are somehow toxic to neurons, causing neuronal
dysfunction, neurodegeneration and dementia (for review
see Tanzi and Bertram, 2001). For this reason, the majority
of studies have focused on the neurotoxic consequences
of the aggregation of extracellular A fragments in AD and
in AD animal models. However, there is no unequivocal
correlation between plaque load and the degree of dementia (Hardy and Higgins, 1992) suggesting that other extracellular oligomeric forms of A other than the extracellular
aggregated peptide could also play a role in AD pathology
(for review see Walsh et al., 2002). Furthermore, there is
evidence that A is generated and sequestered intracellularly in the early AD pathology (for reviews see Wilson et
al., 1999; Hartmann, 1999; Echeverria and Cuello, 2002).
A is derived by the sequential cleavage of APP in a
number of sub-cellular organelles including the endoplasmic reticulum, Golgi and endosomal/lysosomal system
(Dickson et al., 1995). It has been shown that A is present
intracellularly in Down syndrome preceding the appearance of amyloid plaques (Mori et al., 2002). A number of
studies have also suggested that intracellular A accumulation would precede plaque formation in AD patients
(DAndrea et al., 2001; Takahashi et al., 2002) and a
similar sequence has also been reported in transgenic (Tg)
mice overexpressing AD-related genes (Gouras et al.,
2000). Despite the mounting evidence for intracellular
amyloid-induced cell dysfunction in vitro (Grant et al.,
1999b; Glabe, 2001; Zhang et al., 2002), the pathological

Neurology and Neurosurgery, McGill University, Montreal, Canada

H3G 1Y6

d
Brain Repair Group, School of Biosciences, Cardiff University,
Cardiff, Wales, UK
e

A. I. Virtanen Institute, University of Kuopio, Kuopio, Finland

Centro de Biologa Molecular Severo Ochoa, CSIC-Universidad

Autnoma de Madrid, Madrid, Spain
g

Center for Molecular Biology, University of Heidelberg, Heidelberg,

Germany

AbstractThe pathological significance of intracellular A

accumulation in vivo is not yet fully understood. To address
this, we have studied transgenic rats expressing Alzheimersrelated transgenes that accumulate A intraneuronally in the
cerebral and hippocampal cortices but do not develop extracellular amyloid plaques. In these rats, the presence of intraneuronal A is sufficient to provoke up-regulation of the
phosphorylated form of extracellular-regulated kinase (ERK)
2 and its enzymatic activity in the hippocampus while no
changes were observed in the activity or phosphorylation
status of other putative tau kinases such as p38, glycogen
synthase kinase 3, and cycline-dependent kinase 5. The increase in active phospho-ERK2 was accompanied by increased levels of tau phosphorylation at S396 and S404 ERK2
sites and a decrease in the phosphorylation of the CREB
kinase p90RSK. In a water maze paradigm, male transgenic
rats displayed a mild spatial learning deficit relative to control
littermates. Our results suggest that in the absence of
plaques, intraneuronal accumulation of A peptide correlates
*Correspondence to: A. C. Cuello, Department of Pharmacology and
Therapeutics, McGill University, 3655 Promenade Sir-William-Osler,
Montreal, Quebec, Canada H3G 1Y6. Tel: 1-514-398-3618; fax:
1-514-398-8317.
E-mail address: claudio.cuello@mcgill.ca (A. C. Cuello).
Abbreviations: A, -amyloid; AD, Alzheimers disease; APP, amyloid
precursor protein; CDK5, cycline-dependent kinase 5; ERK, extracellularregulated kinase; GSK3, glycogen synthase kinase 3; IR, immunoreactive; mAb, monoclonal antibody; MAPK, mitogen-activated protein kinase;
NFT, neurofibrillary tangles; PBS, phosphate-buffered saline; PBST, 5%
non-fat milk in phosphate-buffered saline, 0.2% Tween-20; PHF, paired
helical filaments; PS1, Presenlin-1; SDS, sodium dodecyl sulfate; Tg,
transgenic.

0306-4522/04$30.000.00 2004 IBRO. Published by Elsevier Ltd. All rights reserved.

doi:10.1016/j.neuroscience.2004.07.036

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V. Echeverria et al. / Neuroscience 129 (2004) 583592

significance of in vivo intracellular A accumulation is not

yet fully understood. This question can be answered in a
model that presents this early AD-related pathology without amyloid plaque formation, thus avoiding the confounding downstream effects of intracellular versus extracellular
A aggregation.
We have taken advantage of a Tg rat line expressing
mutated forms of the human transgenes APP and
Presenlin-1 (PS1), with a phenotype limited to the intracellular accumulation of A immunoreactive (IR) material in neurons of the cerebral cortex and hippocampus
(Echeverria et al., 2003), to investigate the consequences of such a phenotype on the phosphorylation
status of protein kinases relevant to the AD neuropathology, the phosphorylation status of tau proteins and the
long term behavioral consequences of such changes.

EXPERIMENTAL PROCEDURES
Expression constructs and generation of Tg rats
The generation of the Tg constructs and generation of the Tg rat
lines was done as described (Echeverria et al., 2003). Briefly, the
two Tg constructs used in this study incorporated a plateletderived growth factor and contained the human APP751 cDNA
with the Swedish (K670N; M671L; Citron et al., 1992) and the
Indiana mutations (V717F; Fidani et al., 1992) or the human PS1
gene with the Finn mutation (M146V; Sherrington et al., 1995).
The Tg rats were generated by the simultaneous pronuclear injection of HsdBrl:WH Wistar rats embryos (Hogan et al., 1986). To
identify positive founder rats from F1 offspring, genomic DNA was
prepared from kidney and analyzed by Southern blotting (Blin and
Stafford, 1976). The probes were digoxigeninUTP-labeled PCR
products, corresponding to transgene DNA sequences. Transgene mRNA expression in the Tg rat cortex was confirmed by
RT-PCR (Halmekyto et al., 1991) as described (Echeverria et al.,
2003).

Antibodies
For the identification of APP products by immunoelectrophoresis, we applied a monoclonal antibody (coded WO-2) directed to
amino acids 4 10 of the human A fragment. WO-2 does not
cross-react with the rat form of A but it does with the holo APP
protein and the guinea-pig A molecule, which is identical to
human A (The Genetics Company, Zurich, Switzerland). We
used an array of different antibodies to detect A in immunohistochemical studies: the mouse monoclonal antibody (mAb),
McSA1, that recognizes A epitopes 112, which displays 1000fold higher affinity for the human A than for the rat form of the
peptide and does not recognize the APP holoprotein by immunohistochemistry (Grant et al., 2000), and the mAb 6E10 recognizes A epitopes 117 (Chemicon). To detect tau phosphorylation, we used the AT8 (Innogenetics, Gent, Belgium), Alz 50 and
PHF1 antibodies (Gifts from Dr. P. Davies), the latter of which
recognizes tau phosphorylated at residues 396 and 404. We also
used: polyclonal antibodies against extracellular-regulated kinase
(ERK)1/2; Mabs against phospho-ERK2, phospho-glycogen synthase kinase 3 (GSK3)/ at Y279/Y216 and phospho-p90RSK at
S380 (Cell Signaling Technology, Beverly, MA, USA); a polyclonal
antibody (H-76) to detect GSK3 / and p35 protein levels (Santa
Cruz Biotech, Santa Cruz, CA, USA); tau-1 and tau-5 antibodies,
which recognize total tau protein (Laboratory Vision, Fremont, CA,
USA); and the mAb Jonas and 22C11 antibodies (Roche Molecular Biochemicals, Laval, Canada) which recognize the carboxy
and amino-terminal portions of APP protein respectively. Finally,

anti-tubulin III antibodies (Promega, Madison, WI, USA) were

used as a reference.

Immunohistochemistry
Tg and littermate control rats were anesthetized with Equithesin
(2.5 ml kg1 i.p.) and fixed by intranscardial perfusion (Ct et al.,
1993). Brains were postfixed for 3 h at room temperature and then
kept in 30% sucrose in 0.1 M phosphate buffer at 4 C. Forty
micrometer sections were cut from the relevant brain area using a
microtome and transferred into phosphate-buffered saline (PBS)
with 0.2% Triton X-100. Free-floating immunohistochemical staining was performed as previously described (Ct et al., 1993). In
brief, sections were incubated for 30 min in a blocking solution
containing 5% of the appropriate normal serum in PBS, after
which they were incubated overnight at 4 C with primary antibodies. The sections were then incubated with anti-IgG speciesspecific biotinylated IgGs and visualized using ABC kits according
to the manufacturers instructions (Vector, Burlingame, CA, USA).

Western blotting
Tg and littermate controls rats were killed, and rat brains were
removed, placed on ice and the hippocampus and the cerebellum
tissues were dissected out and stored at 80 C until used.
Hippocampus and cerebellum tissue samples were homogenized
in 10 volumes of homogenization buffer: 50 mM TrisHCl, pH
7.4/100 mM NaCl/5 mM EGTA/1% Triton X-100/1 mM Na3VO4/
100 mM NaF/250 nM okadaic acid/1 complete protease inhibitor
mixture (Roche Molecular Biochemicals). The homogenates were
then centrifuged at 16,000 r.p.m. for 30 min at 4 C. The supernatants were collected, protein concentrations were determined,
mixed with 5 sodium dodecyl sulfate (SDS) sample buffer, and
then denatured by boiling for 10 min. Next, 2550 g of total
proteins were separated on SDSpolyacrylamide gel and transferred to a nitrocellulose filter. The A control used was the
supernatant of a human neuroblastoma cell line culture stably
transfected with APP (SpA4CT; Grimm et al., 2003). Nitrocellulose sheets were blocked with 5% non-fat milk in PBS, 0.2%
Tween-20 (PBST). The nitrocellulose was then incubated with
primary antibodies overnight at 4 C. Filters were rinsed three
times in PBST buffer, and then incubated with the corresponding
peroxidase-conjugated secondary antibody (diluted 1: 5000; Promega) for 1 h at room temperature. Immunoreactivity was visualized by the use of an enhanced chemiluminescence detection
system (ECL; Amersham/Pharmacia Biotec, Canada). Band intensity was quantified from film exposures (X-Omat LS; Kodak,
Rochester, NY, USA) using densitometry (Fidani et al., 1992).
Group values were obtained simultaneously and normalized with
respect to total protein or tubuline III immunoreactivity.

ERK2 activity assay

ERK2 activity was analyzed using four controls and six Tg postnuclear hippocampal and cerebellum extracts applying the nonradioactive ERK1/2 activity assay kit (Cell Signaling Technology)
according to the manufacturers instructions. Briefly ERK2 was
immunoprecipitated from the post-nuclear hippocampus extracts
(200 g total protein) and incubated for 30 min at 30 C with an
excess of Elk-agarose matrix immobilized substrate. The levels of
phosphorylated substrate were determined by immuno-Western
blotting and the band intensities were quantified from films by
densitometry.

Behavioral analysis
All behavioral testing was carried out during the light phase of a
12-h light/dark cycle. All experimental procedures were approved
by the McGill University Animal Care Committee and were con-

V. Echeverria et al. / Neuroscience 129 (2004) 583592

ducted in accordance with the Canadian Council on Animal Cares
guidelines. Efforts were made to minimize the number of animals
and their suffering.
Twelve Tg rats (six male, six female) and 12 age- and sexmatched littermate controls were analyzed using the Morris water
maze (Morris et al., 1982), at 7 and 16 months of age. For each
swim, rats were placed into the pool at one of four starting positions in pseudorandom order. Trials lasted for a maximum of 120 s
and 10 min was allowed between trials. Rats were given eight
trials per day for 14 days. For the first 13 days the platform was in
the same location but on the 14th day its location was changed.
On the last day of each experiment, rats were given four trials on
a visible platform task with the platform in a different position for
each of the trials. Swim path lengths, latencies and speeds were
measured in all trials.

Statistical analysis
Unpaired Students t-test was performed on immunoblot results.
Behavioral data were analyzed using two-way repeated-measures
ANOVA. Differences were considered significant when P0.05.

RESULTS
We have reported the expression of mutated forms of
human APP and PS1 transgenes in several heterozygous and homozygous, single and double, Tg rat lines, and
the presence of their corresponding mRNA in cortical tissue as well as the expression of the resulting proteins
(Echeverria et al., 2004). In this study, we confirmed the
occurrence of a cytological phenotype of A-IR material
abnormally accumulated within a large number of pyramidal neurons of the neocortex, in the near totality of pyramidal neurons of the CA3 and hylar regions of the hippocampal complex, and, to a much lesser extent, in pyramidal neurons of CA2 and CA1 regions in the
heterozygous double Tg APPPS1 rat, coded UKUR25.
The A-IR material appeared granular in aspect, and was
distributed evenly in the neuronal cell somata and occasionally was observed in dendritic extensions and proximal
axonal portions of cortical pyramidal neurons. Fig. 1 illustrates these microanatomical and cytological features.
These Tg rats did not display amyloid plaques or detectable extracellular A-IR material. The cytological phenotype appeared as early as 6 months of age and remained stable for at least 2 years of age (the last time point
investigated). The incidence of pyramidal neurons displaying intracellular A-IR material was larger in the cingulate
cortex than other cortical areas. The A-IR material was
revealed immunocytochemically applying different, wellcharacterized mAb. This reaction was more intense with
the McSA1 mAb, which does not recognize the holo APP
protein immunohistochemically (Grant et al., 2000), directed to the N-terminal portion of A. No such immunoreactions were found in non-Tg littermate rats with any of
the antibodies applied.
In order to better establish the nature of the intracellular A-IR material found in the cerebral cortex and hippocampus, we proceeded to investigate the A molecular
species detected from the areas of the CNS expressing the
above phenotype (cerebral cortex and hippocampus) and
not expressing intracellular A-IR material (cerebellum)
using electrophoresis followed by immunoblotting. For this

585

we applied the well-characterized and highly human specific WO-2 mAb (The Genetics Company) for the Western
blot analysis of soluble protein extracts from the above
CNS regions obtained from control littermates and
UKUR25 Tg rats. This demonstrated distinctive IR bands
corresponding to authentic human APP and A standards present only in the cerebral cortex and hippocampus
of Tg rats, but not in the cerebellum or in any of these
regions in extracts from non-Tg littermate rats (see Fig. 2).
This pattern corresponded well with the distribution of
A-IR neurons in the Tg rats.
As we have previously observed a dysregulation of
mitogen-activated protein kinases (MAPK) in transfected
cell lines stably overexpressing wild human APP and
displaying abundant intracellular A immunoreactivity
(Grant et al., 1999a) as well as in a APP (SWE717)
homozygous Tg rat line (Echeverria et al., 2003), we proceeded to investigate further this aspect in the Tg rat line
UKUR25 here described. Western blotting analysis revealed a 40% selective increase in phosphorylated ERK2
in the hippocampus of 9-month old UKUR25 Tg rats when
compared with age and sex-matched littermate controls
(see Fig. 3A, B). No similar upregulation of ERK2 phosphorylation levels was found in other brain areas of the
UKUR25 Tg rat line, such as cerebellum, in which no
intracellular A immunoreactivity was observed at all. As
ERK2 is a putative kinase for the microtubule associated
protein tau (Avila et al., 2002; Pei et al., 2002; Stamer et
al., 2002), we investigated whether other previously reported tau kinases were also activated. In these Tg rats, of
the putative tau kinases analyzed, only ERK2 showed a
clear increase in the level of its phosphorylated form, as no
changes in the levels or phosphorylation status of the p38
or GSK3/ protein kinases or in the cycline-dependent
kinase 5 (CDK5) regulatory subunit p35 were observed
(data not shown). The observed changes appear regulatory in nature, as the overall level of the ERK2 protein
remained unchanged in comparison to expression levels of
the cytoskeletal protein tubulin III in Tg and littermate
control rats (Fig. 3A). This reinforces the notion that the
elevated phosphorylation levels had a functional impact,
as hippocampal extracts from Tg rats displayed a clear-cut
increase in their phosphorylating activity on ERK2 substrates as compared with control littermate rats (Fig. 3C).
This increased ERK2 kinase activity was not observed in
the cerebellum of the Tg rats (Fig. 3D).
Since ERK2 is known to phosphorylate tau at epitopes
S396/S402, we utilized the well-characterized PHF1 mAb
(courtesy of Dr Peter Davis, NY, USA) to determine the
phosphorylation state of tau proteins at these sites on
Western blots. We found an increased level of tau phosphorylation at PHF1 sites in extracts from the hippocampus of the double Tg rat UKUR25 (see Fig. 4) while the
total tau protein levels remained constant. No other
changes in tau immunoreactivity were detected using other
antibodies, such as AT8 and CP13, which recognize tau
phosphorylated at the p38 kinase sites S199 and S202,
nor with Tau-1 (data not shown). Furthermore, and consistent with the Western blot evidence, we observed in

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Fig. 1. Intracellular A accumulation and immunoreactivity in 9 month-old APP/PS1 (UKUR25) Tg rats. The A-IR localization as revealed using the
monoclonal antibodies McSA1 (AC) and 6E10 (D, E). (A) Frontal cerebral cortex (CCX) and hippocampus. Note the heavy localization of human (h-)
Tg A-IR material in CA2, CA3 and hilar (H) regions of the hippocampal complex. CC, crus cerebri, DG, dentate gyrus. Dark dots in the cerebral cortex
denote A-IR pyramidal neurons. (B) Non Tg littermate control. Scale bar300 m for A, B. Inset in A denotes a large pyramidal neuron from lamina
V loaded with h-A-IR material, some of which is present in the proximal portions of the apical dendrite (ad). Scale bar25 m. (C) Higher
magnification micrograph denotes intracellular h-A-IR material in nearly all CA3 pyramidal and H neurons in the UKUR25. Tg rat. Scale bar100 m.
Inset: details of the granular appearance of the h-A-IR in cytoplasm of two pyramidal neurons. Scale bar20 m. D and E depict distribution of A-IR
neurons as revealed with mAb 6E10. Similar field of the hippocampal formation to that represented in A and B. (D) UKUR25 Tg rat; (E) non-Tg
littermate control. Scale bar300 m for D and E. (F, G) Western blot analysis showing the immunoreactivity of monoclonal antibodies 6E10 (F) and
McSA1 (G) to human A peptides 1 42 and 1 40.

UKUR25 Tg rats a higher level of PHF1 immunoreactivity

in neurites of the CA2, CA3 and hilar hippocampal regions,
in close correspondence with neurons displaying intracellular A-IR material (Fig. 4B, C).
As ERK2 is involved in the activation of the CREB kinase
p90RSK, a protein involved in several plasticity processes

including learning and memory (Frodin and Gammeltoft,

1999), we investigated the levels of phosphorylated p90RSK
at the activation residue serine 380 in UKUR25 rats using
Western blot analysis. Contrary to the expected outcome, in
these Tg rats, which display intraneuronal A-IR material, the
increased ERK2 phosphorylation and activity resulted in an

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587

Fig. 2. Expression of human APP transgene and human A peptide

in the hippocampus, cortex and cerebellum of UKUR25 (APP/PS1)
and control littermate non-Tg rats. Immunoblot analysis of hAPP
expression from the hippocampus of 9 month-old UKUR25 Tg (n5)
and age-matched littermate controls (n3) as described in Experimental Procedures. Postnuclear extracts of the rat tissues were subjected
to immunoprecipitation and Western blotting with anti-A WO-2 antibody that recognizes hAPP and human A but does not recognize
the corresponding rat forms. Note the marked expression of the human Tg APP holoprotein in the cortex and hippocampus of the
UKUR25 Tg rat, and its absent in non-Tg littermate controls. In addition, monomeric A-IR material (lower band) and oligomeric A forms
(*) of the peptide are observed in the hippocampus and cerebral cortex
of Tg rats but not in non-Tg littermate controls. Arrows indicate IR
bands corresponding to APP and A standards.

apparent downregulation of p90RSK activity, as the Western

blot analysis revealed a marked diminution of its phosphorylated form in the hippocampus of the Tg rats as compared
with littermate controls (Fig. 5).
In order to establish whether this limited cortical and
hippocampal A neuropathology, in the absence of plaques,
could lead to disruption of higher CNS functions, we investigated the spatial learning ability of the UKUR25 Tg and
littermate rats in the water maze task. Rats were analyzed at

Fig. 3. Increased phospho-ERK2 levels in the hippocampus of Tg rats

displaying intraneuronal A. (A) Western blot analysis revealed increased levels of ERK2 phosphorylation at T202/Y204 sites in the
hippocampus of Tg rats (a) in relation to control littermates, while no
change in the overall ERK2 protein levels were detected (b) in relation
to tubulin III (c). (B) Normalized activity levels representing the ratio
of immunoreactivity of phospho-MAPK/unphosphorylated MAPK. Optical density (OD). * Indicates P0.05 unpaired t-test. (C) ERK2 enzymatic activity. Immunoprecipitated ERK2 from hippocampal homogenates of Tg rats (UKUR25) revealed higher levels of phosphorylated
products than in non-Tg littermate controls. * P0.05 unpaired t-test.

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V. Echeverria et al. / Neuroscience 129 (2004) 583592

Fig. 4. Increased tau phosphorylation in the hippocampus of Tg rats displaying intraneuronal A. (A) Immunoblot analysis of tau phosphorylation in
the hippocampus of 9 month-old rats with the site-specific phospho-tau antibody PHF1 revealing an increment of phosphor-tau-IR material in Tg rats
(n8) in relation to non-Tg littermates (n4). Immunoreactivity was normalized to tubulin III immunoreactivity (* P0.01). Controls, littermate non-Tg
rats; ptau, tau phosphorylated at residues S396 and S404. Micrographs illustrate moderate PHF-1 immunoreactivity in neurites of the stratum radiatum
(SR) of the CA2 region of non-Tg rats (B) and a more intense immuno-reaction in neurites of the same hippocampal region in UKUR25 Tg rats (C);
PCL, pyramidal cell layer. Scale bar50 m for B and C.

7 and 16 months of age, representing the initial and advanced stages of the A intraneuronal accumulation phenotype. The animals were given eight trials per day for 13 days,
with the platform in a fixed location, and the swim distance
and latencies to find the platform were recorded. No significant differences were found in the escape latency nor in the
path length between Tg and non-Tg rats at 7 months of age
or in female rats at 16 months of age. However, as shown in
Fig. 6A, from the third day of testing there was an overall
significant increase in the distance swam by the 16 monthold male Tg rats to find the escape platform in comparison
to their littermate controls (Fig. 6A; TransgeneDays,

F12, 2332.02, P0.05). The escape latency analysis also

indicated that male Tg rats took longer to locate the escape
platform (Fig. 6B; TransgeneDays, F12, 2332.16, P0.05).
On the 14th day, the platform was moved to the opposite end
of the pool and rats were given eight trials over which to learn
this new location. Whereas the control males showed clear
improvements in performance across trials (trial 1:
1057218 cm; trial 8: 25088 cm), the Tg rats showed no
overall improvement (trial 1: 596184 cm; trial 8:
573355 cm) but this difference was not statistically significant (TransgeneTrial, F7,700.85, ns) probably due to the
high variability in the Tg group. At the end of the study, the

Fig. 5. Decreased phospho-p90RSK in UKUR25 Tg rat hippocampi. The analysis of p90RSK phosphorylation at S380 in the hippocampus of the
UKUR25 double Tg rats (9 months) by Western blotting revealed a 3 4-fold decrease in the phospho-p90RSK immunoreactivity (pp90RSK) in relation
to control non-Tg littermates. (A) Histogram illustrating the relative phospho-p90RSK immunoreactivity, represented as relative optical density (OD)
of the IR phospho-p90RSK band normalized to tubulin III-IR. (B) Autoradiography showing the phospho-p90RSK-IR bands obtained after Western
blotting analysis of hippocampal post nuclear extracts of littermate controls and UKUR25 double Tg rats. Phospho-p90RSK, p90RSK phosphorylated
at residues S380. * P0.015, unpaired t-test.

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589

Fig. 6. Impaired performance of male UKUR25 rats in a Morris water maze task. Sixteen month-old male UKUR25 Tg rats were impaired relative to
littermate controls in their ability to locate the escape platform during training to a fixed platform location. (A) Indicates path length and (B) indicates
latency from days 113. There was no difference between Tg rats and non-Tg littermate controls in latency or swim speed to a visible platform (C,
D). Data in (A) and (B) represent the mean of eight daily trials. Open squares are non-Tg littermate controls, filled squares are UKUR25 rats; n6 per
group.

rats were given four trials on a visible platform task. Both

groups of rats performed equally well in this task (Fig. 6C; all
main effects and interaction n.s.), indicating that neither overt
visual nor motor impairments were responsible for the deficits
observed during training in the water maze task. Interestingly,
there was no significant difference in swim speed during
performance of cued trials (Fig. 6D), indicating that the differences observed during hidden platform testing may have
reflected different search strategies used or stress levels
associated with finding the hidden platform, as the male rats
displayed higher swimming velocity than controls during the
experimental trials (Fig. 6B, inset).

DISCUSSION
There is increasing evidence which suggests that intraneuronal A accumulation may be an early event in AD and
Down syndrome pathogenesis (Gouras et al., 2000;
DAndrea et al., 2001; Takahashi et al., 2002; Mori et al.,
2002). In this study, we investigate the consequences of
the intraneuronal accumulation of this peptide in hippocampal neurons on the activity of protein kinases, potentially relevant to early AD pathology, as well as the
possible behavioral consequences of this cytological and
biochemical phenotype.
In this rat Tg model of intracellular A peptide accumulation, the A-IR products appeared preferentially in
large pyramidal neurons of the cerebral cortex and hippocampus in a particulate form. This is in line with the

recent ultra-structural demonstration of preferential intracellular A localization in multivesicular bodies in Tg mice

(Takahashi et al., 2002) and also in AD brains (Gouras et
al., 2000). In a previous mass spectrometric study, we
described the upregulation in the hippocampus of Tg rats
of peptides on the 4.5 kDa bands, probably corresponding
to the Tg A peptides (Echeverria et al., 2003). In the
present study, Western blot analysis confirmed both the
presence of the holo-APP as well as the Tg human A
fragments in the cerebral and hippocampal cortices (Fig.
2). As the monoclonal McSA1 does not reveal holo-APP
immunocytochemically, but only the A material as found
in Alzheimers tissue (Grant et al., 2000) or in amyloid
plaques of Tg animals (Wong et al., 1999; Hernandez et
al., 2001), it can be assumed that the intracellular A
immunoreactivity detected in pyramidal neurons of the
cerebral cortex and hippocampus largely represents authentic A fragments. The fact that the A band observed
in the Western blots from Tg rat hippocampi and cortices is
from a detergent soluble extract also rules out possible
contamination with extracellular, aggregated material.
The intracellular accumulation of A fragments has
been reported in different AD Tg models besides the
present one. It has been observed in the hippocampus of
aged Tg mice expressing the carboxy-terminal portion of
hAPP containing the V717F London mutation (Li et al.,
1999), in APP and PS1 double Tg mice before plaque
formation (Wirths et al., 2001), and recently in the hip-

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pocampi of a single APP Tg mice before plaque formation

(Shie et al., 2003). It has also been shown in the hippocampus and neocortex of a triple Tg mouse expressing
the APPsw, PS1 finnm146v and Tau p301L proteins
before amyloid plaques develop (Oddo et al., 2003). Most
importantly, intracellular A accumulation has been observed in aged and AD human brains (Gouras et al., 2000).
According to Chui et al. (2001), its presence correlates with
neuronal apoptosis markers in neurons of AD brains before plaque formation. Furthermore there is evidence that
A42 accumulates intracellularly prior to extracellular A
deposition in Down syndrome (Mori et al., 2002).
Besides the amyloid plaques, the other salient pathological characteristic of AD is the presence of neurofibrillary tangles (NFTs) in the cell bodies of affected neurons
(Terry et al., 1991). NFTs are composed of paired helical
filaments (PHFs) of abnormally phosphorylated tau (for
reviews see Iqbal et al., 2000; Avila et al., 2002; Barghorn
and Mandelkow, 2002). Although the exact relationship
between plaques and NFTs remains unknown, proponents
of the amyloid hypothesis argue that the extracellular deposition of amyloid precedes the abnormal phosphorylation
of tau and the maturation of the AD tauopathy (for review
see Tanzi and Bertram, 2001). In support of this view is the
observation that the injection of A material in mice overexpressing the tau p301L transgene (Gotz et al., 2001) or
crossing Tg mice expressing the same tau mutation with
Tg mice overexpressing APP familial AD mutations
(Lewis et al., 2001) results in the appearance of NFTs
similar to those observed in AD.
As in most Tg animal models, the Tg rats discussed
here do not display NFTs; nevertheless they display altered ERK2 tau kinase activity as well as tau hyperphosphorylation along with intracellular A accumulation.
These findings suggest that intracellular A may also contribute to a tau pathogenic cascade initiated at early stages
of AD neupathology. This is of interest as it has been
reported that tau dysfunction affects neuronal organelles
and protein transport, both essential for cell survival and
resistance to oxidative and toxic insults (Stamer et al.,
2002). The fact that other kinases known to phosphorylate
tau in vivo, such as GSK3 or CDK5, were not activated by
intracellular A, supports the view that abnormal tau phosphorylation may occur in a sequential manner, in which tau
ERK2 sites are among the first to be phosphorylated. A
similar situation seems to occur in the human as Ferrer et
al. (2001) have found an analogous ERK2 activation in AD
brains associated with early tau deposition in neurons. In
addition, Zhu et al. (2001) found a differential pattern of
activation of MAPKs in AD pathology. These authors found
that some protein kinases are activated in mild and severe
AD cases, but not in non-demented cases with mild CNS
pathology. An up-regulation of the active form of ERK1/2,
p-ERK1/2, has also been reported in the initial stages of
neurofibrillary degeneration in projecting neurons of the
transentorhinal region in the brains of AD patients with
neurofibrillary neurodegeneration Braak stages III, but
which are devoid of amyloid deposition (Pei et al., 2002).
Taken together, these observations and the present find-

ings point to an early and not well-characterized protein

dysregulation in AD pathology.
Although no aggregated extracellular A was observed in
UKUR25 Tg rats throughout their enter life cycle, 16 monthold male rats were significantly impaired in their ability to learn
the location of the hidden escape platform in a Morris water
maze task. Thus, the accumulation of A within neurons of
the cortex and hippocampus seems to be sufficient to disrupt
some higher-order cognitive processes. Female rats at the
same age demonstrated some impairment, but these did not
reach significance levels as their training patterns were also
slower. On the basis of our present data, it is difficult to
speculate on this gender differential behavior, as further work
is necessary to rule out confounding factors such as hormonal differences. The present observations would reinforce
the concept that intracellular A can disrupt, at least in male
rats, neuronal function sufficiently to provoke cognitive impairment. The behavioral impairments observed in our male
Tg rats could tentatively be related to the early tau dysmetabolism as it has been reported that the onset of clinical
symptoms is associated with the appearance of abnormally
phosphorylated tau in the axons of hippocampal neurons
(Halmekyto et al., 1991). As tau is a microtubule-associated
protein, it is reasonable to propose that tau dysfunction might
destabilize neuronal cellular homeostasis and synaptic function resulting in the cognitive impairments observed here.
Another possibility to consider is that this cognitive
impairment was induced directly by the increased levels of
extracellular soluble A not detectable by current methods.
In other models, low APP expression levels did not result
in the formation of amyloid plaques, but did elicit spatial
learning and memory deficits and synaptotoxicity in mice
expressing human wild type or mutated A forms (Moran
et al., 1995; DHooge et al., 1996; Mucke et al., 2000;
Koistinaho et al., 2001). In other animal models it has been
shown that behavioral impairments (Holcomb et al., 1999;
Kumar-Singh et al., 2000) and synaptic transmission deficits (Hsia et al., 1999) could precede plaque formation.
These results suggest that plaque formation may not be
the only factor contributing to the development of behavioral and/or cognitive deficits. However, the cognitive impairment of the Tg rats is unlikely to be due to an APP
over-expression, as the cognitive ability of an APP-YAC
Tg mice was unaffected by similar over-expression of
hAPPs (Murai et al., 1998).
The importance of intracellular A in AD pathology has
been further defined in the behavioral and physiological
characterization of the triple-Tg mice model expressing
APP, PS1 and tau transgenes (Oddo et al., 2003). In this
study, they found that synaptic dysfunction was an early
change that precedes plaque and NFT formation, and that
the occurrence of intraneuronal A immunoreactivity in
CA1 pyramidal neurons correlates with impairments in
synaptic plasticity.
A further explanation for the observed behavioral impairment could be a dysregulation of the MAPK/CREB
signaling pathway, which is known to be involved in neural
plasticity (Abel and Kandel, 1998). In this regard, it is worth
noting that the UKUR25 Tg rats presented a p90RSK

V. Echeverria et al. / Neuroscience 129 (2004) 583592

downregulation, despite the ERK2 hyperactivity. We hypothesize that the phospho-p90RSK downregulation resulted from a sustained, anomalous ERK2 alteration, and
that this had an impact on cognitive functions. In this
regard, it has been observed that the p90RSK gene is
mutated in CoffinLowry syndrome, a condition which
leads to mental retardation (Merienne et al., 1999). A
disconnect between ERK2 and CREB signaling would be
compatible with the observation that sustained ERK activation could be harmful to neuronal subsets (Runden et al.,
1998). Furthermore, dysregulation of ERK/CREB signaling
has been demonstrated in a Tg AD mouse model displaying extracellular A accumulation (Dineley et al., 2001).
In summary, our rat Tg model revealed that A intracellular accumulation is sufficient to alter ERK/CREB signaling, affect the state of tau phosphorylation and induced
behavioral impairment in male rats. We postulate that this
ERK, tau and CREB dysregulation may lead to the behavioral impairments observed in the UKUR25 Tg male rats.
We hypothesize that similar biochemical changes might
contribute to the mild cognitive impairment observed at the
prodromic, preclinical stages of AD.
AcknowledgmentsThis research was supported by grants from
the US Alzheimers Association (IIRG-00-1964) and the CIHR
(MOP-37996) to A.C.C., and by a Wellcome Traveling Fellowship
to E.D. The authors are grateful for the financial assistance provided by the Dean of the Faculty of Medicine, McGill University,
toward the maintenance of the Tg rat colony in its early stages.
The authors would like to thank Dr P. Davies for the generous gift
of antibodies used in these studies. The authors would also like to
thank S. Ct and E. Tsang Pun Yin for their technical assistance
in preparing histochemical materials, Alan Forster for photographic expertise and Sid Parkinson for editorial assistance.
A.C.C. was the recipient of a Visiting Professorship from the
Iberdrola Foundation (Spain), which made some of this collaborative research possible. A.C.C. is the holder of the McGill University Charles E. Frosst Merck Chair in Pharmacology.

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(Accepted 13 July 2004)