Postsynaptic dysfunction is associated with spatial and object recognition memory loss in a natural model of Alzheimer’s disease
ABSTRACT Alzheimer’s disease (AD) is an age-related neurodegenerative disorder associated with progressive memory loss, severe dementia,
and hallmark neuropathological markers, such as deposition of amyloid-β (Aβ) peptides in senile plaques and accumulation of
hyperphosphorylated tau proteins in neurofibrillary tangles. Recent evidence obtained from transgenic mouse models suggests
that soluble, nonfibrillar Aβ oligomers may induce synaptic failure early in AD. Despite their undoubted value, these transgenic
models rely on genetic manipulations that represent the inherited and familial, but not the most abundant, sporadic form of
AD. A nontransgenic animal model that still develops hallmarks of AD would be an important step toward understanding how sporadic
AD is initiated. Here we show that starting between 12 and 36 mo of age, the rodent Octodon degus naturally develops neuropathological signs of AD, such as accumulation of Aβ oligomers and phosphorylated tau proteins. Moreover,
age-related changes in Aβ oligomers and tau phosphorylation levels are correlated with decreases in spatial and object recognition
memory, postsynaptic function, and synaptic plasticity. These findings validate O. degus as a suitable natural model for studying how sporadic AD may be initiated.
- SourceAvailable from: Rosario Socas[Show abstract] [Hide abstract]
ABSTRACT: The most popular animal models of Alzheimer's disease (AD) are transgenic mice expressing human genes with known mutations which do not represent the most abundant sporadic form of the disease. An increasing number of genetic, vascular and psychosocial data strongly support that the Octodon degus, a moderate-sized and diurnal precocial rodent, provides a naturalistic model for the study of the early neurodegenerative process associated with sporadic AD. In this minireview we describe and analyze the risk factors that contribute to Alzheimer-like characteristics in the degus, following recent publications, and establish some guidelines for future studies in this model of natural aging associated with the disease. Given the heterogeneity of current data derived from the diverse transgenic animal models of AD, now may be the time for the degus to become a strong attractor for academic research labs and companies involved with AD. This may help to understand the mechanisms responsible for the early neurodegenerative process associated with this devastating disease.Autonomic neuroscience: basic & clinical 01/2013; 4(1):91-6. · 1.82 Impact Factor
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ABSTRACT: The threshold for bidirectional modification of synaptic plasticity is known to be controlled by several factors, including the balance between protein phosphorylation and dephosphorylation, postsynaptic free Ca(2+) concentration and NMDA receptor (NMDAR) composition of GluN2 subunits. Pannexin 1 (Panx1), a member of the integral membrane protein family, has been shown to form non-selective channels and to regulate the induction of synaptic plasticity as well as hippocampal-dependent learning. Although Panx1 channels have been suggested to play a role in excitatory long-term potentiation (LTP), it remains unknown whether these channels also modulate long-term depression (LTD) or the balance between both types of synaptic plasticity. To study how Panx1 contributes to excitatory synaptic efficacy, we examined the age-dependent effects of eliminating or blocking Panx1 channels on excitatory synaptic plasticity within the CA1 region of the mouse hippocampus. By using different protocols to induce bidirectional synaptic plasticity, Panx1 channel blockade or lack of Panx1 were found to enhance LTP, whereas both conditions precluded the induction of LTD in adults, but not in young animals. These findings suggest that Panx1 channels restrain the sliding threshold for the induction of synaptic plasticity and underlying brain mechanisms of learning and memory.Frontiers in Cellular Neuroscience 01/2014; 8:326. · 4.18 Impact Factor
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ABSTRACT: Alzheimer's disease (AD) is the most common cause of dementia, affecting more than 36 million people worldwide. AD is characterized by a progressive loss of cognitive functions. For years, it has been thought that age is the main risk factor for AD. Recent studies suggest that life style factors, including nutritional behaviors, play a critical role in the onset of dementia. Evidence about the relationship between nutritional behavior and AD includes the role of conditions such as obesity, hypertension, dyslipidemia and elevated glucose levels. The coexistence of some of these cardio-metabolic risk factors is generally known as metabolic syndrome (MS). Some clinical studies support the role of MS in the onset of AD. However, the cross-talk between the molecular signaling implicated in these disorders is unknown. In the present review, we focus on the molecular correlates that support the relationship between MS and the onset of AD. We also discuss relevant issues such as the role of leptin, insulin and renin-angiotensin signaling in the brain and the possible role of Wnt signaling in both MS and AD. We discuss the evidence supporting the use of ob/ob mice, high-fructose diets, aortic coarctation-induced hypertension and Octodon degus, which spontaneously develops β-amyloid deposits and metabolic derangements, as suitable animal models to address the relationships between MS and AD. Finally, we examine emergent data supporting the role of Wnt signaling in the modulation of AD and MS, implicating this pathway as a therapeutic target in both conditions.Progress in Neurobiology 07/2014; · 10.30 Impact Factor
Postsynaptic dysfunction is associated with spatial
and object recognition memory loss in a natural
model of Alzheimer’s disease
Álvaro O. Ardilesa, Cheril C. Tapia-Rojasb, Madhuchhanda Mandalc, Frédéric Alexandred, Alfredo Kirkwoodc,1,
Nibaldo C. Inestrosab, and Adrian G. Palaciosa,1
aCentro Interdisciplinario de Neurociencia de Valparaíso, Facultad de Ciencias, Universidad de Valparaíso, 2360102 Valparaíso, Chile;bCentro de
Envejecimiento y Regeneración, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, 8331010 Santiago, Chile;cMind/Brain Institute
and Department of Neurosciences, Johns Hopkins University, Baltimore, MD 21218; anddInstitut des Maladies Neurodégénératives, Department Mnemosyne,
Centre de Recherche Inria Bordeaux, F-33076 Bordeaux, France
Edited by Robert C. Malenka, Stanford University School of Medicine, Stanford, CA, and approved July 6, 2012 (received for review January 23, 2012)
Alzheimer’s disease (AD) is an age-related neurodegenerative
disorder associated with progressive memory loss, severe demen-
tia, and hallmark neuropathological markers, such as deposition
of amyloid-β (Aβ) peptides in senile plaques and accumulation of
hyperphosphorylated tau proteins in neurofibrillary tangles. Recent
evidence obtained from transgenic mouse models suggests that
soluble, nonfibrillar Aβ oligomers may induce synaptic failure early
in AD. Despite their undoubted value, these transgenic models rely
on genetic manipulations that represent the inherited and familial,
but not the most abundant, sporadic form of AD. A nontransgenic
animal model that still develops hallmarks of AD would be an
important step toward understanding how sporadic AD is initiated.
Here we show that starting between 12 and 36 mo of age, the
rodent Octodon degus naturally develops neuropathological signs
of AD, such as accumulation of Aβ oligomers and phosphorylated
tau proteins. Moreover, age-related changes in Aβ oligomers and
tau phosphorylation levels are correlated with decreases in spatial
and object recognition memory, postsynaptic function, and synap-
tic plasticity. These findings validate O. degus as a suitable natural
model for studying how sporadic AD may be initiated.
memory dysfunction|neural plasticity|aging|T-maze|hippocampus
processed proteins in neurofibrillary tangles (NFTs) and senile
plaques (1). These lesions are present in both familial and spo-
radic forms of AD. Familial AD is linked to inherited mutations
in AD-related genes and represents a small percentage of AD
cases, whereas sporadic AD represents the vast majority of cases
and is not inherited. Results from transgenic mice bearing mu-
tations in APP, PSEN1/2, and TAU show synaptic dysfunction in
early stages of AD, before overt neurodegeneration (2, 3). More
recent studies have demonstrated a critical role for soluble Aβ
oligomers as an early trigger for AD, as well as associations with
memory and neural plasticity loss (4–8).
Although transgenic mice have been extremely useful in elu-
cidating the pathological mechanisms of AD, they have some
substantial limitations. Examples include the absence of tau
mutations linked to AD except for a triple transgenic mouse
3xTg-AD, bearing mutations for APP, PSEN1/2, and TAU (9);
inability to develop the whole spectrum of the disease; over-
expression of transgenes into a nonphysiological scenario; and
the fact that the manipulated genes represent only familial, not
sporadic forms of AD (10, 11). It would be highly desirable to
have a nontransgenic model of AD to complement the existing
models. Several species naturally develop features of AD with
age; however, the usefulness of these species is limited, because
none exhibits the full spectrum of AD-related alterations (12–14).
For example, the Aβ peptide sequences of Cavia porcellus (guinea
pig) and Microcebus murinus are similar to that of human (15, 16),
but the first fails to develop senile plaques and NFTs (15), and
lzheimer’s disease (AD) is an age-related neurodegenerative
disorder characterized by the accumulation of abnormally
experiments examining synaptic function and memory have not
been carried out in such models. A promising candidate model
for sporadic AD is the rodent Octodon degus (degus), which
naturally develops the histochemical hallmarks of AD, including
intracellular and extracellular accumulation of amyloid plaques,
tau deposition in NFT (17), and hippocampal disconnection and
brain parenchyma pathology (18). Prompted by these preliminary
observations, we examined the neuropathological spectrum of
AD in degus. Here we report that degus exhibits an age-related
accumulation of soluble Aβ oligomers and tau protein phosphor-
ylation that correlates with cognitive decline in spatial memory
(T-maze) and object recognition memory (ORM), as well as
synaptic and neural plasticity dysfunction. Based on these find-
ings, we propose that (i) Aβ dodecamers (Aβ*56) may associate
with phosphorylated tau proteins, constituting an early candidate
for the neural toxicity and synaptic dysfunction that occurs be-
fore the appearance of fibrillar forms of Aβ, which are common
to familial and sporadic forms of AD, and (ii) degus is a suitable
nontransgenic model of sporadic AD.
We evaluated the degree of neuropathology at the behavioral,
synaptic, and molecular levels in degus at different ages. This
approach allowed us to establish interpretative correlations to
identify those animals suffering from AD (Table S1).
Age-Related Cognitive Decline with Aging in O. degus. We first
evaluated memory capacity with ORM and T-maze tests in 6-,
12-, 36-, and 60-mo-old degus. The results showed no significant
difference between 6- and 12-mo-old degus and between 36- and
60-mo-old degus, and thus we classified the animals as either
young (6 and 12 mo-old) or aged (36 and 60 mo-old) (Fig. 1 B
and D). In the T-maze, the aged degus had poorer performance
than the young degus (Fig. 1 A and B; P < 0.0001, ANOVA).
On the ORM test, the aged degus explored less (total time and
number of visits) and had longer latency than the younger degus
(Table S2). Moreover, unlike young degus, aged degus did not
demonstrate a preference between new objects and familiar objects
(Fig. 1 C and D; P < 0.0001, t test). In general, we observed an
age-dependent decline in memory performance beginning at
36 mo-old and persisting through 60 mo-old (Fig. 1B).
Author contributions: A.O.A., C.C.T.-R., A.K., N.C.I., and A.G.P. designed research; A.O.A.,
C.C.T.-R., and M.M. performed research; A.K., N.C.I., and A.G.P. contributed new reagents/
analytic tools; A.O.A., C.C.T.-R., F.A., A.K., N.C.I., and A.G.P. analyzed data; and A.O.A.,
A.K., and A.G.P. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1To whom correspondence may be addressed. E-mail: email@example.com or adrian.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| August 21, 2012
| vol. 109
| no. 34
Selective Postsynaptic Dysfunction Induces Impairments in Synaptic
Transmission and Plasticity. We next examined the synaptic basis
of these learning deficits by evaluating the strength and plasticity
of the CA3–CA1 synapses in hippocampal slices prepared from
behaviorally tested animals. Basal excitatory synaptic transmission
was reduced in aged degus compared with young degus (Fig. 2A1;
P < 0.0001, ANOVA). Importantly, the field excitatory post-
synaptic potential (fEPSP) slopes, but not fiber volley amplitude,
were significantly reduced in aged animals (Fig. 2 A2 and A3;
P < 0.0001, repeated-measures ANOVA). This is consistent with
findings reported in AD mouse models overexpressing mutant
forms of amyloid precursor protein (APP) (19, 20; reviewed in
ref. 21). The reduced transmission could be due to a reduced
postsynaptic responsiveness or to a decreased probability of
neurotransmitter release. We ruled out the latter possibility,
because we found no difference in the paired-pulse facilitation
ratio (Fig. 2 B1 and B2; P > 0.05, two-way ANOVA). To confirm
the likely postsynaptic basis for the reduced fEPSPs, we recorded
AMPA receptor (AMPAR)-mediated miniature excitatory post-
synaptic currents (mEPSCs). Consistent with previous reports
in AD models (22, 23), we found that the significantly reduced
amplitude, but not frequency, of mEPSCs in aged degus (Fig. 2
C1–C3; P < 0.01, t test), suggesting a change in AMPAR func-
tion or number. The electrophysiological analysis indicated that
aging preferentially affects postsynaptic processes in degus. Thus,
we further assessed the integrity of presynaptic and postsynaptic
elements by measuring the levels of critical synaptic proteins
extracted from hippocampal slices by Western blot analysis. We
found a selective reduction in PSD-95, AMPAR subunit GluR2,
and NMDA receptor (NMDAR) subunit NR2B expression (Fig.
3 A and B; P < 0.05, ANOVA), but not of synaptophysin, in ac-
cordance with the observation that measures of presynaptic func-
tion (fiber volley and paired pulse facilitation) are not affected in
aged degus (Fig. 2 A2 and B1). These data support the observation
of more vulnerable postsynaptic integrity in aged degus.
We next assessed whether synaptic plasticity was affected at
the CA3–CA1 synapses of the aged degus. We measured long-
term potentiation (LTP) induced with theta-burst stimulation
(TBS) and long-term depression (LTD) induced with paired-
pulse low-frequency stimulation (ppLFS). The LTP amplitude
was significantly decreased after 60 min in aged degus compared
with young degus (Fig. 4A; P < 0.0001, two-way ANOVA), whereas
LTD was slightly but significantly increased in aged degus com-
pared with young degus (Fig. 4B; P < 0.0001, two-way ANOVA).
Prompted by the fact that neural plasticity was similar in the
36- and 60-mo-old degus, suggesting slowed progression of neu-
rodegeneration, we examined an additional group of 72-mo-old
degus. At this age, LTP continued to decrease and LTD to increase
(Fig. S1A), indicating further progression of the neuropathology
associated with AD.
Synaptic plasticity measurements were recorded in behaviorally
characterized degus, raising the possibility that synaptic mod-
ifications induced during the learning tasks might have affected
the subsequent induction of LTP and LTD in hippocampal slices
(24). However, the magnitude of LTP and LTD were similar
Average number of correct choices per day in the T-maze test. Two-way
ANOVA [F(11,33)= 34.59, *P < 0.0001], followed by the Bonferroni post hoc
test (P < 0.05) in the last 3 d for aged (36–60 mo-old) vs. young (6–12 mo-old).
(B) Average correct choices at the end of the experimental phase for 6-mo-
old (white), 12-mo-old (gray), 36-mo-old (blue), and 60-mo-old (red) degus.
One-way ANOVA [F(3,26)= 6.509, *P = 0.002], followed by Tukey´s post hoc
test (P < 0.05). (C) Preference index for object recognition. Paired two-tailed
t test (*P < 0.01), novel vs. familiar objects. (D) Average exploration time for
novel vs. familiar objects. Paired two-tailed t test (*P < 0.01) vs. familiar
objects. The values in parentheses indicate the number of animals.
Age-dependent decline in cognitive performance in O. degus. (A)
collateral–CA1 pathway. Representative traces of fEPSP at different stimulus
intensities from 6-mo-old (white), 12 mo-old (gray), 36-mo-old (blue), and 60-
mo-old (red) degus. (Scale bars: 1 mV, 10 ms.) AMPAR-mediated input–output
curves from 6-mo-old (white), 12-mo-old (gray), 36-mo-old (blue), and 60-
mo-old (red) degus. One-way ANOVA [F(3,58)= 157.3, *P < 0.0001], followed
by Tukey’s post hoc test (P < 0.05). (A2 and A3) Relationship between stimulus
intensity and fiber volley amplitude (A2) and fEPSP slope (A3) in 6-mo-old
(white), 12-mo-old (gray), 36-mo-old (blue), and 60-mo-old (red) degus. Re-
peated-measures ANOVA [F(15,45) = 61.06, *P < 0.0001], followed by the
Bonferroni post hoc test (P < 0.05). (B1) Normal paired-pulse facilitation be-
tween groups. (B2) Representative traces at interstimulus intervals of 50 ms
are shown. (Scale bars: 1 mV, 20 ms.) (C1) Amplitudes and frequencies of
AMPAR-mediated mEPSCs from young (white) and aged (red) degus. (C2)
Cumulative probability plots for mEPSC amplitude size. (Inset) Representative
superposed events. Calibration: 5 pA, 20 ms. (C3) Representative traces of
mEPSCs. (Scale bars: 20 pA, 2 s.) Calibration: 1 mV, 10 ms. Unpaired two-tailed
t test (*P < 0.01) for young vs. aged. The values in parentheses indicate the
number of hippocampal slices (first number) and the number of animals
(second number) used.
Altered synaptic transmission and postsynaptic deficits in the Schaffer
| www.pnas.org/cgi/doi/10.1073/pnas.1201209109Ardiles et al.
(Fig. S2) in slices from naïve and trained degus, indicating that the
effects of previous behavioral testing were minimal.
Increased Soluble Aβ Oligomers and Tau Phosphorylation in Aged
Degus. Accumulating evidence indicates that soluble Aβ oligom-
ers are involved in synapse destruction and memory impairment in
AD (5–7). To identify the nature of different types of Aβ species
in hippocampal extracts from young and aged degus, we used
specific mouse anti-β peptide 4G8, followed by immunoblot
analysis (7). Young and aged degus exhibited similar forms of
small Aβ peptides, including trimers (∼12 kDa), tetramers (∼16
kDa), and hexamers (∼27 kDa), whereas aged degus displayed
higher levels of Aβ dodecamers (∼56 kDa) (Fig. 5 A1 and A2).
The presence of these Aβ dodecamers in the hippocampus was
confirmed by a slot-blot assay using the specific antioligomeric
antibody A11, which detects soluble amyloid assemblies larger than
40 kDa (25). Levels of A11 were higher in the aged degus (Fig. 5B).
The number and localization of NFT, but not of senile pla-
ques, have been correlated with the level of dementia in patients
with AD (26). To identify the presence of NFTs, we measured
tau phosphorylation at sites known to be present in paired helical
filaments (PHFs). The PHF-1 antibody detects tau phosphory-
lation at serine residues 396 and 404 (27). Compared with young
degus, aged degus exhibited increased phosphorylation levels
detected by this antibody (Fig. 5C). A similar result was obtained
using AT8 antibody, which detects tau phosphorylation at serine
202 and threonine 205 (28). Tau phosphorylation at these sites
was only slightly greater in aged degus, including 72-mo-old
degus (Fig. S1 B and C), compared with young degus (Fig. 5C).
The increased tau phosphorylation was not simply related to
an increase in tau protein levels, given that the total tau level
(Tau-5) remains unchanged up to age 72 mo (Fig. S1 B and C).
Furthermore, pathological phosphorylated tau begins to appear
first in the cortex and then in the hippocampus, where neurons
containing hyperphosphorylated tau were preferentially detected
in aged degus (see Fig. 7; also see figure 1 in ref. 17).
Increased Levels of Soluble Aβ Oligomers and Tau Phosphorylation
Plasticity. Table S1 shows that the 12mer dodecamer (Aβ*56)
is highly negatively correlated with LTP magnitude (r2= 0.31,
P = 0.0012; Fig. 6A) and performance on T-maze (r2= 0.24, P =
0.0048; Fig. 6B) and ORM (r2= 0.34, P = 0.0006; Fig. 6C) tasks.
Similarly, higher levels of PHF-1 are correlated with lower levels
of LTP (r2= 0.28, P = 0.0020; Fig. 6D), poorer ORM test
performance (r2= 0.37, P = 0.0003; Fig. 6F), and, to a lesser
degree, poorer T-maze performance (r2= 0.19, P = 0.0138; Fig.
6E). Comparable correlations were obtained when A11 and AT8
were compared with LTP values and T-maze and ORM test
performance (Fig. S4). Further analysis using Principal Com-
ponent Analysis is presented in supplementary information.
Although these results suggest a similar time course and plau-
sible causal links among the increases in soluble Aβ oligomers and
phosphorylated tau and the incidence of synaptic and cognitive
alterations, we cannot rule out the possibility that insoluble amy-
loid fibrils also participate in this cascade of neurotoxic events.
For this reason, we analyzed the presence of amyloid deposi-
tion by immunohistochemistry and thioflavin S (ThS) staining
(Fig. 7 and Fig. S3). Amyloid plaques were almost absent in
young degus and began to appear in the cortex of aged degus at
36–60 mo-old, whereas plaques did not appear in the hippo-
campus until after 60 mo-old (Fig. S3). Just as with ThS-positive
plaques, extracellular Aβ immunoreactivity (Fig. 7I) and patho-
logical phosphorylated tau were first observed in the cortex at
36 mo-old and in the hippocampus after 60 mo-old (Fig. 7J).
Our findings indicate that degus develop a neurodegenerative
AD-like condition with aging. In the initial stages, increased
Aβ oligomers and phosphorylated tau levels could explain the
impairments in learning, memory, and neural plasticity capacity.
In later stages (after 72 mo-old), progressive deposition of pla-
ques and tangles, likely leading to neurodegeneration, may ag-
gravate or accelerate the symptomatology of AD in degus (Fig. 7
and Figs. S1 and S3).
Throughout this study, we noted that the behavioral, func-
tional, and molecular alterations did not proceed at a uniform
pace during aging. Rather, most of the changes occurred between
12 and 36 mo-old. To identify the most critical factors involved
in the progression of AD, we performed a principal components
analysis using variables with correlation ≥0.50: LTP, T-maze,
ORM, Aβ*56, PHF-1, GluR2, and NR2b (Table S1). Interest-
ingly, the plot in the space of the first two principal components
(PC1 vs. PC2) clearly segregates two different classes of indi-
viduals: young degus and aged degus (Table S3–S5 and Fig. S5),
supporting the importance of the variables examined here. Finally,
it is important to note that although most of the deficits develop
sentative blot of synaptic proteins from hippocampus extracts (age indicated
above lanes). Arrows indicate respective migration positions. (B) Relative levels
of synaptophysin (SYP), PSD-95, GluR2-AMPAR subunit, and NR2b-NMDAR
subunit in the hippocampus from 6-mo-old (white), 12-mo-old (gray), 36-mo-
old (blue), and 60-mo-old (red) degus. Mean values of synaptic proteins
are relative to β-tubulin levels. One-way ANOVA [F(3,20)= 6.626, *P = 0.0027
for PSD-95; F(3,20)= 3.968, *P = 0.0227 for GluR2; F(3,21)= 2.662, *P = 0.0745
for NR2b], followed by Tukey´s post hoc test (P < 0.05), young vs. aged.
Postsynaptic proteins affect synapse during degus aging. (A) Repre-
induced LTP in the Schaffer collateral–CA1 synapse. (Left) Representative
fEPSPs recorded 1 min before TBS (1) and 60 min after TBS (2). LTP protocol
was delivered at the time indicated by the arrow. Averaged LTP magnitudes
during the last 10 min of recording in 6-mo-old (white), 12-mo-old (gray), 36-
mo-old (blue), and 60-mo-old (red) degus are shown as well. Two-way
ANOVA [F(1,19)= 1841, *P < 0.0001], followed by the Bonferroni post hoc test
(P < 0.05) in the last 10 min, for aged vs. young. (B) ppLFS-induced LTD in the
Schaffer collateral–CA1 synapse. (Left) Representative fEPSPs recorded 1 min
before ppLFS (1) and 60 min after ppLFS (2). LTD protocol was delivered at
the time indicated by the horizontal open bar. Averaged LTD magnitudes
during the last 10 min of recording for 6-mo-old (white), 12-mo-old (gray),
36-mo-old (blue), and 60-mo-old (red) degus are also as well. Two-way
ANOVA [F(1,19)= 435.6, *P < 0.0001], followed by the Bonferroni post hoc
test (P < 0.05) in the last 10 min for aged vs. young. (Scale bars: 1 mV, 10 ms.)
The values in parentheses indicate the number of hippocampal slices (first
number) and the number of animals (second number) used.
Impaired hippocampal synaptic plasticity in aged O. degus. (A) TBS-
Ardiles et al. PNAS
| August 21, 2012
| vol. 109
| no. 34
between 12 and 36 mo-old, the changes are not homogeneous in
this population. Approximately 25% of individuals aged 36 mo
exhibit “unimpaired” performance on either in the T-maze or
ORM test.* This variability is expected in a group of non-
transgenic animals with genetic backgrounds from a controlled
but natural population.
Synaptic and cognitive dysfunction in AD models has revealed
significant impairments before neurodegeneration becomes evi-
dent (19, 20, 22, 23). We have shown a clear correlation between
high levels of Aβ*56 oligomers and tau phosphorylation and
reductions in synaptic strength and plasticity (Fig. 6 and Fig. S4);
however, the exact mechanism by which Aβ oligomers or phos-
phorylated tau might impair synaptic function remains under
debate. Some previous studies have reported that the addition
of Aβ oligomers is sufficient to affect synaptic function in vitro
(5, 29, 30) and also alter cognitive function (7, 31). Other studies
have provided evidence suggesting that chemical reactions
occurring during the process of Aβ aggregation produce toxic
species, such as reactive oxygen species (32, 33). In that
context, degus could provide a valuable “natural model” for
testing toxicity mechanisms.
Recent evidence suggests that soluble Aβ oligomers (also re-
ferred to as ADDLs) may induce synaptic failure as an early event
related to memory deficits in AD (6, 34). Indeed, soluble Aβ has
been found to affect synapses by selectively targeting postsynaptic
components (34, 35). More recently, it has been reported that
cellular prion protein functions as a receptor for Aβ oligomers
(36). Once bound to the membrane, these oligomers tend to ac-
cumulate at excitatory synapses, forming clusters with metabo-
tropic glutamate receptors (mGluR5) and causing impairments in
synaptic plasticity (37). Many different lengths and conformational
states of Aβ peptide are generated during its biosynthesis, in-
cluding highly mobile soluble Aβ oligomers and prefibrillar and
fibrillar aggregates. These diverse assemblies have been associated
with the disruption of memory and synaptic plasticity. For in-
stance, dimers (8, 38), trimers (39), and dodecamers (7) derived
from diverse sources (including chemical synthesis, transfected
cells, and mouse and human AD brains) potently impair synaptic
plasticity and memory. However, it has been shown that compared
with other assemblies, Aβ*56 has the greatest effect on memory
(7, 40, 41) and has been proposed as the key neurotoxic non-
fibrillar assembly in AD because it is highly stable and prone to
aggregation (42). In the same way, degus exhibited a stronger
inverse relationship between higher Aβ*56 and LTP levels and
improved T-maze and ORM test performance, which increases
with aging (Fig. 6). Strikingly, aged degus also exhibited increased
levels of phosphorylated tau (Fig. 5C and Fig. S3), suggesting a
functional link between Aβ processing and tau phosphorylation,
given that phosphorylated tau residues are also negatively corre-
lated with LTP and cognitive impairment (Fig. 6). Several pre-
vious studies have shown that in mouse hippocampus, an increase
in soluble Aβ by local administration increases the level of phos-
phorylated tau proteins, producing cognitive impairment (43–45).
Furthermore, when the levels of both proteins decreased, recovery
of cognitive abilities was observed (44). Interestingly, direct inter-
action between tau proteins and Aβ peptides induces tau aggre-
gation and hyperphosphorylation (45). Furthermore, Aβ oligomers
have been related to missorting and phosphorylation of tau (46),
tion during aging. (A1) Representative blot for amyloid oligomers using anti-
Aβ peptide antibody 4G8. Arrows indicate respective migration positions of
hexamers (6-mer), nonamers (9-mer), and dodecamers (12-mer). Synthetic
Aβ42 peptide was used as size marker and positive control (right lane). (A2)
Identification and relative levels of different Aβ oligomeric associations in
hippocampal extracts from 6-mo-old (white), 12-mo-old (gray), 36-mo-old
(blue), and 60-mo-old (red) degus. One-way ANOVA [F(3,21)= 17.21, *P <
0.0001 for 6-mer; F(3,19)= 5.439, *P = 0.0026 for 12-mer], followed by Tukey’s
post hoc test (P < 0.05) for aged vs. young. (B) Relative levels of soluble Aβ
oligomers. (Inset) Representative slot blot from hippocampal extracts using
antioligomeric antibody A11. One-way ANOVA [F(3,19)= 5.439, P = 0.0072],
followed by Tukey’s post hoc test (P < 0.05) for aged vs. young. (C) De-
termination of phosphorylated tau protein levels using PHF-1 and AT8
antibodies. (Inset) Representative blot for PHF-1, AT8, and β-actin. One-way
ANOVA [F(3,20)= 23.21, *P < 0.0001 for PHF-1; F(3,20)= 5.95, *P = 0.0059 for
AT8], followed by Tukey´s post hoc test (P < 0.05) for aged vs. young. The
values in parentheses indicate the number of animals used.
Accumulation of large soluble Aβ oligomers and tau phosphoryla-
memory impairments. (A–C) Relationships between soluble dodecamer
(Aβ*56) level and LTP magnitude (A), T-maze (B), and ORM (C) in young and
aged degus. (D–F) Relationships between PHF-1 epitope tau phosphorylated
level and LTP magnitude (D), T-maze (E), and ORM (F) in young and aged
degus. High correlation for LTPand ORM can be seen. The values in parentheses
indicate the number of animals used.
Large Aβ oligomers and tau phosphorylation correlate with LTP and
*Ponce A, Cerpa W, Inestrosa N, Palacios AG, Aging and spatial memory in the rodent
Octodon degus. Annual Meeting of the Chilean Neuroscience Society, September 27,
2006, Curico, Chile.
| www.pnas.org/cgi/doi/10.1073/pnas.1201209109 Ardiles et al.
destabilization of microtubules, and disruption of axonal trans-
port (47). On the other hand, Aβ-induced impairments in LTP
are mediated by tau phosphorylation, suggesting that tau proteins
are required for the synaptotoxic effects of Aβ oligomers (48).
Age-related reductions in spontaneous and evoked AMPAR-
mediated currents also have been reported in AD models, attrib-
uted to a reduced number of these receptors (22, 23). We found
no differences in the frequency of mEPSCs, suggesting that the
number of functional synapses is not reduced in 60-mo-old degus.
A possible explanation for the observed decrease in mEPSC am-
plitude is that the content of AMPARs available for trafficking is
impaired in aged degus. Certainly, Aβ peptides prevent recruit-
mentandanchoringofAMPARstothe postsynaptic compartment
by reducing CAMKII activation and distribution as well as PSD-
95 levels (49–51). Taken together, these results suggest that post-
synaptic compartments are more susceptible to the effects of Aβ
peptides compared with presynaptic elements, considering that
both PSD-95 and glutamate receptors were reduced in aged degus
(Figs. 2 and 4). However, presynaptic mechanisms cannot be to-
tallyexcluded, given that APP (23),Aβ (52), and presenilin 1 and 2
(53) have been reported to be localized presynaptically.
An interesting comparison can be made between degus and
3xTg-AD mice (9). In both models, the decline in synaptic plas-
ticity may be associated with an increase in Aβ peptides before the
increase in tau phosphorylation. Because this process occurs
naturally in degus, it represents a unique opportunity to examine
physiological mechanisms and evaluate rescue therapies during
Another motivating comparison here is with Microcebus murinus,
a small nocturnal primate that lives to age 8–14 y in captivity, in
which 20% of elderly adults exhibit neurodegenerative hall-
marks of spontaneous AD, including brain amyloid plaques,
tau pathology, decreased number of ACh neurons, and behavioral
changes, with loss of sensory and cognitive functions (reviewed
in ref. 16). Like Microcebus, 72-mo-old degus develop a discrete
number of Aβ deposits, detectable first in the cortex and later in
the hippocampus (Fig. 7 and Fig. S3; also see figure 1 in ref. 17).
We have established changes in neural plasticity (LTP and LTD)
in degus that correlate with the presence of soluble Aβ oligomers
and phosphorylated tau proteins.
A recurrent and difficult question to address while studying
neurodegeneration is its close association with the natural aging
process. For example, aged rats show modest decreases in basal
synaptic transmission, NMDAR-mediated response, and deficits in
synaptic plasticity (54–56) similar to the decreases in both synaptic
transmission and plasticity seen in degus (Figs. 2 and 3). However,
aged rats do not demonstrate decreased spatial (T-maze) (56) or
ORM test performance (57, 58), in clear contrast to degus (Fig. 1).
Taken together, our findings suggest that degus provides a
strong and naturalistic model for the study of early neurode-
generative process associated with sporadic AD. Of note, degus
can live approximately 9–10 y in captivity, and so the present
work represents only half of the degus lifespan. Finally, although
the precise mechanism remains unclear, our data are consistent
with the concept that soluble Aβ oligomers at prefibrillar stages
can act as toxic ligands at postsynaptic compartments, driving the
synaptic and memory disruption seen in early AD models.
Materials and Methods
Animals. O. degus were obtained from a breeding colony at the animal fa-
cility of the University of Valparaiso. All experiments were approved by the
bioethics committee of the Universidad de Valparaiso and complied with
the international NIH Approved Animal Welfare Assurance A5823-01.
Protocols. More detailed information on the study procedures is provided in
SI Materials and Methods. The different groups of degus were submitted
to a complete characterization including behavioral tests, electrophysio-
logical recordings, and biochemical measures. All animals received the same
manipulations, regardless of age. Before evaluation of cognitive capacity,
the animals were habituated to an open field over 5 consecutive days. Then
they were submitted to the ORM test for 5 d, followed by the T-maze test
for 17 d. Once the behavioral characterization was complete, degus were
killed to obtain hippocampal slices in which to study synaptic transmission
and plasticity. After completion of the electrophysiological experiments,
the hippocampal slices were immediately frozen for biochemical character-
ization. Finally, the tissue was collected to obtain homogenates from the
hippocampus to quantify different proteins levels by immunoblot analysis.
Behavioral Tests. ORM was assessed using an open-field arena constructed
of black Plexiglas (50 cm × 40 cm × 63 cm) over 5 d. Each ORM session con-
sisted of three phases (180 s each): (i) familiarization, where degus explored
a pair of identical objects; (ii) retention, where degus were removed for
object cleaning and changing; and (iii) recognition, where degus explored
a pair of different objects: a familiar object (FO) (extra copy of familiar
object) and a novel object (NO). To quantitate OMR, a preference index (PI)
was calculated as PI = NO/NO + FO.
Spatial working memory was assessed in a T-maze task using a training
protocol to search for a reward over 12 consecutive days. Each session con-
sisted of 10 trials composed of three parts (60 s each): (i) forced choice
memory learning, with no food reward and one arm remaining closed;
(ii) retention time, where the animal was removed for cleaning and the
closed arm opened; (iii) free choice for memory recognition, where the
two arms were open and the reward was placed in the previously closed
arm. A correct response corresponded to a visit to the closed arm during
part (i), in which case the animal was rewarded with a sunflower seed.
Electrophysiological Assessment. Extracellular and whole-cell patch-clamp
recordings were performed in hippocampal slices obtained from behaviorally
characterized degus as described previously (54, 55).
Immunoblot Analysis. Proteins were run on gradient denaturing gels, blotted,
and probed with appropriate antibodies.
cortex of O. degus. (A–D) Immunoreactivity for Aβ peptides using the specific
antibody 6E10 showing extracellular (black arrows) staining in the hippo-
campus and the cerebral cortex in degus. A greater number of extracellular
insoluble deposits were observed in aged degus (72 mo-old; C and D) com-
pared with young degus (12 mo-old; A and B). (E–H) Immunodetection of
pathological phosphorylated tau using the specific antibody AT8 in the
hippocampus and cerebral cortex from degus. A greater number of posi-
tively stained somas (black arrows) were observed in aged degus (G and H)
compared with young degus (E and F). (I and J) Quantification of Aβ burden
(I; percentage of area occupied by 6E10-positive plaques) and the number of
AT8-positive cells (J; number of neurons per area) in 6-mo-old (white, n = 2),
12-mo-old (gray, n = 2), 36-mo-old (blue, n = 2), 60-mo-old (red, n = 2), and
72-mo-old (black, n = 2) degus showing a significant increase after 36 mo in
cortex and after 60 mo in hippocampus. One-way ANOVA [F(4,88)= 238.8,
*P < 0.0001 for 6E10-Hip; F(4,88)= 646.4, *P < 0.0001 for 6E10-Cx; F(4,88)=
266.0, *P < 0.0001 for AT8-Hip; F(4,88)= 134.9, *P < 0.0001 for AT8-Cx], fol-
lowed by Tukey’s post hoc test (P < 0.05) compared with 6 mo-old.
Amyloid deposition and tau phosphorylation begins in the cerebral
Ardiles et al. PNAS
| August 21, 2012
| vol. 109
| no. 34
Histology. PFA-fixed brains were sectioned into 20-μm slices, and free-
floating slides were processed following immunohistochemical and ThS
Statistics. All data are presented as mean ± SE or deviation of the mean (SEM
or SD). Data were analyzed using Prism software (GraphPad).
ACKNOWLEDGMENTS. We thank H. K. Lee and A. Megill (Johns Hopkins
University), J. Ewer (Universidad de Valparaíso), A. Chavez (Albert Einstein
University), A. Reichenbach (University of Leipzig), and L. Peichl (Max
Planck University) for discussion and comments; C. Elgueta (University of
Freiburg) for software assistance; and T. Lee (University of Michigan) for
degus specimens from the United States. This work was supported by Na-
tional Institutes of Health Fogarty International Research Collaboration
Awards R03 TW007171-01A1 and R01 AG034606 (to A.K.); Chilean National
Commission for Scientific and Technological Research Grant ANR-47 (to A.G.P.
and F.A.), Grant PFB 12/2007 (to N.C.I.), and Fellowship AT-24091109 (to
A.O.A.); and Interdisciplinary Center for Neuroscience of Valparaiso Millen-
ium Scientific Initiative P09-022-F (to A.G.P.).
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