Neurobiology of Aging 26 (2005) 10231028

Brief communication

Human-like rodent amyloid--peptide determines Alzheimer pathology

in aged wild-type Octodon degu
Nibaldo C. Inestrosaa, , Ariel E. Reyesa , Marcelo A. Chacona , Waldo Cerpaa , Aldo Villalonb ,
Juan Montielb , Genevieve Merabachvilia , Rebeca Aldunatea ,
Francisco Bozinovicc , Francisco Aboitizb
a

Centro FONDAP de Regulacion Celular y Patologa Joaqun V. Luco, MIFAB, Facultad de Ciencias Biologicas, P. Universidad Catolica de Chile,
Alameda 340, Santiago, Chile
b Departamento de Psiquiatra y Centro de Investigaciones M
edicas, Facultad de Medicina, Chile
Centro de Estudios Avanzados en Ecologa y Biodiversidad (CASEB), Facultad de Ciencias Biologicas, Ponticia Universidad Catolica de Chile, Chile
Received 29 March 2004; received in revised form 16 August 2004; accepted 23 September 2004

Abstract
It is generally accepted that human Alzheimers disease (AD) neuropathology markers are completely absent in rodent brains. We report
here that an aged wild-type South American rodent, Octodon degu, expresses neuronal -amyloid precursor protein (-APP695) displaying
both intracellular and extracellular deposits of amyloid--peptide (A), intracellular accumulations of tau-protein and ubiquitin, a strong
astrocytic response and acetylcholinesterase (AChE)-rich pyramidal neurons. The high amino acid homology (97.5%) between deguA and
humanA sequences is probably a major factor in the appearance of AD markers in this aged rodent. Our results indicate that aged O. degu
constitutes the first wild-type rodent model for neurodegenerative processes associated to AD.
2004 Elsevier Inc. All rights reserved.
Keywords: Alzheimers disease; Alzheimer model; Neuropathology; Amyloid--peptide; APP; Octodon degu

1. Introduction
Alzheimers disease (AD) is an age-related and progressive neurological disorder characterized by neuropathological hallmarks such as excessive deposition of amyloid-peptide (A) and accumulation of neurofibrillary tangles
(NTFs) in the hippocampal region and cortical parenchyma
[14,21]. Studies performed on humans, monkeys, rats
and other mammals [4,6,13,22] have demonstrated that
Alzheimers pathology markers are absent in wild-type rodents, being necessary to generate transgenic animals overexpressing human -amyloid precursor protein (-APP)
[8,11,17] or to perform intracerebral injections of A aggregates [7,18] to generate in vivo models as paradigms for
AD studies. Nevertheless, none of the transgenic or stereotaxic models of AD recapitulate all the features of the dis

Corresponding author. Tel.: +56 2 686 2724; fax: +56 2 686 2959.
E-mail address: ninestr@genes.bio.puc.cl (N.C. Inestrosa).

0197-4580/$ see front matter 2004 Elsevier Inc. All rights reserved.
doi:10.1016/j.neurobiolaging.2004.09.016

ease [11]. In this article, we report that a South American

rodent, Octodon degu, spontaneously develops neuropathological signs of AD in old age. This may be related to the
high degree of homology between human and O. degu A.
Furthermore, like the human and contrary to the mouse and
rat, this rodent shows the presence of acetylcholinesteraserich neurons (AChERN) in the adult cerebral cortex [9,16].
In the human, these neurons show a noticeable decline with
the progression of AD. Together, these results suggest that
O. degu may be considered as a natural model for the study
of AD pathology.

2. Materials and methods

2.1. Animals
O. degu, an exclusive Chilean rodent [19], was obtained
from the central region of Chile. Animals were separated in

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two groups, 1-year-old young animals (born captive) (n = 21)

and old wild-type animals (over 3-years-old) that present
cataracts (n = 5). The animals were servants in captivity and
the age of the older animals was known. Ten young animal
brains were used for RNA extraction, RT-PCR and sequence
analysis. All other animals were used for histological characterization. The life span of these animals is 10 years, its age
is equivalents to 120 human years [15].
2.2. Perfusion

procedures and criteria described by Mesulam and Geula

[16].
2.5. Thioavin-S uorescence staining
Mounted sections were defatted in xylene and hydrated
in ethanol and water series. Thioflavin-S (ThS) staining
was carried out as described by Elghetany and Saleem
[5].

Old and young animals were anesthesized with Equitesin

(2.5 ml/kg, i.p.) and injected with heparin (4 USP/kg, i.p.) before perfusion. Then, they were perfused through the heart
with perfusion buffer containing 0.1% sodium nitrite, followed by fixation with 4% parafolmaldehyde in 0.l M phosphate buffer (PB) for 30 min. Brains were removed from their
skulls and post-fixed in the same fixative for 3 h at room temperature, followed by 10% sucrose in PB at 4 C overnight.
After fixation, brains were cooled to ensure unbiased processing and analysis [3]. The brains were subdivided in three
coronal main parts (approximately 3 mm): frontal, medial,
and caudal slides. Then each slide was cut into 36 coronal
sections of 50 m with a cryostat at 20 C.

2.6. RNA extraction

2.3. Immunocytochemistry

Stock PCR reaction mixtures (25 l) were prepared on

ice and contained 50 M dCTP, 100 M each of dGTP,
dATP, and dTTP, 10 Ci of dCTP (3000 Ci/mmol), 1.5 mM
MgCl2 , 1 PCR buffer (Life Technologies), 1 M of each
primer, 1 U of Taq polymerase (Life Technologies), and
25 ng of cDNA synthesized in the reverse transcription.
Then, mix solutions were subjected to PCR reaction (PerkinElmer Termocycler GeneAmp PCR system 2400). Analysis of -APP brain isoforms was performed with an APP forward primer: 5 -CACCACAGAGTCTGTGGAAG3 ; (APPf-primer) and an APP reverse (APPr-primer) primer:
5 -AGGTGTCTCGAGATACTTGT-3 (these primers flank
an alternative splice site such that APP695 produces
an 87 bp product, APP751 produces a 255 bp fragment,
and APP770 produces a 312 bp product) [10]. Besides,
we designed two other primers: A forward (Af)
primer 5 -GATGTCTTGGCCAACATGAT-3 and A reverse (Ar) primer 5 -CATCTGCTCAAAGAACTTGTA3 (these primers flank the region of rat cDNA that contains the A sequence and amplify a 620-bp fragment).
We used the following amplification protocol: thermal cycling for cDNA synthesis and pre-denaturation: 1 cycle of
50 C for 30 min, 94 C for 2 min. PCR amplification 40
cycles of denaturation at 94 C for 15 s; anneal 58 C for
30 s and extend 70 C for 1 min. Final extension of 70 C
for 7 min (in these experiments we used a high fidelity
Taq polymerase pfu (Roche, Mannheim, Germany). The
fragments were isolated by Quiagen II agarose Gel Extraction kit as manufacture recommended (QiaGen Gmbh,
Hilden, Germany). The isolated fragment was sequenced
directly.

Free-floating sections for immunohistochemical procedures were performed as previously described [1,3]. Washing
and dilution of immunoreagents were performed with PBS
plus 0.2% Triton X-100 (PBS + T) throughout the experiments, and two PBS + T washes were performed between
antibody incubations. A-peptide and glial fibrillar acidic
protein (GFAP) immunodetection was performed using antihuman-A (1:500) (Sigma, St. Louis, MO) and anti-cowGFAP (1:500) rabbit polyclonal antibodies (DAKO, Carpentera, CA). Tau and ubiquitin protein immunodetection was
performed with anti-human-tau and anti-human-ubiquitin
rabbit polyclonal antibodies (DAKO, Carpentera, CA), a
samples were incubated overnight at 4 C. Antibody detection was made using the peroxidaseanti-peroxidase method
(PAP) with an anti-rabbit-IgG diluted 1:30 (DAKO) and after
wash, incubated with PAP (1:100), 30 min each at room temperature. Staining was developed by incubating the slices for
15 min in 0.6% diaminobenzidine followed by the addition
of H2 O2 for 4 min.
2.4. AChE histochemistry
Seven specimens of adult O. degu, three SpragueDawley
rats, and four human brains with no signs of neurological disease were used for AChE histochemistry, following the methods described by Mesulam and Geula [16].
The density and size of AChE-rich neurons were calculated
by counting the total neurons in transversal sections, using a computer image analysis system and following the

Total RNA was extracted from rat and O. degu brain tissues
using Trizol reagent according to the manufacturers protocol
(Life Technologies, Frederick, MD).
2.7. cDNA preparation
SuperScript preamplification system (Life Technologies)
was used to prepare cDNA for reverse transcription.
2.8. PCR amplication

N.C. Inestrosa et al. / Neurobiology of Aging 26 (2005) 10231028

2.9. Semi-quantitative PCR

Stock mix PCR reactions (25 l) were prepared on ice and
contained 100 M dNTPs mix, 1.5 mM MgCl2, 1 PCR
buffer (Life Technologies), 1 M APPf-primer, 1 M APPrprimer, 1 U of Taq polymerase (Life Technologies). The concentration of cDNA and the quantity on water depended of
the saturation of the reaction; in this case, for O. degu cDNA,
the concentration of cDNA was 5, 10 and 20 ng for the amplification of the -APP fragments and we tried 24, 26, 28, 29
and 30 cycles of PCR amplification. As standard RNA amplification, primers to amplify a tubulin fragment of 320 bp were
used on separate tube reactions, using the same amplification
protocol.

3. Results
We analyzed different regions from the cerebral cortex
(frontal, parietal, temporal and entorhinal) and hippocampus
of young and old wild-type O. degu brains to investigate the
presence of A-peptide deposits. In old O. degu brains, immunohistochemical analysis using specific anti-human Apeptide (140) antibodies, revealed prominent localizations
of extracellular and intracellular A deposits in both cerebral
cortex (Fig. 1C) and hippocampus (Fig. 1F). The extracellular
deposits were ubiquitous in all cortical layers, while the intracellular aggregates were restricted to the second and the third
cortical layers. In the molecular layer of the dorsal hippocampus, it was possible to detect immunopositive intraneuronal
aggregates (Fig. 1F). Interestingly, in young animals it was
not possible to find A aggregates (Fig. 1A).
Next, we examined the amyloid character of these A
deposits accumulated in old O. degu brains, using the
amyloid fluorescent dye thioflavine-S. Under confocal microscopy, we observed localized ThS-positive amyloid deposits (Fig. 1DH) in frontal (Fig. 1D and E), parietal and
entorhinal cortices and hippocampus (Fig. 1G and H), areas
which are strongly deteriorated in AD patients [14,20,23].
Moreover, we found diffuse smaller aggregates in the entorhinal cortex, which were immunoreactive to anti-A but
not ThS-positive. These aggregates probably correspond to
early stages of amyloid core formation (data not shown).
Again, amyloid cores and diffuse amyloid aggregates were
almost absent in the cortex and hippocampus of young animals (Fig. 1B), indicating that age plays a determinant role
in the appearance and formation of amyloid aggregates.
Also, we were able to detect other types of intracellular
aggregates in the cortex and hippocampus of old animals, using specific antibodies directed against neurofibrillary tangles
(Fig. 1J) and ubiquitin (Fig. 1L). No reaction was observed for
either tau or ubiquitin in young animals (Fig. 1I and K). Since
the amyloid enriched brain areas are expected to present high
densities of glial-activated cells, we performed an immunohistochemical analysis using an anti-GFAP antibody [2]. In
old O. degu, this revealed an intense, extended astrogliosis

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throughout the cortex and hippocampus, including blood vessels walls (Fig. 1N), compared to the weak immunoreactivity
observed in the brains from young animals (Fig. 1M).
In addition, in the cortex of adult O. degu we observed
scattered clusters of AChE-rich pyramidal neurons; these had
some differences with those found in humans (Fig. 1P versus Q). In adult O. degu, they were located only in layer
IIIc, but in humans these neurons were located in layers III
and V. The neurons have a similar morphology, but they differ in size (346 67 m2 in the human, and 188 35 m2
in O. degu) and density (0.18 0.01 neurons/mm2 in humans and 0.15 0.02 neurons/mm2 in O. degu) (Fig. 1R and
S). AChE-rich neurons were absent from the rat cerebral
cortex.
Since both -APP expression and the deguA peptide
sequence could be determinant in the development of the
amyloid plaques observed in old degu, we analyzed these
two variables in this rodent. RT-PCR amplification was performed using mRNA extracted from rat and O. degu brains
using a combination of two specific primers. One of them was
complementary to an -APP splicing site, and was used in
order to analyze the expression of -APP forms; the other
primers flanked the A region to determine the deguA
sequence. The expression levels of -APP isoforms (i.e.,
APP770 , APP751 and APP695 ) in O. degu brain were studied
using RT-PCR in total RNA extracted from young animals.
The expected fragments of 362-, 255- and 87-bp-long fragments were consistent with the sizes of corresponding APP
subregions in the human cDNA and were detected in rat and
O. degu brain tissue (Fig. 2A). The resultant PCR data indicates that the major form of -APP expressed in the O.
degu brain is APP695 like in rat (Fig. 2A and B, lane 3). We
also analyzed the -APP nucleotide sequence in a region that
flanks the A cDNA. The expected fragment 620-bp-long
fragment was also consistent with the size of corresponding
-APP subregion in the human cDNA and was detected in
both rat and O. degu brain tissue. After amplification, the
fragment was isolated and sequenced, first using the same
PCR primers and then using nested primers deduced from
the first sequencing.
Studies on exons 1718 indicate that the deguA-peptide
sequence shares an important degree of homology with the
human sequences (97.5%) (Fig. 2C). Compared with the rat
A which presents three amino acids substitutions [4,6], the
degu A presents only one amino acid substitution with respect to the human sequence.

4. Discussion
Overall, our findings indicate that O. degu aged brains
present molecular markers strongly reminiscent of AD neuropathology, and suggest that O. degu is probably the first
wild-type rodent model to study AD. Moreover, this work
establishes that the human-like amyloidogenic properties of
deguA peptide, may triggers neurodegenerative processes

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Fig. 1. Left panel: aged O. degu present A amyloid brain deposits. Brain A immunodetection in young (A) and old animals (C, F). (C) Extracellular aggregates
(white arrows) in frontal cortex and (F) intracellular A aggregates (black arrow) in hippocampus of old animals (bar = 50 m). In situ amyloid detection of
young (B) and old animals (D, E, G, H) using ThS-fluorescence observed under confocal microscope. (B) ThS-fluorescence in frontal cortex of young animals
and (D, E) frontal cortex and (G, H) dorsal hippocampus of old animals. Each image corresponds to a projection composed by 25 optical adjacent sections
(2 m each) projected in the plane. Both (E) and (H), represent transversal sections in z-axis in the zone that contains the extracellular aggregates observed in
(D) and intracellular amyloid observed (G), respectively. Right panel: neurofibrillary tangles, ubiquitin aggregates and astrocytic response are present in aged
O. degu brains. Anti-tau immunohistochemistry in parietal cortex of: (I) young and (J) old animals. Anti-ubiquitin immunohistochemistry in parietal cortex of:
(K) young and (L) old animals. Anti-GFAP immunohistochemistry evidenced in hippocampus of: old young (M) and old animals (N). (figure bar 100 m and
inset bar 10 m). Lower panel: sections of the frontal cortex of adult rat, O. degu (O) and human. Rat cortex (1.5 years old) was used as negative control (see
text). In Octodon, we have found AChE-rich neurons in layer IIIc (P). In humans, we found this type of neurons in layers III and V (scale bar = 100 m). (Q)
The neurons in O. degu have a morphology similar to those in humans, although they differ in size and density (scale bar = 50 m). (R) Correspond to O. degu
AChE-rich neurons, at high magnification (scale bar = 50 M). (S) Human AChE-rich neurons, at high magnification (scale bar = 50 M).

N.C. Inestrosa et al. / Neurobiology of Aging 26 (2005) 10231028

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Fig. 2. O. degu brain expresses APP containing an amyloidogenic peptide sequence with high homology to human A peptide. (A) Autoradiography of RT-PCR
amplified fragments from rat and O. degu brain mRNAs, using primers that flank splicing regions of APP. (B) RT-PCR amplified fragments from rat and O.
degu brain mRNAs, using primers that flank the A codifying region into APP cDNA. Rat (lanes 1 and 2) and O. degu (3 and 4); APP (lanes 1 and 3) and
tubulin (lanes 2 and 4). (C) Amino acid homology of deduced deguA sequence with human and ratA sequences.

including tau positive structure (possible NFT), ubiquitin aggregates and astrocytic response.
An interesting feature of the Alzheimers-like changes observed in O. degu brains is that they occur in aged animals
and so far have never been detected in young animals (1year-old). This suggests that amyloid and tau deposition are
age-dependent, as also occurs in AD patients [20,21,23] and
in some of the more successful transgenic mice studied so far
[17].
Furthermore, adult O. degu displays AChE-rich cortical
neurons similar to those in human. AChE-rich pyramidal neurons described in supragranular and infragranular layers of
the primate cerebral cortex. These neurons have been observed to decline in numbers during the progression of AD,
and this decrease has been claimed to be partly responsible
for the memory deficits in Alzheimer patients [16]. In rats,
only a transient population of AChE-rich neurons has been
described during the early postnatal period, which declines
in the late postnatal period and becomes absent in the adult
cortex [9]. On the other hand, in O. degu we observed humanlike AChE-rich neurons in layer III of the cerebral cortex. It
will have to be determined if, during ontogeny, these neurons
decline concomitantly with the appearance of the neuropathological signs of AD as they do in the human.
Our molecular analysis of the O. degu APP, particularly in exons 1718, indicate that the degu A presents
only one amino acid substitution with respect to the human sequence (Arg13 His, respectively). Mice and rats
do not present Alzheimers-like pathology, and their A
presents three amino acid substitutions with respect to
humans (Gly5 Arg; Phe10 Tyr; Arg13 His) [4,6].
Therefore, it is likely that the high homology (97.5%) be-

tween the human and degu A plays a major role in the appearance of AD markers in this rodent, including the presence
of intracellular amyloid deposits [17,21] which seem to precede the extracellular deposits that will eventually form the
senile plaques [14,20].
Therefore, O. degu and the degu A peptide may be considered good models to study AD. In addition, our results
are consistent with the amyloid cascade hypothesis [21], by
which either a genetic or an environmental factor facilitates
amyloid formation and deposition, and starts the neurodegenerative process associated to AD [12,22].
Acknowledgments
This work was supported by grants from FONDAPBiomedicine (No. 13980001), from the Millennium Institute
of Fundamental and Applied Biology (MIFAB) (No. P99007-F) and from the Millenium Center for Integrative Neuroscience (ICM P01-007-F).
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at 10.1016/j.neurobiolaging.
2004.09.016.
References
[1] Chacon MA, Barra MI, Lorca R, Huidobro-Toro JP, Inestrosa NC.
A human prion protein peptide (PrP(59-91)) protects against copper
neurotoxicity. Mol Psychiatry 2003;8(10):85362.

1028

N.C. Inestrosa et al. / Neurobiology of Aging 26 (2005) 10231028

[2] Chacon MA, Reyes AE, Inestrosa NC. Acetylcholinesterase induces

neuronal cell loss, astrocyte hypertrophy and behavioral deficits in
mammalian hippocampus. J Neurochem 2003;87(1):195204.
[3] Cote SL. Immunocytochemistry II. NY: John Willey & Sons; 1993.
[4] De Strooper B, Simons M, Multhaup G, Van Leuven F, Beyreuther
K, Dotti CG. Production of intracellular amyloid-containing fragments in hippocampal neurons expressing human amyloid precursor
protein and protection against amyloidogenesis by subtle amino acid
substitutions in the rodent sequence. EMBO J 1995;14(20):49328.
[5] Elghetany MT, Saleem A. Methods for staining amyloid in tissues:
a review. Stain Technol 1988;63(4):20112.
[6] Fraser PE, Nguyen JT, Inouye H, Surewicz WK, Selkoe DJ,
Podlisny MB, et al. Fibril formation by primate, rodent, and Dutchhemorrhagic analogues of Alzheimer amyloid -protein. Biochemistry 1992;31(44):1071623.
[7] Frautschy SA, Baird A, Cole GM. Effects of injected Alzheimer
-amyloid cores in rat brain. Proc Natl Acad Sci USA
1991;88(19):83626.
[8] Games D, Adams D, Alessandrini R, Barbour R, Berthelette P,
Blackwell C, et al. Alzheimer-type neuropathology in transgenic
mice overexpressing V717F -amyloid precursor protein. Nature
1995;373(6514):5237.
[9] Geula C, Mesulam MM, Kuo CC, Tokuno H. Postnatal development of cortical acetylcholinesterase-rich neurons in the rat brain:
permanent and transient patterns. Exp Neurol 1995;134(2):15778.
[10] Golde TE, Estus S, Usiak M, Younkin LH, Younkin SG. Expression
of -amyloid protein precursor mRNAs: recognition of a novel alternatively spliced form and quantitation in Alzheimers disease using
PCR. Neuron 1990;4(2):25367.
[11] Hock Jr BJ, Lamb B. Transgenic mouse models of Alzheimers
disease. Trends Genet 2001;17(10):S712.
[12] Inestrosa NC, Alvarez A, Perez CA, Moreno RD, Vicente M, Linker
C, et al. Acetylcholinesterase accelerates assembly of amyloid-peptides into Alzheimers fibrils: possible role of the peripheral site
of the enzyme. Neuron 1996;16(4):88191.
[13] Johnstone EM, Chaney MO, Norris FH, Pascual R, Little SP. Conservation of the sequence of the Alzheimers disease amyloid pep-

[14]
[15]

[16]

[17]

[18]

[19]

[20]

[21]
[22]

[23]

tide in dog, polar bear and five other mammals by cross-species

polymerase chain reaction analysis. Brain Res Mol Brain Res
1991;10(4):299305.
Katzman R, Saitoh T. Advances in Alzheimers disease. FASEB J
1991;5(3):27886.
Meserve PL. Trophic relationships among small mammals in
a chilean semiarid thorn scrub community. J Mammalogy
1981;62:30414.
Mesulam MM, Geula C. Acetylcholinesterase-rich pyramidal neurons in the human neocortex and hippocampus: absence at birth,
development during the life span, and dissolution in Alzheimers
disease. Ann Neurol 1988;24(6):76573.
Oddo S, Caccamo A, Shepherd JD, Murphy MP, Golde TE, Kayed
R, et al. Triple-transgenic model of Alzheimers disease with plaques
and tangles: intracellular A and synaptic dysfunction. Neuron
2003;39(3):40921.
Reyes AE, Chacon MA, Dinamarca MC, Cerpa W, Morgan C, Inestrosa NC. Acetylcholinesterase-A complexes are more toxic than
A fibrils in rat hippocampus: effect on rat -amyloid aggregation,
laminin expression, reactive astrocytosis and neuronal cell loss. Am
J Pathol 2004;164(6):216374.
Rundel PW. The matorral zone of Central Chile. In: DiCastri F,
Goodall DW, Specht RL, editors. Mediterranean Type Shrublands.
Amsterdam: Elsevier; 1981. p. 175201.
Samuel W, Terry RD, DeTeresa R, Butters N, Masliah E. Clinical
correlates of cortical and nucleus basalis pathology in Alzheimer
dementia. Arch Neurol 1994;51(8):7728.
Selkoe DJ. Alzheimers disease: genes, proteins, and therapy. Physiol
Rev 2001;81(2):74166.
Soto C, Branes MC, Alvarez J, Inestrosa NC. Structural determinants of the Alzheimers amyloid -peptide. J Neurochem
1994;63(4):11918.
Terry RD, Masliah E, Salmon DP, Butters N, DeTeresa R, Hill R,
et al. Physical basis of cognitive alterations in Alzheimers disease:
synapse loss is the major correlate of cognitive impairment. Ann
Neurol 1991;30(4):57280.