Beruflich Dokumente
Kultur Dokumente
Brief communication
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
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2.3. Immunocytochemistry
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
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).
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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.
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