New Insights into the Spontaneous Human Alzheimer’s Disease-Like Model Octodon degus : Unraveling Amyloid-β Peptide Aggregation and Age-Related Amyloid Pathology

Abstract

Alzheimer’s disease (AD) is the most common cause of dementia worldwide. Despite advances in our understanding of the molecular milieu driving AD pathophysiology, no effective therapy is currently available. Moreover, various clinical trials have continued to fail, suggesting that our approach to AD must be revised. Accordingly, the development and validation of new models are highly desirable. Over the last decade, we have been working with Octodon degus (degu), a Chilean rodent, which spontaneously develops AD-like neuropathology, including increased amyloid-β (Aβ) aggregates, tau hyperphosphorylation, and postsynaptic dysfunction. However, for proper validation of degu as an AD model, the aggregation properties of its Aβ peptide must be analyzed. Thus, in this study, we examined the capacity of the degu Aβ peptide to aggregate in vitro. Then, we analyzed the age-dependent variation in soluble Aβ levels in the hippocampus and cortex of third- to fifth-generation captive-born degu. We also assessed the appearance and spatial distribution of amyloid plaques in O. degus and compared them with the plaques in two AD transgenic mouse models. In agreement with our previous studies, degu Aβ was able to aggregate, forming fibrillar species in vitro. Furthermore, amyloid plaques appeared in the anterior brain structures of O. degus at approximately 32 months of age and in the whole brain at 56 months, along with concomitant increases in Aβ levels and the Aβ₄₂/Aβ₄₀ ratio, indicating that O. degus spontaneously develops AD-like pathology earlier than other spontaneous models. Based on these results, we can confirm that O. degus constitutes a valuable model to improve AD research.

Keywords

Alzheimer’s disease Aβ peptide natural model senile plaques

INTRODUCTION

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by the progressive deterioration of cognitive functions caused by synaptic dysfunction and damage to specific brain regions [1 –4]. The distinctive histopathological hallmarks of AD are extracellular senile plaques, which are composed of extracellular deposits of amyloid-β (Aβ) aggregates, and intraneuronal neurofibrillary tangles (NFTs), containing hyperphosphorylated tau protein, in the brains of patients with AD [4 –7]. Aggregated forms of Aβ₁_–₄₂, especially oligomers, are the relevant neurotoxic species and are the main factor responsible for alterations in hippocampal synaptic plasticity due to the inhibition of long-term potentiation, increased long-term depression, and a reduction in the spine density on hippocampal neurons in vivo [8 –10].

Despite the development of several models for the study of AD, which have improved our knowledge regarding the molecular cascade triggered during the pathophysiology of the disease, various clinical trials have continued to fail, suggesting that our approach and the tools used to study AD must be revised [6 , 11–14].

In recent years, a South American rodent endemic to the central region of Chile, Octodon degus (degu), has been proposed as a natural model of AD [15, 16]. Degu is a diurnal, vision-dependent (i.e., excellent perceptual system), and highly social rodent already used in social and neuroaffective research [17 –19]. O. degus has excellent cognitive abilities, including social and spatial recognition as well as a complex vocal communication system, which decline with aging, similar to patterns observed in humans [19 –21]. This caviomorph lives for an average of 7 years in captivity [22, 23], making it a useful model for longitudinal studies. For example, we recently demonstrated that long-term sugar consumption (from pup to adulthood) affected the normal aging process in degus, resulting in reduced synaptic plasticity and cognitive impairment [24].

Interestingly, O. degus expresses an Aβ peptide that is highly homologous to the human form, differing only in a single amino acid (aa); perhaps because of this specific feature, aged degu individuals naturally develop an AD-like pathology [6 , 25–27]. Indeed, our laboratory first identified degu as a natural rodent model of AD that spontaneously develops the characteristic neuropathological hallmarks of the disease, including Aβ plaques, tau hyperphosphorylation, and cognitive decline, after only 3–4 years of age [6 , 28]. In this regard, given that memory impairment is the main symptom of AD pathology and the most prominent of its observable consequences, one of the requirements that O. degus fulfills is enabling the discrimination of the different memory deficits classically impaired in AD through different cognitive tasks [21]. In this context, we have demonstrated that aged degu (56 months old) showed impaired performance in spatial and recognition memory tasks, as evaluated by means of the novel location/object recognition task and the Barnes maze [27, 29], together with a reduction in synaptic function in old animals compared with that in young animals [27, 29].

However, an additional study regarding the aggregation properties of the degu Aβ peptide is required to properly validate this natural model. Thus, in this study, we examined the capacity of degu Aβ peptide to aggregate in vitro and to form Aβ fibrils. We analyzed the age-dependent production of soluble Aβ₄₀ and Aβ₄₂ peptides, as well as the variation in the Aβ₄₂/Aβ₄₀ peptide ratio in the hippocampus and cortex of third- to fifth-generation captive-born degu. Moreover, we evaluated the appearance and distribution of amyloid plaques in the brains of aging animals using three complementary stains. Furthermore, we compared the overall distribution and 3D structure of the amyloid plaques in the brains of aged degu with those in two well-known transgenic animal models of AD [30].

In addition to further confirming our previous findings [28 , 31], we demonstrated here that 1) O. degus Aβ peptide aggregates in vitro, similar to the human Aβ-peptide; 2) plaque deposition occurs in an age-dependent manner from the anterior (32 months) to the posterior brain (56 months) and is closely related to the increases in the levels of Aβ₄₀ and Aβ₄₂ peptides as well as in the Aβ₄₂/Aβ₄₀ ratio in both the hippocampus and cortex; and 3) O. degus exhibits fewer but larger plaques than transgenic models. In conclusion, we believe that the Chilean rodent O. degus is a natural model of AD that represents both the molecular and the cognitive alterations that together define this pathology and compared with other alternatives, exhibits a reasonable time lapse to the onset of the pathological hallmarks of the disease. Based on the available information, degu is one of the most valuable animal models for exploring the mechanisms underlying the behavioral and cognitive deficits resulting from healthy and pathological aging processes and for investigating the endogenous/environmental factors that promote AD.

MATERIALS AND METHODS

Animals

Adult female degu aged 19, 32, 44, and 56 months (n = 3 animals per age group) were obtained from our colony at the Faculty of Biological Sciences, Pontificia Universidad Católica de Chile. These animals were all derived from third- to fifth-generation captive-born animals. Related and unrelated pairs of degu females were housed in clear acrylic cages (length x height x depth: 50 x 35 x 23 cm) with hardwood chip bedding. Water and food (commercial rabbit pellets; Champion, Santiago, Chile) were available ad libitum. Additionally, each cage contained one nestbox of clear acrylic (22 x 12 x 15 cm). The animals were housed in a ventilated room with a natural photoperiod and a controlled temperature (yearly minimum = 13.4±0.2°C; yearly maximum = 24.9±0.2°C).

The chemical composition of the diet was dry matter = 90.6%, ash = 10.8%, crude fiber = 16.5%, neutral detergent fiber = 37.8%, acid detergent fiber = 19.8%, lipids = 3.0%, proteins (N×6.25) = 20.0%, carbohydrates = 40.3%, and total energy content = 18.4±0.5 kJ g^–1 [24, 32]. In addition, all animals received pieces of wood or cardboard twice per week for minimal physical stimulation. For comparison purposes regarding plaque accumulation and Aβ levels, we included data from male degu up to 84 months old. Similarly, additional female degu were considered in each group (resulting a total of 19 months old, n = 5; 32 months old, n = 5; 44 months old, n = 4; 56 months old, n = 6) to improve the quantification of the Thioflavin S (ThS)-positive plaque frequency. These animals were also third- to fifth-generation captive-born animals from our colony and were maintained in similar housing conditions. All experiments followed the guidelines of the American Society of Mammalogists and the National Institutes of Health (NIH, Baltimore, MD). All procedures were approved by the Bioethical and Biosafety Committee of the Faculty of Biological Sciences of the Pontificia Universidad Católica de Chile (CBB-121-2013 and 170113007). All efforts were made to minimize animal distress and suffering, as well as to minimize the number of animals used.

Preparation of the stock 1 mM Aβ desiccated film and 5 mM Aβ solution in DMSO

Human Aβ₁_–42 and Aβ₁_–40 peptides were purchased from Bachem and Genemed (95% purity; Bachem Americas Inc., Torrance, CA; Genemed Biotechnologies Inc., Torrance, CA), and the O. degus Aβ₁_–40 peptide was synthetized by Bachem (95% purity). Lyophilized Aβ was dissolved in hexafluoroisopropanol (HFIP) (Sigma-Aldrich Corp., St Louis, MO) to a final concentration of 1 mM, and 10-μl aliquots were placed in microcentrifuge tubes. HFIP was evaporated overnight under a fume hood, followed by 1 h of additional evaporation using a vacuum desiccator. At the end of this process, thin transparent films were obtained at the bottom of the microcentrifuge tubes and stored at –20°C. Prior to use, the films were allowed to reach room temperature and then dissolved in 2 μl DMSO to obtain a 5 mM Aβ solution [33].

Preparation of Aβ aggregated species

In this work, we used two different methods to study Aβ aggregation. In the first method, human Aβ₁_–42 was used as an aggregation reference. Human Aβ₁_–42 oligomers and fibrils were prepared according to the methods reported by Stine [33], with slight modifications. Briefly, the 5 mM Aβ solution in DMSO was diluted in 98 μl cold phosphate-buffered saline (PBS) (final concentration of 100 μM Aβ) and incubated at 4°C for 24 h to obtain Aβ oligomers. The 5 mM Aβ DMSO solution was diluted with 10 μM HCl (final concentration of 100 μM Aβ) and incubated at 37°C for 24 h to obtain Aβ fibrils. In the second method, human and degu Aβ₁_–40 fibrils were prepared using previously reported methods [33 –36]. Briefly, 5 mM Aβ in DMSO was diluted with cold PBS to a final concentration of 100 μM and agitated at 240 rpm on an orbital shaker at room temperature. The method described by Stine [33] was also used to prepare degu fibrils. In the latter case, 5 mM degu Aβ₁_–40 in DMSO was diluted with 10 μM HCl to a final concentration of 100 μM and incubated at 37°C for 24 h. A scrambled sequence of the degu Aβ1–40 peptide was used as a control for the normal degu Aβ1–40 peptide. Additionally, 100 μM solutions of human and degu Aβ₁_–40 in PBS were shaken at 240 rpm, and we monitored the aggregation dynamics by measuring the absorbance at 405 nm for up to 27 h.

Transmission electron microscopy (TEM) of amyloid fibrils

Amyloid fibrils were negatively stained with uranyl acetate for a TEM study [37 –39]. Briefly, 15 μl of each sample was incubated with 300 μm Formvar/carbon copper grids (Tedpella Inc., Altadena, CA) for 1 min. Then, the sample drop was removed using filter paper and further incubated with 15 μl uranyl acetate for 1 min. At the end of the incubation, the uranyl acetate was removed using filter paper, and the sample was washed with 15 μl double-distilled water, which was then immediately removed using filter paper. The grids were dried for at least 5 min at 37°C. Images were captured at different magnifications using a Philips Tecnai 12 microscope (Eindhoven, Netherlands) at 80 kV equipped with a SIS CCD Megaview G2 camera and iTEM Olympus Soft Imaging Solutions software at the Advanced Microscopy Unit of the Faculty of Biological Sciences of our university.

Detection and quantification of Aβ peptide levels

Two sandwich ELISAs specific for Aβ₁_–40 and Aβ₁_–42 were employed to determine the concentrations of Aβ peptides, as previously described [40, 41]. Hippocampal and cortical homogenates from each animal were diluted to 1 μg/ μl in homogenization buffer containing protease and phosphatase inhibitors. Diluted homogenates (100 μl) were prepared to measure Aβ₁_–40/Aβ₁_–42 levels, according to the manufacturer’s instructions. Plates were read at the corresponding wavelengths on a Metertech 960 ELISA Analyzer. Aβ₁_–40 and Aβ₁_–42 relative levels within the hippocampus and cortex were calculated considering the levels of the younger animals as “background” which was subtracted from the levels corresponding to each age.

Protein (100 μg) from the hippocampal and cortical total protein lysates was separated on gels using a Tris–Tricine buffer system [0.2 M Tris (pH 8.9) as the anode buffer and 0.1 M Tris, 0.1 M Tricine, and 0.1% SDS (pH 8.25) as the cathode buffer] and was then transferred to PVDF membranes to detect soluble Aβ oligomers by using western blot analysis. The membranes were incubated with a primary anti-Aβ-4G8 antibody (Biolegend, San Diego, CA) and developed using an ECL Kit (Luminata Forte Western HRP Substrate, Millipore Corporation, Burlington, MA).

Western blot analysis

The hippocampus and entorhinal cortex of degu (19, 32, and 56 months old) were dissected on ice and immediately frozen at –150°C or processed as previously described [41]. Briefly, hippocampal tissues were homogenized in RIPA buffer (50 mM Tris-Cl, pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, and 1% SDS) supplemented with a protease inhibitor cocktail (Sigma-Aldrich, P8340) and phosphatase inhibitors (50 mM NaF, 1 mM Na₃VO₄, and 30 μM Na₄P₂0₇) using a Potter homogenizer and then sequentially passed through different caliber syringes. Protein samples were centrifuged at 14,000 rpm for 20 min at 4°C. The total soluble protein concentration was determined using a BCA Protein Assay Kit (Pierce Biotechnology, Rockford, IL). A total of 240 μg of protein samples were resolved on 10% SDS-PAGE gels and transferred to PVDF membranes. After incubation with different antibodies, the membranes were developed using an ECL Kit (Western Lightning Plus ECL, PerkinElmer). For analysis, all target protein signals were normalized to the loading control (α-tubulin or β-actin).

Brain sample preparation

Animals were euthanized by isoflurane overdose, followed by cervical dislocation. The brains were surgically removed and postfixed with 4% p-formaldehyde in 0.1 M phosphate buffer (PB) for 3 h at room temperature, followed by storage in 10% sucrose in PBS at 4°C overnight. After fixation, the brains were cooled to ensure unbiased processing and analysis. The brains were subdivided into the following three coronal regions (approximately 10 mm in size): the frontal, medial, and caudal regions. Each region was sectioned into 36 coronal sections (40 μm in thickness) using a Leica CM 1850 cryostat (Leica Biosystems, Nussloch, Germany) at –20°C. We used the stereotaxic map for the brain of degu as a reference [42]. We analyzed 20 slices from each region, including anterior (+5 to 0), middle (0 to –5) and posterior (–5 to –10).

Thioflavin-S staining for amyloid plaques

We compared our data with two transgenic mouse models of AD to validate our results. We used brain samples from both models obtained at 9 months of age (n = 5 per model). One of the models was the double-transgenic APPswe/PS1dE9 (APP/PS1) mouse, which coexpresses the Swedish mutation (K594M/N595 L) of a chimeric mouse/human APP (Mo/HuAPP695swe) together with the human exon 9-deleted variant of PS1 (PS1dE9) and secretes elevated amounts of human Aβ peptide [43]. The second model was the APPSwInd transgenic (J20) mouse, which expresses a mutant form of human APP bearing both the Swedish (K670 N/M671 L) and Indiana (V717F) mutations (APPSwInd) [41]. Both strains were obtained from The Jackson Laboratory (Bar Harbor, ME).

ThS staining was performed on sections mounted on gelatin-coated slides. After dehydration and rehydration in ethanol and xylene, slides were incubated in distilled water for 10 min and then immersed in ThS solution (0.1% ThS in 70% ethanol) for 5 min. The slides were then washed twice with 70% ethanol for 30 s, and the sections were covered with a coverslip and antifade mounting medium in the dark, as previously described by our laboratory [41, 44].

Immunofluorescence staining

Immunofluorescence staining of brain sections was performed as previously described [44]. After PBS and PBS-T (PBS plus Tween 20, 0.1%) washes, brain sections were incubated with 0.15 mM glycine and 10 mg/ml NaBH₄ to diminish background autofluorescence. The sections were washed with PBS and PBS-T and blocked with 3% bovine serum albumin (BSA) at room temperature for 1.5 h to avoid nonspecific binding. The target protein was detected using the corresponding primary antibody after overnight incubation at 4°C in PBS-T containing 0.5% BSA. Then, sections were washed with PBS-T and incubated with Alexa-conjugated secondary antibodies (Invitrogen, Carlsbad, CA) in PBS-T containing 3% BSA for 2 h at 37°C. The sections were washed with PBS-T, PBS, and water and mounted on gelatin-coated slides. The sections were sealed with coverslips and fluorescent mounting medium. The following primary antibodies were used: anti-Aβ-4G8 (Cat. 800701, against aas 17–24) and anti-Aβ-6E10 (Cat. 803015, against aas 1–16), which were both obtained from Biolegend.

Image analysis

Stained brain sections were photographed using an Olympus BX51 microscope equipped with a MicroPublisher 3.3 RTV camera (QImaging). The luminescence of the incident light and the time of exposure were calibrated to assign pixel values ranging from 0 to 255 in RGB images (no-light to full-light transmission) in all preparations. Images were loaded into ImageJ v.1.40 g (NIH, Bethesda, MD, USA) for analysis. The areas for measurement were selected by performing a manual threshold adjustment or direct manual selection of regions of interest (ROIs) in heterogeneously stained sections [31]. Images of immunofluorescence staining in neurons were captured with a Zeiss LSM 5 Pascal confocal microscope. Series of 15–20 confocal layers representing fluorescence data from each ROI were examined. The 3D reconstruction was performed using Imaris v 9.1 software.

Preparation of images

Digital images were processed using Adobe Photoshop 7.0 and converted to figures. General image adjustments included color, contrast and brightness corrections to improve visualization but not for analysis.

Statistical analysis

All data are presented as the means±SEM. Comparisons between groups were performed using one-way ANOVA followed by Bonferroni’s post hoc test. Significance was set to p = 0.05. The assumptions of a normal distribution and homogeneity of variances were confirmed using a fitting test. Accordingly, nonparametric analyses (Mann-Whitney and Kruskal-Wallis) were used when the data did not meet these assumptions.

RESULTS

Aβ₁_–40 aggregation: similarities between human and O. degus Aβ_1–40

As reported elsewhere, the O. degus Aβ peptide sequence differs from the human sequence in only one aa [6, 40]. Specifically, at the 13^th aa residue, human Aβ possesses a histidine that is replaced by an arginine in the degu protein (Fig. 1A). Due to the sequence similarity between the two Aβ sequences and the evidence that the brain of degu spontaneously develops Aβ plaques resembling those present in the brains of patients with AD, we decided to evaluate the putative in vitro aggregation of the degu Aβ₁_–40 peptide. Turbidimetry revealed the aggregation of both human and degu Aβ₁_–40 over time, reaching a plateau at approximately 25 h of incubation compared with that of the control scrambled degu Aβ₁_–40 peptide sequence (Fig. 1B). In fact, no amyloidogenic structures containing the scrambled Aβ₁_–40 degu peptide were observed under an electron microscope after 24 h of incubation (Fig. 2Be). Moreover, using the human Aβ₁_–42 peptide as an aggregation reference for the formation of oligomeric and fibrillary species (Fig. 2Ac-e, respectively), we verified that human Aβ₁_–40 tended to form fibrils after 24 h of incubation under the same experimental conditions (Fig. 2Aa–b). Similarly, the degu Aβ₁_–40 peptide formed fibrillary structures after 24 h of incubation under the following different conditions: 1) a low-pH environment at 37°C [35] and 2) PBS plus agitation on an orbital shaker at room temperature [34, 37] (Fig. 2Ba–d). Interestingly, both methods produced twisted structures of the amyloid fibrils (Fig. 2Ad and 2Bd, white arrows). Concomitantly, oligomeric species were also generated by both human and degu Aβ₁_–40 peptide (Fig. 2Ac, human; and Fig. 2Bc, degu). These results confirmed that O. degus Aβ forms molecular aggregates in vitro.

Fig. 1.

A) O. degus Aβ shares high homology with the human Aβ peptide, suggesting that the degu peptide has the capacity to form Aβ aggregates. B) Results of a turbidity assay used to compare the aggregation capacity of degu and human Aβ peptides and the degu scrambled Aβ peptide (control). The formation of aggregates was monitored in a spectrophotometer at 405 nm for 25 h.

Fig. 2.

Human and O. degus Aβ_{1 - 40} and Aβ_{1 - 42} peptide aggregates form oligomeric and fibrillar species under different in vitro conditions. A) Photomicrographs showing the human Aβ_{1 - 40} fibrillar species obtained using different protocols (a, b). c-e) Human Aβ_{1 - 42} aggregated species obtained using the method reported by Stine [33]. c) Oligomeric species (160 kX magnified and digitally zoomed). d,e) Fibrillar species (160 kX magnification). B) Photomicrographs show the degu Aβ_{1 - 40} fibrillar species obtained using the reported protocol. a) Fibrils at 60 kX magnification. b) Fibrillar species at 160 kX magnification. c,d) High-magnification digital images of oligomeric and fibril sections obtained at 160 kX magnification. d) Shows the twisted structures of the human (Ad) and degu (Bd) Aβ_{1 - 40} fibrils. The arrows indicate the twisted shape of the amyloid fibril. The white bars in each photomicrograph represent 200 nm.

Quantification of Aβ₁_–40 and Aβ₁_–42 levels in the brains of aging O. degus

Next, we used specific ELISAs to evaluate the levels of Aβ₁_–40 and Aβ₁_–42 in the hippocampus and cortex of female degu at different ages. For these experiments, we used animals aged 19 (young), 32 (adult), and 56 months old (old). We detected very low levels of Aβ₁_–42 in the hippocampus of young animals, with a concentration of up to 0.1 pmol/l. In 32 months old animals, we observed a slight increase in Aβ₁_–42 levels up to 0.5 pmol/l. However, in old animals, we observed a significant increase in Aβ₁_–42 levels up to 3.5 pmol/l, representing 35-fold and 7-fold increases compared with those in young and adult animals, respectively (Fig. 3A). Similar results were measured in the cortex where we detected levels of 0.1 pmol/l, 0.7 pmol/l, and 3.0 pmol/l in young, adult and old animals, respectively (Fig. 3B).

Fig. 3.

Using a specific ELISA for Aβ_{1 - 40} or Aβ_{1 - 42}, we measured Aβ_{1 - 42} (A, B) and Aβ_{1 - 40} levels (C, D) in the hippocampus and cortex of female degu and calculated the ratio between the two forms (Aβ₄₂/Aβ₄₀). Aβ_{1 - 40} and Aβ_{1 - 42} relative levels within the hippocampus and cortex (A-D) were calculated considering the levels of the younger animals as “background” which was subtracted from the levels corresponding to each age. This value is an important marker of the progression of AD and was calculated for both the hippocampus and the cortex (E and F, respectively). Asterisks over bars indicate significant differences (*p<0.05, **p<0.01, **p<0.001) determined using one-way ANOVA with Bonferroni’s post hoc comparison. Each bar corresponds to a single group, and the data are presented as the means±SEM.

Regarding Aβ₁_–40, we detected levels of 1 pmol/l in the young animals, 20 pmol/l in the adults, and 80 pmol/l in the old animals, representing 20-fold and 80-fold increases in the levels of Aβ_{1 - 40} in the hippocampus of old animals compared with those in young and adult animals, respectively (Fig. 3 C). In the cortex, we observed a similar result, but a more substantial increase in Aβ₁_–40 levels was observed during the aging of degu. Indeed, in the young animals, we detected levels of 1 pmol/l, while in the adult and old animals, we observed increases up to 30 pmol/l and 85 pmol/l, respectively (Fig. 3D).

In addition to the total increase in Aβ species levels, an increase in the ratio between Aβ₁_–42 and Aβ₁_–40 is relevant to AD pathophysiology [45 –47]. In both the cortex and hippocampus, the Aβ₁_–42/Aβ₁_–40 ratio increased significantly with age, consistent with the increased levels in each species. In the hippocampus, we observed 1.5- and 2-fold increases when young animals were compared with adult and old animals, respectively (Fig. 3E). This change was more significant in the cortex, where a more substantial increase was observed across the three age groups. Compared with younger animals, adults and older animals exhibited 2.7- and 4.4-fold increases, respectively (Fig. 3F).

In addition to the abovementioned ratio, the presence and load of oligomeric forms of Aβ constitute the main molecular structure that triggers the pathological alterations observed in AD. Notably, using an anti-Aβ antibody (4G8), we observed a significantly higher levels of 100 kDa Aβ-positive peptides in the hippocampus of the 56 months old group than in the young animal group (Fig. 4A, B); however, no significant differences were observed in the levels of the 56 kDa (Fig. 4A, D). In contrast to the hippocampus, significantly higher levels of 56 kDa Aβ-positive species were observed in the cortex of the 56 months old group than in the 19 months old group; however, the levels of the 100 kDa Aβ-positive peptides were not significantly different (Fig. 4 C). No low-molecular-weight Aβ-positive species were observed under our extraction conditions. Although the 4G8 antibody detects an epitope that can be present in the full-length AβPP as well as in the immature- and/or cleaved-AβPP forms, this result suggest an increase in the Aβ-containing species within the brain of the O. degus which changes according animal age.

Fig. 4.

Detection of Aβ-positive species in the hippocampus and cortex of female O. degus. Western blots of hippocampal (A) and cortical (C) total protein lysates separated on Tris–Tricine gels and incubated with an anti-Aβ 4G8 antibody (arrows indicate the species that were quantified). The results of the densitometry analysis of the blots shown in A are presented in B. The results of the densitometry analysis of the blots shown in C are presented in D. Asterisks over bars indicate significant differences (*p<0.05 and **p<0.01) determined using one-way ANOVA with Bonferroni’s post hoc comparison. Each bar corresponds to a single group, and the data are presented as the means±SEM.

Together, these results confirm the presence of Aβ₁_–40 and Aβ₁_–42 in the cortex and hippocampus of O. degus. Their levels increased in an age-dependent manner, and aggregated oligomeric species also formed and exhibited a different distribution throughout the brain, suggesting that the progression of amyloid pathology from the hippocampus to the cortex occurs through an age-dependent process.

Localization of Aβ aggregates in the brain of O. degus

Once we revealed the presence of Aβ species in the brain of degu and the increased levels of these species during aging, the next critical step for any model of AD was to examine the presence of extracellular Aβ aggregates. Accordingly, we used different approaches to detect the presence of these aggregates. Three different markers were used to detect the presence of Aβ peptide aggregates, ThS, and two specific antibodies, 4G8 and 6E10, which detect different epitopes of the Aβ peptide. For these experiments, we divided each brain into three regions (anterior, middle, and posterior), and each region was analyzed for the presence or absence of Aβ aggregates (Fig. 5A). ThS staining did not reveal Aβ aggregates in any region in 19 months old animals. However, at 32 months, we observed the appearance of Aβ aggregates exclusively in the anterior region (Fig. 5B). At 44 months, Aβ aggregates were observed in both the anterior and middle regions but not in the posterior region. Finally, in animals aged 56 months, we observed Aβ aggregates in all three regions and an increase in the total number of amyloid aggregates (Fig. 5B).

Fig. 5.

Distribution of Aβ aggregates in the brains of female O. degus. A) We obtained brains from degu that were processed and cut into several slices representing the anterior, middle and posterior regions. We performed ThS staining in 20 slices from each region in each animal. B) Representative images of ThS-positive areas in the brains of degu at different ages. Aβ aggregates accumulated from the anterior to the posterior region with aging, n = 3 animals per group. Scale bar: 100 μm.

Moreover, using two specific antibodies against Aβ, we evaluated the presence of Aβ aggregates in the hippocampal region, one of the first and most relevant brain areas in AD pathology [4]. The 4G8 and 6E10 antibodies did not detect aggregates in the hippocampus of young animals, whereas a slight but not significant signal was observed at 32 months old. However, in animals aged 44 and 56 months, clear Aβ aggregates were observed (Fig. 6). Together with the ThS staining results, the current findings confirm our previous data and indicate that the brain of O. degus spontaneously develops Aβ aggregates, which were detected using the same molecular tools used in AD research. Moreover, we confirmed that the aggregates were closely related to the age of the animals, naturally resembling the pathophysiology of AD observed in patients.

Next, we performed a 3D reconstruction of several plaques observed in the brains of 56 months old O. degus to further characterize the Aβ aggregates. According to the plaque descriptions reported by different research groups [25, 48], the Aβ aggregates located in the anterior region of the brain were mainly type 2b, with an average volume of 7734 μm³, while the aggregates in the middle region of the brain were mainly type 2c, with an average volume of 4989 μm³. Finally, the Aβ aggregates located in the posterior region were the smallest and mainly corresponded to type 1, with an average volume of 2550 μm³ (Fig. 7). Importantly, characteristics of plaques other than their appearance can provide valuable information about the progression of the disease. As reported in a study by Bussière [48], the morphology and density of the aggregates are also relevant to the pathology. The compactness of the plaques constitutes the main factor differentiating type 1 and type 2. While type 1 corresponds to loosened aggregates without a central core, type 2 and its subcategories a to c represent denser structures and the increased development of a compact core. Because O. degus exhibited different pathological plaque types, this animal displays the natural development of several characteristics closely related to AD pathology.

Fig. 6.

a-d) Localization of Aβ deposits using 4G8 immunostaining in the hippocampus of female O. degus. e-h) Detection of Aβ deposits with 6E10 immunostaining in the hippocampus of aging degu, n = 3 animals per group. Scale bar: 50 μm.

Fig. 7.

We selected several images from the hippocampus of 56-month-old female O. degus stained with the 4G8 antibody (a-c) and generated representative 3D reconstructions of these images using Imaris software. From these reconstructions, we calculated the volume and types of amyloid plaques (d-i).

Histological characterization of the plaque load in the O. degus brain

After characterizing the Aβ plaques, we conducted a histological evaluation of the frequency and total plaque burden within the brains of degu, similar to the quantification of several parameters described in assessments of the pathology in AD patients. In this analysis, we included samples from two commonly used transgenic AD-like mouse models. ThS staining clearly shows greater numbers of Aβ plaques in the brains of both transgenic models than in the brains of 56 months old O. degus (Fig. 8A–C). Moreover, in the degu model, the number of plaques increased with age. Indeed, although we did not observe plaques in the brains of 19 months old animals, the brains of 32 months old animals contained 3 plaques per 10000 μm². The number of Aβ aggregates increased to 10 and 16 per 10000 μm² in animals aged 44 and 56 months, respectively (Fig. 8D). As expected, the transgenic mouse models presented more plaques than the natural model, with up to 25–28 plaques per 10000 μm² (Fig. 8D).

Fig. 8.

a-c) ThS-stained Aβ aggregates in the brains of two transgenic mouse models (2xTg APPswe/PS1dE9, J20 APPSwInd) and female O. degus aged 56 months. d) Quantification of ThS-, 4G8-, and 6E10-labeled Aβ aggregates per 10000 μm² in the brains of both transgenic mice and degu. Data are presented as the means±SEM. Significant differences were calculated using ANOVA, followed by Bonferroni’s post hoc test. Asterisks indicate statistically significant differences (*p<0.05) (19 months old, n = 3; 32 months old, n = 5; 44 months old, n = 4; 56 months old, n = 6; Tg, n = 5 each).

To complete our analysis, we also assessed the age-related frequency and distribution of the Aβ pathology in the degu brain. Accordingly, the plaque/no-plaque ratio increases with the age of the animals. Although plaque-positive degus were not detected at a young age (19 months old) and slightly increased at older ages (32 and 44 months), at 56 months old, half of the animals exhibited a clear Aβ pathology (Fig. 9A). Moreover, using ThS staining, we described the appearance of the Aβ aggregates in a region-specific manner. In 32-month-old animals, a few Aβ aggregates were present, mainly in the anterior region. However, in animals aged 44 and 56 months, we observed increased Aβ burden with higher levels detected in the anterior region and lower levels in the posterior region (Fig. 9B).

Fig. 9.

A) Age-related absolute frequency of plaque/no-plaque female degus. Grey section represents absence of plaque; Black section represents plaque presence. B) We counted the number of Aβ aggregates per 10000 μm² in different regions of the degu brain during aging and in the brains of both transgenic mouse models. C) Plot of the cumulative distribution of amyloid plaque size in the brains of female degu and both transgenic mouse models. Data are presented as the means±SEM. Significant differences were calculated using ANOVA followed by Bonferroni’s post hoc test. Asterisks indicate statistically significant differences (*p<0.05) (19 months old, n = 5; 32 months old, n = 5; 44 months old, n = 4; 56 months old, n = 6; Tg, n = 5 each).

An interesting observation emerged when we examined the size of the plaques in terms of the cumulative area covered by Aβ aggregates. The cumulative frequency plot (Fig. 9 C) shows that the total plaque number in aged degu was lower than in the other transgenic models, but the size of the aggregates compensated for this shortcoming, leading to comparable Aβ-compromised areas of the brain (in terms of surface area).

Relevantly, for comparison purposes, we also studied the Aβ dynamics in the brains of male O. degus (Fig. 10). Although amyloid pathology was more severe among females than males, at 84 months old, male degu exhibited very large plaques across the three main brain areas (anterior, middle and posterior) (Fig. 10A–C; A’–C’). Moreover, the Aβ₄₂/Aβ₄₀ ratio increased and was markedly higher at this age than at 12 and 36 months, suggesting that both Aβ species continued to accumulate within the brain (Fig. 10D). Indeed, from 36 to 80 months old, the rate of increase in both Aβ species became dramatically greater (Fig. 10E, F). We observed the same results when we analyzed the cortex of male degu (data not shown), suggesting that amyloid pathology not only increases with age but also involves the whole brain, as reported in the human pathology.

Fig. 10.

Representative photomicrographs of amyloid plaques found in anterior, middle and posterior regions of very old male brains (84 months old) (A, anterior; B, middle; C, posterior). The white bar represents 50 μm. 3D reconstruction from the representative photomicrographs (A’, anterior; B’, middle; C’, posterior). The white bar represents 20 μm. D) Age-related variation in the Aβ₄₂/Aβ₄₀ ratio in the hippocampus of male brains. E, F) Age-related variation in Aβ₄₂ and Aβ₄₀ levels in the hippocampus of male brains, evaluated by ELISA. Data are presented as the means±SEM. Significant differences were calculated using ANOVA followed by Bonferroni’s post hoc test. Asterisks indicate statistically significant differences (*p<0.05, ***p<0.001; n = 3 animals per group).

DISCUSSION

AD is the most common form of dementia and affects nearly 10% of individuals aged > 65 years and approximately 50% of individuals over the age of 85 [1]. The increased longevity of the population, combined with the high incidence of AD in older adults, will exacerbate the global impact of this disease on public health [4 , 50]. Regrettably, the continuous failure of multiple clinical trials has forced us to reevaluate our approach to AD research [6]. In this context, several disease models have been used to improve the reliability of AD investigations. Although some spontaneous AD-like models are available, including guinea pigs, dogs, dolphins, and nonhuman primates, each model has its own particularities to be considered in terms of practicality and reliability [6]. Moreover, some of these models fail to exhibit all the various aspects of the pathology or require a long time to show the pathological hallmarks of AD [11, 12]. Transgenic mice have been the most useful tool in studies of the pathological mechanisms of AD and have rapidly improved our knowledge regarding the molecular events related to the establishment and progression of this pathology [15]. Unfortunately, some of these models do not recapitulate the spectrum of lesions present in the brains of patients with AD [15 , 51–54]. Moreover, the overexpression of human transgenes in a non-physiological scenario strongly influences the onset of histopathological features and the cognitive decline observed in patients with AD [15 , 55]. In this context, the poor reliability of the currently available AD models limits our understanding of the pathophysiology of AD and might be the basis of the unsatisfactory translation of preclinical data to human clinical trials. Thus, considering the urgent need for new AD therapies [14], the identification and validation of a natural, wild-type AD model that mimics the pathological hallmarks observed in patients with AD would be very useful for identifying and validating potential therapeutic targets.

Over the last decade, we have been working to show the potential of O. degus, an endemic rodent from Mediterranean region of Chile, as a natural and reliable model of AD pathology. We have systematically described several histopathological hallmarks of AD that develop spontaneously in aged degu, including neuroinflammatory markers, amyloid plaque formation, and tau phosphorylation, together with social impairments and cognitive deficits [26 , 56]. Moreover, we determined that aging is a primary contributor to the impaired cognitive function exhibited by degu and is related to the decrease in synaptic function and to the appearance of the pathological hallmarks of AD [6 , 29]. Although some controversies have arisen [57, 58], the observed correlation between the molecular alterations and behavioral performance, along with the natural age-related development of the pathology, strongly support the hypothesis that O. degus might constitute a valuable model for AD research [6, 15, 26]. Accordingly, to elucidate the capacity of degu Aβ to aggregate as well as to resolve some discrepancies regarding the model outcomes, we conceived a systematic approach to demonstrate that compared with the currently available models, O. degus is a reliable and valuable AD-like model.

Since the degu Aβ sequence differs from the human Aβ sequence in one amino acid only, the first question that we attempted to answer was whether degu Aβ₁_–40 was able to aggregate. Using methods commonly utilized to prepare different aggregated species of human Aβ in vitro [35, 36], we confirmed that degu Aβ₁_–40 formed different Aβ species, including oligomers and fibrils and that these aggregated forms were comparable to human Aβ₁_–40 and Aβ₁_–42. Moreover, because Aβ₁_–42 tends to aggregate more easily than Aβ₁_–40, the observation that degu Aβ₁_–40 formed oligomeric and fibrillar structures in vitro is highly relevant to estimate aggregation phenomena within the brain. Thus, to evaluate whether the levels of Aβ₁_–40 and Aβ₁_–42 show similar behavior to the human pathology favoring the aggregation dynamic, we quantified the levels of both peptides within the brains of O. degus and evaluated whether these values were affected by the age of the animals. Consistent with several reports showing an age-dependent increase in the levels of both Aβ₁_–40 and Aβ₁_–42, as well as an increase in the Aβ₁_–42/Aβ₁_–40 ratio [59, 60], we observed increases of up to 37- and 80-fold in the levels of Aβ₁_–40 and Aβ₁_–42, respectively, when young (19 months old) and aged (56 months old) female animals were compared. These differences were observed in both the cortex and the hippocampus, confirming that the amyloid pathology compromises the whole brain in an age-dependent manner and that the increase in peptide levels is an initial required step prior to amyloid aggregation and plaque formation [6]. Indeed, when we evaluated the presence and characteristics of amyloid plaques in the brains of O. degus, we observed that older animals presented larger and more numerous ThS-, 4G8-, and 6E10-positive aggregates than did younger animals. More importantly, the compromise of the three main brain areas (anterior, middle, and posterior) was related to the age of the animals. The anterior regions of the brain (bregma+5) were the first to exhibit plaque deposition in adult animals (32 months old), followed by the middle (bregma –5) (44 months old) and posterior brain regions (bregma –5, –10) in older animals (56 months old) (Fig. 3B). We observed the same timing for the development of amyloid plaques within the hippocampus, the critical brain structure involved in memory and cognitive performance. Although young animals did not exhibit amyloid plaques, the number of these aggregates significantly increased in older animals. Moreover, an age-dependent compromise of the hippocampus was evident. Relevantly, when we studied the age-related progression of amyloid pathology within the brains of male degu, we observed not only the same distribution and increased levels of both Aβ species but also a dramatic increase in the rate of accumulation at older ages (84 months old), with values almost double those reported in the younger group. In particular, these findings are highly relevant because they allow us to perfectly connect our previously observed age-dependent impairments in cognitive performance with the timing of the development of the molecular alterations within the brain, particularly within the hippocampus [7 , 61]. Moreover, our current findings demonstrate that O. degus develops amyloid plaques and that these plaques vary in shape, size and internal morphology; these findings are strongly correlated with the expected development of the AD pathology described elsewhere [6, 7].

Finally, to further validate our model, we decided to compare degu with the commercially available APPswe/PS1dE9 and J20 (APPSwInd) transgenic murine models of AD. Although at an older age (56 months old), only half of O. degus exhibited a clear Aβ pathology compared with the AD commercial models (transgenic mice reached 100%), O. degus plaques were larger, ultimately exhibited the same cumulative area in terms of ThS staining and importantly, followed the same age-dependent distribution within the different brain areas. This latter finding further confirms that degu exhibits clear differences from genetically engineered models, but overall, it recapitulates the main molecular events of AD pathology in the absence of any dramatic intervention [6, 15], suggesting that the development of the pathophysiological process is closely related to both individual variability and environmental conditions.

In this regard, as noted previously, some controversies have emerged regarding the development of certain molecular hallmarks of AD in aged O. degus [57, 58]. For example, some authors have suggested that the arginine substitution present in the degu Aβ peptide limits its aggregation capacity because the histidine present in the human peptide interacts with the metals in the microenvironment, causing its final aggregation and the formation of amyloid plaques [58]. Although this situation is widely known, different studies have indicated that sequence similarity is only one of multiple elements to be considered when approaching plaque formations. Indeed, dogs, rabbits and guinea pigs share the same peptide sequence as humans, but only dogs spontaneously develop the amyloid pathology observed in humans [58 , 62–65]. Thus, plaque formation and AD hallmarks extend beyond the amyloid sequence. Importantly, in this context, AD is a complex disease, and lifestyle and environmental conditions play a fundamental role in its development [6, 66]. Moreover, voluntary exercise, diet, and social activity, among other factors, affect the establishment and progression of the disease in both humans and AD-like models [66 –70]. The modulation of these factors has been suggested to constitute a relevant strategy to prevent cognitive decline [71, 72]. As shown in our recent study, early intervention involving voluntary exercise on a running wheel significantly modulates the pathological alterations observed in the double-transgenic APPswe/PS1 mouse model of AD, which was used in this study as a comparative model. Among the various beneficial effects of exercise, we observed a reduced Aβ load in the brain, decreased levels of neuroinflammatory markers and reduced amyloid plaque deposition. Moreover, we observed better cognitive performance in animals that used the running wheel than in sedentary transgenic animals [70]. Degu is not an exception to this rule. Among these highly social and diurnal animals, limited social interactions and restricted housing conditions are necessary requirements to promote the development of AD-like pathology [27]. Therefore, when approaching a “natural” model, a proper understanding of the ethology of the species used is mandatory. Otherwise, the use of inappropriate housing conditions with different enrichment elements available in the environment, including access to running wheels for voluntary exercise, can prevent the development of AD-like pathology. An additional feature of degu is its tendency to develop diabetes, a condition that seems to be strongly related to the increased risk of developing AD [15, 16]. In fact, we initially observed eye cataracts (implying glycation of the crystalline proteins) and postulated that aged degu had amyloid aggregates in the brain in early 2005 [15,16, 15,16]. Although further research is needed, the possibility that this condition became critical for the subsequent development of AD-like pathology or that the environmental conditions provided in the experimental laboratories, such as voluntary exercise, prevent the development of diabetes and subsequent neurodegeneration might constitute an additional explanation for the differences in experimental outcomes using this model.

In this regard, together with our previous studies [16 , 29], our current findings demonstrate that amyloid plaques are not detected in young degu (19 months old) and that they appear in the anterior brain structures at approximately 32 months, in the anterior-medial area at 44 months, and in the anterior, medial and posterior regions at 56 months. Moreover, we demonstrate that these changes occur with concomitant increases in the levels of Aβ₄₀ and Aβ₄₂ peptides, as well as in the Aβ₄₂/Aβ₄₀ ratio, in both the hippocampus and cortex. Additionally, analysis of the 3D structure revealed larger amyloid plaques in the anterior region than in other regions of the brain. Even when the total number of plaques was lower than that in both transgenic models, the plaques were larger in the aged degu brain than in the transgenic mouse brain, covering the same cumulative area. Importantly, in both degu and mice, the plaques exhibited the same distribution from the anterior to the posterior regions of the brain.

We have previously suggested that the Chilean rodent O. degus represents a potentially novel natural model for research on the onset and progression of AD [23 , 56]. According to our current findings and over ten years of experience working with this long-lived rodent, O. degus truly develops a natural condition that resembles the pathology of AD in humans and constitutes an invaluable tool for AD research because it can develop the full pathological markers of the disease without genetic manipulation, drug administration, or invasive experimental interventions [12 , 29]. Moreover, compared with other “natural” models, degu offers better practicability and reduced time to the onset of pathological hallmarks, allowing the acceleration of AD research. Indeed, we have recently demonstrated that andrographolide, a natural compound obtained from Andrographis paniculata, rescues cognitive impairment, prevents synaptic protein loss, reduces soluble Aβ₄₀ and Aβ₄₂ peptide levels, and reduces Aβ aggregate maturation in aged degu and the double-transgenic APPswe/PS1 mouse model of AD, as detected by ThS, anti-Aβ 6E10 and 4G8 antibody staining [29 , 73]. Moreover, we are developing an exhaustive study to evaluate the effects of physical and cognitive stimulation (measured through the use of an exercise wheel) in addition to long-term andrographolide administration in aged (60 months old) and young (12 months old) O. degus. In parallel, we are also studying the effect of social stress in the form of social isolation throughout the lifespan on the progression of AD hallmarks. Based on our preliminary data, this approach will provide us with the opportunity to thoroughly understand and explore the role of environmental influences on cognitive and behavioral processes and brain plasticity during aging.

Footnotes

ACKNOWLEDGMENTS

This work was supported by grants from the Basal Center of Excellence in Aging and Regeneration AFB 170005; FONDECYT (no. 1160724) to N.C.I. and FONDECYT (no. 11160651) to P.C. In addition, a grant from CAPES-CONICYT FB 0002-2014 (Line 3) was awarded to F.B. We also thank the Centro de Microscopía Avanzada, CMA BIO-BIO, and Proyecto CONICYT PIA ECM-12. Finally, we also acknowledge funding from a special grant “Lithium in Health and Disease” from the Sociedad Química y Minera de Chile (SQM).

Authors’ disclosures available online ().

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New Insights into the Spontaneous Human Alzheimer’s Disease-Like Model Octodon degus : Unraveling Amyloid-β Peptide Aggregation and Age-Related Amyloid Pathology

Abstract

Keywords

INTRODUCTION

MATERIALS AND METHODS

Animals

Preparation of the stock 1 mM Aβ desiccated film and 5 mM Aβ solution in DMSO

Preparation of Aβ aggregated species

Transmission electron microscopy (TEM) of amyloid fibrils

Detection and quantification of Aβ peptide levels

Western blot analysis

Brain sample preparation

Thioflavin-S staining for amyloid plaques

Immunofluorescence staining

Image analysis

Preparation of images

Statistical analysis

RESULTS

Aβ1–40 aggregation: similarities between human and O. degus Aβ1–40

Quantification of Aβ1–40 and Aβ1–42 levels in the brains of aging O. degus

Localization of Aβ aggregates in the brain of O. degus

Histological characterization of the plaque load in the O. degus brain

DISCUSSION

Footnotes

ACKNOWLEDGMENTS

References

Aβ₁_–40 aggregation: similarities between human and O. degus Aβ_1–40

Quantification of Aβ₁_–40 and Aβ₁_–42 levels in the brains of aging O. degus