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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
J Neurosci. Author manuscript; available in PMC 2013 September 10.
Published in final edited form as:
PMCID: PMC3769160

Synergistic effects of long-term antioxidant diet and behavioral enrichment on beta-amyloid load and non-amyloidogenic processing in aged canines


A long-term intervention (2.69 years) with an antioxidant diet, behavioral enrichment, or the combined treatment preserved and improved cognitive function in aged canines. While each intervention alone provided cognitive benefits, the combination treatment was additive. We evaluate the hypothesis that antioxidants, enrichment, or the combination intervention reduces age-related beta-amyloid (Aβ) neuropathology, as one mechanism mediating observed functional improvements. Measures assessed were Aβ neuropathology in plaques, biochemically extractable Aβ40 and Aβ42 species, soluble oligomeric forms of Aβ, and various proteins in the beta-amyloid precursor protein (APP) processing pathway. The strongest and most consistent effects on Aβ pathology were observed in animals receiving the combined antioxidant and enrichment treatment. Specifically, Aβ plaque load was significantly decreased in several brain regions, soluble Aβ42 was decreased selectively in the frontal cortex, and a trend for lower Aβ oligomer levels was found in the parietal cortex. Reductions in Aβ may be related to shifted APP processing towards the non-amyloidogenic pathway, as alpha-secretase enzymatic activity was increased, in the absence of changes in beta-secretase activity. While enrichment alone had no significant effects on Aβ, reduced Aβ load and plaque maturation occurred in animals receiving antioxidants as a component of treatment. AB measures did not correlate with cognitive performance on any of the 6 tasks assessed, suggesting that modulation of AB alone may be a relatively minor mechanism mediating cognitive benefits of the interventions. Overall, the data indicate that multi-domain treatments may be a valuable intervention strategy to reduce neuropathology and improve cognitive function in humans.

Keywords: antioxidant, enrichment, beta amyloid, oligomer, canine, secretase


Increasing evidence from human studies suggests that dietary and environmental interventions provide cognitive benefits and improve brain health, particularly in aged populations. For example, age-related cognitive decline was lessened by increased physical activity (Erickson and Kramer, 2009) or an “enriched environment” such as provided by cognitive training (Willis et al., 2006). Similarly, various dietary interventions rich in antioxidants may slow age-related cognitive decline and reduce the risk of Alzheimer's disease (AD) (Zandi et al., 2004) (Gray et al., 2003; Dai et al., 2006).

Studies in animal models provide several advantages over human studies, in that precise dietary and behavioral protocols can be maintained over an extended time-period, and effects on both cognitive function as well as underlying brain mechanisms can be assessed. Aged canines provide a valuable model for investigating mechanisms that underlie cognitive impairment. Like humans, aged beagles undergo cognitive decline in multiple cognitive domains that correlate with neuropathological changes (Cummings et al., 1996; Head et al., 1998; Head et al., 2000). In parallel, aged beagles naturally accumulate several types of neuropathology that are consistent with human brain aging and AD, including cortical atrophy (Tapp et al., 2004), neuron loss (Siwak-Tapp et al., 2008), declines in neurogenesis (Siwak-Tapp et al., 2007), reduced brain-derived neurotrophic factor (BDNF) (Fahnestock, in press), increased oxidative damage (Head et al., 2002; Skoumalova et al., 2003), and increased Aβ accumulation (Cummings et al., 1996; Head et al., 1998; Head et al., 2000).

Previously, we demonstrated that an antioxidant diet (AOX), behavioral enrichment (ENR), or the combination treatment improves cognition and prevents age-related cognitive decline in aged canines (Cotman et al., 2002; Milgram et al., 2002b; Milgram et al., 2002a). Mechanisms identified include the brain's improved capacity to counteract oxidative stress (Opii et al., 2008), improved mitochondrial function (Head et al., 2009), reduced neuron loss (Siwak-Tapp et al., 2007, 2008), and increased availability of growth factors such as BDNF (Fahnestock, in press). The combination of AOXs and ENR consistently produced greater benefits to cognition and neurobiological outcome measures than either treatment alone.

In this study, we hypothesized that AOX, ENR, or the combined treatment may reduce Aβ neuropathology in the canine brain. Previous studies have demonstrated that Aβ is reduced and cognition improved in transgenic AD mice by exposure to exercise (Adlard et al., 2005), ENR (Lazarov et al., 2005; Ambree et al., 2006; Costa et al., 2007), and select dietary compounds (Frautschy et al., 2001; Lim et al., 2001; Wang et al., 2008). However, other studies in transgenic AD mice have demonstrated improved cognition with ENR despite increased Aβ pathology (Jankowsky et al., 2005),and improved cognition in transgenic AD mice with AOX dietary supplementation without an affect on Aβ (Joseph et al., 2003; Sung et al., 2004; Quinn et al., 2007). Thus, the relationship between improved cognitive function and reductions in Aβ pathology is not clear-cut. To investigate if cognitive improvements in the current study are paralleled by decreased Aβ pathology, we analyzed treatment effects on Aβ neuropathology and the amyloid precursor protein (APP) processing pathway in the aged canine.



Twenty-four beagles ranging aged 8.05 to 12.35 yrs at the start of the study (mean=10.69 years, SEM=0.25) were obtained from Lovelace Respiratory Research Institute (Albuquerque, NM) (Supplementary Table 1). Animals were bred and maintained in the same environment and all had documented dates of birth and comprehensive medical histories. All study dogs underwent extensive baseline cognitive testing as described previously (Milgram et al., 2002a). Animals were subsequently ranked based on cognitive test scores and assigned to one of the following four treatment conditions: CC – control environment/control diet, EC – behavioral enrichment/control diet, CA – control environment/antioxidant diet, EA – behavioral enrichment/antioxidant diet. All animals, regardless of treatment condition, were evaluated annually on tests of visuospatial memory (Chan et al., 2002), object recognition memory (Callahan et al., 2000) and either size discrimination and reversal learning (Tapp et al., 2003), or black/white discrimination and reversal on consecutive years (Milgram et al., 2005). Cognitive results have been published for these animals (Cotman et al., 2002; Milgram et al., 2002b; Milgram et al., 2002a). At the time of euthanasia, of the 24 aged animals that began the study, 23 dogs had received the intervention for over 2 years (mean=2.69yrs, SEM=0.04) and ranged in age from 10.71 to 15.01 yrs (mean=13.31years, SEM=0.26) with one animal not completing the baseline phase of the study (Supplemental Table 1). All research was conducted in accordance with approved IACUC protocols.

Diet Treatment

The control and AOX test diets were formulated to meet the nutrient profile recommendations for adult dogs from the American Association of Feed Control Officials (AAFCO). Control and test diets were identical in composition, other than inclusion of a broad-based cellular AOX and mitochondrial cofactor supplementation in the AOX test diet (Supplemental Table 2). The control and test diet had the following differences in formulation on an as fed basis, respectively: dl-alpha-tocopherol acetate (120 vs 1050 ppm), ascorbic acid as Stay-C (30 vs 80 ppm), acetyl-l-carnitine (20 vs 260 ppm), dl-alpha-lipoic acid (20 vs 128 ppm). Based on an average weight of 10 kg per animal, the daily doses for each compound were 800IU or 210 mg/day (21 mg/kg/day) of vitamin E, 16 mg/day (1.6 mg/kg/day) of vitamin C, 52 mg/day (5.2 mg/kg/day) of acetyl-l-carnitine, and 26 mg/day (2.6 mg/kg/day) of lipoic acid (Supplemental Table 2). The AOX diet used compounds and doses that have shown some efficacy in epidemiological studies. However, human trials have often used much higher doses than those used in the current study, with AD patients receiving acetyl-l-carnitine at either 1 g/day (14.3-16.6 mg/kg/day assuming average weight of 60-70 kg) (Bonavita, 1986) or 2 g/day (Rai et al., 1990), and lipoic acid at dose levels up to 600 mg/day (8.5-10 mg/kg/day assuming average weight of 60-70 kg) (Hager et al., 2001). Fruits and vegetables were also incorporated at a 1:1 exchange ratio for corn, resulting in 1% inclusions of each of the following: spinach flakes, tomato pomace, grape pomace, carrot granules, and citrus pulp (Supplemental Tables 2 and 3).

Behavioral Enrichment Treatment

The behavioral ENR protocol consisted of four components: “social ENR” by housing animals in pairs, “environmental ENR” by providing novel play toys, “physical ENR” by providing two 20-minute outdoor walks/week, and “cognitive ENR” through continuous cognitive testing consisting of 20-30 minutes/day. The “cognitive ENR” consisted of a landmark discrimination task, an oddity discrimination task (Milgram et al., 2002b), and size concept learning (Tapp et al., 2003) as described previously (Cotman et al., 2002; Milgram et al., 2002b; Milgram et al., 2002a).

Brain Tissue

Twenty minutes before induction of general anesthesia, animals were sedated by subcutaneous injection with 0.2mg/kg acepromazine. Subsequently, surgery-level general anesthesia was induced by inhalation with 5% isoflurane and animals were exsanguinated. The brain was removed and sectioned midsagitally, with the entire left hemisphere immediately placed in 4% paraformaldehyde for 48-72 hours at 4°C prior to long-term storage in phosphate buffered saline (PBS) with 0.05% sodium azide at 4°C. The remaining right hemisphere was sectioned coronally (1cm thick sections) and flash frozen at -80°C. The dissection procedure was completed within 20 minutes. The post mortem interval for all animals was 35-45 minutes.

Aβ Immunohistochemistry & Quantification

Following previously published protocols, (Head et al., 2008), tissue from the left hemisphere was sectioned at 40μm by Neuroscience Associates (Knoxville, TN), followed by immunohistochemical processing. Briefly, free-floating sections containing the prefrontal, parietal, cingulate, entorhinal, and occipital cortices were pretreated with 90% formic acid, and Aβ plaques were detected with anti-Aβ1-17 (mouse monoclonal 6E10 antibody, 1:5000, Signet Labs. Inc., Dedham, MA) and visualized with DAB (Vector Labs., Burlingame, CA). Control experiments where primary or secondary antibody was omitted resulted in negative staining. The procedure for quantifying Aβ loads has been reported previously (Head et al., 2000). Briefly, ten images were captured using a 20× objective in each brain region and the area occupied by Aβ was quantified using gray scale thresholding (NIH Image) to obtain “Aβ loads”. Results were confirmed with an additional set of sections at least 200μm away.

Maturation gradient of Aβ Deposition (Aβ Type)

Aβ deposition in the canine frontal cortex is a progressive age-related process beginning with diffuse deposits in the deep cortical layers followed by the development of deposits in outer cortical layers. To assess the severity of Aβ pathology across treatment groups, we characterized Aβ maturation patterns in the same brain regions where Aβ load was assessed, using methods described previously (Satou et al., 1997). Briefly, Type 0 described regions without any Aβ plaque pathology; Type 1 was characterized by small, faint, round Aβ deposits (less than 180μm in diameter) usually in deep cortical layers V and VI; Type 2 were diffuse, cloud-like deposits that often fused together in the deep layers, and occasionally spread to the superficial layers; Type 3 had both diffuse deposits like those of Type 2 in deep layers, and dense, round plaques (less than 120μm but sometimes over 200μm) in superficial layers; Type 4 consisted solely of dense, round plaques throughout all layers of cortex. Sections were analyzed at several magnifications as needed for clarity, and then assigned the correct type. All cases with Aβ type 0, 1, or 2 were considered “early stage” pathology while all cases with Aβ type 3 or 4 contained plaques in the superficial layers and were considered “late stage” pathology.

Aβ Elisa

To detect differences among treatment groups in the prefrontal, parietal, temporal, and occipital cortices, we used previously published methods (Head et al., 2008). Briefly, frozen cortical samples were sequentially extracted in radioimmunoprecipitation assay (RIPA) buffer (pH=8, 50mM Tris-HCl, 150mM NaCl, 0.5% deoxycholate, 0.1% SDS, 1% Triton X-100, protease inhibitor cocktail (MP Biomedicals, Costa Mesa, CA)) to obtain a soluble RIPA fraction and a RIPA pellet, which was resuspended in a 70% formic acid buffer (FA) to measure insoluble Aβ. All FA samples were neutralized in neutralization buffer, and RIPA and FA preparations were run in triplicate on ELISA plates coated with a monoclonal anti-Aβ1-16 antibody (kindly provided by Dr. William Van Nostrand, Stony Brook University, Stony Brook, NY). Detection was by monoclonal HRP-conjugated antibodies anti-Aβx-40 (MM32-13.1.1) or anti-Aβx-42 (MM40-21.3.1) (both antibodies kindly provided by Dr. Christopher Eckman, Mayo Clinic Jacksonville, Jacksonville, CA) (Kukar et al., 2005; McGowan et al., 2005). For standards, dilutions of Aβ1-40 and Aβ1-42 peptides (Bachem California, Inc., Torrance, CA) were used after a pretreatment with hexafluoroisopropanol (HFIP) to prevent fibril formation. The inclusion of a series of controls to test the absorbance of buffers, samples, and antibodies yielded negative results.

Western Blot

Parietal cortex was used for analysis of Aβ oligomer levels and proteins in the APP processing pathway (TACE, ADAM10, and c-terminal fragments (CTFs)) as this region showed decreased Aβ plaque load in response to treatments. Protein levels of APP, insulin degrading enzyme (IDE), and neprilysin were quantified in the parietal, prefrontal, hippocampal, and occipital regions. For oligomer studies, pulverized tissue samples were extracted in PBS buffer (powder packet from Sigma-Aldrich, St. Louis, MO, pH 7.4, 0.2% NaN3 with Complete Mini protease inhibitor (Roche Diagnostics, Indianapolis, IN)). For all other proteins, frozen tissue was homogenized in RIPA buffer at 1 mL buffer/150mg pulverized tissue weight. All samples were centrifuged at 100,000g for 1 hour at 4C, and soluble fractions recovered and brought to an equal protein concentration by BCA (Pierce Biotechnology Inc., Rockford, IL). For the detection of the 56kDa Aβ aggregate, Aβ was immunoprecipitated from soluble PBS samples by overnight incubation with Protein A/G PLUS-Agarose bead complex (Santa Cruz Biotechnology, Santa Cruz, CA) and 5μl of the anti-Aβ 6E10 antibody (mouse monoclonal for Aβ1-17, Covance, CA). Unbound proteins were removed by washing with PBS buffer, and samples were centrifuged at 2500 rpm for 5min at 4C to pellet the bead-antibody-protein complex. Loading buffer (2.5mM Tris pH 6.8, 2% SDS, 0.007% bromophenol blue, 4% beta-mercaptoethanol, 10% glycerol) was added to each sample, followed by boiling at 100C for 5 minutes. For Western blots, equal protein amounts were loaded for each sample on 4-20% Tris-HCl Criterion gels (BioRad Laboratories, Hercules, CA), followed by transfer to sequi-blot PVDF membranes (Biorad Laboratories, Hercules, CA). For the oligomer studies, incubations were in Tris Buffered Saline with 0.01% Tween-20 (TTBS), specifically using 3% BSA/TTBS for all blocking and antibody incubations with β-actin (mouse, 1:5000, Abcam Inc., Cambridge, MA) and A11 antibodies (rabbit, 1:1000, Chemicon, Temecula, CA) (Kayed et al., 2003). For other protein analyses, incubations were in 5% milk/TTBS. The antibodies used in Western blots were β-actin (rabbit, 1:5000, Abcam Inc., Cambridge, MA), GAPDH (rabbit, 1:10,000; Chemicon, Temecula, CA), the N-terminal anti-Aβ antibody 6E10 for APP (mouse, 1:5000, Signet Labs. Inc., Dedham, MA), the C-terminal anti-Aβ antibody CT20 for full length APP and CTF alpha (CTFα) and beta (CTFβ) (rabbit, 1:2000, raised against the C-terminal 20 amino acids of APP), antibodies to clearance enzymes IDE (mouse, 1:250, Covance) and neprilysin (rabbit, 1:50, Abcam Inc., Cambridge, MA), and an antibody to the precursor and mature form of the alpha secretase (αSEC) enzyme TACE/ADAM17 (rabbit, 1:2000, Chemicon, Temecula, CA) or ADAM10 (rabbit, 1:500, Chemicon, Temecula, CA). Secondary antibodies were HRP-conjugated IgG anti-mouse (goat anti-mouse, 1:5000, BioRad Laboratories, Hercules, CA) or anti-rabbit (goat anti-rabbit, 1:10,000 from BioRad Laboratories, Hercules, CA or Rockland Immunochemicals, Gilbertsville, PA) as needed. Supersignal Chemiluminscent Substrate (Pierce Biotechnology Inc., Rockford, IL) was used to visualize HRP activity on Hyperfilm ECL (Amersham Bioscience, Piscataway, NJ). The omission of primary antibody resulted in negative staining. Immunoblots were quantified using NIH Image J software, with optical density (OD) measures adjusted for individual β-actin OD levels.

Alpha & Beta Secretase Activity Assays

These protocols have been published previously (Nistor et al., 2007). Briefly, frozen tissue was prepared according to protocols provided by the commercial supplier of the Secretase Activity Kits (R&D Systems, Minneapolis, MN). A total of 125 μg protein (2.5μg/μl in 50μl total per well, OD read at 2 hours) was used for αSEC, and a total of 7.5μg protein (0.15μg/μl in 50μl total per well, OD read at 30 min.) was used for the beta secretase (βSEC) assay. All samples were run in triplicate and assays were replicated to confirm results.

Statistical Analyses

To preclude bias, all data were collected while blind with respect to the experimental conditions. All statistical analyses were performed using SPSS for Windows, SYSTAT, or SAS, and graphs were produced using Sigma Plot. Generalized estimating equations (Zeger S, 1986) and ANOVAs were used to compare mean Aβ across treatment groups. Post-hoc analyses considered the main effect of the AOX diet (comparing the CC+EC groups versus the CA+EA groups) and the behavioral ENR (comparing the CC+CA groups versus the EC+EA groups). In data measures where normality and variance assumptions were violated and not rectifiable by converting raw data to Log10 scores, group differences were assessed according to the nonparametric Kruskal-Wallis analysis for multiple samples and the Mann-Whitney U analysis for two independent samples. Correlations were assessed using the Pearson coefficient or Spearman rank nonparametric statistic as needed.


In order to define the effects of AOX, ENR, or the combined intervention on Aβ pathology and Aβ processing in the canine brain, we analyzed treatment effects on extracellular Aβ plaque load, levels of biochemically extractable Aβ40 and Aβ42 species, soluble oligomeric forms of Aβ, and the APP processing pathway.

Treatment effects on extracellular plaque pathology

Aβ plaque immunostaining with the 6E10 antibody was quantified in the prefrontal, cingulate, parietal, entorhinal, and occipital cortices, revealing a differential treatment and brain region effect (Figure 1). When all brain regions were considered together, Aβ plaque load was significantly reduced in the combined EA treatment group to 19% of untreated control (CC) levels (p=0.01), while AOX treatment alone reduced Aβ plaque load in the CA group to 41% of untreated control (CC) levels (p=0.084) (Figure 1A). In individual brain regions, the EA treatment group reduced Aβ plaque load to 18% of control (CC) levels in the cingulate cortex (p=0.021), to 13% of control (CC) levels in the parietal cortex (p=0.093), and to 10% of control (CC) levels in the entorhinal cortex (p=0.077) (Figure 1B). Representative images from the parietal and entorhinal cortex are shown in Figures 1C and 1D, respectively.

Figure 1
Aβ load quantification across treatment groups

Because the EA treatment group showed the largest overall effect on reducing Aβ plaque load, treatment groups were pooled according to AOX diet or ENR condition to determine the possible relative contributions of each intervention strategy. Thus, the pooled AOX analysis consisted of control diet groups (CC+EC) compared to the AOX diet groups (CA+EA), while the pooled ENR analysis compared the control environment groups (CC+CA) versus the behavioral enrichment groups (EC+EA). Significantly lower Aβ load was detected in animals receiving the AOX diet (Figure 2A and 2B), but not in animals receiving ENR treatment (Figure 2C and 2D). The pooled AOX groups showed significantly lower Aβ load in the total brain analysis (p=0.004) (Figure 2A), as well as in the cingulate cortex (p=0.032) and the parietal cortex (p=0.043) (Figure 2B). These data indicate that the reduction in Aβ plaques is stronger in animals receiving the AOX diet as a component of treatment, than in animals receiving ENR as a component of treatment.

Figure 2
Aβ load pooled according to diet or environment

Because the duration of treatment spanned an extensive time period (2.69 years), we hypothesized that the treatment effects may reflect a change in the maturation pattern of Aβ accumulation. With age, Aβ accumulates in a stereotypical laminar pattern within the cortex, reflecting a maturation gradient with early stage deposits in the deep layers and late stage deposits occupying more superficial layers (Satou et al., 1997) (Figure 3A). To determine if AOX or ENR conditions influence plaque maturation, the percentage of animals exhibiting either “early” or “late” stage Aβ plaque pathology was calculated in the pooled AOX or ENR groups. Fewer late stage deposits were observed in the prefrontal, cingulate, parietal, and entorhinal cortices in animals receiving the AOX diet as a component of treatment (Figure 3B). In contrast, no consistent difference in early and late stage plaque pathology was observed in groups receiving ENR as a component of treatment (p>0.05, data not shown). These results suggest that the AOX diet may slow the progression of Aβ accumulation within individual brain regions.

Figure 3
Aβ plaque pathology

Treatment effects on Aβ40, Aβ42, and Aβ oligomers

To determine if the different interventions affected amyloid species and assembly states, we assessed the levels of soluble and insoluble Aβ40 and Aβ42 species, and levels of Aβ oligomers. Soluble and insoluble Aβ40 or Aβ42 species were measured by ELISA in the prefrontal, parietal, temporal, and occipital cortices (Figure 4). Mean Aβ40 was lowest in groups receiving AOXs, with the largest observed difference occurring in the parietal region, however, no significant differences in soluble Aβ40 (Figure 4A) or insoluble Aβ40 (Figure 4B) were detected in any brain region. Significantly lower levels of soluble Aβ42 were detected selectively in the prefrontal cortex of the EA group compared to CC controls (p=0.032) (Figure 4C) while no significant changes in insoluble Aβ42 were observed in any region (Figure 4D). Soluble and insoluble Aβ42/40 ratios showed no significant differences between groups in any brain region (p>0.05, data not shown).

Figure 4
Soluble and insoluble Aβ40 and Aβ42 species by ELISA

We next assessed if Aβ oligomers are present in the canine brain and if oligomer levels were modulated by AOX, ENR, or the combination treatment. Oligomeric proteins were assayed in the parietal cortex, as this region showed marked reductions in Aβ plaque load following treatment. An oligomeric protein migrating at 56kDa was detected (Figure 5A) that appears similar to the oligomeric species previously reported in transgenic mouse models of AD. Quantification of the 56kDa band revealed a ~50% reduction (non-significant trend) and reduced within-group variability selectively in the combination EA treatment group (Figure 5B).

Figure 5
Aβ oligomeric proteins

These results indicate that the AOX or ENR interventions alone had little effect on steady-state levels of Aβ40 and Aβ42 species and oligomeric assembly states. However, the combined EA treatment resulted in lower levels of soluble Aβ42 in the prefrontal cortex, and a trend for reduced levels of the 56kDa oligomer in the parietal cortex.

Changes in the APP processing pathway

Taken together, the above data reveal a robust decrease in Aβ load in the canine brain, particularly in response to the combined EA treatment. Several mechanisms may underlie the reduction in Aβ load, including increased clearance by Aβ degrading enzymes, decreased availability of APP, or altered APP processing favoring the non-amyloidogenic pathway.

Levels of Aβ degrading enzymes and APP protein were analyzed by western blot in the prefrontal, parietal, hippocampal, and occipital brain regions. The amyloid clearance proteins neprilysin and IDE remained steady across all treatment groups in all brain regions examined (p>0.05, data not shown). Similarly, there were no trends or significant differences in steady state levels of total APP protein levels in any brain regions, as assessed using both an N-terminal or a C-terminal anti-Aβ antibody (p>0.05, data not shown). These results indicate that neither increased availability of clearance proteins nor decreased availability of APP protein appear to contribute to the observed reductions in Aβ load following treatment with AOX, ENR, or the combined intervention.

Because Aβ accumulation likely reflects a balance between production and clearance mechanisms, we next examined whether differential APP cleavage might explain the reductions in Aβ due to enhanced non-amyloidogenic processing. Key proteins in the APP processing pathway were assessed by western blot in the parietal cortex, a region where Aβ was decreased in response to treatments. CTFα and CTFβ, from APP cleavage by αSEC and βSEC, respectively, remained unchanged with any treatment. Similarly, there were no significant differences in βSEC enzymatic activity (p>0.05, data not shown). However, αSEC enzyme activity, indicative of non-amyloidogenic APP processing, was significantly increased in the combined EA treatment group (20%, p<0.05), but not in the individual AOX or ENR treatment groups (Figure 6A). βSEC activity was unchanged in all treatment groups. To determine which αSEC protein might underlie increases in enzymatic activity, protein levels of the major αSEC candidates, TACE and ADAM10, were quantified. TACE levels were not significantly altered among groups (p>0.05, data not shown), but levels of the precursor to ADAM10 showed a non-significant increase in groups receiving the AOX diet (CA and EA) but not enrichment alone (EC) (Figure 6B and 6C). Protein levels of the mature form of ADAM10 remained unchanged.

Figure 6
Non-amyloidogenic APP processing

Taken together, these results indicate that the treatment interventions do not alter levels of APP or 2 of the major clearance proteases, neprilysin and IDE. However, combined EA treatment appears to increase αSEC enzymatic activity, in the absence of a significant change in βSEC, suggesting a shift to the non-amyloidogenic pathway, consistent with the robust reductions in Aβ plaque deposition observed in the EA treatment group.

Relationship of cognitive performance with measures of Aβ pathology

We next assessed if changes in Aβ pathology correlated with the improved cognitive performance that was previously reported in these dogs in response to ENR, AOX, or the combination of the interventions. Six performance measures had been previously evaluated across all 4 groups of dogs: discrimination errors and discrimination reversal errors in year 2 of treatment, spatial memory phase 10 errors (years 2 and 3 of treatment), and black/white intensity discrimination errors and reversal errors (year 3 of treatment). Two-tailed pearson correlations were calculated for performance and the following variables for the 23 dogs: total Aβ load across brain regions, Aβ load in individual brain regions, levels of Aβ40, Aβ42, oligomers, and αSEC activity. No significant correlation or trend for a correlation was found between any cognitive measure and any measure of Aβ pathology or APP processing. For example, looking at Aβ load across brain regions and behavioral task performance in year 3 of treatment, pearson correlations were as follows: spatial memory performance (r=0.198, p=0.366), black/white intensity discrimination errors (r=-0.096, p=0.662), black/white intensity discrimination reversal errors (r=-0.134, p=0.543). These results suggest that improvements in cognition in response to treatments were minimally related to changes in Aβ pathology.


Previously we have shown that a long-term intervention (2.69 years) with an AOX diet, behavioral ENR, or the combined EA treatment preserves and improves cognitive function in aged canines. While each intervention alone provided benefits, the combination treatment had additive benefits for cognition (Cotman et al., 2002; Milgram et al., 2002b; Milgram et al., 2002a). Here we evaluated the hypothesis that AOX and ENR interventions reduce age-related Aβ neuropathology, as one mechanism that may contribute to the observed cognitive improvement.

The combination of dietary AOXs and behavioral ENR interventions is optimal

Overall, across the treatment groups, the strongest and most consistent effects on Aβ pathology were observed in canines receiving the combined EA intervention. Following combined EA treatment, Aβ load was significantly decreased in several brain regions, soluble Aβ42 was decreased selectively in the frontal cortex, and the Aβ56kDa oligomer showed a trend for lower levels and reduced variability in the parietal cortex. The increase in αSEC activity, in the absence of changes in βSEC activity, suggests that reductions in Aβ may be related to a shift in APP processing toward the non-amyloidogenic pathway. Previously, increased αSEC activity in the absence of changed beta or gamma secretase activity has been linked to moderate decreases in Aβ40 and Aβ42 in calorie restricted primates (Qin et al., 2006), suggesting that shifted processing to the non-amyloidogenic pathway may serve to reduce Aβ in higher animal models. In addition, our findings are consistent with effects of other natural compounds that reduced Aβ and increased αSEC activity, such as ginkgo biloba (Colciaghi et al., 2004) and green tea (Levites et al., 2003; Rezai-Zadeh et al., 2005; Obregon et al., 2006). The absence of a detectable increase in the CTFα fragment concomitant with the 20% increase in αSEC activity may reflect an effect on APP processing that is below detection or that the CTFα fragment is rapidly degraded.

In order to estimate the relative contribution of either AOX or ENR treatment to reductions in Aβ load, treatment groups were pooled according to the inclusion of AOX or ENR as a component of treatment. This analysis revealed significantly reduced Aβ load when animals were pooled according to the presence of AOXs (~50% reduction) but not when pooled according to ENR (~20% reduction). Similarly, the pooled AOX groups (but not the ENR groups) exhibited more early stage deposits and fewer late stage deposits, indicating a slower Aβ plaque maturation and accumulation process. These data indicate that the reduction in Aβ plaques is stronger in animals that received the AOX diet as a component of treatment, as compared with ENR. Interestingly, the effect of the AOX intervention on reducing Aβ load and maturation are enhanced when combined with ENR, resulting in a 80% reduction in amyloid plaque load, even though ENR on its own had little effect.

The concept that combined dietary and behavioral interventions can have superior effects to either approach alone builds upon previous literature. For example, in mice, the effects of voluntary exercise (a component of the ENR paradigm) was improved when exercise was combined with dietary administration of the plant flavonol, epicatechin (van Praag et al., 2007). In addition, in rats, combining exercise with the omega-3 fatty acid docosahexaenoic acid (DHA) resulted in synergistic effects on improving cognition and enhancing synaptic plasticity in the BDNF pathway (Wu et al., 2008). Similarly, we demonstrate that combined dietary and behavioral interventions are more effective at reducing amyloid pathology than either intervention alone.

Does Aβ accumulation influence cognitive decline in higher animal models?

Our data indicate that the combination of AOX and ENR significantly reduced Aβ load in the brain, with only modest effects on levels of soluble and insoluble Aβ species and assembly states. On one hand, the reduction in amyloid load in the absence of reductions in steady-state amyloid levels seems paradoxical. However, Aβ immunization, which decreases Aβ load, has been associated with unchanged or even increased steady-state Aβ levels in humans (Patton et al., 2006). The observed reductions in Aβ load and more immature plaque distribution with EA treatment may reflect a compartmentalization of amyloid, such that with reduced amyloid burden overall, fewer cellular elements are exposed to extracellular amyloid. The more immature plaque distribution pattern may further allow select brain regions to function in a more youthful state even with minimal changes in amyloid levels.

The importance of Aβ accumulation in driving cognitive decline in higher animal models of aging is under ongoing debate. It is well accepted that Aβ impairs cognition in transgenic mouse models of AD, but the case is less clear in higher animal models with a natural course of Aβ accumulation. For example, in aged canines, while Aβ immunization substantially reduced Aβ plaque load and Aβ levels, cognitive performance was largely unaffected, with the exception of some improvement and maintenance in executive function on a reversal learning task after a 2+ year treatment (Head et al., 2008). By comparison, in aged canines given AOX and ENR treatments and administered the same cognitive tasks as immunized animals, there were robust cognitive improvements across multiple measures of learning and memory (Cotman et al., 2002; Milgram et al., 2002b; Milgram et al., 2002a). Similar to the canine immunization results, in recent human clinical trials, immunization of AD patients reduced Aβ load but had only minor effects on cognitive improvement and did not slow disease progression (Gilman et al., 2005; Holmes et al., 2008; Kokjohn and Roher, 2009). This literature suggests that factors in addition to Aβ neuropathology may be important determinants of cognitive performance and the rate of cognitive decline.

The modest effects of AOX, ENR, or the combination intervention on Aβ40 and Aβ42 levels in canines may provide insight into human brain aging. In humans, various lifestyle factors (e.g., education, social networks, and activity participation) can help build cognitive reserve, allowing the brain to tolerate more amyloid pathology, and maintain intact cognitive function (Bennett et al., 2006; Roe et al., 2008b; Roe et al., 2008a).

Multiple mechanisms may underlie the cognitive benefits of AOX and ENR interventions

Multiple mechanisms may ultimately determine the capacity of the AOX and ENR interventions to improve cognitive function. Several mechanisms have been identified, including improved capacity to counteract oxidative stress (Opii et al., 2008), improved mitochondrial function (Head et al., 2009), preserved neuron number (Siwak-Tapp et al., 2008), and increased availability of growth factors such as BDNF (Fahnestock, in press). Interestingly, the combined EA treatment appears to have additive or synergistic effects on several neurobiological endpoints. The molecular endpoint showing the greatest effects of the interventions to date has been an improved capacity to counteract age-related oxidative damage (Opii et al., 2008). In canines, the AOX, ENR or combined EA intervention reduced the extent of oxidative damage and improved the AOX reserve system, by decreasing levels of oxidative stress biomarkers and increasing activity of key enzymes involved in energy metabolism and regulation of oxidative stress (e.g., Cu-Zn superoxide dismutase (SOD) and glutathione-s-transferase (GST)) (Opii et al., 2008). The increases in SOD and GST enzymatic activity were positively correlated with improvements in cognitive performance (Opii et al., 2008). Similar to the correlation between oxidative enzymatic activity and cognitive performance, the extent of neurogenesis correlated with cognitive score across treatment groups, with the strongest relationship in the combined intervention (Siwak-Tapp et al., 2007). In contrast, changes in Aβ pathology observed in the current study did not correlate with improvements in cognitive performance, based on the same cognitive tasks. This literature suggests that the AOX and ENR interventions may engage compensatory molecular mechanisms that build cognitive reserve, allowing the canine to maintain intact cognitive abilities despite the continued presence of Aβ in the brain (Bennett et al., 2006; Roe et al., 2008b; Roe et al., 2008a).


Our data adds to a growing body of knowledge that changes in cognitive function in higher animal models and humans are not consistently linked to changes in amyloid neuropathology. In this study, treatment of aged dogs with AOX, ENR or the combined intervention strongly improved cognitive function, even though amyloid pathology overall was only moderately reduced. While Aβ load was decreased, particularly with the combined intervention, steady state levels of Aβ40 and Aβ42 and oligomeric assembly states were only modestly reduced, despite a shift of APP processing to the non-amyloidogenic pathway. Recent data confirm that multiple mechanisms, perhaps in parallel with modest reductions in Aβ, may contribute to the cognitive health and functional benefits provided by the interventions. Importantly, across all molecular mechanisms, including the Aβ pathway, the strongest effects consistently occurred in the combined EA treatment group, which also sustained the greatest cognitive benefits. These data suggest that multi-domain treatments may be a valuable intervention strategy to reduce pathology and improve cognitive function in humans.

Supplementary Material



The authors would like to thank the following people: Mihaela Nistor and Floyd Sarsoza for immunohistochemistry, Dr. Kim Green for ELISA, Drs. Paul Adlard and Wayne Poon for enzyme activity assays, and Dr. Lori-Ann Christie for manuscripts edits. Funding provided by NIH/NIA AG12694, AG17066 and U. S. Department of the Army, Contract No. DAMD17-98-1-8622.


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