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

Abstract

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. Although 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 (Abeta) neuropathology, as one mechanism mediating observed functional improvements. Measures assessed were Abeta neuropathology in plaques, biochemically extractable Abeta(40) and Abeta(42) species, soluble oligomeric forms of Abeta, and various proteins in the beta-amyloid precursor protein (APP) processing pathway. The strongest and most consistent effects on Abeta pathology were observed in animals receiving the combined antioxidant and enrichment treatment. Specifically, Abeta plaque load was significantly decreased in several brain regions, soluble Abeta(42) was decreased selectively in the frontal cortex, and a trend for lower Abeta oligomer levels was found in the parietal cortex. Reductions in Abeta may be related to shifted APP processing toward the non-amyloidogenic pathway, because alpha-secretase enzymatic activity was increased in the absence of changes in beta-secretase activity. Although enrichment alone had no significant effects on Abeta, reduced Abeta load and plaque maturation occurred in animals receiving antioxidants as a component of treatment. Abeta measures did not correlate with cognitive performance on any of the six tasks assessed, suggesting that modulation of Abeta alone may be a relatively minor mechanism mediating cognitive benefits of the interventions. Overall, the data indicate that multidomain treatments may be a valuable intervention strategy to reduce neuropathology and improve cognitive function in humans.

Figure 1.

Aβ load quantification across treatment groups. A, Mean Aβ plaque accumulation across all brain regions shows selective reductions in the CA and EA groups receiving the antioxidant diet but not with behavioral enrichment alone. B, Aβ load is selectively decreased in the combination treatment EA group in the cingulate cortex, with trends for reduced levels in the parietal and entorhinal regions and no changes in the prefrontal or occipital cortex. Representative images are shown for each of the four treatment groups from the parietal (C) and entorhinal (D) cortices. PFR, Prefrontal; CNG, cingulate; PAR, parietal; ENT, entorhinal; OCC, occipital. Scale bar, 500 μm. ^#p < 0.09 from CC controls; *p < 0.05 from CC controls.

Figure 2.

Aβ load pooled according to diet or environment. A, B, When groups are separated according to diet, significantly lower Aβ load is found in response to antioxidants in the total brain (A) and individual brain region (B) analysis in the cingulate and parietal cortex. C, D, When groups are separated according to environment, there are no significant changes in the total brain (C) or individual region (B) analysis. PFR, Prefrontal; CNG, cingulate; PAR, parietal; ENT, entorhinal; OCC, occipital, Control in A, B, control diet groups CC and EC; Control in C, D, control environment groups CC and CA; AOX, antioxidant diet groups CA and EA; ENR, behavioral enrichment groups EC and EA. *p < 0.05 from Control.

Figure 3.

Aβ plaque pathology. A, Sample images of the possible categorical Aβ plaque types 0, 1, 2, 3, or 4. Early-stage patterns (type 0, 1, or 2) reflect cortical involvement of the deep layers, whereas late stages (type 3 or 4) exhibit superficial layer involvement. B, When groups are separated according to diet, animals receiving antioxidants have fewer late-stage deposits in the superficial cortical layers, including the prefrontal cortex, which did not show significant reductions in Aβ load with treatments. Scale bar, 500 μm. Ctl, Control diet groups CC and EC; AOX, antioxidant diet groups CA and EA.

Figure 4.

Soluble and insoluble Aβ₄₀ and Aβ₄₂ species by ELISA. In general, insoluble Aβ was higher than soluble levels, and more Aβ was detected in rostral versus caudal regions. There were no significant treatment effects in any brain regions in soluble Aβ₄₀ (A) or insoluble Aβ₄₀ (B). C, D,With the exception of significantly lower Aβ₄₂ in the EA group of the frontal cortex, there were no significant changes in soluble Aβ₄₂ (C) or insoluble Aβ₄₂ (D). PFR, Prefrontal cortex; PAR, parietal cortex; TMP, temporal cortex; OCC, occipital cortex. Error bars indicate group mean ± SEM. *p < 0.05 from CC controls.

Figure 5.

Aβ oligomeric proteins. A, Representative samples from the canine parietal cortex show the anti-oligomer A11 antibody detecting the Aβ oligomeric protein at 56 kDa. B, Lower levels and reduced variability of the 56 kDa aggregate are seen in the combination treatment group EA despite a large amount of variability within the groups. IP, Immunoprecipitated canine sample with anti-Aβ_1-16 6E10 antibody. Numbers on left in A indicate kilodalton marker; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 6.

Non-amyloidogenic APP processing. A, α-Secretase activity is selectively increased in the parietal cortex of the combination EA group. B, Representative samples show ADAM10 precursor and mature protein in the parietal cortex of canines. C, Quantification of proteins in B shows a trend toward increased ADAM10 precursor levels in the CA and EA groups. βac, β-Actin; P, ADAM10 precursor protein; M, ADAM10 mature protein. Error bars indicate group mean ± SEM; circles indicate individual data points. *p < 0.05 compared with CC and EC.