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 (). 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) (). 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) (). Representative images from the parietal and entorhinal cortex are shown in , respectively.
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 (), but not in animals receiving ENR treatment (). The pooled AOX groups showed significantly lower Aβ load in the total brain analysis (p=0.004) (), as well as in the cingulate cortex (p=0.032) and the parietal cortex (p=0.043) (). 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.
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
) (). 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 (). 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.
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 (). 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 () or insoluble Aβ40 () 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) () while no significant changes in insoluble Aβ42 were observed in any region (). Soluble and insoluble Aβ42/40 ratios showed no significant differences between groups in any brain region (p>0.05, data not shown).
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 () 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 ().
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 (). 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) (). Protein levels of the mature form of ADAM10 remained unchanged.
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.