3.1 Changes in Aβ with age
For initial analyses dogs were divided into young (mean=4.12 +/− 0.19 SEM yrs) and old (mean=13.95 +/− 0.30 SEM yrs) groups, and Aβ extracellular plaques were quantified as a measure of Aβ load in the prefrontal, parietal, cingulate, entorhinal, and occipital cortices. Representative sections of Aβ immunostaining from the old and young beagles () demonstrate overall increases in Aβ plaques across brain regions and cortical layers with age. Aβ plaque load was significantly increased across all cortical layers (), specifically in the prefrontal (p=0.006), parietal (p=0.006), cingulate (p=0.006), entorhinal (p=0.028), and occipital cortices (p=0.045).
To detect changes in Aβ species, we measured soluble and insoluble levels of Aβ40 and Aβ42 in the young and old dogs in the prefrontal, parietal, temporal, and occipital cortices. Soluble levels were obtained using RIPA buffer and insoluble Aβ levels were from the FA buffer fraction. There were general increases in Aβ with age across most brain regions and tissue fractions (). The frontal cortex showed the most consistent increases with age, having significantly more soluble Aβ40 (p=0.011), soluble Aβ42 (p=0.044), insoluble Aβ40 (p=0.006), and insoluble Aβ42 (p=0.006) compared to the young animals. The parietal cortex also showed significant increases in soluble Aβ40 and Aβ42 (p=0.028 and p=0.045, respectively) and insoluble Aβ42 (p=0.006), but no significant changes for insoluble Aβ40 (p>0.1). Aβ levels in the temporal and occipital cortices were very low overall in comparison to other regions. Insoluble Aβ42 levels in the temporal cortex were significantly increased with age (p=0.028), with no changes in soluble levels of Aβ40 or Aβ42, or insoluble Aβ40 (p>0.1 for all) as measured by ELISA. Similarly, occipital levels of insoluble Aβ42 were increased with age (p=0.018), but no changes were found in soluble levels of Aβ40 or Aβ42 (p>0.1 for both). Marginal increases for insoluble Aβ40 were found in the occipital cortex (p=0.068). The results from the ELISA suggest that deposition of Aβ42 occurs prior to Aβ40, and the emergence of Aβ first in the RIPA soluble fractions and then in the FA insoluble pool.
Soluble and insoluble Aβ40 and Aβ42 in young and old canines
To obtain a more precise measure of changes in Aβ profiles in the temporal cortex, we quantified Aβ in a larger group of canines with ages ranging from 1 to 16 years old. We obtained a chronological profile of soluble and insoluble Aβ accumulation in the lateral temporal cortex by ELISA (). Aβ40 and Aβ42 were detected in cellular fractions obtained by serial extraction with PBS (), SDS (), and FA buffers (). There was a significant linear relationship with age in soluble levels of Aβ40 (r=0.469, p=0.007, ) and in Aβ42 (r=0.563, p=0.001, ) extracted in PBS buffer. In SDS buffer, Aβ40 was associated with increased age (r=0.339, p=0.058, ) and Aβ42 was significantly increased with age (r=0.651, p<0.001, ). In the insoluble FA fraction, Aβ levels remained stable until 12 years of age, at which point dogs exhibited a spike in Aβ accumulation with a high amount of individual variability with an increase in Aβ40 (r=0.348, p=0.051, ) and a significant increase in Aβ42 (r=0.502, p=0.003, ).
Soluble and insoluble Aβ40 and Aβ42 in canines across the lifespan
3.2 Detection and quantification of oligomeric proteins
We next hypothesized that increases in Aβ in the soluble fraction reflected increases in soluble oligomeric proteins, we proceeded to find Aβ-specific oligomers in the canine brain. An initial study determined that oligomeric proteins were most abundant in the soluble PBS cellular fraction as detected by an anti-oligomer antibody A11 (Supplemental Figure 1
). A follow-up experiment using immunoprecipitation with anti-Aβ antibodies prior to immunoblotting with A11 selectively revealed Aβ oligomers with a prominent band at 56kDa (Supplemental Figure 2
). We also confirmed increases in oligomeric proteins with age using dot blots of the soluble PBS tissue fraction of young and old dogs, and detected significant increases with the anti-oligomer antibodies M204 (p=0.051) and I11 (p=0.004) (Supplemental Figure 3
We proceeded to look at age-related changes in early Aβ assembly states in the larger cohort of animals. We first analyzed the Aβ oligomer at 56kDa using the PBS fraction of the lateral temporal cortex from 34 dogs ranging in age from 1 to 16 years old. Quantification revealed significant increases with age in the 56kDa Aβ oligomer (r=0.41, p=0.02, ).along with a large amount of individual variability. In addition, parietal cortex available from a subset of the same animals was grouped into young (mean=2.61 +/− 0.62 SEM yrs), middle age (mean=6.98 +/− 0.49 SEM yrs), and old groups (mean age=14.39 +/− 0.58 SEM yrs). Following size fractionation, aliquots were analyzed for total Aβ by ELISA and showed a differential age profile for Aβ monomers and Aβ aggregates (). Young dogs showed high levels of monomeric Aβ and almost no aggregated protein, while middle-aged dogs showed the opposite trend with low levels of monomeric Aβ and increased Aβ aggregates. Old animals exhibited both Aβ pools, with prominent peaks for monomeric and aggregated Aβ assembly states. Thus, we detected Aβ oligomeric proteins in canines and show that they gradually accumulate beginning in middle age and throughout the rest of the lifespan.
Age-related changes in Aβ assembly states
3.3 Changes in the APP processing pathway with age
Several mechanisms may underlie the accumulation of Aβ with age, including increased availability of APP, decreased clearance by Aβ degrading enzymes, or altered processing favoring the amyloidogenic pathway. To determine whether age-related increases in Aβ, regardless of its assembly state, are due to changes in the APP processing pathway, we used cortical tissue from young and old canines.
To determine whether increases in APP might account for increases in Aβ, we measured total APP protein levels by Western blot () in tissue available from the prefrontal, parietal, hippocampal, and occipital cortex of young and old canines. Unexpectedly, total APP was significantly decreased with age in all examined regions. Significant decreases were found in the prefrontal cortex (p=0.008, and F), parietal cortex (p=0.005, and F), hippocampus (p=0.013, and F), and occipital cortex (p=0.0001, and F). These results indicate that that increased availability of APP does not account for increased Aβ accumulation with age.
We next investigated whether decreased Aβ clearance may be responsible for increased age-related accumulation of Aβ. We measured protein levels of NEP and IDE by Western blot in tissue available from the parietal cortex of young and old canines (figure not shown). No significant changes were detected with age in NEP (p>0.1) or IDE (p>0.1), suggesting that decreased enzymatic clearance by lower protein levels of NEP or IDE does not account for increased Aβ accumulation with age.
Because Aβ accumulation likely reflects a balance between production and clearance mechanisms, we next asked if differential APP cleavage might explain our previous findings. Using commercially available αSEC and βSEC enzyme activity kits, we found that old animals displayed a significant 20% decrease in αSEC activity (p=0.004, ) indicative of decreased non-amyloidogenic APP processing with age. In addition, aged dogs showed a 24% increase in (SEC activity with age (p=0.024, ) indicative of increased amyloidogenic APP processing. With changes in APP enzymatic cleavage indicating a significant shift towards amyloidogenic processing in aged dogs, we expected that the APP CTF proteins would show a similar trend (). In agreement with increased (SEC activity, aged dogs displayed significantly higher levels of CTF(protein compared to young animals in the parietal cortex (p=0.02, ) and significantly higher levels of the (-secretase protein BACE1 in the parietal cortex (p=0.05, ). However, we found no significant changes in CTF(protein (p>0.1, ) and thus proceeded to look at the protein levels of one of the candidate (SEC enzymes, ADAM10. The antibody detected both the precursor and mature forms of ADAM10 in the parietal cortex of young and old canines (). Quantification of both proteins revealed a significant decrease in the ADAM10 precursor (p=0.045, ) with very low levels in aged animals. Likewise, the mature form of the protein was significantly decreased in aged animals compared to young (p=0.0001, ). Overall, these findings indicate a shift in APP processing that favors amyloidogenesis with age.
Enzyme activity levels of alpha and beta secretase
Protein levels of C-terminal fragments alpha and beta
Levels of BACE1 protein as a function of age
Levels of ADAM10 precursor and mature protein