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. 2008 Jan;29(1):51-70.
doi: 10.1016/j.neurobiolaging.2006.09.012. Epub 2006 Oct 20.

Proteomic identification of brain proteins in the canine model of human aging following a long-term treatment with antioxidants and a program of behavioral enrichment: relevance to Alzheimer's disease

Wycliffe O Opii  1 , Gururaj Joshi, Elizabeth Head, N William Milgram, Bruce A Muggenburg, Jon B Klein, William M Pierce, Carl W Cotman, D Allan Butterfield
Affiliations

Proteomic identification of brain proteins in the canine model of human aging following a long-term treatment with antioxidants and a program of behavioral enrichment: relevance to Alzheimer's disease

Wycliffe O Opii et al. Neurobiol Aging. 2008 Jan.

Abstract

Aging and age-related disorders such as Alzheimer's disease (AD) are usually accompanied by oxidative stress as one of the main mechanisms contributing to neurodegeneration and cognitive decline. Aging canines develop cognitive dysfunction and neuropathology similar to those seen in humans, and the use of antioxidants results in reductions in oxidative damage and in improvement in cognitive function in this canine model of human aging. In the present study, the effect of a long-term treatment with an antioxidant-fortified diet and a program of behavioral enrichment on oxidative damage was studied in aged canines. To identify the neurobiological mechanisms underlying these treatment effects, the parietal cortex from 23 beagle dogs (8.1-12.4 years) were treated for 2.8 years in one of four treatment groups: i.e., control food-control behavioral enrichment (CC); control food-behavioral enrichment (CE); antioxidant food-control behavioral enrichment (CA); enriched environment-antioxidant-fortified food (EA). We analyzed the levels of the oxidative stress biomarkers, i.e., protein carbonyls, 3-nitrotyrosine (3-NT), and the lipid peroxidation product, 4-hydroxynonenal (HNE), and observed a decrease in their levels on all treatments when compared to control, with the most significant effects found in the combined treatment, EA. Since EA treatment was most effective, we also carried out a comparative proteomics study to identify specific brain proteins that were differentially expressed and used a parallel redox proteomics approach to identify specific brain proteins that were less oxidized following EA. The specific protein carbonyl levels of glutamate dehydrogenase [NAD (P)], glyceraldehyde-3-phosphate dehydrogenase (GAPDH), alpha-enolase, neurofilament triplet L protein, glutathione-S-transferase (GST) and fascin actin bundling protein were significantly reduced in brain of EA-treated dogs compared to control. We also observed significant increases in expression of Cu/Zn superoxide dismutase, fructose-bisphosphate aldolase C, creatine kinase, glutamate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase. The increased expression of these proteins and in particular Cu/Zn SOD correlated with improved cognitive function. In addition, there was a significant increase in the enzymatic activities of glutathione-S-transferase (GST) and total superoxide dismutase (SOD), and significant increase in the protein levels of heme oxygenase (HO-1) in EA treated dogs compared to control. These findings suggest that the combined treatment reduces the levels of oxidative damage and improves the antioxidant reserve systems in the aging canine brain, and may contribute to improvements in learning and memory. These observations provide insights into a possible neurobiological mechanism underlying the effects of the combined treatment. These results support the combination treatments as a possible therapeutic approach that could be translated to the aging human population who are at risk for age-related neurodegenerative disorders, including Alzheimer's disease.

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Figures

Figure 1
Figure 1
Changes in protein carbonyls (A), 3NT (B) and HNE (C) levels in canine brain homogenate samples following treatment. There was a decrease in the levels of protein carbonyls, 3NT and HNE measured from the various treatments, i.e. EC, CA and EA compared to the control group CC. Data are represented as % control ± SEM for animals in each treatment group. Measured values are normalized to the CC values (n=6) * p < 0.05 for canines on EA treatment.
Figure 2
Figure 2
Combined treatment of aged dogs with an antioxidant enriched diet and behavioral enrichment leads to reduced protein oxidation. Carbonyl immunoblots showing proteins with less oxidation in the parietal cortex of canines given a combined treatment with an antioxidant fortified diet and an exposure to a behavioral enrichment programme EA) (B) as compared to control (CC) (A).
Figure 3
Figure 3
SYPRO Ruby-stained 2D-gels maps, (A) CC vs. CE, (B) CC vs. CA and (C) CC vs. EA of canine parietal cortex homogenates samples from the CC CE,CA and EA treated animals are presented. Proteins identified by mass spectrometry are presented as the boxed spots.
Figure 4
Figure 4
Validation of proteins identified by proteomics. (A) shows immunoblot probed with anti GADPH antibody. Lanes 1-3 represent CC, while lanes 4-6 represent EA, and a graphical quantification of the blot also is shown. (B) shows an immuno-oxyblot of GAPDH and a graphical representation, validating the identification of reduced oxidation of GAPDH (n=3).
Figure 5
Figure 5
Validation of reduced oxidation of proteins (CuZnSOD) identified by proteomics. An immunoblot of the expression of Cu-Zn SOD, blots probed with anti CuZnSOD is shown. Lanes 1-3 represent CC, while lanes 4-6 represent EA. A graphical quantification of the blot also is shown. (n=3).
Figure 6
Figure 6
SOD and GST activity are significant increased in response to treatment in aged dogs. Dogs provided with the combination of an antioxidant-fortified diet and behavioral enrichment show significantly increased GST and total SOD enzyme activity relative to controls. Activities of GST and SOD are expressed as units per milligram of protein and data are presented as % control ± SEM for animals in each treatment group, (n=5) * p <0.05.
Figure 7
Figure 7
HO-1 protein levels increase in response to treatment in aged canines. Western immunoblot analysis and quantification of canine brain homogenates samples containing 50μg of protein loaded onto 10% SDS-PAGE gels were completed using an anti-HO-1 antibody. A representative immunoblot (A) with lanes (1-6) representing the treatment group EA, and lanes 7-12 representing the control group CC is shown. GAPDH was used as a control for equal loading of protein. Densitometric values are plotted as a function of treatment group (B) showing a significant increase in HO-1 expression following the combined treatment of enriched environment and antioxidant-fortified food (EA) compared to control (CC). GAPDH densitometric data are represented as % control; ± SEM for each group. (n=6), * p <0.05.
Figure 8
Figure 8
Individual error scores are plotted as a function of CuZnSOD protein levels in the parietal cortex. A. Black/white reversal learning was poorer in animals with lower levels of SOD. B. Spatial learning was also impaired in animals with lower SOD protein levels. Line represents the results of a linear regression analysis.
Figure 9
Figure 9
Association between cognitive test scores and measures of oxidative damage in treated animals. Shows error scores on individual cognitive tasks associated with reduced oxidative damage or increased antioxidant enzyme/protein levels. Higher error scores on tests of black/white discrimination, black/white reversal and spatial memory were associated with higher levels of oxidative damage. Higher error scores on a reversal learning task (A) and on a visuospatial memory task (B) were correlated with 3-NT. Discrimination learning ability was inversely associated with GST activity (C). Reversal learning error scores were also negatively associated with HO-1, with higher levels of HO-1 associated with better cognition (D).
Figure 10
Figure 10
Schematic diagram of a functional interacteome of all parietal cortex proteins identified to be significantly less oxidatively modified following the combined treatment of the enriched environment and antioxidant-fortified food (EA). This diagram was generated by the interaction explorer ™ Pathway Module (Stratagene), indicating that all the proteins directly or indirectly is associated with cellular process shown.
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