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

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), α-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.

Introduction

Aged dogs naturally develop cognitive deficits and accumulate brain pathology that is similar to aging humans providing a useful model for studying the neurobiological mechanisms underlying age-related cognitive dysfunction [52], [53]. Aged canines show reduced cerebral volume, cortical atrophy and ventricular widening by in vivo magnetic resonance imaging [103], [110], [111]. The aging canine also shows impairments in visuospatial working memory and executive function [36], [102], [108]. Aged beagle brain accumulates amyloid-β-peptide (Aβ) that is of the same sequence as humans [63], [96] and is correlated with decline in cognitive function with age [43], [51]. Beagle dogs are accessible, easy to handle, capable of learning a broad repertoire of cognitive tasks, do not need food deprivation to be motivated and absorb dietary nutrients in similar ways as humans, hence making them a good model for dietary treatments [44]. The deposition of Aβ could play a significant role in molecular pathways involving free radical generation and oxidative stress as previously shown in AD-related studies from our laboratory [18], [23].

The brain is particularly vulnerable to oxidative damage due to its relative lack of antioxidant capacity, high concentration of unsaturated fatty acids, and high consumption rate of oxygen [74]. Oxidative stress leads to damaged to DNA, proteins and lipids that may consequently lead to dysfunction in various proteins or enzymes involved in several neurodegenerative disorders [16], [71], [76].

The aging process is associated with a progressive accumulation of oxidative damage that could play a role in the development or accumulation of neuropathology typically observed in age-related neurodegenerative disorders like AD [17], [57], [73], [74]. When compared to age-matched controls, the AD brain shows a higher levels of protein and DNA oxidation, and lipid peroxidation leading to loss of function of key enzymes [56], [73], [99]. In various AD studies from our laboratory, we have shown that Aβ1–42 plays a central role in the oxidative stress observed and that the key to this link is a key amino acid residue methionine 35 [18], [23]. Similar events may also occur in the canine model of aging as deposits of Aβ1–42 may account for increased oxidative damage, a decline in glutathione content and decreased glutamine synthetase (GS) activity reported previously [54].

The use of antioxidants and/or related compounds reduces the level of oxidative damage and delays or reduces age-related cognitive decline in both animal models and in humans [10], [64], [78]. Previous studies in aged canines show that oxidative damage may be critically involved in the maintenance of cognitive function and long-term treatment with antioxidants and a program of behavioral enrichment reduces cognitive decline [40], [54], [79], [80]. In the canine model of human aging, short term and long-term treatment with a diet rich in a broad spectrum of antioxidants leads to rapid and sustained learning ability and improved spatial attention; these effects were further enhanced with the addition of behavioral enrichment [78], [40]. However, the neurobiological changes elicited by these two interventions alone or in combination have yet to be established.

In the present study, we hypothesized that a possible mechanism for the improvement of cognition in aged treated animals may be mediated through the protection of neuronal function as a consequence of reduced oxidative damage and improved antioxidant reserves and possibly an increase in the expression of key brain proteins associated with neuronal improvement. We report that the use of antioxidants composed of mitochondrial cofactors and cellular antioxidants and a program of behavioral enrichment in the present study could potentially protect proteins from oxidative damage and enhance mitochondrial function leading to the observed improved memory and cognitive function in this model.