Oxidative stress impairs learning and memory in apoE knockout mice

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Abstract

Cardiovascular risk factors, such as oxidative stress and elevated lipids, are linked to the development of cognitive impairment. A mediator common to both stressors is the apolipoprotein E (apoE). The objectives of this study are to determine the effects of apoE deficiency and diet-induced systemic oxidative stress in mice on vascular expression of inflammatory proteins and on cognitive function. Mice are placed on a diet enriched in homocysteine for fifteen weeks and then assessed for spatial learning using an eight-arm radial maze and for inflammatory protein expression by immunohistochemistry. Our results show that diet-induced oxidative stress does not affect cognitive function in normal mice. In contrast, apoE−/− mice on the homocysteine diet show significantly impaired (p < 0. 001) maze performance. ApoE−/− mice also have high cholesterol levels. There is no expression of inflammatory proteins IL-6 and IL-8 in the vasculature of control mice on normal or homocysteine diet and little in apoE−/− mice on normal diet. In contrast, apoE−/− mice on homocysteine diet show pronounced vascular reactivity to IL-6 and IL-8 antibodies. These data show that systemic oxidative stress correlates with expression of inflammatory proteins in the cerebral vasculature and impaired cognitive function. These results are consistent with the hypothesis that an oxidative-inflammatory cycle in the cerebral vasculature could have deleterious consequences for cognition.

Introduction

Increasing evidence links cardiovascular-associated risk factors to the development of cognitive impairment, especially in Alzheimer's disease (AD). Both oxidative stress and elevated lipids correlate with increased risk of developing dementia (Herrmann and Knapp, 2002; Rojo et al., 2006). A mediator common to both stressors is the lipid-carrying protein apolipoprotein E (apoE). Apolipoprotein E is a vital component in lipoprotein metabolism (Hirsch-Reinshagen et al., 2009), but may also underlie biological processes and pathologic conditions independent of lipid metabolism, including AD, cognitive function, and immunoregulation (Mahley and Rall, 2000). After liver, the highest levels of apoE expression are found in the nervous system (Boyles et al., 1985, Elshourbagy et al., 1985), supporting an important role for this protein in neural function. Inheritance of the apoE allele 4 increases the risk of developing late-onset AD (Lendon et al., 2000, Wakutani et al., 2002). Impairments in cognitive performance have been observed in aged apoE-deficient (apoE−/−) mice (Champagne et al., 2002, Grootendorst et al., 2005, Troen et al., 2006, van Meer et al., 2007, van Praag et al., 2005, Zhou et al., 1998). Absence of apoE also correlates with diminished anti-oxidative capacity in animals (Law et al., 2003). Finally, in aged mice lack of apoE has a destabilizing effect on the cerebral microcirculation leading to blood-brain barrier leakage (Hafezi-Moghadam et al., 2007).

Consistently, studies report a correlation between cognitive function and the serum concentrations of folate (Mihalick et al., 2003, Morris, 2002), vitamin B-12 (Selhub, 1999), vitamin B-6 (Borroni et al., 2002, Miller et al., 2002), and more recently homocysteine (Auer et al., 2002, Herrmann & Knapp, 2002, Korczyn, 2002, McIlroy et al., 2002, Mizrahi et al., 2002, Nilsson et al., 2002, Nourhashemi et al., 2000, Prins et al., 2002, Reutens & Sachdev, 2002; Shea & Rogers, 2002, Sheehan & Fazel, 2002, Vermeer et al., 2002). Hyperhomocysteinemia promotes oxidative stress by increasing formation of reactive oxygen species (Papatheodorou and Weiss, 2007).The damaging effects of homocysteine may influence normal development as well as age-related decline. Addition of methionine to the diet of pregnant rats induces hyperhomocysteinemia and leads to the formation of sustained oxidative stress in the brains of progeny (Makhro et al., 2008). Newborn animals are characterized by lower body weight, cerebral superoxide dismutase deficiency, and impaired cognitive capacity. In aging, plasma levels of homocysteine are predictive of cognitive decline (Herrmann et al., 2007). Hyperhomocystemia has been implicated in the pathogenesis of neurodegenerative and neuropsychiatric disorders including AD, stroke and vascular dementia (Reutens & Sachdev, 2002, Kronenberg et al., 2009). Finally, because homocysteine can damage vascular endothelial cells (Papatheodorou and Weiss, 2007), homocysteine may contribute to the pathogenesis of CNS disorders by causing both cerebrovascular injury as well as direct neurotoxicity.

The brain microvasculature could be a convergence point for the effects of oxidative stress and lipid injury in health and disease. In AD, we have shown that brain microvessels express oxidized proteins and release high levels, compared to control-derived vessels, of a number of inflammatory proteins including tumor necrosis factor-α (TNFα), interleukin (IL) IL-1β, IL-6, and IL-8 (Grammas & Ovase, 2001, Grammas et al., 2006). Recent brain imaging data from humans and animals suggest that cerebrovascular dysfunction may precede cognitive decline and neurodegeneration in AD and AD animal models (Bell and Zlokovic, 2009). The effects of oxidative stress in apoE knockout mice on brain vascular inflammation and cognition are unknown.

In humans, assessment of vascular inflammation in the context of cognitive function is not possible. However, behavioral experiments in rodents can be utilized to model memory and cognitive functions, while assessing vascular inflammation. In this regard, the 8-arm radial maze (RAM), developed by Olton and Samuelson (1976), is a widely used measure of cognitive function that captures spatial learning and working memory in rodents (Hodges, 1996).

The objectives of this study are to determine the effects of apoE deficiency and diet-induced systemic oxidative stress in mice on vascular expression of inflammatory proteins and on cognitive function, as assessed by behavioral experiments in the radial arm maze.

Section snippets

Animals and diet

Animal procedures were performed in accordance with NIH “Guide for the Care and Use of Laboratory Animals” and Texas Tech University Health Sciences Center Institutional Animal Care and Use Committee (IACUC) guidelines. Due to limited availability at the time of the study, ApoE knockout (−/−) mice were bred in our research facility. One male and three female breeders (4–6 weeks old) were purchased from Jackson laboratory (Bal Harbor, Maine 04609). ApoE (−/−) were homozygous for the ApoE tm1Unc

Results

Analysis of blood lipids in control and apoE−/− mice showed that baseline total cholesterol was significantly elevated in apoE−/− mice compared to levels in control mice (Fig. 1). Placement of animals on the homocysteine diet for 2 months did not affect total cholesterol levels in either group (Fig. 1). At the start of experiments (block 1), groups did not differ with regard to accuracy of maze performance (p = 0.14). However, as training progressed, the level of performance dissociated across

Discussion

ApoE knockout mice are a widely used model of hypercholesterolemia. These animals, on standard chow diet, spontaneously develop atherosclerosis and have total cholesterol levels in excess of 500 mg/dL (Meir and Leitersdorf, 2004). In the current study, these animals demonstrate high cholesterol levels and show modest impairment of cognitive function. These data are consistent with a body of literature that demonstrates a link between hypercholesterolemia and brain function. In a cholesterol-fed

Acknowledgments

This work was supported in part by grants from the National Institutes of Health (AG15964, AG020569 and AG028367). Dr. Grammas is the recipient of the Shirley and Mildred Garrison Chair in Aging. The authors gratefully acknowledge the secretarial assistance of Terri Stahl.

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