Changes in cerebral blood volume and amyloid pathology in aged Alzheimer APP/PS1 mice on a docosahexaenoic acid (DHA) diet or cholesterol enriched Typical Western Diet (TWD)
Introduction
Worldwide, 15 million people suffer from Alzheimer’s disease (AD). This number will continue to grow as a result of our aging population. Our western population is not only getting older, but has also developed a less healthy lifestyle with high cholesterol and high caloric diets, resulting in overweight with its associated disorders. Hypercholesterolemia for example is an important risk factor in vascular disorders, and a link between vascular risk factors, hypercholesterolemia and AD has been suggested (Breteler, 2000, Casserly and Topol, 2004, de la Torre, 2002b, Kivipelto et al., 2001, Luchsinger et al., 2005). Epidemiological studies have indicated that cholesterol lowering statins diminish the prevalence of AD (Jick et al., 2000) and cause less deterioration of cognitive functions (Sparks et al., 2005). Identification of the cholesterol transporter apolipoprotein E4 as a major genetic risk factor for hypercholesterolemia, vascular dementia and sporadic AD (Corder et al., 1994, Poirier et al., 1993, Strittmatter et al., 1993), also reinforces the relationship between cholesterol and AD. Further support for the link between high cholesterol intake and AD comes from experimental animal studies. In cell culture studies, reduction of intracellular cholesterol levels in rat hippocampal neurons inhibits the formation of amyloid beta (Aβ) (Simons et al., 1998) and animal studies with double transgenic (amyloid precursor protein (APP)–presenilin (PS)) mice have shown that high dietary cholesterol increases Aβ accumulation (Refolo et al., 2000) and cholesterol lowering agents increase processing of APP through the non-amyloidogenic α-secretase pathway in different cell lines via increased membrane fluidity (Kojro et al., 2001).
Membrane fluidity can be influenced by alterations in membrane phospholipid and cholesterol content (Kirsch et al., 2003, Wolozin, 2001, Yeagle, 1991), which in turn, can modulate the activity of membrane-bound enzymes (Criado et al., 1982, Mitchell et al., 1990, Oner et al., 1994, Sinha et al., 1977) such as the main enzymes cleaving the transmembrane protein APP to generate Aβ (β- and γ-secretase). It has been suggested that these secretases require cholesterol-rich lipid domains (lipid rafts) within the membrane (Fassbender et al., 2001, Wahrle et al., 2002), whereas the non-amyloidogenic pathway by α-secretases needs cholesterol-poor membranes, such as phospholipid domains which are more fluid (Wolozin, 2001, Wolozin, 2004).
Important components of membrane phospholipids that contribute to membrane fluidity are the omega-3 long chain poly-unsaturated fatty acids (n-3 LCPUFAs), like docosahexaenoic acid (DHA) and the omega-6 (n-6) LCPUFAs (such as arachidonic acid). Further, increased DHA levels in membranes are known to augment membrane fluidity (Hashimoto et al., 2006). Epidemiological studies show that sufficient DHA intake reduces the risk of developing AD (Barberger-Gateau et al., 2002, Kalmijn et al., 1997, Morris et al., 2003). In support of this notion, two recent experimental studies reported decreased brain Aβ levels in APP transgenic mice after dietary DHA supplementation (Lim et al., 2005, Oksman et al., 2006). In addition to its influence on Aβ metabolism, DHA has also a positive effect on vascular health (Breslow, 2006, de Wilde et al., 2002, de Wilde et al., 2003).
Taken together, these data indicate that high dietary cholesterol levels or low DHA levels are risk factors in AD. However, it remains unclear how these dietary factors may influence the development of AD. It can be hypothesized that high cholesterol intake impairs the vasculature causing hypoperfusion of vulnerable brain areas, ultimately leading to an overproduction of Aβ. The increased Aβ production may also result in cerebral amyloid angiopathy (CAA) further exacerbating cerebrovascular degeneration (Bennett et al., 2000, Lin et al., 1999, van Groen et al., 2005). In contrast, high intake of DHA may have a protective effect by improving vascular health and accordingly cerebral perfusion.
In a recently published study of our collaborators (Oksman et al., 2006) it was found that a Typical Western Diet (TWD) containing 1% cholesterol increases hippocampal Aβ levels in the double transgenic APP/PS1 mice at 10 months of age. In contrast, a DHA-enriched diet decreased hippocampal Aβ levels of the same mice compared to the TWD group. However, although these in vivo dietary effects could be detected with ELISA, immunohistochemically determined Aβ plaque load was unaffected. These findings raise the obvious question whether life-long dietary factors may have an even stronger impact on Aβ pathology.
In the present study we used the same APP/PS1 transgenic mice and the same effective dietary manipulations as Oksman et al. to study the long-term effects of dietary lipids on Aβ accumulation and brain circulation. This mouse line has rapid age-dependent accumulation of Aβ into extracellular plaques but relatively little vascular deposition, which allows the assessment of dietary lipids on both parenchymal Aβ accumulation and vascular function. We started the dietary manipulation at 6 months of age and continued up to 18 months. To address the diet effects on brain circulation we measured relative cerebral blood volume (rCBV) and cerebral blood flow (CBF). Both parameters have been shown to be affected in AD (Harris et al., 1998, Iadecola, 2003, Tohgi et al., 1998). We used 2H MR spectroscopy with the freely diffusible tracer deuterium oxide, and susceptibility enhanced MRI, a technique that has been widely used in experimental stroke studies in rats but never applied to APP transgenic mice before. In addition immunohistochemical analysis of neuropathology and a chemical assessment of sterol and fatty acid profiles of the brain were performed. The findings indicate that dietary lipids can impact both brain circulation and deposition of Aβ in plaques and blood vessel walls.
Section snippets
Animals and diets
The APPswe/PS1dE9 founders were obtained from Johns Hopkins University, Baltimore, MD, USA (D. Borchelt and J. Jankowsky, Dept. Pathology) and a colony was established at the University of Kuopio Finland. In short, mice were created by co-injection of chimeric mouse/human APPswe (mouse APP695 harboring a human Aβ domain and mutations K595N and M596L linked to Swedish familial AD pedigrees) and human PS1-dE9 (deletion of exon 9) vectors controlled by independent mouse prion protein promoter
Body and brain weight
Mice were weighed before assigning them to the different diet groups. The body weight did not differ between the groups neither at the start (STD(wt) 32.1 ± 1.0; STD(tg) 31.9 ± 1.0; TWD(tg) 32.2 ± 1.2; DHA(tg) 32.4 ± 1.1) nor at the end of the experiment (STD(wt) 40.7 ± 1.7; STD(tg) 43.5 ± 2.5; TWD(tg) 41.0 ± 2.0; DHA(tg) 45.0 ± 2.4). Thus, body weight was not affected by genotype (p = 0.36) or dietary manipulations (p = 0.56). Also brain weight did not differ between the genotypes (p = 0.71) or diet groups (p = 0.19;
Discussion
In this study we investigated the long-term effects of dietary cholesterol and docosahexaenoic acid (DHA) in the development of AD-like pathology and brain circulation in transgenic APP/PS1 mice. Both cholesterol and DHA may affect the degenerative processes in AD by influencing the Aβ metabolism via indirect effects on vasculature. High serum cholesterol may contribute to the development of AD via hypoperfusion of the brain leading to the elevated production of Aβ (Bennett et al., 2000, de la
Acknowledgments
We would like to thank David Borchelt and Janina Jankowsky (Johns Hopkins University, Baltimore) for providing us with the mice and Henk Arnts for his excellent care giving of our animals. The laboratory work on sterol analysis of Anja Kerksiek and Sylvia Friedrichs from the Department of Clinical Pharmacology, University of Bonn, Germany, is also gratefully acknowledged. Martin Balvers at Numico Research, Wageningen, skillfully performed all PL and FA analyses. This study is partly supported
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