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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online May 21, 2002 as doi:10.1096/fj.02-0012fje. |
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Center for Experimental Therapeutics and Department of Pharmacology and
* Center for Neurodegenerative Disease Research and Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA
2Correspondence Center for Experimental Therapeutics, Department of Pharmacology, University of Pennsylvania, BRB2/3, room 812, 421, Curie Blvd., Philadelphia, PA 19104, USA. E-mail: domenico{at}spirit.gcrc.upenn.edu
SPECIFIC AIM
Aluminum (Al) exposure has been implicated in the Alzheimer’s disease (AD) pathogenesis and its known capacity to exacerbate oxidative events has been suggested as one possible mechanism of its neurotoxicity. To test this hypothesis, we fed transgenic mice overexpressing human amyloid precursor protein (Tg 2576) and wild-type (WT) littermates with a diet enriched in Al alone or in combination with the natural antioxidant vitamin E. Brain levels of the isoprostane 8,12-iso-iPF2
-VI, a specific marker of in vivo lipid peroxidation, as well as amyloid ß peptide and deposition were measured.
PRINCIPAL FINDINGS
1. Dietary aluminum increases in vivo lipid peroxidation
Tg 2576 receiving a diet supplemented with Al showed an increase in 8,12-iso-iPF2-VI urinary levels that occurred earlier and attained higher levels than Tg mice on regular chow. This increase was already significant after 6 months on the Al diet (3.1±0.1 vs. 2.2±0.06 ng/mg of creatinine, P<0.01) and was nearly doubled by 12 months of age (5.3±0.2 vs. 2.9±0.1 ng/mg of creatinine, P<0.01). Compared with baseline, WT receiving an Al-enriched diet also showed a slight increase in 8,12-iso-iPF2-VI levels. However, the values were much lower than the ones observed in Tg animals on chow (0.6±0.02 vs. 1.2±0.1 ng/mg of creatinine, P<0.05). Similar effects of Al were observed for plasma 8,12-iso-iPF2-VI levels in Tg 2576 and WT (not shown). A direct correlation was found between urinary and plasma levels of 8,12-iso-iPF2-VI (r2=0.95, P<0.001). Animals were killed at 12 months of age, neocortices and hippocampi were dissected, and 8,12-iso-iPF2-VI levels were assessed. Analysis showed that Tg animals receiving Al-supplemented chow had significantly higher levels of 8,12-iso-iPF2-VI in cortex and hippocampus compared with Tg animals on regular chow (Fig. 1
). WT receiving an Al-supplemented diet had a slight increase in brain iP levels, which were always lower than in Tg mice on chow (21±3 vs. 38±2 pg/mg, P<0.05). No signs of somatic toxicity (weight loss, red eyes, fur loss) or neurotoxicity (circling) were observed during the entire study in any animal group.
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2. Dietary aluminum accelerates amyloid ß peptide formation and plaque deposition
We next assessed Aß levels and deposition in brains harvested from 12-month-old mice. Levels of Aß1–40 and Aß1–42 in the soluble and insoluble fractions of brain homogenates were measured by a sensitive Aß sandwich ELISA. Significantly, Tg mice on Al-supplemented diet had elevated levels of soluble and insoluble Aß1–40 and Aß1–42 in cortex and hippocampus when compared with Tg animals on chow (Fig. 2
). A direct correlation was observed between insoluble Aß1–40, Aß1–42 and 8,12-iso-iPF2-VI levels (r2=0.68, r2=0.65, respectively; P<0.01 for both). WT on the Al-enriched diet did not show any detectable levels of Aß1–40 or Aß1–42.
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Analysis of the amount of Aß brain deposition (i.e., amyloid plaque burden) using immunohistochemistry further confirmed these observations. Although significant biochemical changes in Aß levels occurred in Tg 2576 mice at 12 months of age (Fig. 2)
, only minimal histological evidence of Aß deposition occurred in these mice on chow. By contrast, Tg 2576 mice receiving chow supplemented with Al showed an appreciable increase in the amyloid plaque burden in the neocortex and hippocampus. Quantitative analyses showed consistently that the amyloid plaque burden was higher in Tg animals receiving a diet supplemented with Al than regular chow in all of the three brain regions examined (somatosensory cortex, peri-hippocampal cortex, and hippocampus). As predicted, no positive reaction was observed in any WT controls (not shown).
3. Vitamin E reverses the effect of dietary aluminum on lipid peroxidation, amyloid ß peptide formation, and plaque deposition
Tg 2576 mice receiving vitamin E supplementation with the diet containing Al showed significantly reduced urinary 8,12-iso-iPF2-VI levels comparable to values of Tg animals on regular chow (5.3±0.2 vs. 2.8±0.1 ng/mg of creatinine, P<0.01). Similar effects of vitamin E were observed for plasma 8,12-iso-iPF2-VI levels (not shown). Vitamin E treatment reduced isoprostane levels in WT receiving Al (0.85±0.03 ng/mg of creatinine, P<0.05). Compliance with the vitamin E supplementation was documented by analyzing plasma levels of this vitamin, which increased above control levels by the end of the study (21±2 vs. 65±4 µM, P<0.001). Inverse correlations existed between plasma vitamin E and urinary (r20.81, P<0.001) and plasma 8,12-iso-iPF2-VI levels (r20.68, P<0.001). Tg 2576 mice receiving vitamin E supplementation with the Al-enriched diet had reduced brain 8,12-iso-iPF2-VI levels similar to those observed in Tg 2576 on regular chow (Fig. 1)
. Vitamin E reduced by 30% brain isoprostane levels in WT.
Tg mice receiving Al plus vitamin E-supplemented chow showed no significant increase in soluble or insoluble Aß levels (Fig. 2)
. They had a significant reduction of the amyloid plaque burden similar to the one observed in animals on chow (not shown).
CONCLUSIONS AND SIGNIFICANCE
Our study provides compelling evidence that chronic dietary administration of Al increases Aß levels and accelerates plaque deposition in a model of AD-like amyloidosis. These effects are consistent with an exacerbation of brain lipid peroxidation since inclusion of Al in the diet increased brain isoprostanes and Aß levels as well as Aß deposition. Conversely, addition of the antioxidant vitamin E reduced isoprostane levels and decreased Aß levels and deposition.
Different lines of evidence have implicated a role for Al exposure in AD pathogenesis: observational, in vitro, and epidemiological studies. However, other studies have failed to confirm this. Because of these conflicting results, the issue of whether Al plays a role in the pathogenesis of AD is controversial and unresolved. Our results provide the first in vivo evidence that dietary Al can promote Aß accumulation and accelerate amyloid pathology in an animal model of AD-like amyloidosis. The effect of Al on amyloidosis in Tg 2576 mice is consistent with increased brain oxidative stress, since it directly correlated with the increase of brain isoprostane 8,12-iso-iPF2-VI levels, a specific marker of lipid peroxidation. Abundant data in the literature have linked oxidative stress to the pathogenesis of AD. However, the ability to implicate this mechanism directly to the disease has been hampered by a lack of animal models that reproduce the disease and the nonspecific analytical approaches used. Although not perfect, today there are several mouse models that mimic some aspects of the human disease. Tg 2576 is probably one of the best-characterized mouse models of AD-like amyloidosis. It is widely accepted that isoprostanes, stable end products of free radical oxidation of polyunsaturated fatty acids, are sensitive and specific marker of in vivo lipid peroxidation. We have shown previously that they are increased in postmortem AD brains as well as in living patients with a clinical diagnosis of AD, where their levels correlated with disease severity. These findings support the hypothesis that oxidative stress occurs in AD and provide evidence that isoprostanes are reliable markers of brain oxidative damage. In our study, the inclusion of vitamin E, at a dosage that suppresses lipid peroxidation, reduces Aß levels, and amyloid deposition induced by dietary Al, significantly supports this idea and provides further insight into the role of brain oxidative stress in AD amyloidosis.
Our results could also have some relevance to Al exposure in humans. Aluminum, one of the most abundant metals in the crust of the earth, has for a long time been considered to exist predominantly in forms not biologically available to humans and animals. During the last decade, however, it has been recognized that acid rain has markedly augmented the mobilization of this metal into surface waters. This has greatly increased its availability in biological ecosystems. We are aware of the limitations of our study, some of which are related to the imperfect animal model we used and some to the high dosage of Al in the diet. Therefore, our results are not immediately transferable to human AD, and further studies are warranted. Considering the variables known or suspected that can influence an individual’s susceptibility to AD, such as apolipoprotein E allele status, traumatic brain injury, and family history, we speculate that in these conditions a high Al exposure could result in its accumulation in the central nervous system, where it induces the formation of reactive oxygen species. This would result in an increase of brain oxidative stress and lipid peroxidation that would promote Aß formation, deposition, and, with time, AD-like amyloidosis (Fig. 3
).
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0012fje; to cite this article, use FASEB J. (May 21, 2002) 10.1096/fj.02-0012fje. 
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