Chronic exposure to high levels of zinc or copper has little effect on brain metal homeostasis or Abeta accumulation in transgenic APP-C100 mice

Abstract

Aberrant metal homeostasis may enhance the formation of reactive oxygen species and Abeta oligomerization and may therefore be a contributing factor in Alzheimer's disease. This study investigated the effect of chronic high intake of dietary Zn or Cu on brain metal levels and the accumulation and solubility of Abeta in vivo, using a transgenic mouse model that over expresses the C-terminal containing Abeta fragment of human amyloid precursor protein but does not develop amyloid deposits. Exposure to chronic high Zn or Cu in the drinking water resulted in only slight elevations of the respective metals in the brain. Total Abeta levels were unchanged although soluble Abeta levels were slightly decreased, without visible plaque formation, enhanced gliosis, antioxidant upregulation or neuronal loss. This study indicates that brain metal levels are only marginally altered by long term oral exposure to extremely high Cu or Zn levels, and that this does not induce Abeta-amyloid formation in human Abeta expressing, amyloid-free mice, although this is sufficient to modulate Abeta solubility in vivo.

Fig. 1

High dietary copper intake increases brain copper levels. Brain Zn and Cu levels were measured in TgC100 and NTg (BL6/DBA) mice fed various high Zn diets [300–1,000 ppm] (a) and high Cu diets [100–150 ppm] (b) and compared with age-matched control drinking water mice. Metal levels are expressed as ‘percentage difference from control feeding group’, in box and whisker plots. Significant differences between mice fed high metal diets and controls ■ were determined by ANOVA with genotype as an independent variable, and values are indicated above each box. Post-hoc Scheffé tests were used to test for significant differences in TgC100 and NTg mouse lines individually (*P < 0.05). Boxes represent average ±SEM, and whiskers represent 1.96*SEM

Fig. 2

ELISA detection of Aβ40 in mice fed high zinc or copper diets. a Aβ40 levels were measured in total brain homogenate and PBS-soluble fractions of TgC100 mice fed either 500 ppm Zn, 100 ppm Cu or pure dH₂O up to 17 months of age. Aβ40 concentration is reported as ng Aβ40 per g total protein. Asterisk represents significant difference from water control by two-tailed t-test (*P < 0.05). Error bars represent SEM

Fig. 3

Protein markers in the brains of 17-month TgC100 mice fed high zinc or copper. Brain homogenates from 17-month TgC100 mice fed 500 ppm Zn, 100 ppm Cu or pure dH₂O were immunoblotted for detection of NF200, GFAP, SOD1, APP and C100. β-tubulin was measured as a loading control. a Representative lanes of Western blot from mice fed water control, Zn 500 ppm (Zn) and Cu 100 ppm (Cu) diets. Densitometric analysis was performed on band intensities from mice of both sexes fed 500 ppm Zn (n=11) (b), and 100 ppm Cu (n=9) (c) compared with water controls (n=7). No significant differences were found in any of the proteins by t-test. Error bars represent SEM

Fig. 4

Aβ accumulation and GFAP distribution are not affected by zinc or copper feeding in mouse brain. TgC100 mice were given pure dH₂O containing no additives (left), 500 ppm Zn (centre) or 100 ppm Cu (right) as their sole source of drinking water, from the age of 2 months until euthanisia at 17 months. Brain sections show representative female mice from each feeding group, immunostained with WO2 (a and b) or anti-GFAP (c and d). a 20× magnification of hippocampus (CA2 region). b 20× magnification of striatum. Bars, 0.1 mm. NTg brain shows no WO2 immunoreactivity (not shown). Brain sections of NTg (c) and TgC100 (d) mice immunostained with GFAP antibody. Brain sections in figures show representative sections from mice examined in each group (BL6/DBA n=3, 4 and 4, TgC100 n=2, 5 and 4 in water control, Zn, and Cu fed groups, respectively). Bar, 2 mm