Dysregulation of Na+/K+ ATPase by amyloid in APP+PS1 transgenic mice

Abstract

Background: The pathology of Alzheimer's disease (AD) is comprised of extracellular amyloid plaques, intracellular tau tangles, dystrophic neurites and neurodegeneration. The mechanisms by which these various pathological features arise are under intense investigation. Here, expanding upon pilot gene expression studies, we have further analyzed the relationship between Na+/K+ ATPase and amyloid using APP+PS1 transgenic mice, a model that develops amyloid plaques and memory deficits in the absence of tangle formation and neuronal or synaptic loss.

Results: We report that in addition to decreased mRNA expression, there was decreased overall Na+/K+ ATPase enzyme activity in the amyloid-containing hippocampi of the APP+PS1 mice (although not in the amyloid-free cerebellum). In addition, dual immunolabeling revealed an absence of Na+/K+ ATPase staining in a zone surrounding congophilic plaques that was occupied by dystrophic neurites. We also demonstrate that cerebral Na+/K+ ATPase activity can be directly inhibited by high concentrations of soluble Abeta.

Conclusions: The data suggest that the reductions in Na+/K+ ATPase activity in Alzheimer tissue may not be purely secondary to neuronal loss, but may results from direct effects of amyloid on this enzyme. This disruption of ion homeostasis and osmotic balance may interfere with normal electrotonic properties of dendrites, blocking intraneuronal signal processing, and contribute to neuritic dystrophia. These results suggest that therapies aimed at enhancing Na+/K+ ATPase activity in AD may improve symptoms and/or delay disease progression.

Figure 1

Reduced activity and protein levels of the Na+/K+ ATPase enzyme in APP+PS1 mice. Activity of total (A) and ouabain-sensitive (B) ATPase was assayed colorimetrically in APP+PS1 mice tissue (n = 8, open bars) and non-transgenic littermate tissue (n = 8, solid bars), presented here as μmols of phosphate liberated by ATPase per mg of protein per hour. In the amyloid-containing hippocampus, there was a significant decrease in the specific activity of ouabain-sensitive ATPase in APP+PS1 mice compared to non-transgenic mice, but there was no decrease in cerebellar Na+/K+ ATPase activity. Cerebellar activity was 20% of that seen in non-transgenic hippocampus. Panel C shows the quantitation of the optical density of bands corresponding to the molecular weight of Na+/K+ ATPase using standard Western blot technique. This reveals a trend for decreased protein levels. *** indicates significant differences between APP+PS1 mice and non-transgenic littermates (p < 0.001) when measured by one-way ANOVA.

Figure 2

Verification of Na+/K+ ATPase αIII antibody specificity by immunohistochemistry following pre-incubation with purified protein and Western blotting. Horizontal sections were immunostained for Na+/K+ ATPase αIII with (Panel B) and without (Panel A) pre-incubation with 70 μunits of purified enzyme followed by Congo red staining. The pre-incubation significantly decreased staining, confirming antibody specificity. Scale bar = 50 μm.

Figure 3

Hippocampal and cortical immunohistochemistry for Na+/K+ ATPase in APP+PS1 mice and non-transgenic littermates. Horizontal hippocampal and cortical sections were immunostained for Na+/K+ ATPase αII. The left panels depict hippocampus, while the right panels encompass the cerebral cortex. Panels A & B: high power magnification (Scale bar = 8.33 μm) revealed membrane-localized staining for Na+/K+ ATPase in the insular cortex (Panel B) and CA3 of the hippocampus (Panel A) of non-transgenic mice. Panel C: hippocampal staining of non-transgenic mice showed a ubiquitous distribution of Na+/K+ ATPase throughout the neuropil, while less staining was apparent along the pyramidal layer of Ammon's horn, the hilus and the granular layer of the dentate gyrus. Panel D: cortical staining was also substantial with slight lightening in layers 1 and 2. White matter in both structures remained unstained. A similar staining pattern was observed in the APP+PS1 mice with the exception of focal non-stained areas in the gray matter, presumably where amyloid plaques are located (Panel E [hippocampus] & F [cortex]). Immunostaining followed by Congo red histochemistry confirmed that the Na+/K+ ATPase staining was absent not only immediately where amyloid was located, but also in a zone surrounding the plaque (Panels G [hippocampus] & H [cortex]). Scale bar for panels C-H = 120 μm.

Figure 4

Dual Immunostaining of Na+/K+ ATPase αIII and dystrophic neurites. Horizontal sections were immunostained for Na+/K+ ATPase using DAB with nickel intensification followed by Congo red staining for Aβ plaques. A discernable circumferential void in ATPase staining surrounding the plaque was observed (Panel A, scale bar = 50 μm; Panel B, scale bar = 16.67 μm). Immunofluorescent staining of dystrophic neurites using the anti-phosphorylated neurofilament antibody SMI-312 followed by Congo red staining demonstrate a close relationship between amyloid plaques and dystrophic neurites (Panel C; white arrows demarcate the neurites). Congo red yellow fluorescence was digitally suppressed to more clearly reveal the green-stained neurites (scale bar = 16.67 μm). Panel D is a digital overlay of the fluorescein-labeled dystrophic neurite image onto the bright field peroxidase-labeled Na+/K+ ATPase + Congo red image which demonstrates that dystrophic neurites are present predominantly in the zone devoid of Na+/K+ ATPase staining surrounding the congophilic plaques (scale bar = 16.67 μm).

Figure 5

Amyloid-beta 1–42 peptide inhibits Na+/K+ ATPase activity. Purified cerebral Na+/K+ ATPase was pre-incubated with vehicle or Aβ 1–42, and enzyme activity percentages of the vehicle (20 μmols Pi/mg protein/hour) are presented (mean ± SEM). "◊" indicates the values for the soluble (DMSO+water) Aβ suspension. "○" indicates the values for the fibrillar (DMSO+neutralized HCl) Aβ suspension. At the highest concentrations, the soluble preparation dramatically reduces Na+/K+ ATPase activity compared to both vehicle and the fibrillar Aβ preparation. * (p < 0.05) and *** (p < 0.001) indicates significant differences between Aβ and vehicle alone when measured by one-way ANOVA.