Chronic nicotine administration exacerbates tau pathology in a transgenic model of Alzheimer's disease

Abstract

The association between nicotinic acetylcholine receptor (nAChR) dysfunction and cognitive decline in Alzheimer's disease (AD) has been widely exploited for its therapeutic potential. The effects of chronic nicotine exposure on Abeta accumulation have been studied in both humans and animal models, but its therapeutic efficacy for AD neuropathology is still unresolved. To date, no in vivo studies have addressed the consequences of activating nAChRs on tau pathology. To determine the effects of chronic nicotine administration on Abeta and tau pathology, we chronically administrated nicotine to a transgenic model of AD (3xTg-AD) in their drinking water. Here, we show that chronic nicotine intake causes an up-regulation of nicotinic receptors, which correlated with a marked increase in the aggregation and phosphorylation state of tau. These data show that nicotine exacerbates tau pathology in vivo. The increase in tau phosphorylation appears to be due to the activation of p38-mitogen-activated protein kinase, which is known to phosphorylate tau in vivo and in vitro. We also show that the 3xTg-AD mice have an age-dependent reduction of alpha7nAChRs compared with age-matched nontransgenic mice in specific brain regions. The reduction of alpha7nAChRs is first apparent at 6 months of age and is restricted to brain regions that show intraneuronal Abeta(42) accumulation. Finally, this study highlights the importance of testing compounds designed to ameliorate AD pathology in a model with both neuropathological lesions because of the differential effects it can have on either Abeta or tau.

Fig. 1.

Reduction in α7nAChRs levels in selective brain regions correlates with intraneuronal Aβ. (A) Steady-state levels of α4β2nAChRs were not significantly altered between untreated NonTg and 3xTg-AD mice in all brain regions analyzed, except the thalamus, where we found significantly more receptors in the 3xTg-AD mice (P = 0.011). Chronic nicotine administration significantly increased α4β2 levels in all brain regions in the 3xTg-AD and NonTg mice. (B) Steady-state levels of α7nAChRs were significantly lower in untreated 6-month-old 3xTg-AD compared with age-matched untreated NonTg mice. This effect was evident only in brain regions that accumulate intraneuronal Aβ, such as hippocampus, retrosplenial and parietal cortices, and thalamus (P < 0.05). No statistically significant alteration was apparent in the auditory cortex, which does not show any intraneuronal Aβ accumulation at this age (P > 0.05). Chronic nicotine administration did not alter the steady-state levels of α7nAChRs because the levels between treated and untreated mice were not statistically significant. (C–H) Immunohistochemical analysis by using an anti-Aβ₄₂ specific antibody shows the buildup of intraneuronal Aβ in the hippocampus (C), the retrosplenial and parietal cortices (D–F) and the thalamus (G). Intraneuronal Aβ accumulation is not apparent in the auditory cortex at this age (H). E and F show a higher magnification of the retrosplenial and parietal cortex, respectively. Hi, hippocampus; Th, thalamus; Re, retrosplenial cortex; Pa, parietal cortex; Au, auditory cortex; rf, rhinal fissure.

Fig. 2.

Age-dependent reduction in α7nAChRs levels in the hippocampus of 3xTg-AD mice. (A) Quantitative analysis of α-bungarotoxin binding reveals an age-related decrease in α7nAChR steady-state levels in the hippocampus of 3xTg-AD mice beginning at 6 months of age (P < 0.001). At 2 and 4 months of age, no difference in the α7nAChRs steady-state levels is apparent between 3xTg-AD and NonTg mice, indicating that the 3xTg-AD mice are not born with this deficit (P = 0.208 and 0.087 for 2 and 4 month olds, respectively). The reduction in α7nAChRs correlates with intraneuronal Aβ accumulation because 2- and 4-month-old 3xTg-AD mice do not show any immunoreactivity with an anti-Aβ antibody. (B) A representative microphotograph of the hippocampus of a 4-month-old hemizygous 3xTg-AD mouse stained with an Aβ₄₂ specific-antibody. Note the lack of immunostaining in the CA1 pyramidal neurons defined by the arrows. (C) By 6 months of age, hemizygous 3xTg-AD mice show prominent intraneuronal Aβ₄₂ buildup in CA1 pyramidal neurons after staining with an anti-Aβ specific antibody.

Fig. 3.

APP processing and Aβ deposition are not altered after chronic nicotine administration. (A) Immunoblot shows that APP and C99 levels are not significantly different between treated and untreated 3xTg-AD mice. (B) Quantitative analysis of blots in A after normalizing to β-actin shows that nicotine administration did not significantly alter the steady-state levels of APP or C99. (C and D) Immunohistochemical analysis by using an anti-Aβ₄₂ specific antibody shows that Aβ deposition was not altered after chronic nicotine administration. (E) Densitometric analysis of C and D did not reveal any significant change in the Aβ load in the hippocampus of treated versus untreated 3xTg-AD mice. (F) Sandwich ELISA revealed that Aβ₄₀ and Aβ₄₂ steady-state levels were unaltered after chronic nicotine administration (n = 5 per group). Although there appears to be reduced Aβ₄₀ levels in the treated mice, it did not achieve significance (P = 0.332 and 0.676 for Aβ₄₀ and Aβ₄₂, respectively). (G) The ratio of Aβ₄₂/Aβ₄₀ was also unchanged by the nicotine administration (P = 0.198).

Fig. 4.

Phosphorylation and aggregation of tau are enhanced after chronic nicotine administration. Immunohistochemical analysis uses the human-specific anti-tau antibody, HT7 (A and B) and AT270, which recognizes tau phosphorylated at Thr-181 (C and D). Chronic nicotine administration exacerbates tau pathology in the hippocampus of the 3xTg-AD mice because both HT7 and AT270 immunoreactivity is increased. Note the increase in the number of HT7- and AT270-positive neurons in the CA1 subfield in B and D compared with A and C. (E) Immunoblot analysis by using the phospho-specific tau antibody AT270 shows increased tau phosphorylation in the brains of treated versus untreated 3xTg-AD mice. The arrow in E points to the 64-kDa band, which indicates a pathological aggregated form of tau. Note the shift from faster migrating tau species to the slower 64-kDa band, consistent with an increase in the tau aggregation state induced by chronic nicotine administration. (F) Quantitative analysis indicates a significant increase in the 64-kDa band in the brains of treated versus untreated mice (P < 0.005). (G and H) The increase in tau phosphorylation was confirmed by using another phospho-specific tau antibody, AT8, which also shows a significant increase in tau phosphorylation at Ser-202 (P < 0.05).

Fig. 5.

Chronic nicotine exposure selectively increases the steady-state levels of activated p38-MAP kinase. To determine which kinase is responsible for the increase in tau phosphorylation after nicotine administration, the levels of several candidate tau kinases were measured by quantitative Western blot analysis. A and B show that the steady-state levels of GSK3α and GSK3β are not significantly altered between treated and untreated 3xTg-AD mice. C and D show that the activated form of p38-MAP kinase is significantly increased in 3xTg-AD mice after chronic nicotine administration (P < 0.05).