Cognitive impairment in humanized APP×PS1 mice is linked to Aβ(1-42) and NOX activation

Abstract

Cognitive impairment in Alzheimer's disease (AD) is strongly associated with both extensive deposition of amyloid β peptides and oxidative stress, but the exact role of these indices in the development of dementia is not clear. This study was designed to determine the relationship between cognitive impairment, activation of the free radical producing enzyme NADPH oxidase (NOX), and progressive changes in Aβ deposition and solubility in humanized APP×PS1 knock-in mice of increasing age. Data show that cognitive performance and expression of key synaptic proteins were progressively decreased in aging APP×PS1 mice. Likewise, NOX activity and expression of the specific NOX subunit NOX4 were significantly increased in APP×PS1 mice in an age-dependent manner, and NOX activity and cognitive impairment shared a significant linear relationship. Data further show that age-dependent increases in Aβ(1-42) had a significant linear relationship with both NOX activity and cognitive performance in APP×PS1 knock-in mice. Collectively, these data show that NOX expression and activity are significantly upregulated with age in this humanized model of Aβ pathogenesis, and suggest that NOX-associated redox pathways are intimately linked to both the loss of cognitive function and the deposition of Aβ(1-42).

Figure 1. Age-related impairment in cognitive ability in APP × PS1 mice

Male APP × PS1 and WT mice were tested behaviorally in the Stone T-maze as described in Methods. (A) Procedural memory was evaluated in aged (16-19 month-old) APP × PS1 and WT mice by quantifying the number of errors committed over 15 trials of maze acquisition training. Data are means ± S.E.M. of average errors accrued over 3-trial blocks and were generated from 9-12 mice per group. Data were analyzed by 2-way ANOVA, and * indicates significant (p<0.05) increases in errors made by APP × PS1 mice. (B) Procedural memory in young (4-6 month-old) mice was measured by quantifying the number of errors committed over 15 trials of maze acquisition training. Data are means ± S.E.M. of average errors accrued over 3-trial blocks and were generated from 9-12 mice per group, and were analyzed by 2-way ANOVA. (C) Memory retention was tested in aged (16-19 month-old) APP × PS1 and WT mice by quantifying the number of errors committed over 5 additional trials in the maze 7 days following acquisition training. Data are means ± S.E.M. of average errors accrued over 5 individual trails, and were analyzed by 2-way ANOVA. * indicates significant (p<0.05) increases in errors made by APP × PS1 mice. (D) Memory retention in young (4-6 month-old) APP × PS1 and WT mice was measured by 5 additional trials in the maze 7 days following acquisition training. Data are means ± S.E.M. of average errors accrued over 5 individual trails, and were analyzed by 2-way ANOVA. * indicates significant (p<0.05) increases in errors made by APP × PS1 mice.

Figure 2. Age-related decreases in synaptic marker expression in APP × PS1 mice

(A) Expression of the post-synaptic protein synapse associated protein 97 (SAP97) was evaluated in tissue homogenates prepared from the frontal cortex of young (4-6 month–old) and aged (16-19 month–old) APP × PS1 and WT mice by Western blot as described in Methods. For consistent quantification across multiple blots, individual samples were normalized first to tubulin and then to levels in WT mice, as described in Methods. Data are mean ± SEM SAP97 expression in 19-20 mice/group, and were analyzed by 2-tailed, unpaired t-tests. * and ** indicate significant (p < 0.05 and 0.01, respectively) decreases in SAP97 expression detected in young and aged APP × PS1 mice, respectively. (B) Expression of the pre-synaptic protein synapsin 1 (SYN1) was evaluated in tissue homogenates prepared from the frontal cortex of young (4-6 month–old) and aged (16-19 month–old) APP × PS1 and WT mice as described in Methods. Data are mean ± SEM SYN1 expression in 19-20 mice/group. (C) Expression of phosphorylated synapsin 1 (phos-SYN1) was evaluated in tissue homogenates prepared from the frontal cortex of young (4-6 month–old) and aged (16-19 month–old) APP × PS1 and WT mice as described in Methods. Data are mean ± SEM phos-SYN1 expression in 19-20 mice/group, and were analyzed by 2-tailed, unpaired t-tests. *** indicates significant (p < 0.001) decreases in phos-SYN1 expression in aged APP × PS1 mice as compared to aged WT mice. (D) Representative images depict the pattern of phos-SYN1 expression in young and aged APP × PS1 and WT mice.

Figure 3. Age-related increases in NOX activity in APP × PS1 mice

NOX activity was measured in tissue homogenates prepared from the frontal cortex of young (4-6 month–old) and aged (16-19 month–old) APP × PS1 and WT mice using lucigenin as described in Methods. Data were calculated as counts per minute (CPM) per microgram total protein, and are presented as mean ± SEM. 2-way ANOVA indicated an overall significant main effect of age and genotype on NOX activity, and also a significant interaction of age and genotype on NOX activity (text insert). *** indicates significant (p < 0.001) increases in NOX activity in aged APP × PS1 mice as compared to aged WT mice.

Figure 4. NOX subunit expression in young and aged WT and APP × PS1 mice

(A) Expression of the regulatory subunit p47phox (p47) was evaluated in tissue homogenates prepared from the frontal cortex of young (4-6 month–old) and aged (16-19 month–old) APP × PS1 and WT mice by Western blot as described in Methods. For consistent quantification across multiple blots, individual samples were normalized first to tubulin and then to levels in WT mice, as described in Methods. Data are mean ± SEM p47expression in 19-20 mice/group, and were analyzed by 2-tailed, unpaired t-tests. (B) Expression of the catalytic subunit NOX2 was evaluated in tissue homogenates prepared from the frontal cortex of young (4-6 month–old) and aged (16-19 month–old) APP × PS1 and WT mice as described in Methods. Data are mean ± SEM NOX2 expression in 19-20 mice/group. (C) Expression of NOX2 homolog NOX4 was evaluated in tissue homogenates prepared from the frontal cortex of young (4-6 month–old) and aged (16-19 month–old) APP × PS1 and WT mice as described in Methods. Data are mean ± SEM NOX4 expression in 19-20 mice/group, and were analyzed by 2-tailed, unpaired t-tests. ** indicates significant (p < 0.01) increases in NOX4 expression in aged APP × PS1 mice as compared to aged WT mice. (D) Representative images depict the pattern of NOX4 expression in young and aged APP × PS1 and WT mice.

Figure 5. Linear relationship between maze performance and NOX activity in young and aged WT and APP × PS1 mice

(A) Scatter plots show the relationship between errors in acquisition trail block 4-6 (errors in ATB 4-6) and NOX activity in aged (16-19 month-old) APP × PS1 (open squares) and WT (closed circles) mice. Each point represents an individual subject, and the line depicts the degree of linear regression. (B) Scatter plots show the relationship between errors in acquisition trail block 4-6 (errors in ATB 4-6) and NOX activity in young (4-6 month-old) APP × PS1 (open squares) and WT (closed circles) mice. Each point represents an individual subject, and the line depicts the degree of linear regression.

Figure 6. Linear relationship between Aβ_1-42 and both maze performance and NOX activity in aged APP × PS1 mice

(A) Scatter plots show the relationship between errors in acquisition trail block 4-6 (errors in ATB 4-6) and SDS-soluble Aβ_1-42 levels in 16-19 month-old APP × PS1 mice. Each point represents an individual subject, and the line depicts the degree of linear regression. (B) Scatter plots show the relationship between errors in acquisition trial block 4-6 (errors in ATB 4-6) and total Aβ_1-42 levels in 16-19 month-old APP × PS1 mice. Each point represents an individual subject, and the line depicts the degree of linear regression. (C) Scatter plots show the relationship between NOX activity and SDS-soluble Aβ_1-42 levels in 16-19 month-old APP × PS1 mice. Each point represents an individual subject, and the line depicts the degree of linear regression. (D) Scatter plots show the relationship between NOX activity and total Aβ_1-42 levels in 16-19 month-old APP × PS1 mice. Each point represents an individual subject, and the line depicts the degree of linear regression.