Changes in canonical Wnt components during aging and in AD-like pathology
To determine the activation status of canonical Wnt signaling in the hippocampus during aging and throughout the progression of AD-type pathology, we analyzed the levels of the canonical antagonist and agonist Dkk-1 and Wnt7a in female WT (C57BL/6) and 3xTg-AD mice at 4 different ages: young (3 months old), mature (6 months old), middle aged (9–12 months old) and old (18 months old). As shown in Fig. 1, in WT mice, Dkk-1 levels tended to increase with age, although the difference did not reach statistical significance. In contrast, in the hippocampus of 3xTg-AD female mice, Dkk-1 levels were significantly greater at all ages, starting at young age and compared with those in the respective age groups of WT animals (p < 0.05, p < 0.01) (Fig. 1A), starting at 3 months of age. Interestingly, the significant increase in Dkk-1 at early ages in 3xTg-AD mice occurred before the appearance of the histopathological hallmarks of AD (increased Aβ and p-tau accumulation, cognitive deficit, and gliosis), as previously reported in this transgenic model [31]. Dkk-1 was found localized mainly in the hippocampal neuronal somata and particularly in the CA3 region in 18-month-old 3xTg-AD female mice compared to WT mice of the same age (Fig. 1B). In transgenic mice, more intense staining was observed, and clusters were formed inside the neuronal somata (Fig. 1B, arrows).
To analyze the balance between the effects of the Wnt antagonist and agonist, we measured the concentration of the agonist Wnt7a. In the case of female 3xTg-AD mice, low levels of Wnt7a were present during the ages tested, but only at middle-aged were significantly lower than those in the WT mice (p < 0.05) (Fig. 2A). Given that a reduction in LRP6 has been associated with aging and the late onset of AD (De Ferrari et al., 2007; Jones, et al., 2023), the LRP6 protein level was quantified at different ages and during Alzheimer disease-like progression. A significant reduction of nearly 40% (p < 0.05) was found at 18 months of age compared with that in 3-month-old WT mice, and this reduction was greatly exacerbated by almost 60% in the 3xTg-AD mice at 18 months compared with that in the WT mice at 18 months (p < 0.01) (Fig. 2B). These results suggest that canonical Wnt signaling activation is compromised beginning in the early stage of AD-like pathology in the 3xTg-AD model, starting with the upregulation of Dkk-1 and, later, with a decrease in the expression of the Wnt7a agonist and the LRP6 coreceptor.
GSK3β activation correlates with the extent of tau phosphorylation in 3xTg-AD mice
To determine the involvement of the downstream regulator of Wnt signaling, the kinase GSK3β, we analyzed the active form of the enzyme through the measurement of the activation residue p-Tyr216-GSK3β and the inhibitory site p-S9-GSK3β. The aging of mice presented an activation profile of GSK3β, as revealed by a reduction in the p-S9 concentration and an increase in the p-Tyr216. A significant decrease in p-Ser9 was observed in WT animals compared young (3 months) and middle-aged (9–12 months) mice. This activation was exacerbated in the 3xTg-AD mice, which exhibited significant activation beginning at 3 months after the appearance of AD markers and remained activated at all analyzed ages (Fig. 3A). A similar pattern of activation of GSK3β was observed through quantification of the p-Tyr216 site (Fig. 3B). At all ages, the total kinase levels did not significantly change (Fig. 3C).
Because GSK3β is one of the main kinases implicated in tau phosphorylation, we measured the levels of the GSK3β-dependent phosphoepitope S202/Thr205 of tau protein. Although no significant changes in the total content of this epitope were observed in WT mice during aging, clearly reduced tau electrophoretic mobility was present, indicating an increase in total tau phosphorylation. Similarly, reduced tau electrophoretic mobility was observed in 3xTg-AD mice, and this change was accompanied by a significant increase in the total p-S202/Thr205 tau concentration, particularly beginning at 6 months (Fig. 4A). To analyze the relationship between Dkk-1 levels and tau phosphorylation, we performed an index that revealed a significant (R = 0.54) correlation between Dkk-1 and tau phosphorylation in the transgenic mice but not in the WT mice (Fig. 4B). To summarize the progression of the different Wnt signaling components and tau phosphorylation we plotted the average value of Dkk-1 levels, GSK3β activation, and the appearance of p-tau throughout the aging process and disease progression in the hippocampus of 3xTg-AD female mice compared with those in the control group (Fig. 4C).
BDNF and pro-BDNF expression changes during aging and in the 3xTg-AD model
BDNF has been implicated in neurodegenerative diseases, and its expression is regulated by the canonical Wnt pathway [9]. Thus, we analyzed whether the content of this neurotrophin changed in the hippocampi of WT and 3xTg-AD mice during aging. We observed a progressive increase in pro-BDNF levels throughout aging in the WT mice but not in the 3xTg-AD mice, in which the levels remained lower at all ages tested (Fig. 5A). Contrary to the pro-BDNF findings, no changes in the BDNF content were observed at any age or in any of the WT or transgenic mice (Fig. 5B). Interestingly, an increase in the proBDNF/BDNF ratio was observed during aging only in WT animals but not in 3XTg-AD animals.
months and remained at the same level during aging. BDNF expression was higher in the 3xTg-AD mice than in the WT mice at 3 months (b) The pro-BDNF/BDNF ratio increased during aging in the WT animals and remained low in the 3xTg-AD mice (c). Representative Western blot and densitometric analysis data from 4–6 independent experiments are shown. The data are presented as the mean ± SEM. *p < 0.05 (one-way ANOVA).
Dkk-1 and p-tau levels are significantly greater in females than in males in the 3xTg-AD model
Because sex-related differences in AD are more common and more severe in females than in males [32], we explored whether the increase in Dkk-1 levels differs between males and females in the WT and 3xTg-AD models with age. As expected, Dkk-1 was more highly expressed in female 3xTg-AD mice than in male mice, but interestingly, Dkk-1 was only highly expressed in middle-aged and aged animals (Fig. 6A). In young mice, significantly lower Dkk-1 levels were observed in females than in males, suggesting a possible protective effect of female hormones, as has been previously reported in a model of cerebral ischemia and Dkk-1 expression [33]. Consistent with Dkk-1 levels, tau hyperphosphorylation was also significantly greater in females than in males, particularly at advanced ages (p < 0.01) (Fig. 6B).