In this study, we found that Dkk-1 and Wnt7a are differentially expressed during normal aging (in WT mice) compared to pathological aging (in 3xTg-AD mice). Aging was accompanied by a gradual increase in Dkk-1 in WT mice, which was strongly exacerbated since young in 3xTg-AD mice correlating with early activation of the kinase GSK3β before tau phosphorylation. Dysregulation of Wnt signaling molecules have been reported in postmortem tissue from AD patients, suggesting that the pathway is impaired at late stages of the disease [7, 28]. Similarly, it has been reported that inhibition of the canonical Wnt pathway triggers the appearance of AD hallmarks in vitro [11, 12, 28] and in vivo [34, 35]. However, the questions of whether in vivo Wnt dysregulation occurs prior to the appearance of disease hallmarks and whether this dysregulation is a consequence or cause of Wnt signaling changes remain open. Herein, we found that the canonical Wnt pathway seems to be impaired beginning in the early stages of pathology prior to the accumulation of p-S202/Thr205-tau, one of the hallmarks that defines AD pathology. Later, during the progression of the pathology, we also found a decrease in the levels of the canonical agonist Wnt7a and at more advances ages, the coreceptor LRP6. Although these changes also occur during normal aging, as reported in other aged species [26, 27], in the 3xTg-AD mice, the downregulation of these Wnt components occurred earlier than in the control mice and was particularly severe. In addition to the negative effects of Dkk-1 and the decrease in the agonist Wnt7a, we found that the symptomatic stage of pathology in transgenic mice could worsen synaptic function because of its role in promoting synaptic plasticity events [36, 37].

The mechanisms involved in Dkk-1 upregulation during aging and pathology are not known; however, Dkk-1 transcription is modulated by the tumor suppressor protein p53, which responds to cellular damage [38]. Thus, the signals that may promote the early accumulation of Dkk-1 in transgenic mice prior to the pathological accumulation of p-tau can be associated with the induction of several biochemical stressors that occur early in pathology but that only appear in WT mice at more advanced ages. Another mechanism underlying the early upregulation of Dkk-1 may be associated in part with the activation of the c-Jun N-terminal kinase (JNK) pathway, which senses harmful stimuli in cells, such as oxidative stress [39]. Accordingly, JNK has been found to be activated in human postmortem AD brain samples [40] and in presymptomatic stages in 2.5-month-old 3xTg-AD mice [41]. In the brains of young 3xTg-AD mice, the activation of JNK could be triggered by several stressors, such as reactive oxygen species, as mentioned above, by hypoxia, and by the early presence of oligomeric forms of Aβ [42]. Notably, the upregulation of Dkk-1 revealed important sexual dimorphism in the WT and AD model. The differences were marked according to animal age: there was a trend toward lower Dkk-1 content in young females but a greater increase in Dkk-1 in old females than in males, suggesting a lack of protective female hormone effects. Sexual dimorphism is gaining interest, as clinical data show that women are at greater risk of developing AD and having worse pathology and faster cognitive decline. In this sense, the impact of female hormones, such as progesterone, on Dkk-1 expression was reported [43]. It was also found that 17β-estradiol downregulates Dkk-1 in a model of global cerebral ischemia [33]. Interestingly, compared with WT females, 3xTg-AD females are known to have a shorter reproductive window, which could suggest that transgenic females experience an early decrease in the neuroprotective effects of estrogens and progesterone on Dkk-1 expression. Unlike women, female rodents do not experience menopause; however, between the ages of 11 and 16 months, they undergo a transition period characterized by an altered estrous cycle [44], corresponding to the age at which Dkk-1 upregulation was prominent in the female WT mice.

Notably, female 3xTg-AD mice displayed more prominent amyloid plaques, neurofibrillary tangles, neuroinflammation, and spatial cognitive deficits than male 3xTg-AD mice, as has been widely documented [45,46,47]. These differences may explain the lower content of p-tau observed in male 3xTg-AD model mice.

Although the 3xTg-AD model has the advantage that pathology occurs in an age-dependent manner, some of the molecules in the Wnt pathway that are altered and the expression of the hallmark p-tau that are commonly observed in females can be associated with the presence of the Thy1 promoter transgene cassette for mutated APP and tau. This promoter contains an estrogen response-like element that, as suggested, can explain the higher expression of transgenes in females [47, 48]. However, more research is needed to fully explain these sex differences because the age-associated increases in the Dkk-1 and p-tau levels in aged females were maintained at the age at which estrogen levels tend to decline.

As mentioned above, we identified 2 impairments in the Wnt signaling pathway throughout the pathology. The second impairment was the downregulation of the Wnt7a agonist and the coreceptor LRP6 that occurred in the symptomatic stage of the pathology in the 3xTg-AD mice but also occurred in the older WT animals. Recent research has shown that Wnt signaling activation can also be regulated by signalosome endocytosis of the Wnt receptor complex [49]. Notably, Dkk-1 induces the internalization of LRP6 together with the protein Kremen, inhibiting the canonical Wnt pathway [50] and promoting LRP6 degradation. This mechanism supports a model in which the upregulation of Dkk-1 during aging and incipient AD-like pathology negatively regulates Wnt signaling and subsequently reduces the content of the LRP6 coreceptor, as we found here, worsening the Wnt molecular pathway that participates in synapse assembly and function in the mature brain [51, 52]. GSK3β is considered an important player in aging studies because it is a central node for the control of multiple pathways involved in cellular metabolism, growth, survival, inflammation, and senescence, among others [24, 53, 54]. As part of the first impairment in the Wnt signaling pathway reported in this study, enhanced GSK3β kinase activation was found beginning at the presymptomatic stage of the pathology in 2-3-month-old 3xTg-AD mice, similar in magnitude to what occurred until 9–12 months of age in the WT animals, correlating with the induction of the Wnt antagonist Dkk-1. These observations point to a role of Dkk-1 in inducing the hallmark of AD, at least through the phosphorylation of tau in the 3xTg-AD model. In agreement with the present results, the activity of GSK3β was found to increase prior to the formation of nurofibrillary tangles in the brains of AD patients [55], and the overexpression of this kinase was also found in the prefrontal cortex and was associated with cognitive decline in AD patients [7]. Taken together, these results reveal important changes in GSK3β activity in middle-aged WT animals, which were significantly exacerbated in young 3xTg-AD mice, suggesting a cascade of events that starts with Dkk-1 overproduction as a key factor associated with the development of AD pathology.

Although the BDNF gene promoter contains binding motifs for Wnt-dependent TCF/LEF transcription factors [9], we did not observe significant changes in the protein content of this neurotrophin in the WT or in the 3xTg-AD mice, in agreement with other works in which 3xTg-AD mice were found to have BDNF levels that are comparable to those of WT controls [56]. However, a significant increase in pro-BDNF levels was observed in WT animals during aging, while BDNF levels remained stable relative to those in young adults according to previous work [57, 58]. These results suggest that changes in gene expression do not correlate with changes in protein levels or that abnormalities in the production of mature BDNF or other components of the BDNF signaling pathway, such as TrkB or p75NTR, may be involved in aging and AD pathology.