Memory loss is a common characteristic of normal aging (Leal et al., 2017) which gets greatly accelerated in some neurodegenerative diseases. Alzheimer’s disease (AD) is the most frequent cause of memory loss and other dementia symptoms among elderly patients (Hickman et al., 2016). AD is an irreversible degenerative pathology of the brain characterized by progressive deterioration of cognitive functions, affecting mainly neurons in the hippocampus and the cerebral cortex. The histological hallmarks of AD include senile plaques, made up by accumulations of β-amyloid (Aβ) peptide, and neurofibrillary tangles, which are deposits of Tau, a microtubule associated protein (MAP), which gets abnormally phosphorylated (pTau; Hernandez et al., 2013). Pathological accumulation of pTau, which compromises synaptic transmission and neuronal viability by contributing to cytoskeleton collapse, correlates better with the severity of cognitive decline in AD than senile plaques (Nelson et al., 2012).

The causes of memory loss during normal aging are not completely understood. Atrophy of some brain areas has been shown in normal aging (Pini et al., 2016) and changes in intrinsic neural electrical excitability associated with oxidative stress have been hypothesized as potential causes (Hermann et al., 2014). Subtle perturbations in stabilization of neuronal cytoskeleton, reminiscent of those occurring during AD neurodegeneration, may also be an important underlying cause of age-associated neuronal dysfunction and cognitive decline (Savva et al., 2009). In this line, modifications on pTau expression and status (tauopathies) are also typical of normal aging (Delacourte et al., 2002) and their distribution pattern correlates with memory capabilities (Guillozet et al., 2003).

Some interventions have been proposed to delay aging-related cognitive deficits and thus improve the quality of life in older people. Some of these interventions include mental stimulation and physical activity (Collette and Salmon, 2014) but a better knowledge of the mechanisms underlying the loss of memory may help in devising novel pharmaceutical approaches.

In the search for predictive blood biomarkers of AD cognitive decline, some studies have found that mid-regional proadrenomedullin is elevated in the plasma of AD patients and that the concentration of this peptide could have predictive value in the progression from predementia to clinical AD (Buerger et al., 2011; Henriksen et al., 2014), although a recent study on a Swedish population found no correlation (Holm et al., 2017). The proadrenomedullin gene, adm, codes for a 185 amino acid preprohormone which, after post-translational modifications, generates two biologically active peptides: proadrenomedullin N-terminal 20 peptide (PAMP) and adrenomedullin (AM). Both peptides are amidated at their carboxy terminus and their 3-dimensional structure is based on a central α-helix (Pérez-Castells et al., 2012). Expression of these peptides is widespread and several functions have been ascribed to them, including vasodilatation, bronchodilatation, angiogenesis, hormone secretion regulation, growth modulation and antimicrobial activities, among others (López and Martínez, 2002). In the central nervous system (CNS), AM is expressed throughout the whole brain and spinal cord (Serrano et al., 2000) where it acts as a neuromodulator through mechanisms dependent and independent of NMDA receptors (Xu and Krukoff, 2004). It has been shown that plasma levels of AM increase with normal aging (Kato et al., 2002).

Knockout studies have shown that total abrogation of adm results in embryo lethality (Caron and Smithies, 2001). To circumvent this problem, we generated a conditional knockout model where adm was eliminated just from neurons by using Cre/loxP technology, and the physiological consequences of such manipulation have been published (Fernández et al., 2008, 2010; Hurtado et al., 2010).

An intriguing finding from our laboratory showed that both AM and PAMP decorate the microtubules in a variety of cell types, including neurons (Sackett et al., 2008). Yeast-2-hybrid analysis demonstrated that AM binds to several MAPs, whereas PAMP binds directly to tubulin and kinesin. Cell physiology studies point to a direct involvement of PAMP in regulating microtubule dynamics and kinesin speed (Larráyoz and Martínez, 2012). Downregulation of adm expression, through either gene knockdown or targeted knockout, results in a massive hyperpolymerization of the tubulin cytoskeleton, an increase on Glu- and acetylated-tubulin, a reduction of kinesin velocity, and the apparition of actin filopodia in CNS stem/progenitor cells (Sackett et al., 2008; Vergaño-Vera et al., 2010). In addition, AM immunoreactivity increases in the brain of AD patients (Ferrero et al., 2017) and in mouse models of AD where it seems to be associated with activated astrocytes in the vicinity of amyloid plaques (Fernandez et al., 2016).

Taking all this into consideration we decided to investigate the potential connection between adm gene products and normal aging memory loss in a mouse model and the expression of AM in human brains.

In this study we have shown that aged mice that lack neuronal AM have better contextual and recognition memory than their WT littermates. In parallel, the brain cortex and hippocampus of these mice have a lower accumulation of p-Tau, suggesting that p-Tau may be the link between lack of AM and memory preservation, although we cannot rule out other alternative molecular pathways. In addition, we also showed that older human individuals present higher levels of AM and lower levels of acetylated tubulin in their brains than younger controls.

Previous studies had found that plasma AM increases with age (Kato et al., 2002), but a description of the levels of AM in the aging brain was lacking. Here we have demonstrated that normal aging is accompanied by an increase of AM protein expression, at least in the frontal cortex. Previous reports have indicated that elevated AM levels occur in the brain of AD patients (Ferrero et al., 2017) and that plasma levels of the related peptide, mid-regional proadrenomedullin, may constitute an early marker for progression to AD (Buerger et al., 2011; Henriksen et al., 2014). Obviously, patients in their path to develop AD should have a larger AM increase than normally aging individuals. These correlation studies provide important information by identifying potential markers to detect early cases of rapidly declining individuals, but they do not help in understanding the underlying biochemical mechanisms. Our mouse study provides a first insight into the potential mechanisms linking AM levels to contextual memory loss, through the regulation of p-Tau, especially among females. Although a protein-protein contact between Tau and AM was not detected by yeast-2-hybrid experiments (Sackett et al., 2008), these two proteins are MAPs and are located in close proximity on the microtubule surface, so a direct physical interaction cannot be excluded. Alternatively, AM could modulate any of the kinases that phosphorylate Tau (Tell et al., 2016). The exact biochemical mechanism linking AM to Tau phosphorylation should be addressed in future studies.

Two different antibodies were used in this study to asses Tau phosphorylation. AT8 is a phospho-specific antibody that recognizes phosphorylation in Ser202 and Thr205 (Goedert et al., 1995), whereas the PHF1 antibody recognizes phosphorylation on Ser396 and Ser404 of the Tau protein (Otvos et al., 1994). All these phosphorylation sites have been involved in pathological findings (Mondragón-Rodríguez et al., 2014). PHF-1 is generally considered a later marker in the dynamic sequence of Tau phosphorylation events during the evolution of neurofilaments in AD, when compared to AT8 (Oh et al., 2010). Having very similar results with both antibodies indicates that we are detecting highly phosphorylated forms of Tau, which are the hallmarks of AD and other tauopathies related to memory loss (Tepper et al., 2014).

The levels of acetylated tubulin, a post-translational modification of α-tubulin which results in microtubule stability (Strzyz, 2016), were also studied as another potential mechanism of AM-mediated memory preservation. Aged human donors and male mice had lower levels than their younger counterparts, although this did not happen with female mice. In these animals we did not see any difference between genotypes, although we expected them based on previous studies (Sackett et al., 2008). Perhaps this loss of hyperpolymerized microtubules in the neurons occurs late in the aging process, resulting in synaptic disconnection, and is not so prevalent in females, where Tau phosphorylation may be more relevant. Of course, another possibility is that female hormones may have a neuroprotective effect in this context. Other α-tubulin modifications have been also reported to increase in neurodegenerative diseases, including AD (Vu et al., 2017), suggesting an important contribution of the cytoskeleton to the pathogenesis of memory loss.

An intriguing feature is the clear sexual dimorphism we observed in the behavioral and biochemical studies. Our fear conditioning results suggest that aged female mice are more prone to contextual memory loss than their male counterparts, a condition that was compensated by the lack of AM expression in neurons. Albeit using different memory tests, these sexual differences have been also reported in humans where women have an increased risk for memory disorders relative to men later in life (Jacobs et al., 2016; Reed et al., 2017). It seems that this risk depends on sex steroids, whose decline after menopause correlates with more pronounced alterations in hippocampal connectivity (Jacobs et al., 2016). In addition, the physiological effects of AM have shown to be also sex-dependent with females being more susceptible to changes in the levels of AM expression (Martínez-Herrero et al., 2016).

We have previously found that lack of AM has an impact on locomotor activity (Fernández et al., 2008) and pain perception (Fernández et al., 2010) in young mice. These characteristics may have an influence in the way the KO mice respond to the memory tests. To address these issues we performed an open field test in old mice before the NORT and found no differences in motility, as previously shown for mice of this age (Fernández et al., 2008). Since we do not observe any memory differences in younger mice and the older mice do not have locomotor activity differences, we can rule out the influence of this feature on the results of the NORT. Also, to ensure that the different sensitivity to pain did not influence the fear conditioning results, we measured the freezing behavior of mice of either genotype before and after the foot shock. In both cases, the differences were not statistically significant, indicating that this is not an issue for this specific test.

Previous studies have shown that neuronal AM may have a neuroprotective effect, especially in the context of stroke (Hurtado et al., 2010) and exposure to hypobaric environments (Fernández et al., 2008). Nevertheless, we cannot generalize and say that AM is always neuroprotective. For instance, high levels of circulating AM correlate with increased neurological severity in stroke patients (Serrano-Ponz et al., 2016) and endothelial AM seems to have the reverse effect on stroke and brain damage than neuronal AM (Ochoa-Callejero et al., 2016). Until we acquire a more complete understanding on the effects of AM in the CNS, our present data need to be interpreted on their own, indicating that lower levels of AM are beneficial for memory preservation, especially in females.

AM has been shown to bind to complement factor H (CFH) in the blood stream, and each molecule influences the physiological activities of the other (Pio et al., 2001). CFH is also present at high levels in the CNS (Serrano et al., 2003). Although there are no data on the evolution of brain CFH with age, some studies point to a correlation between lower levels of circulating CFH and more severe cognitive impairment during the development of AD, both in patients (Gezen-Ak et al., 2013) and in mouse models (Wang et al., 2016). It would be interesting to investigate the potential impact of CFH on AM-mediated memory loss during normal aging.

Our data suggest that reducing AM/PAMP levels may constitute a novel path to preventing/delaying memory loss. A few years ago, a particular single nucleotide polymorphism (SNP) close to the adm gene was found to be responsible for a natural reduction in the circulating levels of AM (Cheung et al., 2011) and to correlate with cancer susceptibility (Martínez-Herrero and Martínez, 2013). Therefore, it would be interesting to test whether carriers of this SNP are more protected from developing memory impairment. Also, several physiological inhibitors of AM have been proposed for clinical development, including a monoclonal antibody (Martínez et al., 1996), the peptide fragment AM_22–52 (Ishikawa et al., 2003), and a number of small molecules that target either AM or PAMP (Martínez et al., 2004; Roldós et al., 2012). Therefore some of these inhibitors may be used for the pharmacological prevention of age-related memory loss.

In conclusion, normal aging results in higher expression of AM in the brain and AM ablation prevents Tau phosphorylation in female mice and favors memory preservation in advanced age. These observations suggest that a pharmacological inhibition of adm gene products may constitute a novel approach to preventing memory loss in normal aging and in patients suffering moderate AD in high risk of rapid cognitive decline.

AM and MJR conceived of the idea for the study. IML, HF, EM and FJG-B performed experiments and analyzed data. All authors agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All authors contributed intellectually to the revision of the manuscript and approved the final version of the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.