Studies which explicitly examined the impact of years since menopause on the cognitive effects of HT with estradiol are those by Dunkin et al. (2005, 2006). Dunkin et al. (2006) reviewed the literature on the basis of age of the women involved in HT trials and determined that those studies where the women were over the age of 65 have generally failed to find any cognitive benefit, or have found a cognitive disadvantage of HT, whereas those studies using younger participants have been more likely to find positive effects. Indeed, in Dunkin et al.'s randomized placebocontrolled design (Dunkin et al., 2005), these researchers found that recently post-menopausal women benefitted more in terms of executive functioning than women with a longer time since menopause, from a 10 week transdermal estradiol intervention. Some of the latter group actually showed decline in executive function. While these studies showed a significant relationship between years since menopause and cognitive impact of estradiol therapy, with younger women showing a greater positive effect, they did not attempt to disentangle the age and years since menopause confound. Nevertheless, evidence from the Cache County longitudinal study examining HT (Zandi et al., 2002) found that the normally found increased risk of AD in women in comparison to men was not found for women who had begun HT at earlier ages and had used it for around 10 years. The current average age of the women in this study was 73 years, and current users of HT who had not begun it at earlier stages did not show reduced risk of AD.

This accumulation of evidence has led some researchers to propose a critical period for any HT-related neuroprotection (Resnick and Henderson, 2002; Schneider, 2004). Genazzini et al. (2007) suggest a mechanism whereby aging affects ERs, and ER co-activators such as growth factors, neurotransmitters, and neuromodulators. For example, they refer to increased expression of Brain Derived Neurotrophic Factor (BDNF) in young rats in response to estrogens, but decreased BDNF expression in older rats. Given that BDNF has been related to maintenance of brain plasticity in the aging brain in animals and humans, this illustrates the issue. A review by Maki (2013) examines a range of study designs to collate findings on this issue, conducting meta-analyses where appropriate. She concluded that observational studies and neuroimaging studies provide reliable evidence that early use of HT is protective of cognitive function and later use is not, or is detrimental. RCTs showed a benefit or early use of estrogens but not for estrogens + progesterone, regardless of timing and determined that overall there is evidence of both cognitive benefit and reduced risk of AD where the HT is taken in the first 5 years after the last menstrual period when the regimen is not continuous estrogens + progesterone. However, this author also highlighted the possibility that the relationship maybe related to the health of the woman's brain as opposed to their reproductive age, given that data from the Women's Health Initiative Memory Study showed the greatest hippocampal loss in women with initially low cognition scores (Resnick et al., 2009).

Some research has pointed out that, despite the evidence obtained in randomized studies, duration of therapy may have been missed as a variable. Most studies only followed participants for a few years. In response to this uncertainty, the duration of HT was used as a variable in a French cohort assessed for cognitive function. HT was found be associated with better performance in some cognitive domains, such as verbal fluency, visual memory, and psychomotor speed, but only if treatments lasted over 10 years (Ryan et al., 2009). However, if data were adjusted by age, education level, age at menopause, depressive symptoms, etc., none of the effects of HT remained significant. This conclusion is further supported in another recent study in Finland (Imtiaz et al., 2017); in this cohort study, women had received HT from zero (never received it) to more than 5 years and the overall findings provided no strong evidence for a protective effect of estradiol-based HT against cognitive decline.

A further study by Erickson et al. (2007) examined this issue explicitly in relation to cognitive assessments and regional atrophy using detailed voxel based morphometry of MRI scans, enabling analysis of systematic variation of gray and white matter in significant brain regions. They compared HT treatment duration groups of women, balanced for numbers taking opposed or unopposed estrogens, on a test of executive function, the Wisconsin Card Sorting Task, performance on which has been previously shown to be related to prefrontal cortex gray matter. They also used a test of general cognition, the Modified Mini-Mental State Examination. The duration groups were (i) never used HT (ii) up to 10 years (iii) 11–15 years (iv) 16+ years, with a mean age of 69.61 years. Given that both physical exercise and estrogens have similar actions on neurotrophic growth and vascularization, and BDNF actions are affected both by estrogens and exercise in the hippocampus, with humans showing the greatest concentration of BDNF receptors in the frontal lobes, Erickson et al. (2007) hypothesized that estrogens and exercise may interact to positively affect human brain tissue in the frontal lobes and also cognition. They found that HT duration up to 10 years was associated with better performance on the Wisconsin Card Sorting Task and with spared gray matter in the prefrontal cortex, compared with other groups, adjusting for age, age at menopause, socio-economic status and education. However, HT duration longer than 10 years was associated with greater prefrontal deterioration and greater decline in the measures of executive function. Importantly, physical fitness level (measured by VO2 max) interacted with duration such that higher fitness levels ameliorated the negative effects of longer duration of HT and enhanced positive effects of shorter durations in both performance and brain morphometry. This evidence for interactions between estrogen therapy and exercise/fitness on cognitive and brain health is important, and supports the role of healthy lifestyle interventions alongside HT. However, the effects were specific to executive function and did not occur for the general cognition measure.

To summarize, and in consistence with the conclusion from other groups (Henderson and Popat, 2011), the most solid evidence seems to favor the notion that HT is not indicated for treatment of cognitive decline or dementia, and that the inconsistency in results may be related to the control of confounding variables, the timeline (onset and duration) of the use of HT and to the treatment regimen. Maki (2013) suggests that there is tentative support for beneficial effects for hysterectomised women who are treated with estrogens alone, and Erickson et al.'s (2007) suggests durations longer than 10 years are not beneficial but that HT benefit or negative effects interact with physical fitness and health. The concept of a critical period of treatment in early post-menopause seems wellestablished as at least not a disadvantage for cognition. However, studies are still insufficient to be clear on which circumstances, to which women, and for how long HT should be prescribed for maximum cognitive benefit and/or minimum cognitive negative effect. Studies where the underlying brain health or physical fitness of the women is considered as a variable, seem a useful way to move forward.

#### MOOD AND COGNITION

The influence of mood, particularly depressed mood and anxiety, on cognition is supported by considerable evidence at all ages, but a greater impact of depression on cognition is recognized for older people. Indeed, higher levels of depressive and anxiety symptoms are directly related to poorer cognitive performance because both disorders are often accompanied by attention and concentration deficit symptoms. There is considerable support for depression as a risk factor for cognitive impairment, with Chung et al. (2015) finding an association between lifetime history of major depression and amyloid beta deposition in individuals with mild cognitive impairment (MCI). In addition to the influence of depression on cognition, depression is commonly implicated as a significant risk factor for dementia in older adults (da Silva et al., 2013) to the extent that it may be a prodromal symptom of some kinds of dementia (Panza et al., 2010). That is, depression can be both a risk factor for dementia and an early indicator of incipient dementia (Fiske et al., 2009). Both anxiety and depression affect cognitive performance at all ages, with long term anxiety being a risk factor for depression. However, stress also has a long-term negative effect on cognition, particularly executive function. Stress affects similar brain structures and cognitive functions (particularly memory) as advancing age, e.g., the hippocampus or the frontal lobes, with effects of psychological stress mediated by the effects of the action of stress hormones such as glucocorticoids (Bunce et al., 2008).

The correlation of depressed mood with nearly every indicator of memory functioning, for example, in the longitudinal studies discussed above (Woods et al., 2000), raises important questions and opens the door to indirect actions of estrogens on cognition through their well-known relationship with mood states. The influence of ovarian function on mood states has been widely claimed in the literature for a considerable time. The issue is interesting in itself, but also in terms of its effects on cognition in later life. That is, mood state could act as an indirect effect of changes in sex hormones on cognition with increasing age. Thus, cognitive complaints during peri-menopause may at least partially result from the effect of peri-menopausal anxiety or depressive symptoms on the brain (Greendale et al., 2011).

Research has demonstrated that the lifetime prevalence rate of mood disorders is significantly greater in women than in men, approximately two times more frequent (Walf and Frye, 2006; Ter-Horst et al., 2009). Data suggests that estrogens, or their absence, are strongly implicated in the regulation of mood and behavior, as well as in the pathobiology of mood disorders (Halbreich and Kahn, 2001). So the interaction of estrogens with the serotonergic systems (Amin et al., 2006) has been taken to propose estradiol as a protective agent against mood changes related to serotonin withdrawal (Wharton et al., 2012). Due to the correlation between depression and serotonin, estrogen treatment can have beneficial effects to buffer changes in mood.

Clinical observation also shows that estrogens play an essential role in the emergence and course of mood disorders in women. Gender differences in mood disorders are present throughout the lifespan, but periods of hormonal fluctuations or estrogens instability (i.e., pre-menstrually, post-partum, perimenopausally) are related to increased vulnerability to mood disorders among susceptible women (Halbreich and Kahn, 2001).

Mood disorders are prevalent during the menopausal transition, with prevalence figures of 16.5% for depressed mood in women during midlife (Prairie et al., 2015). The SWAN study found that women were two to four times more likely to suffer a depressive episode during the menopausal transition than during the pre-menopausal period (Bromberger et al., 2011). Other investigators have reported similar findings (Soares, 2010). The known interaction of estrogens with serotonin and other monoamines provides biochemical rationality to the clinical findings (Warnock et al., 2017). Mood problems have been found to increase in those with a history of mood disorders, but can also occur de novo as a consequence of the hormonal changes. In most cases, the period of vulnerability to mood problems (Alexander et al., 2007) subsides when hormonal levels stabilize and women enter full menopause. It is understood that it is hormonal fluctuations that more directly determine the mood changes (Wharton et al., 2012).

## HT AND MOOD

The clear hormonal influence in mood fluctuations has prompted the postulation of HT as a useful remedy. One recent central study is the previously mentioned KEEPS-Cog, a multicenter randomized trial that, in addition to cognitive effects, assessed the impact on mood, including depression and anxiety. Oral estrogens (CEE), but not transdermal estradiol, effectively reduced scores of depression and anxiety over the 48 months of treatment (Gleason et al., 2015). The reasons for the difference between the treatments remain elusive, although it is possible that the maintained levels of circulating estradiol downregulates ER in the concerned brain regions. This effect of estrogens in KEEPS-Cog further confirms previous studies of different sizes and relevance, as shown in several reviews (Schmidt et al., 2000; Soares et al., 2001; Schiff et al., 2005). Fischer et al. (2014) reviewed five RCTs which investigated the impact of HT on mood, all of which showed a benefit of treatment. Three of the studies involved only estradiol, whereas two found effects for both estrogens alone or with different amounts of progesterone (MPA) and a further study found reductions in depression and anxiety following 3 months of transdermal estradiol with or without norethisterone. Thus, although there is a suggestion of positive effects on mood of HT, the mechanisms need further investigation, with remaining questions being the extent to which mood improvements are direct effects, or indirect via effects of HT on other distressing menopausal symptoms such as sleep disturbance or hot flushes. Furthermore, the complex relationship of depression with cognition also suggests that the influence of HT on depression as a possible mediator of any observed influence on cognition could also be examined in future research, rather than as separate effects.

## INDIRECT IMPACTS ON COGNITION VIA EFFECTS OF SEX HORMONE REDUCTION ON CARDIOVASCULAR HEALTH

Cardiovascular diseases represent an important health issue all over the world because they are the primary cause of death for both men and women. Morbidity and mortality rates are higher in men before menopause, whereas after this age, these rates converge.

As reviewed in Navarro-Pardo et al. (2017), estrogens act to improve lipid profile, promote vasodilatation and antioxidant activities, supporting vascular health. By contrast, the menopause leads to an overturn of all these effects, increasing cardiovascular risk. Vascular deterioration is an important issue that should be considered when assessing the impact of the menopause on cognition, for example, the vascular changes associated with vasomotor symptoms that take place during and after menopause. The SWAN study evaluated whether the decrease in cognitive processing speed observed during peri-menopause might be influenced by the existence of hot flushes. Moreover, anxiety and depressive symptoms were also considered in the research.

A possible long-term effect has also been raised. The hypothesis that the menopause might accelerate atherosclerosis and consequently increase future cardiovascular risk, has a correlate in terms of cognitive function. Vascular factors are involved in roughly half of all dementia cases, probably because they show the cumulative impact of very different factors along lifespan (Bowler, 2005; Sonnen et al., 2007; Gorelick et al., 2011). Outside of dementia, vascular factors are also associated with cognitive change in otherwise healthy older people. Going back to early longitudinal studies, Wilkie and Eisdorfer (1971) pointed out that "The presence of large numbers of aged with cardiovascular illness suggests that the basis for the cognitive decline associated with aging after maturity should be considered secondary to some pathologic process, and not merely as a "normal" aging process" (p962) and longitudinal studies such as Okonkwo et al. (2011) demonstrated that cardiovascular indices such as variability in blood pressure and cardiac output predicted declines in attention, executive function and psychomotor function over 36 months, but basic resting blood pressure measurements did not. Likewise, Singh-Manoux et al. (2003) demonstrated prediction of poor cognitive function from occurrence of angina pectoris, myocardial infarction, all coronary heart disease and intermittent claudication, 11 years prior to the cognitive measures.

The notion of a critical period for positive cognitive effect or avoidance of a negative impact on cognition or increase in dementia risk is mirrored in investigations of HT where HT is shown to be beneficial in the early peri- and post-menopausal period, but not in older women or those who may already have advanced atherosclerosis (Hodis et al., 2016; Savolainen-Peltonen et al., 2016). These parallel findings further suggest the role of estrogens effects on the cardiovascular system as a mediator in the impact on cognitive function health and changes.

Thus, the relationship between cardiovascular health and cognitive health is well-supported, but studies examining the impact of HT on cardiovascular health as a mediator of any beneficial impact on cognition are needed.

## CONCLUSIONS

The presence of ERs has been confirmed in different brain locations related to cognition, mainly to verbal memory and retrieval, working memory, executive function and attention, such as the hippocampus and the prefrontal cortex, leading to the rationale that changes in levels of estrogens would produce direct effects in the brain and accordingly in cognitive function. Furthermore, the interaction of estrogens with serotonergic pathways also provides a focus for explaining peri-menopausal mood changes.

Menopausal withdrawal of estrogens could also have a specific impact on the vascular tree and thereby have a secondary effect on cognition such as memory declines via vasomotor symptoms.

A range of methodologies have been used to study these effects, such as longitudinal, cross-sectional, and intervention studies, including randomized controlled trials. In reviewing several of these studies, it is concluded that menopause effects on cognitive functioning are not clear because of the series of confounding variables found to have an effect. When confounds such as age are controlled, the most reliable effects are for a measure related to executive function, verbal fluency.

Research on hormonal therapy (HT) in menopausal women has also been reviewed. Differences in HT regarding therapy timeline (onset and duration), composition, manner of administration, and interaction effects with other variables (such as age differences, education, and baseline performance) had significant impacts and made direct comparisons amongst the studies difficult.

It was concluded that HT does not have a clear impact on cognition, including episodic memory or executive functions and is not recommended as a treatment or prevention pathway for cognitive decline or dementia in older women. There is not enough evidence to support the existence of a window of opportunity in the immediate post-menopause, but an HT neutral effect (on cognition) extends for a reasonable number of years post-menopause. The suggestions of beneficial effects on cognitive function in the first few years after menopause may imply some risk reduction regarding cognitive decline and dementias. Although there are useful developments, for

#### REFERENCES

example, the interaction of HT impacts with the benefits of physical exercise, late-life cognitive consequences are still poorly addressed.

On another front, mood disorders are twice as frequent in women than in men, and estrogens changes are probably implicated given that differences appear from menarche through reproductive age, including periods of estrogenic instability. An important component of well-being, self-perceived health, seems to change significantly from pre- to post-menopause.  Related to this depression-serotonin correlation, HT could have a beneficial effect on mood disorders, although the mechanisms need further investigation. Depressed mood and anxiety have a negative influence on cognition, particularly on memory functioning, particularly so for older people. Depression is a well-known risk factor or prodromal symptom for cognitive impairment and dementia.

Cardiovascular effects of reducing estrogen, including increased cardiovascular risk, seem to act as a secondary pathway by which reductions in this sex hormone has an impact on cognition, for example, given that vascular effects are implicated in at least half of all dementia cases and in many other aging processes.

#### AUTHOR CONTRIBUTIONS

EN-P, CH, and AC: have contributed equally to this article. All authors have read and approved the final manuscript.

across the nation (SWAN). J. Womens Health 16, 331–344. doi: 10.1089/jwh. 2006.0057

**Conflict of Interest Statement:** 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.

Copyright © 2018 Navarro-Pardo, Holland and Cano. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

## Adrenomedullin Contributes to Age-Related Memory Loss in Mice and Is Elevated in Aging Human Brains

Ignacio M. Larrayoz 1† , Hilda Ferrero2† , Eva Martisova2 , Francisco J. Gil-Bea2 , María J. Ramírez 2 and Alfredo Martínez 1 \*

1Oncology Area, Center for Biomedical Research of La Rioja (CIBIR), Logroño, Spain, 2Department of Pharmacology and Toxicology, University of Navarra, Pamplona, Spain

Memory decline is common in elderly individuals and is the hallmark of Alzheimer's disease (AD). Memory failure follows the loss of synaptic contacts in the cerebral cortex and hippocampus, caused in part by cytoskeleton disruption. Adrenomedullin (AM) and its gene-related peptide, proadrenomedullin N-terminal 20 peptide (PAMP), are microtubule-associated proteins (MAP) whose expression has been identified as a potential biomarker for predicting progression from predementia to clinical AD. Here we analyze the connection between AM levels and memory preservation. Mice lacking neuronal AM and PAMP (knockout, KO) and their wild type (WT) littermates were subjected, at different ages, to the novel object recognition test and the contextual fear conditioned test. Aged KO mice have significantly better retention memory than their WT counterparts. This feature was more prominent in females than in males. Prefrontal cortex and hippocampus samples from these animals were subjected to Western blotting for phospho-Tau and acetylated tubulin. Aged female KO mice had significantly less accumulation of phospho-Tau than their WT littermates. In addition, protein extracts from the frontal cortex of non-demented mature (65.10 ± 3.86 years) and aged (77.14 ± 2.77 years) human donors were analyzed by Western blotting. Aged human brains had significantly higher levels of AM and lower levels of acetylated tubulin than younger donors. These observations suggest that drugs or interventions that reduce AM/PAMP expression may constitute a new avenue to prevent memory decline during normal aging and in patients suffering moderate AD in high risk of rapid cognitive decline.

Keywords: adrenomedullin, normal aging, Alzheimer's disease, memory loss, microtubules, p-Tau

#### INTRODUCTION

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

#### Edited by:

Luis Miguel Garcia-Segura, Consejo Superior de Investigaciones Científicas (CSIC), Spain

#### Reviewed by:

Jorge Valero, Achucarro Basque Center for Neuroscience/Ikerbasque Basque Foundation for Science, Spain Yolanda Diz-Chaves, Institute of Biomedical Research of Vigo (IBIV), The Biomedical Research Centre (CINBIO), Spain

\*Correspondence:

Alfredo Martínez<EMAIL_ADDRESS>
†These authors have contributed equally to this work.

Received: 04 August 2017 Accepted: 03 November 2017 Published: 15 November 2017

#### Citation:

Larrayoz IM, Ferrero H, Martisova E, Gil-Bea FJ, Ramírez MJ and Martínez A (2017) Adrenomedullin Contributes to Age-Related Memory Loss in Mice and Is Elevated in Aging Human Brains. Front. Mol. Neurosci. 10:384. doi: 10.3389/fnmol.2017.00384

**Abbreviations:** AD, Alzheimer's disease; AM, adrenomedullin; APP, amyloid precursor protein; CFH, complement factor H; CNS, central nervous system; KO, knockout; MAP, microtubule associated protein; NORT, novel object recognition test; PAMP, proadrenomedullin N-terminal 20 peptide; PHF, paired helical filaments; SNP, single nucleotide polymorphism; WT, wild type.

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 agingrelated 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.

## MATERIALS AND METHODS

#### Knockout (KO) Mice Lacking Neuronal AM

Conditional knockout (KO) mice where AM was eliminated from neurons have been previously described (Fernández et al., 2008). KO and wild type (WT) littermates of both sexes were allowed free access to food and water under standard laboratory conditions, with light/dark cycles of 12/12 h, and a constant temperature of 24ºC. All procedures were carried out in accordance with the European Communities Council Directive (86/609/CEE) on animal experiments and with approval from the ethical committees on animal welfare of our institutions (OEBA-CIBIR and University of Navarra protocol 054-12).

## Behavioral Tests

#### Novel Object Recognition Test (NORT)

Test was performed as previously described (Gerenu et al., 2013) in young (3 months old) and old (18 months old) mice. In short, animals (n = 8 per group) were first familiarized with the arena (65 × 65 × 45 cm) for 30 min and after 1 day mice were allowed to explore two identical objects during 5 min (training). Retention was assessed 24 h post-training, when one object was replaced by a novel one. Retention score is expressed as discrimination index (percentage of time exploring the novel object to the total time of object exploration).

#### Contextual Fear Conditioned Test

After 24 h of rest, all mice were subjected to a fear conditioned test. The behavioral procedure involved three phases: habituation, training, and testing. Mice were habituated for 5 min to the context, which consisted in a soundproof box with white walls, light, and a background noise produced by a fan. The training phase was conducted 24 h later, where mice were placed in the same context and allowed to explore for 2 min prior to a 2-s footshock (0.3 mA) stimulus. After 30 s mice were returned to their home cage. 24 h later mice were placed back in the conditioning box and allowed to explore the context for 2 min, during which freezing time was recorded (contextual long-term memory). Freezing behavior was defined as an absence of cage displacement. Freezing scores were expressed as percentages of total freezing time. The conditioning procedure was carried out in a StartFear system (Panlab S.L., Barcelona, Spain) that allows movement recording by a high-sensitivity Weight Transducer system and data analysis by the built-in FREEZING and STARTLE software.

#### Human Brain Tissue

Brain tissues were obtained from the Oxford Project to Investigate Memory and Ageing (OPTIMA, see www.medsci.ox.ac.uk/optima). Subjects for this study constituted a randomly selected subset of the participants, now part of the Thomas Willis Oxford Brain Collection within the Brains for Dementia Research Initiative (BDR). At death, informed consent had been obtained from the patients' next-of-kin before collection of brains and the study was approved by the UK National Research Ethics Service. All cases were selected based on clinic-pathological consensus diagnoses. A total of 12 individuals were included in the study. They included six mature donors (3 men, 3 women, age at death = 65.10 ± 3.86 years; post mortem delay = 34.10 ± 4.91 h) and six older donors (3 men, 3 women, age at death = 77.14 ± 2.77 years; post mortem delay = 49.42 ± 6.82 h). These participants were classified as normal controls, did not have dementia or other neurological diseases, did not meet CERAD criteria for AD diagnosis, and were staged at Braak 0-II. Frontal (Brodmann Area, BA10) cortices were dissected free of meninges. To partially mitigate the possible effects of cause of death on neurochemical

#### TABLE 1 | Antibodies and conditions used in this study.

determinations, brain pH was measured as an index of acidosis associated with terminal coma. Brain pH is used as an indication of tissue quality in post-mortem research, with pH > 6.1 considered acceptable (Bahn et al., 2001; Lewis, 2002). All the tissue used fulfilled this condition. All subsequent analyses were performed blind to clinical information.

#### Western Blotting

Two days after completing behavioral testing, all mice were euthanized and prefrontal cortex and hippocampus were dissected out. Mouse and human samples were homogenized in RIPA buffer (Thermo Scientific, Rockford, IL, USA) containing protease (EDTA-free complete, Roche, Basilea, Switzerland) and phosphatase (PhosStop, Roche) inhibitors. Homogenates were centrifuged for 30 min at 15,000× g and the supernatants collected. Protein concentration was determined by the BCA kit (Pierce, Rockford, IL, USA), with bovine serum albumin as standard, using a spectrophotometer (POLARstar Omega, BMG Labtech, Ortenberg, Germany). Then, 25 µg of each sample were mixed with 4× sample buffer (Invitrogen, Carlsbad, CA, USA) and heated for 10 min at 70◦C. Samples were run on 4%–12% SDS–polyacrylamide gels. Seeblue plus 2 Prestained Standards (Invitrogen) were used as molecular weight markers. Proteins were transferred onto 0.2-µm nitrocellulose membranes (Amersham GE HealthCare, Pittsburgh, PA, USA). Membranes were incubated overnight at 4◦C with primary antibodies followed by peroxidaselabeled secondary antibodies (**Table 1**). Immunoreactive bands were visualized using enhanced chemiluminescence and quantified by an image analyzer (Quantity One, Bio-Rad, Hercules, CA, USA). Membranes were stripped with Restore PLUS Western Blot Stripping Buffer (Thermo Scientific). β-Actin was used as an internal loading control. Results were calculated as the percentage of optical density (OD) values of the WT.

#### Statistical Analysis

Data were analyzed by SPSS for Windows, release 15.0. Normalcy and homoscedasticity were checked by Shapiro–Wilks's and Levene's tests, respectively. Normally distributed data were analyzed by Student's t-test or two-way analysis of variance

(ANOVA) (genotype × age) followed by Tukey's post hoc test. P values lower than 0.05 were considered statistically significant.

#### RESULTS

#### Mouse Experiments

The conditional AM KO animals and their WT littermates were tested in two different cognitive paradigms: the novel object recognition test (NORT) and the fear conditioning test. Animals included were either young (3 months) or aged (18 months) mice. Each age group was composed by KO (n = 8) and WT (n = 8) littermates.

First of all, to ensure that WT and KO mice had no differences in general locomotor activity or other psychological/physiological traits that may interfere with memory determination, their investigative activity was recorded on the first and second day of the experiment. On the first day, in the first 30 min, WT mice traveled 18871 ± 2362 cm whereas KO mice walked for 16486 ± 1666 cm (t test, p > 0.05). On the second day (training), when mice were presented with two identical objects, all groups of mice had a ratio close to 50% and there were no significant differences among them (t test, p > 0.05; **Figures 1A,B**). These results show that genotype does not influence mice ability to move freely or to show curiosity when presented with different objects.

When presented with the novel object, young animals had no significant differences in recognition memory between those carrying adm and the knockouts (**Figures 1C,D**).

Recognition memory was significantly facilitated in aged adm KO animals, both in males (**Figure 1C**, two way ANOVA, significant interaction F = 12.44, p < 0.05, Tukey's multiple comparisons test, p < 0.05) and females (**Figure 1D**, two way ANOVA, significant interaction F = 11.11, p < 0.05, Tukey's multiple comparisons test, p < 0.05).

In the fear conditioning test (contextual learning), first we measured the freezing levels during training and found they were around 40% for both genotypes (t test, p > 0.05) during the first 2.5 min of their stay in the chamber. We also tested foot shock sensitivity by measuring the freezing levels immediately following the foot shock. These levels were close to 80% for both genotypes (t test, p > 0.05) for the 30 s following the shock, indicating that the genotype has no influence in regular freezing behavior or in foot pain sensitivity.

During the memory test period, male mice showed no statistically significant differences in freezing behavior irrespective of age or genotype (**Figure 1E**). In contrast, older female WT mice presented an age-related memory loss as demonstrated by a statistically significant reduction in freezing time when compared with younger females of the same genotype (**Figure 1F**, two way ANOVA, significant interaction F = 9.02, p < 0.05, Tukey's multiple comparisons test, p < 0.05). Interestingly, old female mice lacking neuronal adm showed better memory than their WT littermates (Tukey's multiple comparisons test p < 0.05).

Altogether, both cognitive tests point to a memory protection phenotype in animals lacking neuronal adm.

To investigate the possible mechanism underlying these behavioral observations, changes in pTau expression were checked in the mouse prefrontal cortex and hippocampus using two different antibodies: AT8 (**Figure 2**) and PHF1 (**Figure 3**). In male mice, there was an increased pTau/Tau ratio associated to aging that was not significantly affected by deleting the adm gene (two way ANOVA, main effect

Figure 2 and are repeated here to allow a direct comparison with the total protein and loading controls.

of age, F = 21.50, p < 0.01, **Figures 2A,C**). However, in female mice, a significant interaction between genotype and age was found (**Figures 2B,D**), and the increased expression of pTAU associated to aging was counteracted in mice lacking the adm gene (two way ANOVA, significant interaction F = 16.81, p < 0.05, Tukey's multiple comparisons test, p < 0.05).

For the other antibody, PHF1 (**Figure 3**), results were perfectly parallel to those observed with AT8. There was an increased pTau/Tau ratio associated to aging in males that was not significantly affected by deleting the adm gene (two way ANOVA, main effect of age, F = 28.81, p < 0.05, **Figures 3A,C**). In females, a significant interaction between genotype and age was found (**Figures 3B,D**), and the increased expression of pTAU associated to aging was counteracted in mice lacking the adm gene (two way ANOVA, significant interaction F = 18.24, p < 0.05 Tukey's multiple comparisons test, p < 0.05).

To further study cytoskeleton stability, tubulin acetylation was checked in the frontal cortex and hippocampus of these animals. Aged male mice showed a decrease in the expression of acetylated tubulin that was independent of the genotype (two way ANOVA, main effect of age, F = 39.23, p < 0.01, **Figures 4A,C**). No effect associated to either age or genotype was found in female mice (**Figures 4B,D**).

#### Human Specimens

To test whether similar changes happen in humans, frontal cortex protein extracts were obtained from mature and aged human donors. Western blots for AM showed a significant (p < 0.05) increase in the relative levels of this peptide in aged

brains, which was more than double the levels observed in younger samples (**Figure 5A**). In contrast, the relative levels of acetylated tubulin were significantly (p < 0.05) reduced by aging (**Figure 5B**).

### DISCUSSION

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

#### REFERENCES

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 AM22–52 (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.

#### AUTHOR CONTRIBUTIONS

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.

## FUNDING

IML and AM are funded by Fundación Rioja Salud (FRS). HF is a recipient of a fellowship from Spain's Ministerio de Educación, cultura y Deporte (FPU).

**Conflict of Interest Statement**: 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.

Copyright © 2017 Larrayoz, Ferrero, Martisova, Gil-Bea, Ramírez and Martínez. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

**134**

# Memory Training Program Decreases the Circulating Level of Cortisol and Pro-inflammatory Cytokines in Healthy Older Adults

Mirko Pesce, Raffaella Tatangelo\*, Irene La Fratta, Alessia Rizzuto, Giovanna Campagna, Cinzia Turli, Alessio Ferrone, Sara Franceschelli, Lorenza Speranza, Maria C. Verrocchio, Maria A. De Lutiis, Mario Felaco and Alfredo Grilli

School of Medicine and Health Science, University G. D'Annunzio, Chieti, Italy

#### Edited by:

Julie A. Chowen, Hospital Infantil Universitario Niño Jesús, Spain

#### Reviewed by:

Mariano Ruiz-Gayo, CEU San Pablo University, Spain Vicente Barrios, Hospital Infantil Universitario Niño Jesús, Spain

> \*Correspondence: Raffaella Tatangelo<EMAIL_ADDRESS>
Received: 07 February 2017 Accepted: 06 July 2017 Published: 24 July 2017

#### Citation:

Pesce M, Tatangelo R, La Fratta I, Rizzuto A, Campagna G, Turli C, Ferrone A, Franceschelli S, Speranza L, Verrocchio MC, De Lutiis MA, Felaco M and Grilli A (2017) Memory Training Program Decreases the Circulating Level of Cortisol and Pro-inflammatory Cytokines in Healthy Older Adults. Front. Mol. Neurosci. 10:233. doi: 10.3389/fnmol.2017.00233 Aging cognitive decline has been associated to impairment of the Hypothalamus Pituitary Adrenals (HPA) axis activity and a higher level of the systemic inflammation. However, little is known about the molecules driving this process at peripheral level. In addition, the cognitive function is to some extent modifiable with Memory Training (MT) programs, even among older adults and beyond. The study aims to evaluate whether MT could contribute to ameliorate cognitive performance and modulate the HPA axis activity as well the low level inflammation in the aging phenotype. Whether the phosphatase WIP-1, a negative regulator for inflammation, is involved in this process was also investigated. We recruited 31 young adults (19–28, years of age) and 62 older adults aged over 60. Thirty-two older adults were submitted to 6-months of MT program (EG), and 28 older adults were no treated and used as Control Group (CG). Global cognitive functioning (MMSE score), verbal and visual memory, and attention were assessed at baseline (T0) and after 6-months (T1). At the same time, plasmatic level of Cortisol (C), IL-1β, IL-18, IL-6, and the expression of WIP-1 mRNA and protein in ex vivo Peripheral Blood Mononuclear Cells were analyzed in young adults at T0, as well in older adults at T0 and T1. Together, the results suggest that MT improves the global cognitive functionality, verbal and visual memory, as well as the level of attention. At the same time we observed a decrease of the plasmatic level of C, of the cytokines, and an increase of the expression of mRNA and protein of WIP-1. The analysis of correlations highlighted that the level of the mRNA of WIP-1 was positively associated to the MMSE score, and negatively to the C and cytokine levels. In conclusion, we purpose the MT as tool that could help support successful aging through the improving of memory, attention and global cognitive function performance. Furthermore, this approach could participate to maintain lower the peripheral levels of the C and pro-inflammatory cytokines. The WIP-1 as a potential new target of the pathophysiology of aging is theorized.

Keywords: Memory Training, cortisol, inflammaging, WIP-1, pro-inflammatory cytokines

## INTRODUCTION

fnmol-10-00233 July 21, 2017 Time: 16:29 # 2

During adulthood, the cognitive performance and immunity system begins to decline concurrently prior to the third decade of life suggesting that the mechanisms underlying these processes are inter-related (Rosano et al., 2012). There is comparable evidence indicating that increases in the level of peripheral markers of inflammation are associated with age-related declines in the health of the brain (Franceschi, 2007). Such evidence is parallel to longstanding findings from neurocognitive studies in populations of older adults living in a community, that indicate in a consistent manner an association between higher inflammatory levels and lower cognitive levels and a higher risk of cognitive impairment over time (Gorelick et al., 2014).

The systemic inflammation related to aging involves multiple organs and represents a common mechanism leading to multiple chronic diseases such as dementia, diabetes, atherosclerosis, and arthritis, which has a negative impact on the health span of the elderly (Maggio et al., 2005; Franceschi, 2007; Green et al., 2011). These findings supported the 'inflammaging theory' that hypothesizes a global reduction in the capacity to cope with a variety of stressors and purposes a concomitant progressive cytokines dysregulation as the major characteristics of the aging process (Franceschi and Campisi, 2014).

The modification of the Hypothalamus Pituitary Adrenal (HPA) axis activity represents another hallmark of the aging phenotype (Ferrari and Magri, 2008). Several findings suggest that a higher basal endogenous cortisol (C) concentration, a downstream effect of the increase in basal tonus of the HPA axis activation, is age-related (Deuschle et al., 1997; Giordano et al., 2005). The enhanced HPA axis activity could be explained by a decrease of the hippocampal inhibitory function that leads to an increase of the release of C (De Kloet et al., 1998). Consistently, the aging related higher plasma levels of C have predicted a smaller hippocampal volume, faster hippocampal atrophy (Lupien et al., 1998; O'Hara et al., 2007), that are associated to memory decline, and to general poor cognitive performance (Lupien et al., 2007; Lee et al., 2008; Tatomir et al., 2014; Geerlings et al., 2015; Vogel et al., 2016).

The aging systemic higher exposure to C could be associated with the accelerated features of low-grade inflammation that characterizes older adults. The HPA axis activity exerts a pivotal role for the homeostasis of the immune system. The circulating level of C and those of several cytokines have been concurrently associated with a decreased hippocampal volume and cognitive impairment (Bauer, 2008; Sudheimer et al., 2014; Metti et al., 2015; Chesnokova et al., 2016; Jin et al., 2016). The Glucocorticoids (GCs) and pro-inflammatory cytokines are not independent of each other and they interact on multiple levels. In particular, the GCs that were classically viewed as antiinflammatory could have a dual role, inducing pro-inflammatory effects. Their administration results in an increased systemic trafficking of lymphocytes and monocytes (Bowers et al., 2008), and the exertion of pro-inflammatory actions through synergistic interactions with inflammatory cytokines, such as IL-1β and IL-6 (Langlais et al., 2008; Ding et al., 2010; Busillo et al., 2011).

The mechanisms underlying low-grade systemic inflammation in several organs in the absence of overt infection that was observed during aging are still speculative, and little is known about the "master" molecule involved in this process. The Wild type p53-Induced Phosphatase-1 (WIP-1) is a phosphatase whose expression is induced in response to many types of cellular stress, as γ or UV radiation in a p53-dependent manner (Fiscella et al., 1997). This protein has been associated with cellular senescence (Salminen and Kaarniranta, 2011), and its expression has been showed to decrease during age (Wong et al., 2009). More recently, WIP-1 has been recognized as an intrinsic negative regulator of inflammatory response in the CNS (Tan et al., 2013), and also at the peripheral level where it acts as a negative regulator for neutrophil migration and proinflammatory cytokines production through various pathways, including the feedback regulation of the NF-κB signaling (Sun et al., 2014). It could be hypothesized that WIP-1 could be associated with sterile inflammation that underlines the aging process.

The above mentioned age-related modulations and variations are dynamic and not irreversible processes. The ways that allow modulation of brain plasticity in animal and human using cognitive or physical training have resulted to be of great interest (Curlik and Shors, 2013). Several studies on rodents have shown that Environmental Enrichment (EE) – modification of the environment to enhance the animal physical and psychological well-being – (Baumans, 2005), has been shown to slow down neuronal aging (Kempermann et al., 2002), improve cognition and memory (Jankowsky et al., 2005; Hannan, 2014). Some EE stimuli are able to affect C (Simpson and Kelly, 2011), cytokines and various immune components at a peripheral level, suggesting that this could be a potential mechanism of action in how it could modulate the brain function (Singhal et al., 2014). To this end, within cognitive training procedures, Memory Training (MT) based research generally reveals an improvement in memory performance in older adults through the utilization of mnemonic strategies. However, even cognitive training was successfully included within EE stimuli. To our knowledge, neither study has been carried out to determine whether it reduces the endogenous C levels and the behavioral sequence of chronic inflammatory conditions that characterize the aging phenotype.

Considering the above mentioned in aggregate, we hypothesize that the MT could be considered as a treatment that similar to other stimuli of the EE, could enhance the cognitive and physical well-being of older adults. It is for this reason that this study wants to investigate whether a MT program acts on the plasmatic level of C, the pro-inflammatory cytokines IL-6, IL-1β, and L-18, and the expression of the WIP-1 in ex vivo Peripheral Blood Mononuclear Cells (PBMCs) of apparently healthy elderly (older adults, 65–74 years; De Beni, 2009), relating to their degree of cognitive abilities. We chose these cytokines because all of them had been previously associated with specific features of aging phenotype (Franceschi, 2007), and also their higher levels had been successfully suggested as a marker of poorer cognitive performance in healthy subjects

#### TABLE 1 | Exclusion criteria of all participants.

fnmol-10-00233 July 21, 2017 Time: 16:29 # 3

#### Exclusion criteria

for the IL-6 and the IL-1β (Yirmiya and Goshen, 2011), and in a clinical population for the IL-18 (Bossù et al., 2008; Zhang et al., 2013).

Our results provide important evidence that MT could contribute to the decreasing of the C level, restore the WIP-1 expression and reduce the value of the pro-inflammatory cytokines at a peripheral level, participating in "holding off " the systemic inflammatory condition that characterizes healthy older adults. We first involved WIP-1 deficiency in the pathophysiology of aging.

### MATERIALS AND METHODS

#### Participants

Participants of the study were 62 apparent healthy older adult volunteers (29 female, age range 65–74 years of age), recruited with the use of posters, pamphlets and word of mouth at recreational clubs. Thirty-one apparent healthy young adults (16 female, age 19–28 years of age), were also recruited by word of mouth and with pamphlets at University G. d'Annunzio, CH-PE, Italy. The exclusion criteria regarding all participants were summarized in **Table 1**.

Briefly, subjects with the BMI < 20 and >33 k/m2 were excluded (Travison et al., 2007; Mangnus et al., 2016). Subjects with unusual dietary habits (e.g., vegetarians) were also excluded (Sutliffe et al., 2015). Hematological and biochemical tests needed to be within the normal range. Negative serologies for the HIV and Hepatitis C viruses were verified. To assess the inflammatory status, all recruited subjects underwent the same laboratory blood tests: Erythrocyte Sedimentation Rate (ESR) and C-Reactive Protein (CRP) were measured as non-specific markers for inflammation and were utilized as exclusion criteria. The upper limit of the ESR is 20 mm/hr, for males, and 30 mm/hr for females (Sox and Liang, 1986; Caswell, 1993). To avoid the inclusion of subjects in an acute phase of inflammation (e.g., infection, trauma and tissue necrosis, malignancies, and autoimmune disorders), the subjects with CRP levels > 5 mg/L were also excluded (Gabay and Kashner, 1999; Ablij and Meinders, 2002; Biasucci, 2004).

Habitual smokers were excluded because this factor has already been significantly marked as a modulator of C level and strong pro-inflammatory (Mendelson et al., 2008; Messner and Bernhard, 2014). The participants were invited not to consume alcohol starting 1 week before the sample collection, in order to avoid any effects on C and the systemic inflammation levels (Sher et al., 2007; Morris et al., 2015).

Subjects who had current infections, allergies, or a present and past history of autoimmune disorders, and those on current medication (including herbal remedies or vitamins) such as anti-inflammatory, antiviral agents or immunosuppressive medication that could directly or indirectly affect the systemic inflammatory state were excluded.

Older adults reaching the "exclusion criteria" adapted from the SENIEUR protocol for demographic suitability was excluded from the study (Ligthart et al., 1984). The exclusion criteria for older adults included factors thought to influence the relationship between the cognitive function and chronic inflammation such as the presence of dementia, and depression. Volunteers were invited to a preliminary screening session based on a full medical history and examination including, anthropometric measurements, the assessment of dietary habits, tobacco and alcohol consumption, and screening for cognitive impairment using the Mini-Mental State Examination (MMSE, Folstein et al., 1975), and the Geriatric Depression Scale (GDS), that provides a screening measure of depressive symptoms in adults over 55 years (Yesavage et al., 1982). All participants had their cognitive functions assessed using a neurocognitive battery of tests as described to follow. The neurocognitive battery was designed in order to evaluate the memory and attention which are frequently affected in older persons. The subjects with depression (score ≥ 11 on the GDS) and subjects with dementia (score ≤ 24 on the MMSE) were excluded.

A total of 94 consecutive older adults (39 female) were invited to participate in the study. Ten subjects (4 female) were excluded as they were smokers; 3 male subjects were excluded because they had elevated ESR values, one female subject for an elevated CRP value. Eight male and 10 female were excluded as they were on medication. In the end, 62 consecutive subjects (of which 66% subjects contacted) were included in the study. After having signed the consent, 34 subjects were randomly assigned to an Experimental Group (EG) (16 female) and 28 to a Control Group (CG) (13 female) before the cognitive screening assessment.

A total of 43 consecutive young adults (20 female) were invited to participate in the study. Eight subjects (2 female) were excluded as they were smokers, four (2 female) were excluded as they were on medication. In the end, 31 consecutive subjects (of which 74% of subjects contacted) were included in the study.

The study procedures were described in detail to all participants, a minimum reflection time of 24 h was given before written informed consent was obtained. Participants not having the capacity to consent to research participation and non-Italianspeaking participants (because the inclusion of these participants would have meant not to be able to use standardized assessment techniques) were also excluded from the study.

#### TABLE 2 | Characteristics of older adults at baseline.

fnmol-10-00233 July 21, 2017 Time: 16:29 # 4

aUnpaired Student t-test; bChi-squared test; BMI, Body Mass Index; CRP, C-Reactive Protein.

The study was conducted according to the principles expressed in the Declaration of Helsinki and subsequent revisions and was approved by the Ethics Committee of G. d'Annunzio University, Chieti, Italy.

#### Characteristic of the Sample

Demographic data of the subjects studied are listed in **Table 2**, together with BMI value and plasmatic level of CRP.

The CG included 28 subjects (13 women; age, mean ± SD, 69.0 ± 8.0 years). The EG included 34 subjects (16 women; age, mean ± SD, 70.6 ± 7.0 years).

The two groups were not significantly different at the baseline (T0) in terms of age (p = 0.428), male/female ratio (p = 0.730), BMI (p = 0.841), education (p = 0.681), and plasma CRP (p = 0.530). They did not significantly differ for any neuropsychological scores obtained at the beginning in which all scores were within the normal range. At this period all participants were considered healthy and defined as not suffering from any recent episode that could condition the basal inflammatory level and cognitive performance (i.e., infection and malignancy).

#### Neuropsychological Test Battery

Cognitive functioning in CG and EG was measured using a comprehensive neuropsychological test battery at baseline (T0) and after 6 months (T1), 1 day before blood sample collection. For all subjects and controls, the same test administration order was used. The battery was comprised of tests with strong psychometric properties and assessed the following cognitive domains:

Global Assessment of Cognitive Function. MMSE (Folstein et al., 1975) is the use a psychometric test used to quantify the global cognitive functioning and the cognitive change in population-based longitudinal studies. MMSE consists of a series of questions with the aim of quantifying the global cognitive functioning on a 0–30 scale.

The following instruments were administered (in this fixed order), by a psychologist for the overall assessment of each participant, of which had a duration of approximately 90 min, with breaks provided, as requested by participants:

(1) Global Assessment of Cognitive Function. MMSE (Folstein et al., 1975) is the use a psychometric test used to quantify the global cognitive functioning and the cognitive change in population-based longitudinal studies. MMSE consists of a series of questions with the aim of quantifying the global cognitive functioning on a 0–30 scale.

#### Memory Training

The participants of EG were submitted to MT from T0 to T1. The MT is based on the concept that repeated practice within a specific domain results in gains in both cognitive and behavioral efficiency of the targeted domain, as well as subsequently transferring improvements to untrained domains.

The intervention was conducted in several laboratories. All sessions lasted 1 h and were held twice a week, with a total of 48 laboratories (**Figure 1**).

During MT, two different mnemonics were used. To assess the EG visual mnemonic (Loci) and general strategies (Strategic) were used (Cavallini et al., 2003). The Loci mnemonics is based on participants that generate sequences of visuospatial images of familiar locations, and further produce an image that is more complex, with the initial scene and the name/object to remember. The second training was characterized by the use of disparate strategies in different situations and each of them required a special strategy (generating mental images, association or categorization of items).

Older adults are particularly sensitive to the setting and emotional conditions. For this reason, we used ecological tasks to reproduce daily activities (Baltes and Baltes, 1990).

Story recall. All participants had 5 min to study a short story composed of 22 units. They then had to write what they remembered. Performance was assessed according to the correct number of units they could remember.

Shopping list recall. A shopping list was read to all the participants and they had 5 min to remember and subsequently put it in writing. Performance was evaluated according to the correct number of products which were remembered.

Memory for faces/names. Twelve photographs, each coupled with a name, on a computer screen was presented to all participants for 30 s. The same faces, were then shown without names and participants had to remember the names. Performance was assessed according to the correct face/names associations made.

Memory for places. A map of an Italian city with the name and the position of 10 monuments was presented to all participants for 5 min. They then had to remember and to write the name and the position of the monuments on a blank map. Performance was evaluated according to the correct names and positions which were remembered (Cavallini et al., 2003).

#### Cell Sources and Cultures

Blood samples were collected at T0 and T1. Veni-puncture was performed in the morning between 08.00 and 10.00 am. in order to avoid the effect of diurnal variation, and peripheral blood samples were collected in 4 ml endotoxin-free Heparin tubes (Vacutainer, Becton Dickinson, NJ, United States). Tubes were kept at room temperature and transported to the laboratory for processing within 1 h of collection. The plasma was obtained by blood centrifugation as described previously and was kept frozen at −20◦C (Pesce et al., 2014).

The PBMCs were isolated as described previously and used freshly for the evaluation of the WIP-1 mRNA and protein expressions and for the study of NF-kB signaling (Speranza et al., 2013). Briefly, the PBMCs were isolated by densitygradient centrifugation through Ficoll-Hypaque (Pharmacia), suspended (106 /ml) in RPMI 1640 medium (Sigma-Aldrich, St Louis, MO, United States), containing L-glutamine 1% and antibiotics (penicillin 100 U/ml-streptomycin 100 ug/ml) with

10% heat-inactivated fetal calf serum (Sigma, CA, United States), seeded in polypropylene tubes (Falcon, BD, Lincoln Park, NJ, United States) and incubated at 37◦C in a 95% humidified 5% CO2 cell culture incubator. Cell viability in each culture was assessed by Trypan blue die exclusion. All solutions were prepared using pyrogen-free water and sterile polypropylene plastic-ware and were free of detectable LPS (<0.1 EU/ml), as determined by the Limulus amoebocyte lysate assay (sensitivity limit 12 pg/ml; Associates of Cape Cod, Falmouth, MA, United States). All reagents used were tested before use for Mycoplasma contamination (minimum detection level 0.1 µg/ml) (Whittaker Bioproducts, Walkersville, MD, United States) and found negative. The same batches of serum and medium were used in all experiments. After 24 h incubation, samples were centrifuged at 400 × g for 10 min at room temperature, supernatants were collected and stored at −80◦C pending assay (Pesce et al., 2013). The PBMCs yield per mL of blood was approximately 1 × 106 cells.

## RNA Extraction and the Quantitative Real-Time Polymerase Chain Reaction (qPCR) System

Total RNA was extracted from PBMCs of all subjects using the TRIzol reagent (Invitrogen, Life Technologies, Paisley, United Kingdom) according to the manufacturer's protocol. The RNA concentration and purity were determined by measuring absorbencies at 260 and 280 nm; a 260:280 ratio of 1.9 being considered acceptable for analysis. The RNA concentration was estimated by measuring the absorbance at 260 nm using a Bio-Photometer (Eppendorf AG, Hamburg, Germany), and RNA samples were kept frozen at –80◦C until use. Purified RNA was electrophoresed on a 1% agarose gel to assess the integrity of the purified RNA. The RNA yield was 8.2 ± 2.7 µg/106 cells (mean ± SD).

Before Reverse Transcription, residual genomic DNA was removed from Total RNA using DNaseI (Thermo Scientific). The reaction mix was as follows: 1 µg of Total RNA, 1 µl of 10x Reaction Buffer, 1 U of DNaseI and water up to 10 µl. The mix was incubated at 37◦C for 30 min and stopped by adding 1 µl of EDTA 50 mM and incubating at 65◦C for 10 min. A High Capacity cDNA Reverse Transcription kit (Applied Biosystem) was used for reverse transcription of mRNAs. The reaction mix was prepared as follows: 2 µg of total RNA, 2 µl of 10x RT Buffer, 0.8 µl of dNTP, 2 µl of 10x Random Hexamer, 50 U of MultiScribe Reverse Transcriptase, 1 µl of RNase Inhibitor and water up to 20 µl. cDNA synthesis was performed in a thermal block cycler (Bio-rad) following these steps: priming at 25◦C for 10 min, transcription at 37◦C for 120 min and enzyme inactivation at 85◦C for 5 min. All cDNA samples were stored at −20◦C pending qPCR analysis.

A qPCR assay was carried out in an Eppendorf Mastercycler EP Realplex (Eppendorf AG). Briefly, preliminary PCR reactions were run to optimize the concentration and ratio of each primer set. For all the cDNA templates 2 µL was used in a 20 µL qPCR amplification system of the SYBR Green Real Master Mix Kit. Primers for human WIP-1 gene and GAPDH as reference gene were designed using GeneWorks software (IntelliGenetix, Inc., Mountain View, CA, United States).

The primer pairs used were as follows: WIP-1 (NM003620) forward-50 -TTTTTATATTGTTTTTAGGTTATT-30 and reverse -50 -ATCTATATAAACTTTTAACTCAATC-30 ; GAPDH (NM00- 2046) forward-50 -ACCACCATGGAGAAGGC-30 and reverse-5 0 -GGCATGGACTGTGGTCATGA-30 . The amplification procedures and data computation followed were similar to those described above (Patruno et al., 2012). In this study stability of GAPDH did not vary in the cells whatever the conditions. Thus, the relative expression of WIP-1 was normalized to GAPDH using the 1Cq method [relative expression = 2 −1Cq, where 1Cq = Cq(WIP1) – Cq(GAPDH)].

#### Western Blotting

Peripheral Blood Mononuclear Cells were washed once in cold phosphate-buffered saline (PBS; 0.5 mol/L sodium phosphate, pH 7.5), harvested by gentle scraping, and used to prepare total or nuclear protein extracts. Total protein extracts were prepared as described previously by treating cells with lysis buffer for 30 min at 4◦C (Felaco et al., 2000). Nuclear extracts were prepared as previously described (Speranza et al., 2009). The protein concentrations of the extracts were determined using the Bradford method (Bio-Rad protein assay, Hercules, CA, United States).

For Western blotting (WB) analysis, 50 µg of protein per lane was separated on a 4-12% NuPAGE gradient gel (Gibco Invitrogen), electrotransferred onto a nitrocellulose membrane and blocked with 10% skimmed milk in PBS containing 0.1% Tween-20 (Franceschelli et al., 2011). Blots were probed and incubated overnight at 4◦C with polyclonal rabbit IgG anti-WIP1, and pNF-κB (p-p65Ser536) (Santa Cruz Biotechnology, Santa Cruz, CA, United States), all at 0.2 µg mL−1 in Tris-buffered saline (TBS)/0.1% Tween-20. A mouse antihuman monoclonal antibody recognizing human β-actin (A5441; Sigma–Aldrich) was used as the control in all experiments. A rabbit antihuman antibody for β-tubulin was used as the control in experiments with nuclear extract proteins (Santa Cruz Biotechnology, Santa Cruz, CA, United States). Three microgram of human recombinant the WIP-1 protein was used as positive control (Lifespan Biosciences, Seattle, WA, United States). Fifty microgram of nuclear extract proteins from PBMCs previously treated with the LPS (10 µg/mL) and INFγ (20 ng/mL) was used as positive control for the detection of p-p65Ser536 (Pesce et al., 2014).

Blots were then washed and incubated for 1 h with goat antirabbit-horseradish peroxidase (Pierce Biotechnology, Rockford, IL, United States) diluted 1:10000 in TBS/0.1% Tween-20. Immunoblot signals were developed using Super Signal Ultra chemiluminescence detection reagents (Pierce Biotechnology). The blot images were analyzed by a gel analysis software package (Gel Doc 1000; Bio-Rad, Milan, Italy). Data are expressed as the mean ± SD intensity of optical density.

## Role of WIP-1 in Regulating NF-kB Signaling

In order to detect the role of the WIP-1 in regulating NF-κB signaling, the PBMCs were isolated and cultured during the study, as above described. The cultured cells were pre-treated or not treated with the WIP-1 inhibitor GSK2830371 for 1 h, and later the cells were stimulated with the LPS and INFγ. The LPS was used at 10 µg/mL, IFNγ at 20 ng/mL, and GSK2830371 at 10 µM (Sigma–Aldrich, St Louis, MO, United States) (Pesce et al., 2014).

#### Plasma Sampling and Measurement of C, CRP, IL-1β, IL-18, and IL-6 Levels, and In Vitro Production of Cytokines

Plasma was obtained with blood centrifugation at 1500 × g for 7 min and kept frozen at −20◦C from all participants. The amount of plasma circulating IL-1β, IL-18, IL-6, C, and the CRP was assayed with the use of specific ELISA development systems (Pierce, Rockford, IL, United States) in all individuals. The amount of IL-1β, IL-18, IL-6 from the culture supernatants of the PBMCs was assayed with the use of specific ELISA development systems (Pierce, Rockford, IL, United States) in older adults after 6 months of being on a MT program. The experiments were performed according to the manufacturer's instructions.

Plates were scanned using a specialized Charge Coupled Device cooled tool. The integrated density values of the spots of known standards were used to generate a standard curve. Density values for unknown samples were determined by using the standard curve for each subject in order to calculate the real values in pg/mL. All steps were performed in duplicate and at room temperature. The IL-1β assay sensitivity was ≤1.0 pg/ml, for IL-18 it was ≤12.5 pg/mL, for IL-6 it was ≤1 pg/ml, for C it was ≤56.7 pg/mL, and for CRP ≤ 10.0 pg/ml. The values below the assay sensitivity have been excluded. The intra- and interassay reproducibility was >90%. Duplicate values that differed from the mean by more than 10% were considered suspect and were repeated.

#### Statistical Analysis

The normal distributions of data were tested by the Skewness and Kurtosis measurement. All the variables considered, resulted distributed in a normal manner. The results were reported separately for each group: young adults, total older adults, older adults Control Group (CG) and older adults Experimental Group (EG). The Unpaired Student t-test was used to assess significant differences between the young group and total older adults. The unpaired Student t-test and Chi-squared test were applied to evaluate significant differences between the CG and the EG. The same statistical test was applied to evaluate the differences for biological variables. Pearson's (r) co-efficient correlation was applied to assess the strength of the relationship between the basal levels of age, BMI, WIP-1, C, IL-1β, IL-18, IL-6, CRP and of the MMSE scores in older adult subjects. In order to assess the multicollinearity of the variables, the diagnostic factors, Tolerance and Variance Inflation Factor (VIF) were considered. Accordingly, the MMSE scores were used as an dependent variable in a multiple regression analysis, whereas Age, as well as C, WIP-1, IL-1β, IL-18, and IL-6 were used as independent ones. The differences pre- and post-cognitive training were analyzed by the Paired samples t-test. Statistical analyses were performed using the SPSS 19.0 statistic (SPSS Inc., Chicago, IL, United States) for Windows (IBM). Results are described as means ± SD for each assessment performed in triplicate. All statistical tests were two-tailed and were evaluated at an alpha level of 0.05.

### RESULTS

### The Relationship between Global Cognitive Functioning and Biological Variables in Older Adults

**Table 3** shows the bivariate correlations at T0 between Age, BMI, the plasmatic level of C, CRP, the pro-inflammatory cytokines IL-1β, IL-18, IL-6, the mRNA expression of the WIP-1 from ex vivo PBMCs, and MMSE score of all older adults (n = 62).

The Age parameter correlated significantly as well as negatively with the WIP-1 mRNA expression (r = −0.368, p < 0.01), whereas it correlated positively with the IL-1β (r = 0.320, p < 0.05), the IL-18 (r = 0.371, p < 0.01), and the IL-6 (r = 0.327, p < 0.05). The BMI did not correlate with any variable considered, except for the IL-6 (r = 0.254, p < 0.05).

The MMSE score correlated positively to the WIP-1 (r = 0.322; p < 0.01), and negatively to C (r = 0.340; p < 0.01) and to all the pro-inflammatory cytokines analyzed. Once again, the plasmatic level of C at baseline was significantly as well as negatively associated to the WIP-1 mRNA expression, and positively associated to IL-1β, IL-18, and IL-6. The cytokines were strongly and positively associated with each other. None of the variables considered, correlated significantly with the CRP.

The correlation values obtained at T0 were not confirmed at T1 for Age and the MMSE score, Age and C, and Age as well as immune markers evaluated. The same significant loss also occurred when the correlation were analyzed for the EG at T1.

**Table 4** presents the multiple linear regression analysis results. The MMSE score was considered as a dependent variable. The variables which show significant correlation vs. MMSE score were considered as potential independent variables for the analysis. In order to assess multi-collinearity, the diagnostic factors, Tolerance and VIF were evaluated. As a consequence, none of the variables were excluded. The analysis was carried out using Age, C, WIP-1, IL-1β, IL-18, and IL-6 as independent variables that were added stepwise. The model including Age, C, and amount of the WIP-1 mRNA in ex vivo PBMCs resulted as the most significant, accounting for 13.4% of the MMSE variance [F(1,60) = 4.105; p = 0.010] (Supplementary Table 1).

#### Neuropsychological Test

Cognitive functioning was directly assessed using a neuropsychological test battery. In 62 apparent older adults, the global cognitive function was measured by the MMSE at T0, and after 6 months of the MT program (T1). At the same time, verbal memory was assessed to measure the ability to retain (Short

TABLE 3 | Pearson (r) correlation between Age, Body Mass Index (BMI), Mini Mental State Examination (MMSE) score, Cortisol (C) and immune markers at baseline in apparently healthy older adults (n = 62).

WIP-1, Wild type p53-induced phosphatase 1; IL, Interleukin; CRP, C-reactive protein. ∗p < 0.05; #p < 0.01.

TABLE 4 | Regression analysis summary for biological variables predicting cognitive function.

The MMSE score was regressed on Age, Cortisol (C), Wild type p53-induced phosphatase 1 (WIP-1), IL-1β, IL-18, IL-6 in an stepwise regression analysis (n = 62). All variables were considered at baseline. IL, Interleukin.

Term Memory, STM) and recall (Long Term Memory, LTM) verbal information using the RAVL Test. Visual memory was evaluated through the ROCF Test. Attention was assessed by the TMT A, the TMT B and the test of Attentive Matrices.

The results reported in **Table 4** show that the MMSE score significantly increased at T1 in EG when compared to their baseline score (28.3 ± 1.6 vs. 26.1 ± 1.7, p < 0.01). We also observed that the MMSE score of EG at T1 was significantly higher than those of CG (28.3 ± 1.6 vs. 26.4 ± 1.9, p < 0.01).

With close attention, we observed significant differences preand post- the MT program for attentive test targets. The medium number of targets attempted was augmented in the EG over time (48.8 ± 6.2 vs. 55.7 ± 5.1, p < 0.05), and between the EG and the CG at T1 (55.7 ± 5.1 vs. 50.2 ± 5.0, p < 0.05). The two groups did not differ significantly in their TMT A (p = 0.611) or TMT B (p = 0.684) performance concerning completion time at T0. However, the completion time was different between the two groups at the T1, in which it was lower in the EG both for the TMT A (35.1 ± 8.1 vs. 42.2 ± 11.5, p > 0.05), that the TMT B (66.2 ± 20.7 vs. 78.9 ± 27.7, p > 0.05), but it did not reach the significance. The EG did not show significant variation of scores when compared to themselves at T0 for the TMT A (35.1 ± 8.1 vs. 41.8 ± 10.5, p > 0.05), and B (66.2 ± 20.7 vs. 79.3 ± 28.4, p > 0.05). We again found that the RAVLT score followed the same trend over time observed in the EG for global cognitive assessment. In detail, the STM score significantly increased from 2.5 ± 1.2 to 3.4 ± 1.0 (p < 0.01), and the LTM significantly improved from 2.3 ± 1.2 to 3.3 ± 1.2 (p < 0.01). We did not observe any variation over time in the CG. Nonetheless, the EG showed a significantly higher RAVLT score for the STM at T1 when compared to CG, whereas the groups did not differ significantly for STM and for any neuropsychological measure at the baseline (**Table 5**).

The data obtained for Visual memory through the ROCF Test, suggested that the MT program also significantly improved this function in subjects of the EG over time. We did not observe any difference between groups at the T1 (**Table 5**).

#### Age Dependent WIP-1 mRNA and Protein Expression

The expression of WIP-1 has been shown to decrease during aging in murine model (Wong et al., 2009). In order to confirm on human the differentiated expression of the WIP-1 during age, qPCR, and WB experiments were performed to detect the mRNA and protein level, respectively, on freshly isolated PBMCs from healthy young and older adults. The results showed in **Figure 2**, suggest that older adults were characterized by significantly lower levels of the WIP-1 expression.

### Memory Training Modulation on WIP-1 mRNA and Protein Expression

We determined the expression of the WIP-1 mRNA transcripts in freshly isolated PBMCs from selected healthy older adults by the qPCR at the baseline and after 6 months of the MT program in the CG and in the EG. **Figure 3A** shows the results from subjects of both groups. Compared with the CG, the PBMCs obtained from the EG were found to contain much higher levels of the mRNA for the WIP-1 at T1, determined as its expression

TABLE 5 | Neuropsychological assessment of global cognitive function, attention, verbal and visual memory from a Control Group (CG) and from an Experimental Group (EG) at baseline (T0) and also after 6 months of a Memory Training Program (T1).

The MMSE was used to quantify global cognitive functioning and cognitive change over time. The Attentive Matrices Test (AM) was used to measure selective and sustained attention (max targets = 60). The Trail Making Test (TMT) (Part A and Part B) provides information on visual search, the speed of processing, mental flexibility, selective visual attention and the ability of shifting. The score is represented by the time spent on test completion. The ability to retain (Short Term Memory, STM) and recall (Long Term Memory, LTM) verbal information was measured using the Rey Auditory Verbal Learning Test (RAVLT). The Rey-Osterrieth Complex Figure (ROCF) Test was used to assess visuo-spatial constructional functions, visuo-graphic memory, and a few planning aspects.

ap < 0.05 vs. CG at the same time (Unpaired Student t-test);

bp < 0.05 vs. themselves at T0 (Paired t-test).

relative to the reference gene GAPDH obtained from all subjects at each time. We did not observe any difference between groups at T0. Nevertheless, the WIP-1 mRNA expression was significantly higher in the PBMCs of the EG at T1 when compared to their value at the baseline. In detail, the detected amount of the WIP-1 mRNA in PBMCs from subjects of the EG at T1, is increased by more than 40% when compared to the EG at T0, and the CG at T1.

The levels of the WIP-1 protein in PBMCs were measured by the WB. In accordance with the mRNA levels, the PBMCs from the CG had significantly lower levels of the WIP-1 protein versus the EG after the MT program. The expression of the WIP-1 protein was also significantly increased within the EG over time. According to those reported for the mRNA, the protein level did not significantly differ between groups at T0 (**Figure 3B**).

#### Cortisol, CRP, and Cytokines Production

In order to highlight the effects of the MT on the C level and the low-grade of inflammation which characterizes healthy older adults, the ELISA test was used to detect the plasmatic level of C as well as determining the non-specific marker for inflammation CRP in both groups at baseline and after the MT. Significant variation for CRP was not observed. As reported in **Table 6**, our data suggest that the MT significantly modulates the C plasma level. That level had significantly decreased in the EG at T1 when compared vs. CG at T1 and vs. EG at T0. In order to investigate

any involvement of the MT on cytokine production in healthy older adults, the plasma levels of the IL-1β, the IL-18, and the IL-6 were evaluated. **Table 5** also shows the concentrations of cytokines detected in plasma of both groups at T0 and T1. The levels of the IL-1β, the IL-18 and the IL-6 detected at T0 did not significantly differ between the CG and the EG. On the other hand, the submission to the MT program significantly decreased the plasmatic level of all cytokines analyzed at T1 in the EG. Consequently, the subjects of this group showed a significant decrease over time.

## The Role of WIP-1 in Regulating NF-kB Signaling in PBMCs from Healthy Older Adults

The role of the NF-κB in the expression of pro-inflammatory cytokines has been broadly reviewed (Chung et al., 2011). The post-translational modification of the NF-κB (p65) affects its transcriptional activity; in particular, several reports have documented negative regulation of the NF-κB by the WIP-1, also through the direct dephosphorylation of Ser-536 of p65 (Chew

TABLE 6 | Time-dependent effect of memory training on Cortisol and the plasmatic pro-inflammatory mediators.

Cortisol, IL-1β, IL-18, and IL-6 plasma level are expressed as means ± SD (IL-1β, IL-18, and IL-6 CG at T0 and T1, n = 28; IL-1β, IL-18, and IL-6 EG at T0, n = 34; IL-1β and IL-18 EG at T1, n = 34; IL-6 EG, n = 31). T0, baseline time; T1, 6 months from the baseline time; CG, Control Group; EG, Experimental Group; IL, Interleukin. ap < 0.05 vs. T0; bp < 0.05 vs. CG at the same time.

et al., 2009). We next further performed WB studies to determine the phosphorylation of p65 at Ser-536, using specific phosphop65 antibody on nuclear protein extracts. In the EG, we observed a down-regulation of nuclear translocation of the p-p65Ser536 as compared to the CG group at T1. As a result, the phosphorylated p65 in the EG showed low levels at T1 when compared to their own levels at T0 (**Figure 4A**).

In order to better investigate the role of this phosphatase in regulating the NF-κB nuclear translocation, the PBMCs from the CG and the EG at T0 and T1 were cultured and treated with a selective inhibitor of the WIP-1 (GSK 2830371) and/or LPS + INFγ, while nuclear protein extracts were detected by an antibody specific for the p-p65. As shown in **Figure 4B**, treatment with the LPS + INFγ increased the p-p65Ser536 expression in nuclei. This response was significantly elevated in the PBMCs from the CG as compared to the EG at T1. It should also be noted that the WIP-1 inhibition resulted in a significant increase in the constitutive and the LPS + INFγ-induced p65 phosphorylation and nuclear translocation in both groups. These findings suggested that the WIP-1 expression is significantly involved in the negative regulation of the NF-κB activity.

Furthermore, the inhibition of the activity of the WIP-1 resulted in a significantly higher constitutive and induced the release of the IL-1β, of the IL-18 and of the IL-6 in the medium of cultured cells (**Table 7**). We did not observe any significant differences between cells treated with an inhibitor and a medium alone, either in the p-p65Ser536 nuclear levels (**Figure 4B**) or in cytokines release (**Table 7**).

Altogether, these findings suggest a role for the WIP-1 activity in mediating the NF-κB nuclear levels and pro-inflammatory cytokines released in older adults. Furthermore, the submission to 6 months of the MT significantly restored the WIP-1 inhibitory function.

#### DISCUSSION

In the current study, we assume that the MT through mnemonic strategies could be considered as treatment that similarly to other stimuli of the EE, could enhance the cognitive and physical well-being of older adults. We studied a sample of apparently healthy older adults and used a longitudinal approach to test the hypothesis that the MT program could counteract the higher level of plasmatic C and sterile inflammation that characterizes adulthood as well as a decline in the cognitive function. We also investigated whether the phosphatase WIP-1 in ex vivo PBMCs could be considered a "master" molecule involved in modulating the level of plasmatic pro-inflammatory cytokines IL-6, IL-1β, and IL-18.

Concerning our correlation's data, the associations recorded between cytokines, Age, MMSE score and mRNA of WIP-1 suggested a role of this phosphatase in the inhibition of the release of the IL-6, the IL-1β, and the IL-18. We first purposed a significant connection between global cognitive functioning and the IL-18 in healthy subjects, and highlighted a previously unrecognized relationship between plasmatic C levels and immune markers analyzed. Our results again reinforced the positive correlation between Age and C, as well as the negative association between the global cognitive function and C (Carvalhaes-Neto et al., 2003; Otte et al., 2005). Worth noting is that in a linear regression analysis, Age, C, and the WIP-1 mRNA expression resulted as the best predictors of variance in global cognitive performance and explained approximately the 13% variance in the MMSE score. We were in accordance with previous findings suggesting positive association between the cytokines measured and Age (Dinarello, 2006; Miles et al., 2008), and the IL-6 and BMI (Mohamed-Ali et al., 1997). We also agree with other authors in observing any significant association between the CRP plasmatic level and global cognitive functioning (Elderkin-Thompson et al., 2012).

As far as MT is concerned, accordingly with other studies, our results highlight better performance relating to verbal and visuo-spatial memory after 6 months of laboratory participation. Interestingly, as suggested by a significant decrease in the completion time of attentive matrices, MT contributes to attention performance enhancement. In regards to this, we are in line with the assumption that working memory used effectively is necessary to continuously select what is being represented by paying major attention to the way in which the working memory functions, and to update what is represented according to the relevance of the task (Palladino et al., 2001).

Cognitive improvement was paralleled over time by the lowering of the plasmatic level of the glucocorticoid hormone C, and by a significant reduction in the plasmatic levels of the pro-inflammatory cytokines evaluated. These findings suggest a previously unrecognized beneficial effect of the MT program participation, as increased C levels have been associated to several age-related pathologies that include muscle atrophy (Dohi et al., 2005), osteoporosis/hypercalcemia (Hetyey et al., 2007), hyperglycemia/hyperlipidemia, atherosclerosis, type II diabetes

and major depression (Bindu et al., 2005; Leggio et al., 2005; Bauer, 2008). The peripheral level of the IL-6 contributes to agerelated neural atrophy (Willette et al., 2010), it is considered a marker of increased risk of frailty (Ershler and Keller, 2000), and is associated with reduced muscle strength (Barbieri et al., 2003) during adulthood. No less important, the circulating levels of the IL-1β and of the IL-18 which are considered detrimental for aging successfully were involved in the mediation of several downstream effects of the NLRP3 inflammasome activation (Martinon et al., 2009; Youm et al., 2013). In particular, the IL-1β is thought to play a predominant role in the development of several age-related degenerative diseases including type 2 diabetes and Alzheimer's Disease (AD) (Youm et al., 2011; Heneka et al., 2013). Both cytokines have been suggested to regulate obesityinduced inflammation, insulin resistance, and plaque instability and atherogenesis (Stienstra et al., 2011; Vandanmagsar et al., 2011; Wen et al., 2011; Shi et al., 2015).

At the same time, the MT could contribute to generate a positive loop in which memory performance improvement was further sustained by the lowering of the plasmatic level of C and of pro-inflammatory cytokines. In this respect, the higher level of C was related to the parameters of cognitive

TABLE 7 | Comparison of the IL-1β, of the IL-18, and of the IL-6 release in conditioned medium by non-stimulated and the LPS + INFγ and/or WIP-1 inhibitor (GSK2830371, 10 µM) treated Peripheral Blood Mononuclear Cells (PBMCs) (pg/ml/106 cells) from 28 subjects of Control Group (CG) and 34 of the Experimental Group (EG) after 6 months of being on a Memory Training Program (T1).

Supernatants were collected, stored at −80◦C until analysis, and then measured for IL-1β, IL-18, and IL-6 concentration using a commercial enzyme-linked immunosorbent assay (ELISA). All samples for a given assay were analyzed in duplicate at the same time. The ELISA values have an error range lower than 10%. The variation coefficient both inter-assay and intra-assay was <5%.

Values are presented as means ± SD. ap < 0.05 vs. respective CG; bp < 0.05 vs. respective WIP-1 inhibitor no treated cells.

frailty (Carvalhaes-Neto et al., 2003), and more recently has been directly associated to cognitive performance in a large sample of non-demented subjects aged at least 65 years (Ouanes et al., 2017). Nevertheless, the IL-6 has been associated with poorer performance on tests of encoding and the recall of information and working memory (Elderkin-Thompson et al., 2012). The concentrations of the IL-1β were extensively related to the negative effects on memory in animal and human studies (Yirmiya and Goshen, 2011). To the best of our knowledge, there are no studies that have been previously associated with the IL-18 to some domain of cognition in healthy subjects. However, Zhang et al. (2013) suggests a role of a higher plasmatic level of this cytokine in determining the Visuo-spatial/Constructional deficit that characterizes naïve schizophrenic patients and has resulted in its circulatory level being significantly and positively associated with cognitive impairment in AD patients (Bossù et al., 2008).

The higher plasma levels of the IL-6, the IL-1β, and the IL-18 contribute to explain the more elevated NF-κB activation observed in freshly isolated PBMCs at the baseline vs. the EG after the MT. Our data prompted the fact that the MT inhibition of pro-inflammatory cytokines release from the PBMCs could be influenced by the restoration of the WIP-1 expression.

Our observations on human are consistent with the previous finding on rodents, in which the gene expression of WIP-1 resulted lower during aging (Wong et al., 2009). The PBMCs from older adults at the baseline, in which the WIP-1 expression was lower, were characterized by the higher p65Ser536 nuclear translocation when compared to the PBMCs from older adults treated with the MT in which the WIP-1 expression and activity was enhanced.

The analyses of cultured ex vivo PBMCs have shown that the WIP-1 could suppress the release of targets of the transcription factor NF-κB (i.e., cytokines medium release), with or without the LPS/INFγ stimulation. Our data also suggests that cultured PBMCs from older adults after the MT participation, were less sensitive toward the LPS/INFγ stimulation.

We are in accordance with the previous study which suggested an inhibiting role of the WIP-1 to the NF-κB signaling pathway. As showed by Chew et al. (2009), the over-expression of the WIP-1 or silencing it reduced or increased the NF-κB activation downstream treatment with the IL-1β. Once again, it was purposed that the enhancement of the NF-κB activity by the WIP-1 knockdown was strictly due to the levels of phosphorylation of the p65 on the Ser536. We can conclude that the WIP-1 could reduce the release of these cytokines by the inhibition of the NFκB through direct de-phosphorylation of the p65 sub-unit on the Ser536.

Summarizing, our data confirms the validity of the MT, through mnemonic strategies in counteracting the aging associated decline of verbal, visual memory and of attention. In addition, we first suggested that this method of cognitive training contributes also to improve the other features of the phenotype of older adults by the decreasing level of the circulating C and pro-inflammatory cytokines and inflammation. Nevertheless, we suggest that the effect exerted on cytokines by MT could be due to the restoration of a higher level of the WIP-1 expression and activity.

The main limitation of the study is regards "How" the MT can contribute in restoring the WIP-1 expression and decreasing the pro-inflammatory cytokines level. Considering the associations observed between C, MMSE score, cytokines, and WIP-1 expression, we hypothesized a mechanism in which the lowering of this hormone could represent a potential link between cognitive stimulation and the observed decrease of lowgrade inflammation that characterizes older adults.

The MT is a practice based on working memory tasks, which involves the hippocampus and its connections (Chengyang et al., 2016). At the present, we speculate that the MT could contribute to restore the inhibiting role on the HPA axis activity (Herman et al., 2003). It is indeed known that the hippocampal type I receptors were strictly expressed in the hippocampus and resulted diminished in aging. These receptors mediated the feedback control of the HPA axis, through the activation of tonic inhibitory projections mediated by GABAergic neurons proceeded to the Para Ventricular Nucleus of the hypothalamus (Swanson, 1991; Herman et al., 1996). To follow, the effect of MT on the diminishing C medium level, could determine the modulations on pro-inflammatory cytokines secretion recorded by analysis of ex vivo PBMCs. To this end, recent findings suggested that in vivo administration of C could enhance the migratory response of human monocytes and decrease the mRNA level of the IkBα protein, leading to higher nuclear levels of the p65 sub-unit of NF-kB (Yeager et al., 2016). Other studies

support the existence of a positive feedback between GCs secretion and the activation of the Toll Like Receptor pro-inflammatory signaling pathway that was mediated by the synergistic action of GCs and pro-inflammatory stimuli (Bornstein et al., 2004; Hermoso et al., 2004). GCs have also been shown to induce the expression of NLRP3 inflammasome, in both cultured and primary macrophages, leading to higher secretion of IL1β, and IL-6 (Busillo et al., 2011). These results provide a potential explanation for some of the negative effects of higher level of C in older adults (Cruz-Topete and Cidlowski, 2015).

With regards to the WIP-1 expression, further analysis as to whether its expression in ex vivo PBMCs could be modulated by in vivo administration of C in young and older adults with or without synergy with pro-inflammatory stimulation will be done in the future. Furthermore, it will be of interest to investigate the age-related promoter status methylation of the PPM1D gene to test the hypothesis that an epigenetic mechanism (e.g., hypermethylation of PPM1D promoter κB site) could contribute to the WIP-1 repression, and could explain the progressive long life reduction of the WIP-1 expression previously observed in several cell types by other studies (Wong et al., 2009; Zhu et al., 2009).

### CONCLUSION

Our work provides insight some new properties of the MT in counteracting aging and a purposely planned WIP-1 deficiency as a part of its pathophysiology, bearing in mind that this phosphatase exerts an important role within the mechanisms

#### REFERENCES

that generally act in order to counteract and reduce the negative effects of environmental agents and those which were impaired during adulthood.

### AUTHOR CONTRIBUTIONS

Conceived and designed the experiments: MP, MF, and AG. Performed the experiments: RT, ILF, AR, AF, SF, CT, and GC. Analyzed the data: MP, RT, MCV, MDL, and LS. Wrote the manuscript: MP and RT.

## FUNDING

The Italian Ministry of Education and Research.

#### ACKNOWLEDGMENTS

Dr. Filomena Natarelli for her support in the editing of the paper. The Italian Ministry of Education and Research for its financial support.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fnmol. 2017.00233/full#supplementary-material

refractory to stimulation by mineralocorticoid receptor blockade. J. Clin. Endocrinol. Metab. 90, 5656–5662. doi: 10.1210/jc.2005-0105

predict hippocampal atrophy and memory deficits. Nat. Neurosci. 1, 69–73.

chronic obesity-induced pancreatic damage. Endocrinology 152, 4039–4045. doi: 10.1210/en.2011-1326

**Conflict of Interest Statement:** 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.

The reviewer VB and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.

Copyright © 2017 Pesce, Tatangelo, La Fratta, Rizzuto, Campagna, Turli, Ferrone, Franceschelli, Speranza, Verrocchio, De Lutiis, Felaco and Grilli. This is an openaccess article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

digital media

of impactful research

article's readership