Aging is a multifactorial process characterized by a progressive loss of physiological integrity, resulting in an impaired function and increased vulnerability to death. Physiological brain aging involves cognitive impairment, which includes decreased learning and memory abilities and slower responses to different stimulus [1
]. Indeed, normal aging differs from pathological aging and this might be explained by the lifestyle. Environmental factors drive epigenetic modifications, resulting in phenotypic differences, which alter almost all tissues and organs. Although the brain is one of the most affected structures, such modifications lead to a progressive decline in the cognitive function and create a favourable context for the development of neurodegenerative diseases [2
]. Aging is the most significant risk factor for most chronic diseases, such as age-related cognitive decline and Alzheimer’s disease (AD) [3
]. In fact, AD is the most prevalent dementia and its progression is influenced by both genetic and environmental factors [4
Several studies define that a good environment is essential to enhance learning and cognitive abilities, besides the continued presence of stressors being associated with the opposite effects. Epigenetics refers to potentially heritable and environmentally modifiable changes in gene expression mediated via non-DNA encoded mechanisms [5
]. Recent evidence has demonstrated significant associations between epigenetic alterations and stress [2
]. Potential threats cause a course of action releasing numerous transmitters and hormones throughout our body, particularly catecholamines and glucocorticoids. The interaction of glucocorticoids and adrenergic systems in specific brain regions has proved an essential mediating mechanism for a wide variety of actions displayed by stress on cognition [7
]. Firstly, stress response allows body adaptation. However, when stress is prolonged it has been shown to decrease synaptic plasticity [8
], alter hippocampal volume [9
] and neurotransmitters [11
], and may lead to AD [12
]. In particular, chronic stress is associated with increased Aβ deposits and hyperphosphorylated Tau [13
Oxidative stress (OS) and inflammation are deeply involved in age-related deleterious disorders. Along with the aging process, several factors, such as a naturally decreased capacity of the antioxidant enzymes system, create an imbalance between antioxidant mechanisms and reactive oxygen system (ROS)–production equilibrium, accumulating ROS beyond the detoxifying capacity of the antioxidant system resulting in OS, eventually causing cellular damage that can no longer be repaired by internal mechanisms, and finally causing dysfunction of the system [14
]. In addition, one of the major changes during aging is the dysregulation of the immune response, leading to chronic systemic inflammatory state [15
]. Overall, OS imbalance, mitochondrial dysfunction and the inflammatory response have been linked to accelerated aging and faster progression of neurodegenerative diseases [14
]. Moreover, the accumulation of dysfunctional and damaged cellular proteins and organelles occurs during aging, resulting in a disruption of cellular homeostasis and progressive degeneration and increases the risk of cell death [17
]. Autophagy is particularly important in the maintenance of homeostasis, as it participates in the elimination of disrupted proteins and molecules and helps to maintain a clean cellular environment. It has been widely described that there are alterations in autophagy during aging as well as in age-related cognitive decline, AD and other dementias [18
]. Moderating all these detrimental components is key in the promotion of cell survival and longevity.
The senescence-accelerated mouse (SAM) models include senescence-accelerate prone (SAMP) and senescence-accelerated resistant (SAMR) mice [21
]. SAMP8 strain has been widely used as an aging model for the study of brain aging and age-related pathologies. Its cognitive impairment is associated with alterations in both hippocampal structure and activity, causing oxidative stress imbalance and driving to AD neuropathology, such as tau- and amyloid-related alterations [1
]. On the contrary, SAMR1 strain manifests a normal aging process and is considered as the control strain for SAMP8.
Understanding the epigenetic modifications that a stressful environment triggers in neurodegeneration mechanisms is essential to develop novel therapeutics for age-related cognitive decline. This study aims to determine the effects on brain function of a stressful lifestyle in an animal model with accelerated senescence, SAMP8, and in the resistant senescence strain, SAMR1.
Understanding the mechanisms that define aging and differentiate what determines whether it is pathologic or healthy is one of the challenges that science has faced in recent years. Life expectancy has increased and so has the number of older people. In addition, the life rhythm has changed, becoming increasingly stressful. It has been shown that the environment is extremely important in the development of several diseases, compromising healthy aging; specifically, stressful lifestyle has been identified as an important risk factor for cognitive decline. Therefore, it is crucial to study the effects of stress on cognition and its relationship with aging in order to unveil what challenges we might have to cope with as a society in the not-so-far future. We hypothesize that chronic stress would modulate a large constellation of cellular mechanisms implied in aging and age-related neurodegenerative pathologies.
During aging, there are several epigenetic mechanisms altered, which include DNA methylation, histone modifications, nucleosome remodelling, and miRNA-mediated gene regulation [2
]. In the present study, we evaluated three main epigenetic marks, which modulate chromatin structure and act as platforms for recruitment, assembly or retention of chromatin-associated factors: histone posttranslational modifications, DNA methylation and miRNA-mediated gene regulation. One of the posttranslational modifications of histones is acetylation. This process removes the histone positive charge; thereby, the condensed chromatin (heterochromatin) is transformed into a more relaxed structure (euchromatin) that is associated with greater levels of gene transcription [24
]. However, chromatin condensation depends on different processes including methylation and histone deacetylation. This last process is due to histone deacetylases (HDAC) enzymes. Deregulation of histone acetylation has been related to an increase of the risk of age-dependent memory impairment in mice [4
]. Specifically, histone H4 lysine 12 acetylation alterations causes impaired memory consolidation as well as its restoration reinstates the expression of learning-induced genes and in consequence, cognitive abilities [26
]. In accordance to Cosín-Tomás [28
], we found that CMS increased HDAC2 protein levels only in SAMR1 females, similarly to SAMP8 control mice, which lead us to hypothesize that acetylated histone protein levels were diminished. This was confirmed evaluating acetylated H3 and H4 protein levels. However, while stress produced changes in H3 acetylation, these changes were not observed in H4. Nevertheless, the decrease in H3 lysine 9 (H3K9) acetylation in SAMR1 mice under CMS, similarly to not stressed SAMP8, was correlated with changes on cognition. Other HDACs include sirtuins family; however, it is worth noting that several members of this family do not have deacetylase activity [29
]. Sirt have been linked to aging as they modulate genomic stability, stress resistance and energy metabolism. Activation of Sirt1 enables the deacetylation of a variety of proteins, resulting in a robust, protective cellular response, as it regulates processes such as cell death, metabolism or neurodegeneration [30
]; while Sirt2 has been reported to regulate oxidative stress, genome integrity and myelination and its dysfunction is found in most age-related neurodegenerative disorders such as AD, Parkinson’s disease and Amyotrophic Lateral Sclerosis, as well as in physiological aging [31
]. Accumulated evidence indicates that Sirt6
gene expression is lower in the hippocampus and cerebral cortex of aged mice [32
] and that it is concerned with H3K9 acetylation [32
]. In reference to this family of deacetylases, our results demonstrate decreased Sirt1, Sirt2
gene expression in SAMP8 mice compared to SAMR1 and SAMR1 under CMS. Compelling evidence has proposed that methylation of H3K9 promotes DNA methylation maintenance in mammals and is a hallmark of heterochromatin formation and subsequent gene silencing [2
]. As with histone phosphorylation, CMS treatment increased H3K9 methylation in SAMP8 animals but not in SAMR1, suggesting that even they showed more H3 acetylation, which also resulted in more methylation. Therefore, we demonstrate that CMS favoured chromatin condensation and in consequence, promoted gene silencing.
In reference to other modifications, histone phosphorylation belongs to the cellular response to DNA damage, as phosphorylated histone H2A.X demarcates large chromatin domains around the site of DNA breakage [2
]. Additionally, multiple studies have also shown that histone phosphorylation plays crucial roles in other nuclear processes, such as DNA replication because of apoptosis or DNA damage [36
]. Here CMS raised phosphorylated H2A.X protein level in senescence-accelerated but not in senescence-resistant mice, suggesting that stressful stimuli activate repair/survival mechanisms rather than apoptotic response in SAMP8 mice but not in SAMR1. As two major mechanisms for epigenetic regulation, DNA methylation and histone modifications must act coordinately [34
]. It is well known that DNA methylation at the fifth position of cytosine (5-mC) plays an important role in neuronal gene expression and neural development. Several studies support the idea that dysregulated DNA methylation/demethylation is linked to many neuronal disorders, including AD onset and progression. However, the relationship between AD and altered 5-mC levels is not known [37
]. 5-mC can be further oxidized to 5-hmC, among others, by the TET family of dioxygenases. 5-mC and 5-hmC exert opposite effects on gene expression; the former is in general associated with gene silencing, whereas the latter is mainly involved in up-regulation of gene expression [38
]. In this study, the stressful environment produced lower 5-mC in both mouse strains and differences between strains were observed in 5-hmC marker. Accordingly, TET2 protein levels differed between SAMR1 and SAMP8 animals. Despite our previous results, conversely 5-mC and 5-hmC results appear to contradict the transcriptional access to DNA, but as is known, the term global DNA methylation describes this process across the entire genome and does not represent a precise landscape for specific transcriptional activity; however, global methylation determination is useful as it provides an over-arching picture of methylation status; it is misleading which genes show altered DNA methylation and which do not [39
Furthermore, we evaluated expression changes of miRNAs related to oxidative stress and cellular pathways implicated in AD neuronal death, autophagy and neurodegeneration. Of the 22 miRNA evaluated, those that presented statistically significant changes under experimental conditions were further validated and target genes studied (Figure S2
). Firstly, we studied miR-29c relative expression, which is involved in neural proliferation regulation through Dnmt3a. Dnmt3a
expression was higher in SAMP8 compared to SAMR1 mice correlating with miR-29c-3p gene expression. In our hands, CMS slightly contributed to change Dnmt3a
expression in mice, showing a trend towards an increase in SAMR1. Because recent studies demonstrate that DNMT inhibitors provided neuroprotection in cellular cultures [40
], the increase in Dnmt3a
induced by CMS could mean a deleterious effect on SAMR1 health.
Considering AD neuropathology, we studied miR-431-5p, miR-298-5p, miR-98-5p, and miR-140-5p. It has been described that miR-431-5p cooperates with DKK1 to inhibit Wnt/β-catenin pathway. SAMP8 showed lower relative expression of this miRNA in comparison to SAMR1 mice; changes in β-catenin protein levels were in line with miR-431-5p. Furthermore, there is a tendency to reduce miR-431-5p gene expression and β-catenin protein levels in SAMR1 under CMS. β-catenin is regulated by GSK-3β activity, depending on its phosphorylation. As reported, GSK3-β inactive form (Ser9 phosphorylated) protein levels were lower in SAMP8 in reference to SAMR1 [30
], and also CMS decreased them, especially in SAMP8. It is known that GSK3-β activity is associated with AD neuropathology as it exacerbates cognitive impairment [42
]. In fact, higher activity of this kinase has been found in AD patients and its inhibition restores spatial memory deficits, reduces tau hyperphosphorylation, and decreases reactive gliosis and neuronal death in rodents [42
]. Furthermore, it has been described that GSK3-β inhibition reduces BACE1-mediated cleavage of APP through NF-κB signalling-mediated mechanism, so that it reduces β-amyloid pathology [43
]. Accordingly, we found that CMS increased Ser396 and Ser404 Tau hyperphosphorylation, most pronounced in SAMP8, and promoted amyloid precursor protein (APP) gene expression in both mice strains. In fact, APP processing is regulated by miR-298-5p, which in turn regulates BACE1. miR-298-5p expression differed between SAMR1 and SAMP8 animals, in contrast with BACE1 protein levels. It is reported that BACE1, sAPPβ and β-CTF protein levels were increased when miR-98-5p up-regulated. CMS increased miR-98-5p relative expression and sAPPβ in SAMR1 compared to the control group, but not in SAMP8. However, as mentioned, BACE1 was not modulated under CMS. Taking into account that BACE1 can be up- or down-regulated by different miRNAs, discrepancies can be explained by compensatory responses among different signals. ADAM10 protein levels are controlled by miR-140-5p. Again, huge differences in miR-140-5p gene expression and ADAM10 protein levels were observed between strains. There were differences between control mice under CMS in SAMR1 but not in SAMP8.
miR-181a-5p is involved in the regulation of cell growth, proliferation and survival through mTOR complex 1 (mTORC1) and downstream pathway, in which protein levels were increased in SAMP8 in comparison to SAMR1 mice. In addition, stressful stimuli increased miR-181a-5p relative expression in SAMR1 mice compared to their control littermates and no changes were observed between SAMP8 animals. By contrast, CMS groups in both strains showed higher mTORC1 protein levels than their respective control littermates. Lastly, BCL2 protein levels were controlled by miR-106b-5p. SAMP8 mice had higher miR-106b-5p compared to SAMR1, but no changes were found in BCL2 protein levels. CMS reduced miR-106b-5p gene expression, increasing BCL2 protein levels in SAMP8, without affecting SAMR1 strain.
Autophagy declines during aging, so that it may contribute to the deleterious accumulation of aberrant proteins observed in aged cells. Interestingly, failure of this process has been reported to worsen aging-associated diseases, such as neurodegeneration or cancer [18
]. Pro-autophagy proteins such as Beclin1 and LC3B became decreased after CMS treatment, and in concordance, the p-mTOR ratio was increased. Moreover, we demonstrate huge differences between SAMR1 and SAMP8 control groups, indicating a reduced autophagic flux in senescent mice. CMS-induced impairment in autophagy was more robust and consistent in SAMR1 than in SAMP8, supporting the hypothesis that CMS accelerates the senescence process.
Following the same line about overall processes linked to senescence, it has been widely described that OS possesses a pre-eminent role in pathological senescence and the pathogenesis of AD. In general, cells possess antioxidant mechanisms to cope with OS, such as GPX, Catalase and SOD1, among others, which in turn are regulated by the Nrf2 transcriptional pathway. Herein we found that mice under CMS had lower NRF2 protein levels than control groups and this in turn could explain the lowest protein levels of antioxidant defence as GPX1, SOD1 and Catalase. Consistent with this, CMS promoted an increase in the pro-oxidant enzyme Aox1
gene expression and higher accumulation of ROS. Noteworthy, as described, differences between strains were found in most of the markers evaluated [4
As far as aging and AD are concerned, dysregulation of inflammatory mediators and astrogliosis are major culprits in the development of chronic inflammation and the immunosenescence process, as well as are related to cognitive decline and progression of neurodegenerative diseases [15
]. For instance, our results demonstrate a significant increase in protein levels for NF-κB, transcription factor regulating pro-inflammatory signals, in SAMP8 mice in comparison with SAMR1. One of these signaling pathways includes the insulin and insulin-like growth factor (IGF) pathways; 5’-AMP-activated protein kinase (AMPK)-mechanistic target of rapamycin (mTOR) pathway; and Forkhead box O (FOXO) families, sirtuin (SIRT), and p53-related pathways [15
]. Interestingly, CMS increased proinflammatory pathways in SAMR1 but not in SAMP8. Moreover, we found an increase in Gfap
gene expression in SAMR1 CMS compared to SAMR1 control group. In accordance, recently it has been described that decreasing astrogliosis enhances AD pathology in mice [44
The detrimental effect of stress on psychological well-being and cognitive functioning, emphasizing the relationship between stress and memory, is widely accepted. It is noteworthy that during stressful conditions underlie some of the characteristics described in cognitive decline, either in aging or in neurodegenerative diseases, such as AD [19
]. It has been stated that epigenetic machinery is essential for cognitive function [2
]. Likewise, DNA methylation influences hippocampal memory formation, and growing evidence suggests that the modulation of epigenetic processes by stress, EE and/or hormones is key in regulating memory function.
In line with the molecular results presented, behavioural changes induced by CMS were explored in SAMP8 and SAMR1 mice. Results obtained point out that not only a stressful environment triggers anxiety-like behaviour, but also it mitigates cognitive performance. Locomotor activity, the distance travelled in the central zone and other parameters, indicates well-being/discomfort were altered in mice under CMS. Regarding recognition memory, clear differences were found between the different strains, although we can also assert a detrimental effect caused by CMS, especially on long-term memory. In agreement with these results, learning abilities and spatial memory in SAMR1 and SAMP8 at 6 months of age were significantly different and were negatively affected by the presence of chronic stressors. In stressful situations, glucocorticoids are released into the bloodstream causing, among others, an increase in glucose level in order to have enough energy available to cope with stress. Aberrant glucose metabolism potentiates the aging phenotype and contributes to early stage central nervous system pathology [47
]. According to previous studies, we found differences in blood glucose levels due to stress in SAMR1 mice, exhibiting similar glucose tolerance to SAMP8 control mice. This observation means that the CMS paradigm applied was enough to induce a stressful condition in mice, facilitating an acceleration of the aging process in SAMR1.