Understanding Epigenetics in the Neurodegeneration of Alzheimer’s Disease: SAMP8 Mouse Model

Neurodegeneration in aging

Aging induces a slow decrease in cerebral function, which leads to cognitive impairment, memory loss, motor disorders, and finally, can reach functional decay and death. Among the main features of aging, we might find genomic instability, telomere shortening, epigenetic alterations, loss of proteostasis, nutrient detection deregulation, mitochondrial dysfunction, cellular senescence, stem-cell depletion, and altered intercellular communication [5, 6]. The brain is a high energy-consumption organ that requires about 20% of the body’s total basal oxygen to function [7]. Therefore, alterations in the cerebral energy metabolism lead to disease, from subtle modifications in neuronal function to cellular death and neurodegeneration [8]. As aging progresses, the brain is susceptible to its intrinsic and extrinsic cell signaling and to the systemic environment. Studies performed on the brains of elderly subjects not previously diagnosed with a neurological disease have shown the presence of extracellular amyloid-β (Aβ) and neurofibrillary tangles (NFTs), among other pathological alterations. These features might vary from individual to individual; the cause is not known or whether they could be the precursors of neurodegeneration. Some of them are probably the result of general aging processes. Alternatively, others might indicate a preclinical stage before AD clinical symptoms appear, which might last for decades [9]. In any case, the causes have not been identified that trigger the switch from normal aging to neurodegeneration and AD. Aging is a process regulated by different molecular and genetic mechanisms. There are four molecular longevity pathways known: decrease of insulin signaling; the increase of AMPK (AMP-activated Protein Kinase); the diminishing of TOR (Target of Rapamycin); and sirtuins.

In general, each cell possesses defense mechanisms to cope with oxidative stress (OS). These antioxidant defenses are endogenous enzymes and include superoxide dismutase (SOD), glutathione peroxidase, catalase, glutathione, thioredoxin reductase, and peroxiredoxins. Along with the aging process, several factors, such as a naturally decreased capacity of the antioxidant enzyme 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, finally leading to dysfunction of the system [10].

As previously mentioned, aging and neurodegeneration are closely related. The increase in OS, mitochondrial dysfunction, and the inflammatory response have been linked to accelerated aging and the progression of neurodegenerative diseases [10, 11]. ROS and reactive nitrogen species are the main molecules contributing to OS. The most studied free radicals are superoxide (O₂^-), hydrogen peroxide (H₂O₂), hydroxyl (HO), nitric peroxide (ONO₂^–), and the nitric oxide free radical (NO). OS is the resulting redox state from disequilibrium between the generation and detoxification of ROS. Overproduction of ROS by the dysfunctional mitochondrion, in addition to the imbalance in mitochondrial fission/fusion and mitochondrial DNA mutations, can cause oxidative damage to the cells, inducing alterations to their biochemical and physiological functions and modify the genetic expression profiles inducing apoptosis and, thereafter, neurodegenerative pathologies [7, 13].

Accumulative damage by free radicals over a certain time leads to major brain inflammation. Therefore, stress can also interfere with the homeostatic balance between anti- and proinflammatory cytokines and induce neurodegeneration. The cytokines are signaling proteins secreted by the cell in response to, predominantly, intra- and extraneuronal conditions. Neuroinflammation is a physiological response of the brain to an injury, infection, or disease in order to prevent a potential damage, neuronal destruction, or to dispose of a damaging agent. Several cytokines are small-secreted proteins released by both microglia and astrocytes in the central nervous system (CNS) during the inflammatory response. Stress can increase the levels of proinflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α), interleukin 1beta (IL)-1β, and IL-6 and can decrease the expression of anti-inflammatory cytokines IL-10, IL-4, and the transforming growth factor beta (TGF-β), exerting a harmful effect on neurogenesis, inducing cell death, and causing neurodegeneration [6]. Microglia activation is a key factor in AD progression and leads to increasing the chronic inflammatory state of the CNS during aging, caused by immune pathway dysregulation. Therefore, it is important to maintain control of microglial activation.

Another feature that has appeared as a central regulator of CNS aging is autophagy because it plays a crucial role as a control degradation point by which defective proteins, organelles, and other cellular constituents are sequestered in autophagosomes and delivered to lysosomes for degradation. Many of the detrimental modifications previously mentioned, such as proteostasis loss, nutrient detection alterations, mitochondrial damage, cellular senescence, and stem-cell depletion, could occur by means of basal autophagy deterioration and/or decrease of autophagic degradation activity during cellular stress, aging and neurodegenerative disorders [14, 15].

Hallmarks of Alzheimer’s disease

AD is a progressive, devastating neurodegenerative disorder that leads to behavioral, cognitive, and memory deficits, as well as motor alterations [16]. AD patients suffer from language and learning difficulties, judgment loss, anxiety, depression, and irritability. AD etiology is still not clear, although there are some risk factors, both genetic and environmental. Neuropathological hallmarks of AD include lesions such as the presence of intracellular neurofibrillary tangles and the accumulation of Aβ plaques in specific brain regions and cerebral amyloid angiopathy, reactive gliosis, and neuroinflammation [16], OS, calcium homeostasis disruption, neuronal and synaptic loss, altered functional connectivity among the brain areas, and, finally, neuronal loss and brain atrophy [17].

AD is classified into two types, familial and sporadic, depending on the etiology and the age of the patient when symptoms appeared. Familial AD (FAD) is inherited in an autosomal-dominant pattern with symptoms typically presented in the 4th or 5th decade of life. On the other hand, sporadic late-onset AD (LOAD) usually begins in the 7th-8th decade of life and is the more common of the two. The main risk factor for LOAD is aging [18, 19].

Increasing evidence suggests that AD pathogenesis is not restricted to the neuronal compartment, but includes strong interactions with immunological mechanisms in the brain [20]. However, it should be borne in mind that the elderly also have higher levels of inflammation. Therefore, in the literature, we often find the term inflammaging, although not as much as in AD patients.

Genome-wide association analysis suggests that several genes that increase the risk for sporadic AD encode factors that regulate glial clearance of misfolded proteins and the inflammatory reaction [20]. External factors, including systemic inflammation, changes in intestinal microbiota composition, and obesity, are likely to interfere with immunological processes of the brain and further promote disease progression [20–22]. Regarding FAD, the pathogenic mutations of the APP, PSEN1, and PSEN2 genes cause autosomal-dominant AD represented in less than 0.1% of the total AD cases [23]. The overwhelming majority of AD cases are sporadic and of unknown etiology. Currently, it is accepted that AD is a multifactorial disease; consequently, there is a remarkable effect of environmental factors in the etiology of AD [24]. Therefore, an adequate epigenetic intervention, beginning early in life as well as an elderly stage of life can identify and combat harmful molecular mechanisms through activation of neuroprotective gene expression such as the brain-derived neurotrophic factor (Bdnf), Bcl2, and Bcl-XL, among others, by environmental enrichment (EE) [25].

Methylation of DNA

The only epigenetic mechanism that directly modifies mammalian DNA is methylation of the cytosine carbon ring in the dinucleotides of the CpG islands [32]. Most of the genome does not contain the dinucleotide CpG repeats; however, it concentrates on the so-called CpG islands, located in regulatory regions 5’ of the genes. It is estimated that about 70% of CpG dinucleotides in the genome are methylated in normal somatic cells [33]. DNA methylation has been considered a normal mechanism of gene silencing with a crucial role in the transcriptional repression of repeated and centromeric regions. However, the mechanism is an efficiently regulated dynamic process in which methyl groups might be added or removed from both DNA strands. The result of both actions, methylation, and demethylation, is not permanent, which means that the mechanism of regulation is much more complex. However, some DNA methylation patterns are erased whereas others are maintained in the germline contributing to alter genes expression in a heritable manner without affecting the underlying genomic sequences. It should be kept in mind that most of the constitutive expression genes and a part of the tissue-specific genes and regulatory genes contain CpG islands in their promoter regions. While changes in DNA methylation occurs naturally during development, for example, inactivation of the X chromosome in females or the genomic imprint during development, where it is decided which parental allele will be expressed in the embryo [33], they also arise in response to external factors and aging, and are part of the modifications of various diseases such as fragile X syndrome, cancer, or neurodegenerative diseases, among many others.

DNA methylation is catalyzed by different members of the DNA MethylTransferase family (DNMTs) to form 5’-methyl-cytosine [34]. In mammalian cells, three different enzymes have been identified: DNA MethylTransferase 1 (DNMT1) and two de novo MethylTransferase (DNMT3A and DNMT3B). During the S phase, DNMT1 copies the methylation patterns of the parent strand to the daughter strand of DNA, so that methylation is inherited after cell division [35]. DNMT1 interacts with protein complexes involved in transcriptional repression, which include Histone DeACetylases family (HDACs) [36]. DNMT3A and DNMT3B are able to introduce a methyl group to unmethylated CpG dinucleotides to give methylated CpG. In addition, there are two more methyltransferases, DNMT2 and DNMT3L; DNMT2 is different from all other DNMTs because it methylates cytosine 38 in the anticodon loop of aspartic acid transfer RNA, and DNMT3L differs from other DNMTs because it lacks catalytic motif and does not have methyltransferase activity but it contributes to de novo methylation process interacting with DNMT3A or DNMT3B [37–39]. Promoters that have been silenced by methylation can be reactivated, when demethylated. As for the mechanisms responsible for demethylation, there are two: passive demethylation, the mechanism of which arises by reduction or disappearance of DNMT1 function during replication, and active demethylation: this alternative mechanism is a catalytic removal of the methyl groups carried out by Ten-Eleven Translocation family (TETs) members of DNA hydroxylase family that oxidize the base and the subsequent action of the protein Thymine-DNA-Glycosylase, which will split the oxidized base.

Hydroxymethylation of DNA

5-hydroxymethylCytosine (5-hmC) was initially discovered in the 1950 s in bacteriophage and was observed in mammals in the 1970 s. It is the first oxidative product in the active demethylation of 5-mC. 5-hmC has been referred to as the “sixth base” because of its involvement in gene transcription in a similar way to 5-mC [40]. It has been detected that approximately 1% of cytosines in mammals are hydroxymethylated, although the level of hydroxymethylation varies depending on the tissue, with nervous tissue exhibiting higher levels [41]. Stem cells also have higher levels of 5-hmC decreasing during differentiation [41, 42]. 5-hmC is an intermediary of 5-mC demethylation. This mechanism has been demonstrated by the discoveries of different intermediates 5-formylCytosine (5-fC) and 5-carboxyCytosine (5-caC) in mouse genomic DNA [43]. The hydroxymethylation of the DNA is catalyzed by three enzymes belonging to the TETs. These comprise TET1, TET2, and TET3. This family of enzymes carries out repeated oxidations of 5-hmC into 5-fC generating 5-caC. In addition, TET1 and TET2 have demonstrated to play an important role in gene control and cell death in neurons [44], whereas TET3 is related to early embryonic-development processes [45]. Overall, hydroxymethylation of cytosine in a promoter gene or in CpG islands have been associated with an increase in gene transcription, i.e., it has an opposite effect to that of methylation [46].

In addition, these could affect the activity of different Methyl-CpG-binding domain proteins, thus modifying their recruitment during chromatin regulation or have direct effects on transcription. On the other hand, differential gene expression of TET enzymes was described at distinct stages of development or tissue. Regarding TET1, we found that it is expressed mainly in embryonic stem cells, whereas TET2 and TET3 are more common in all other tissue types [42].

Chromatin remodeling and histone modifications

Chromatin remodeling is a process that controls the dynamic chromatin structure to allow access of the genetic information encoded into the DNA. Such remodeling is principally carried out by post-translational modifications (PTMs), which it requires the interactions between DNA methylation and histone modifications to change the accessibility of the transcriptional machinery. At least 16 histone modifications are known. As a result of a large number of modifications that can be done, there is a great potential for functional control of chromatin structure by histone modifications. However, an extra level exists due to the combination of different modifications give rise to the so-called “code of histones”. This cross-talk can occur via multiple mechanisms [47].

As a consequence, these modifications mediate a variety of biological functions, including recruitment of protein machinery that modulate transcription, repair, and DNA replication. The “writers” are enzymes, which add specific PTMs to histone substrates making the chromatin in compacted or relaxed conformation, and leads to transcriptional repression or activation, respectively, and are divided into classes based on the specific PTM they affect. In contrast, “erasers” are enzymes that remove specific PTMs from histone substrates restoring the interaction between the histone and DNA, and are divided into PTM-specific classes. Finally, “readers” do not alter the histone and recognize either post-translational marks or combination of marks and histone variants. These proteins will act as effectors, being responsible for the activation or repression of transcription and chromatin packaging, among others.

Therefore, a wide variety of modifications exist as a consequence of the enzymatic activity of antagonistic complexes, mainly including acetylation, methylation, phosphorylation, ubiquitination, sumoylation, ADP-ribosylation, and adenylations. These tags participate in the regulation either by altering the interactions of DNA and histones or by generating a recognition platform necessary to recruit to the proteins readers with different activities.

Non-coding RNAs and microRNAs

Analysis of the transcriptome has revealed that, although it depends on the species, only 1.5% of the genome encodes RNA that culminate in protein transcripts, resulting in a broad repertoire of transcripts, more than 65%, of RNA that are not translated into proteins and that are called non-coding RNA (ncRNA). Non-coding RNA are classified depending on their size, as (lncRNA, “Long non-coding RNA") with more than 200 nts, and as (“Short non-coding RNA”, sncRNA), of less than 200 nts. There are several subtypes between sncARN and lncRNA, many of which are involved in the regulation of gene expression and can be grouped according to the genomic origin and biogenic processes [63, 64].

MicroRNAs are small non-coding RNA molecules between 18 and 25 nucleotides that are expressed in the eukaryotic nucleus in both intra- and intergenic regions and that are responsible for the repression of gene expression [65, 66]. miRNAs vary in their mechanism of action depending on the degree of complementarity with their target mRNA, as follows: 1) inhibition of translation; 2) stopping the elongation phase; 3) the premature arrest of ribosomes; and 4) degradation of mRNA at the co-transduction level.

There are 28,645 identified miRNAs precursors expressing approximately 35,828 mature miRNAs in 223 species [66, 67]. It is estimated that between 30 and 90% of human genes might be regulated by miRNAs, and each miRNA can regulate up to 100 genes, which could potentially regulate a large number of protein-coding genes [68].

The first described miRNA was lin-4 in Caenorhabditis elegans. The relevance of this study was its demonstration of the implication of miRNAs in the development of C. elegans. Subsequently, several studies were carried out involving miRNAs in programmed cell death in flies [70], in early and late vertebrate development [71, 72], as well as in certain physiological functions in humans, such as adipocyte differentiation, antiviral defense, neuronal development, or apoptosis [73], with a very profound impact in cardiovascular diseases [65, 74], diabetes [75], schizophrenia [76], and neurodegenerative diseases such as AD [73].

Interactions among epigenetic mechanisms

All of the machinery of histone-modifying enzymes does not act independently, as has been mentioned, but is associated with large multiprotein complexes that, together, modify chromatin structure regulating gene transcription. In turn, miRNAs regulate gene expression in which a profound interaction between different epigenetic modifications is established. Thus, a strong relationship is observed between the methylation of DNA and the modifications of the histones that, acting together, are able to regulate the majority of the transcription process along the whole genome. In addition, histone modifications have been found to play a positive or negative role in controlling the expression of miRNA genes from both normal and pathological cells [77, 78]. On the other hand, miRNAs exert beneficial effects on the regulation of DNA methylation or histone modification through the direct regulation of both histone-modifying enzymes and chromatin-remodeling complexes [79].

SAMP8: Evidence as a sporadic Alzheimer’s disease model

SAMP8 mice exhibit an accelerated aging phenotype with external signs of hair loss and lack of hair glossiness, periophthalmic lesions, increased lordokyphosis, and reduced physical activity [144, 145]. The sensorimotor performance of SAMP8 mice also deteriorated with advancing age at a higher pace than in SAMR1 mice [146]. Other senescence traits include hearing impairment, deficits of immune response, and lifespan shorter than that of control SAMR1 mice [144]; namely, median life expectancy was around 10 months for SAMP8 and 17 months for SAMR1 [147, 148]. At the CNS level, SAMP8 demonstrate deficits in learning and memory and emotional disorders. It is interesting to point out that among the diverse SAMP strains of accelerated senescence, only SAMP8 and SAMP10 showed learning and memory defects [144]. Therefore, the cognitive impairment of these strains might be related to a few recessive genes among the many genes controlling aging of the brain [149]. Furthermore, underlying mechanisms are different for both strains, as SAMP8 mice show AD-related traits, whereas SAMP10 cognitive impairment is related to brain atrophy [150]. Similarly, in humans, old age is a risk factor for AD, but it does not unavoidably lead to the disease in centenarians or, in the case of dementia, it might not progress to AD. Therefore, only SAMP8 mice seems to be accelerated senescence after normal development, but no other senescent strains show progression into AD-like dementia.

Learning and memory impairment of SAMP8 was first demonstrated in passive avoidance task, where mild deficits were already evident at 2 months of age and increased with advancing age [151]. These authors demonstrated only minor impairments in the Morris Water Maze (MWM) test at early age. A number of successive studies confirmed the defective cognitive capacities of SAMP8 mice in a variety of experimental tasks including MWM, radial arm mazes, T/Y-maze, and passive avoidance and active avoidance were clearly established at 8–12 months of age [141, 153]. However, current experimental interests rely on the analysis of early pathogenic mechanisms of age-related neurodegeneration. In AD dementia, the pathological brain processes are known to develop for decades before cognitive impairment can be detected clinically. Therefore, there are clinical interests in moving to the phases of the preclinical AD and early mild cognitive impairment for new clinical trials against AD [154, 155]. Recent studies with SAMP8 mice established the presence of cognitive impairment in these mice compared to SAMR1 mice at the age of 4–6 months in instrumental conditioning (passive avoidance and active avoidance), spatial learning and memory (MWM, radial maze, and object location test), and recognition memory with novel object recognition test (NORT) [156–161].

Therefore, cognitive deficits in SAMP8 mice have been characterized in a range of validated tests and there is an agreement on the early and progressive cognitively impaired responses of these mice [161, 162]. There were reported inconsistencies only in the T-maze test [164, 165]. One of the best-characterized responses is that of spatial learning and memory in MWM. SAMP8 mice demonstrated a deteriorated and progressively impaired response in this hippocampus-dependent task [146]. Indeed, the hippocampus, a brain area that is highly sensitive to AD, is severely affected by pathological alterations in these mice. Furthermore, age-related behavioral and cognitive alterations occurring in the SAMP8 are evident at 4 months in comparison with SAMR1 [165].

Regarding emotional disorders, SAMP8 exhibited reduced anxiety-like behavior in the Plus Maze Test (PMT), lower neophobia, and lower emotionality and higher hyperactivity in the Open Field Test (OFT) than SAMR1 mice [146, 166]. These alterations of non-cognitive behavior were significant in 4-month-old SAMP8 and increased with advancing age. Furthermore, other authors described an enhancement of depression-like behavior in the Forced Swim Test [161, 167] and a decrease of social interaction [168]. Thus, SAMP8 mice show alterations of non-cognitive behavior that do not generally model the behavioral and psychological symptoms of dementia (BPSD) associated with AD. BPSD behaviors are mainly driven by AD pathology in the cortical and basal ganglia areas, where pathology in the SAMP8 mice might be less severe than in the several strains of AD transgenic mice that reproduce these typical neuropathological changes of AD [169].

The analysis of functional alterations underlying the cognitive impairment of SAMP8 mice revealed deficiencies in the mechanism of long-term potentiation (LTP). LTP is based on long-term synaptic plasticity and memory formation [170]. LTP recordings in vivo or in hippocampal slices [171–173] showed a correlation between deficits in synaptic circuitry responses and the cognitive impairment of these mice. The ratio of the electrophysiological recordings of the paired-pulse stimulations, which is on the basis of short-term synaptic plasticity, also demonstrated deficiencies in SAMP8 mice in vivo [158] and in hippocampal slices [173]. Reduction of synaptic markers, such as PSD95 and synaptophysin [174, 175], corroborated the hippocampus plasticity deficiencies of SAMP8 mice. Furthermore, reduced levels of glial cell line-derived neurotrophic factor (GDNF) in the hippocampus of SAMP8 mice were demonstrated transiently at an early age of 2–5 months compared to SAMR1 mice hippocampus [176]. GDNF has been reported as highly neuroprotective against cognitive loss in a mouse model of AD [177]. Another neurotrophic factor, the BDNF, was also reported reduced in the SAMP8 mice hippocampus [174, 179]. Indeed, the reduction of BDNF was concomitant to the decrease of the activation of associated signaling factors Calcium-calModulin-dependent Kinase II (CaMKII) and CREB [171]. The reduced activation of the synaptic plasticity pathway CaMKII-CREB-BDNF in SAMP8 may be derived from a dysfunction of the NMDA receptor in these mice [171]. An age-related loss of cholesterol in the brain was proposed as the cause of the reduction of this pathway [180]. Indeed, a diet supplemented with plant sterols prevented the LTP reduction and cognitive impairment shown by SAMP8 mice [172]. Reduced levels of BDNF and of phosphorylated CaMKII in the hippocampus and in the cortex of AD patients are suggested to contribute to the atrophy and memory impairment in the disease [181, 182]. Additionally, there is a derangement of cholesterol metabolism in the AD brain [183].

SAMP8 mice showed higher levels of Aβ peptides and neurotoxic Aβ oligomers than SAMR1 mice, as demonstrated by ELISA and western blot techniques, respectively [174]. This increase would be caused by an abnormally elevated synthesis of the amyloid-β protein precursor (AβPP) [184, 185]. Strikingly, an increase in the transcription of Aβ(A4)PP gene was detected as early as 2 months of age [184]. Concurrently, increased transcription of Bace1 and decreased transcription of Adam10 would favor the amyloidogenic processing pathway of AβPP [184]. A decrease of the Aβ metabolizing enzyme Insulin-degrading enzyme (Ide) reported at 6 months of age might also contribute to amyloid pathology in SAMP8 mice [174]. Furthermore, age-related increase in presenilin 1 in SAMP8 mice, but not in control strain SAMR1, might also contribute to the imbalance in the AβPP processing [186]. A diminution of Aβ clearance derived from disturbances in the blood-brain barrier [187] is another significant factor leading to amyloid pathology in these mice. The relevance of Aβ neurotoxicity in the SAMP8 phenotype was corroborated by the protection against cognitive loss afforded by the peripheral administration of an antisense oligonucleotide against the Aβ region of AβPP [188] or an antibody against Aβ₄₂ [184]. It is known that the sequence of murine Aβ peptide differs in three amino acid residues from human sequence, this diminishing the capacity to produce amyloid deposits except in the case of murine AβPP-bearing mutations of human familial AD [189]. However, several laboratories have documented the presence of AβPP or Aβ immunoreactive granular structures in the SAMP8 brain, although devoid of the β-pleated sheet of human fibrillary Aβ in AD plaques [190]. Clustered granules devoid of Aβ and containing glycoside components were also identified early in the SAMP8 hippocampus; these attributed to senescence processes [191, 192]. Therefore, amyloid pathology is well established in SAMP8 mice, although the chemical characteristics of murine Aβ do not allow for its deposition in amyloid plaques. In the human disease, Aβ, both by increased accumulation in target brain areas and by the decreased presence in CSF, is the earliest biomarker known for being detected in preclinical phases of AD [193]. Similarly, the accumulation of Aβ in SAMP8 brain comprises an early event in its progression to an AD-like phenotype.

Pathological accumulations of hyperphosphorylated tau (p-tau) were detected in the hippocampus and cortex areas of SAMP8 mice. Analysis of AD-related tau phosphorylation sites revealed increases of p-tau in SAMP8 mice compared to SAMR1 mice aged between 3 and 9 months [174, 194–196]. Tau pathology is induced by the increased activity of specific kinases, as supported by the reported increased levels of activation of the kinases Cdk5 [194–196], GSK3β Tyr216 [178], and JNK1 [174]. It is not known whether tau pathology progresses after amyloid accumulation or whether both are parallel derangements in the SAMP8 mouse brain. However, peripheral administration of an antisense oligonucleotide directed at GSK3β Tyr216 improved learning and memory in SAMP8 [197]. In any case, the SAMP8 mice model spontaneously reproduces both pathologies, similarly to most sporadic AD cases. Contrariwise, AD transgenic mice might bear familial tauopathy genes in addition to the familial AD genes to exhibit significant tau pathology.

OS is a harmful imbalance of redox homeostasis common to both brain aging and AD [198]. Changes in OS including a decrease of antioxidant defense enzymes, an increase of markers of oxidative damage, and an increase of mitochondrial DNA deletions were reported in the brain tissue of SAMP8 mice as early as 2 months of age [199–201]. Signs of OS increased along early and advanced age in SAMP8 mice compared to SAMR1 mice [195, 202–204]. Oxidative alterations were also present in peripheral tissues at a range of age [200, 205–207]. OS might be induced or worsened by mitochondrial alterations. Indeed, brain mitochondrial respiration was dysfunctional at 2 months of age, demonstrating lower activity of complex III [201–207]. Mitochondrial dysfunction plus either increased generation of ROS or lower antioxidant defense was demonstrated in both neurons and astrocytes cultured from SAMP8 embryonic brain tissue [208–210]. SAMP8 liver mitochondria were also dysfunctional but at later age of 6–12 months [203, 211–213]. The activities of Aβ are numerous and yet to be fully elucidated. However, it is well accepted that the imbalanced in production and degradation of Aβ produces the neurodegeneration of AD and provoking physiological effect in the brain [214, 215]. Likewise, the increased burden of Aβ in the SAMP8 brain might contribute to earlier mitochondrial dysfunction and this impairment might be worsened by the presence of p-tau in the SAMP8 brain [216]. Furthermore, in vivo and in vitro studies support that Aβ generates free radicals, inducing protein oxidation, lipid peroxidation, and DNA damage [217–221]. Accordingly, the treatment of 12-month-old SAMP8 with antisense oligonucleotide directed against the Aβ region of APP decreased the levels of both Aβ and oxidative markers [222], improving cognition. Although the interaction among Aβ, OS, and AD pathology is not well understood, a vicious cycle is proposed in which alterations induced by OS would increase accumulation of Aβ and the subsequent AD pathology, the latter in turn increasing mitochondrial dysfunction and OS [223]. Despite the weight of OS in AD, antioxidant therapies were demonstrated ineffective in clinical trials [224]. Therefore, the SAMP8 model with spontaneous OS, mitochondrial dysfunction, and Aβ and p-tau accumulation in the brain makes a useful model defining the mechanisms of free radicals and mitochondrial dysfunction in AD [162].

Neuroinflammation is another age-related process that, together with OS, paves the way for the development of neurodegenerative diseases. Inflammatory signaling might contribute to AD triggering and progression [225, 226]. Phenotypical traits of inflammaging with higher expression of inflammatory mediator molecules were found in the SAMP8 hippocampus at an early age of 2 months [24]. There was also an increased response to acute inflammatory injuries at 3 months of age in SAMP8 compared to SAMR1, as detected by the levels of circulating proinflammatory cytokines [227]. Inflammation was also detected in peripheral tissues such as liver [228]. Inflammatory markers were generally maintained at elevated levels with increasing age in the SAMP8 brain in comparison with SAMR1 [24, 230]. Chronic inflammation in the SAMP8 hippocampus was aggravated by deficiencies in the mechanisms of resolution of inflammation [231]. Therefore, SAMP8 mice could not counteract inflammaging, and this might contribute to the development of its sporadic AD-like phenotype. At the cellular level, reactive astrocytosis was shown in the SAMP8 hippocampus [24, 179]. In this respect, astrogliosis measured the GFAP marker that was present at 2 months of age in SAMP8 mice, and maintained in old age [232], thus confirming the onset of neuroinflammation at early age. A decrease of the inflammatory regulator Rcor2 might mediate the inflamed phenotype of SAMP8 astrocytes [227]. Age-related stress-mediated activation of transcription factor NF-kβ was another proposed molecular mechanism of pathological inflammation in SAMP8 [228, 233]. Although diverse transgenic mice models of AD reproduce OS markers, neuroinflammation and gliosis appear only at the advanced age and is associated with a heavy load of amyloid plaques [139, 140]. Neuron loss, one of the hallmarks of AD, has been described in the hippocampus of 8-month-old SAMP8, paralleling an elevated stage of neurodegeneration [234]. Hippocampus, the most affected brain area in SAMP8, is an essential area for memory function and its functional circuitry is very sensitive to AD pathology. In addition to the AD-like neuropathology discussed earlier, SAMP8 showed a progressive impairment of cholinergic neurotransmission, as reported at 8 months of age in the hippocampus and cerebral cortex [165], similarly to the main neurotransmission defects observed in AD patients [235].

Proteomics, metabolomics, genomics, and transcriptomics studies of SAMP8 brain tissue confirmed the presence of AD-like pathological traits in the brain of SAMP8 mice [236–240]. As a whole, the specific senescent-associated alterations in SAMP8 brain comprise a scenario for the development and progression of an AD-like phenotype including the main AD traits of neuropathology and memory loss. Therefore, SAMP8 is an excellent mice model to analyze mechanisms and therapies for sporadic AD, whose main risk factor is advancing age.

Epigenetics of aging and sporadic Alzheimer’s disease-like phenotype in SAMP8

As mentioned previously, the SAMP8 model has been widely used to study senescence in all systems and from different points of view, including cerebral senescence [139, 144]. SAMP strains were generated by the phenotypic selection of crosses between siblings but, as mentioned extensively in the introduction, the causes of accelerated senescence have not been established.

The participation of OS, the evolution of neuroinflammation, and its correlation with changes in behavior and cognitive impairment, including the alterations in gene expression and protein aberrations associated with aging and neurodegenerative processes, are SAMP8 strain characteristics well reported. The fact that the SAMP8 mouse is a spontaneous animal model and not transgenic mice model of AD suggests that the underlying orchestra mechanisms must be linked to epigenetics. In this respect, several results corroborate the existence of epigenetic modifications related to aging and neurodegeneration in SAMP8 [25, 227]. It is well known that epigenetic machinery is highly modulated by environmental factors such as diet or exercise. Moreover, several studies in SAMP8 demonstrated that exercise [25], EE [233], or resveratrol-enriched diet [148, 241] induced changes in the expression of Sirt1, one of the class III HDACs related to epigenetic marks. Therefore, all this evidence indicated that SAMP8 strain is affected by epigenetic changes in reference to SAMR1 that favors mice to accelerate senescence and present AD-like processes.

For instance, EE increased global 5-mC levels in younger SAMP8 (2 months old), accompanied by reduced 5-hmC levels. Changes in gene expression of Dnmt3b, Hdac1/2, Sirt2, and Sirt6 were also found in SAMP8 under EE. Moreover, increased acetylation in H3 and H4 was found in the same experimental conditions [233].

A recent study showed a reduction in the gene expression of the chromatin regulator Rcor2 in SAMP8, which was found in reference to SAMR1. Reduced levels of Rcor2 complex increases inflammation in the SAMP8 strain by suppression of gene repression, such as Il-6, Ccl2, Ccl3, Cxcl5, Cxcl10, and Mpa2l [227].

The level of seven histone methylation sites, most located on histone H3 (H3K24, H3K27, H3K36, H3K79, H3R128, H4K20 and H2AR89), were found significantly decreased in the SAMP8 brain with respect to SAMR1 [242], providing evidence that posttranslational modification in SAMP8 is linked to senescence process.

It has been published that expression of Dnmt3b was increased in SAMP8 after injection of erythropoietin improving spatial learning and memory, indicating that the methylation state of DNA is necessary for learning and memory, including the memory formation [243]. Epigenetic mechanisms underlying cognitive impairment by accelerated aging in SAMP8 were also found in an AD mice model, as 5xFAD, where Aβ is a key factor in cognitive impairment and neurodegeneration [233, 244].

It has been demonstrated that miRNAs also play an important role in the SAMP8 model [25], corroborating what has already been described concerning the role of miRNAs, both in development and in aging [245]. The first study demonstrated that miR-16 is underexpressed in the SAMP8 strain and that miR-16 reduces the protein levels of AβPP [246]. Other authors demonstrated higher expression of miR-139 in the hippocampus of aged SAMP8 than in SAMR1. When miR-139 is overexpressed in SAMP8, the hippocampus-dependent learning and memory formation by modulating cannabinoid receptor type 2 (CB2) is impaired, whereas downregulation of miR-139 leads to an improvement in learning and memory [247].

Another study showed the abnormal expression of genes (nodes) of certain biological networks related to the AD patients were regulated by miRNAs such as miR-20a; -17, -34a; -155; -18a; -22; -26a; -101; -106b, and -125b and were found changed in SAMP8 in comparison with SAMR1 [248]. Furthermore, there are significant alterations in the expression of various genes of HDACs enzymes, such as Sirt1, Hdac5, and Hdac6, and also of the global levels of acetylation of histones H3 and H4 and of various miRNAs in the hippocampus in the SAMP8 female at 8 months of age, which partially revert with an intervention of voluntary exercise [25]. Finally, the use of the HDAC inhibitor, trichostatin, allowed the reversal of the senescent phenotype and DNA damage by increasing the levels of histone acetylation, and increasing the proliferation of exhausted NSC of SAMP8 in vitro [143].

In addition, a significant reduction in the expression of the miR-9 family in the SAMP8 strain compared to the age-matched SAMR1 control was found; this miR-9 regulated in silico the genes PSEN1, SCN2B, MAP3K3, and BACE1 [249]. This would explain in part the cognitive impairment and pathological process of SAMP8 mice model, confirming its relationship with aging and AD.

Correlating the described modifications with cellular processes mentioned comprises a task that must be carried out in order to further demonstrate that miRNAs can control protein levels and in turn influence brain function and the cognitive abilities in SAMP8. The involvement of epigenetic modulation in the aging process, the neurodegeneration, and the central role of the gene regulation of miRNAs, could explain in part that the senescent phenotype found in the SAMP8 strain is an issue to be developed.

In sum, published results demonstrate the participation of epigenetic modulation in the aging and neurodegenerative processes, and the central role of gene regulation that miRNAs possess, explain in part the senescent phenotype that characterizes SAMP8.