RNA methylation on neurocognitive dysfunctions

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The role for DNA/RNA methylation on neurocognitive dysfunctions

6 Xiangru Xua, b

a

Max Planck Institute for Biology of Ageing, Cologne, Germany ; Department of Anesthesiology, Yale University School of Medicine, New Haven, CT, USAb

1. Brain aging and cognitive dysfunction Learning, memory and cognitive dysfunction is often accompanied by mammalian brain aging. Brain aging is generally defined by the continuing deterioration in different aspects of learning, memory and cognitive performance, brain structures, and neuronal/brain functions. Learning and memory impairment in brain aging is a common feature for elder populations across the spectrum of species from invertebrates, rodents, monkeys, to humans [1]. In the developing world and advanced countries, a growing body of elder populations and their families are suffering from such age-associated learning, memory and cognitive impairments. For example, in the United States, it is anticipated that w12% of people over the age of 65 will suffer from moderate-to-severe memory defects by 2050 [2]. Cognitive dysfunction is often correlated with age-dependent weakening of synaptic functions in the hippocampus and prefrontal cortex that are crucial for memory formation and consolidation. The prefrontal cortex (PFC) in humans plays an important role in complex cognitive behaviors, decision making, personality, and the orchestration of thoughts and actions. Memory and attention deficits are common for people with PFC impairment, yet people largely attribute the age-associated memory decay to the declining functions in the hippocampus. The hippocampus is another crucial section in the brain and is closely associated with the cerebral cortex for learning, memory and cognitive functions. Two major functions of the hippocampus are to store and interpret the spatial information and to facilitate the consolidation of short-term memory into long-term memory. Hippocampus-dependent memory deficits during normal (successful) aging are distinct from the symptoms of devastating neurodegenerative disorders such as Alzheimer’s disease that profoundly impacts memory, but can occur in the absence of massive neuronal death. Instead, subtle changes in the connections and functional integrity of key hippocampal neuronal circuits appear to underlie the memory impairment in elder individuals [3]. In the context of aging, altered transcriptional regulation of genes that promote or are essential for synaptic plasticity is associated with memory impairment in aged mice/rats. Alongside a focus on negative outcomes, there is cumulative recognition that memory decline is not an inevitable consequence of aging as some older individuals maintain excellent memory abilities that match those of younger individuals across the lifespan [4]. It is therefore suggested that identifying the neurobiological mechanisms that regulate differential cognitive outcomes with age is immensely important. Nutritional Epigenomics. https://doi.org/10.1016/B978-0-12-816843-1.00006-0 Copyright © 2019 Elsevier Inc. All rights reserved.

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Before further discussing normal age-associated cognitive changes, it is crucial to mention methodological challenges in human cohort studies regarding normal cognitive dysfunction/brain aging [5]. Selection bias in study participants is the first real challenge; many potential study participants decline enrollment because they are either too healthy/busy or too ill, and people with limited social or financial support and functional limitations may also be less likely to be recruited into a study. This largely compromises the representative soundness in sample selection. Second, results derived from cohort differences may miscalculate the effects of aging as most studies rely on cross-sectional design to compare subjects from different age groups. For instance, a cohort that was born in the 1940’s had a very different life experience from a cohort born in the 1960’s; besides, they may also greatly differ in terms of culture, lifestyle, education, and requirements for success in life. Taken together, these practical challenges in human cognitive dysfunction/brain aging studies can potentially undermine the findings/conclusions. Model organisms, both invertebrate and vertebrate, thus turn out to be a powerful research tool to tackle the neurological mechanism of cognitive dysfunction/brain aging. The neurobiological processes underlying age-associated learning, memory and cognitive deficits include aberrant changes in gene transcription that eventually affects the resilience of the aged brain. Changes in gene expression in active neurons were thought to take place during brain aging, and analysis of regions of the hippocampus and frontal cortex by microarray has confirmed this [6,7]. The molecular mechanisms underlying these changes in gene expression and its regulation are largely unclear. Over the past decade, accumulated evidence has indicated that epigenetic mechanisms may be heavily involved in mediating age-related changes of the brain/cognitive functions. Speaking of epigenetics, the term was first created by Conrad Waddington in the 1940’s to describe the interactions between genes and their environment during development [8]. It has been evolved from a narrative term to a massively studied scientific field in the 21st century. The current meaning of epigenetics contains conformational changes in DNA and/or chromatin without altering the basic genetic code that regulate the sophisticated molecular machinery through which the spatio-temporal dynamics of gene expression are implemented. These mechanisms primarily involve DNA/RNA methylation, histone post-translational modifications and non-coding RNAs. How DNA/RNA methylation affects the cognitive dysfunction/brain aging will be discussed further with details.

2. DNA methylation, brain aging and cognitive dysfunction 2.1 DNA methylation 2.1.1 50 -methylcytosine (5 mC) DNA methylation is the most extensively studied epigenetic mark. It is evolutionarily lost in species including yeast, worm and fly but widespread in plants, rodents, primates and humans, and plays a critical role in regulating gene expression and maintaining genome stability [1,9e10]. DNA methylation involves the addition of a methyl group to the fifth carbon of a cytosine residue to form 50 -methylcytosine (5 mC) by DNA methyltransferases (Dnmts), most frequently in the context of CpG dinucleotides [1,9,10]. This biochemical process creates a relatively stable covalent modification that is traditionally understood to repress gene transcription by promoting closed chromatin states and limiting DNA accessibility to transcriptional activation machinery and/or recruiting of transcriptional repressors [1,11]. CpGs are not uniformly distributed in the genome and tend to be enriched as CpG islands, which are stretches of DNA roughly 1000 base pairs long that have a higher CpG density than

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the rest of the genome [9]. Gene promoters in the genome mostly have an associated CpG island, and, in general, DNA methylation levels at these promoter-associated islands negatively associate with gene transcription levels [9]. However, recent study suggests that the consequences of DNA methylation on transcription can vary considerably depending on a variety of factors including the CpG island locus [11]. The dynamics of genome-wide DNA methylation are regulated by Dnmts, including Dnmt1, Dnmt3a and Dnmt3b. Dnmt1 is abundant in mammalian tissues including the brain. It is responsible for the maintenance of DNA methylation patterns during DNA replication, whereas Dnmt3a and Dnmt3b perform de novo DNA methylation during development and other physiological and pathological conditions [1,11,12]. In addition, another Dnmt family member Dnmt3-Like (Dnmt3L), is homologous to Dnmt3a and Dnmt3b, but lacks a catalytic domain. Dnmt3L, however, can substantially increase activity of other Dnmt enzymes [13]. All Dnmts are intensively involved in embryonic development and their expression is reduced significantly by the time cells reach terminal differentiation. This suggests that the DNA methylation pattern is relatively stable in postmitotic cells such as neurons and cardiocytes. However, in the mature mammalian brain, postmitotic neurons still express substantial levels of Dnmts, raising the possibility that Dnmts and DNA methylation may play more crucial roles in the brain [11]. Indeed, the loss of Dnmt1 and Dnmt3a in the adult brain leads to cognitive deficits in mice [14]; in humans, mutations in Dnmt1 are associated with a form of neurodegenerative disease [15]. These studies exhibited that impairment of DNA methylation may be a fundamental mechanism in regulating mouse learning, memory and cognition. The first suggestion that DNA methylation might play an important biological role was made by Griffith and Mahler in 1969 speculating that DNA methylation could provide a basis for long term memory in the brain [16]. It has since been suggested that the presence of 5 mC in CpG island promoter regions affects the binding of transcription factors, and subsequently gene expression [17,18]. DNA methylation regulates gene expression by recruiting co-repressor complexes (e.g., histone deacetylases (HDACs) and histone methyltransferases) that can sterically block the transcriptional machinery and/or modify nucleosome structure [11]. Such complexes involve several DNA methyl-binding domain proteins (MBDs), which are required for normal cell growth and development. MBDs are expressed at higher levels in brain than in any other tissues, and many MBDs are important for normal neuronal development and function [11,12], such as methyl CpG binding protein2 (MeCP2). MeCP2 is recognized as a transcriptional repressor, and mutations in MeCP2 lead to a neurodevelopment disorder - Rett syndrome [16]. The effects of DNA methylation on gene expression are complex and may vary according to genomic location. Numerous studies suggest that methylation occurring within CpG-rich regions near the transcription start-site of a gene (i.e., CpG islands) tends to have a repressive effect on gene expression across tissues. Gene body DNA methylation is associated with a higher level of gene expression in dividing cells. However, in the murine frontal cortex, gene body methylation of non-CpG sites is negatively correlated with gene expression [19]. The presence of inconsistency in DNA methylation independent of the underlying nucleotide sequence suggests that epigenetic modifications can modulate the impact of genetic variation (i.e., genotypes) on biological processes including brain function. In order to maintain or alter genome-wide DNA methylation patterns in cells, including neurons, in response to environmental changes, both DNA methylation and demethylation have to be active. The process of DNA demethylation is much less understood. Recent studies, imply that key DNA demethylation enzymes are ten-eleven translocation (Tet) family enzymes Tet1, Tet2 and Tet3.

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Tet enzymes are involved in both global and locus-specific DNA demethylation [20e23]. Demethylation often occurs during the process of hippocampal learning, memory and adult neurogenesis, which impacts the olfactory system [24]. Tet1 mutant animals exhibit abnormal hippocampal long-term depression and impaired memory extinction. Learning and memory require neuronal activity, which can strengthen synaptic connections and weaken other synaptic connections through a process of synaptic plasticity [24].

2.1.2 50 - hydroxymethylcytosine (5hmC)

50 - hydroxymethylcytosine (5hmC) is the oxidized form of the canonical 5 mC and was identified in mammalian brain tissue and stem cells [22e25]. Tet enzymes add a hydroxyl group onto the methyl group of 5 mC to form 5hmC and initiate the complete demethylation process [22,23]. Unexpectedly, like 5 mC, 5hmC may also regulate gene expression and/or affect the function of neurons since the conversion of 5 mC to 5hmC impairs the binding of MeCP2 [26]. 5hmC accounts for w40% of modified cytosine in the brain, which is typically 5 to 10 times higher than in any other tissue, and has been implicated in DNA methylationerelated synaptic plasticity [25]. In neuronal cells, 5hmC markedly increases from the early postnatal stage to adulthood, suggesting a strong correlation between 5-hmC and neurodevelopment. Interestingly, 5-hmC is depleted on the X chromosome during postnatal neurodevelopment and aging. Functionally, 5-hmC is associated with actively transcribed genes in adult cerebellum. 5-hmCeregulated regions are dynamically changed during neurodevelopment and aging. 5-hmC is enriched throughout gene bodies in the brain, whereas 5-hmC is also present in embryonic stem cells in the bodies of active genes, although to a lesser degree than that found in the brain. The overall abundance of 5-hmC is negatively correlated with MeCP2 [27]. These findings suggest that 5hmCemediated epigenetic regulation is critical in neurodevelopment, and aging, as well as in other human neurological disorders.

2.1.3 Methods to profile the genome-wide DNA pattern Growing evidence suggested that DNA methylation regulation is critical for maintaining normal brain functions and confers an epigenetic mechanism for learning, memory and cognition [28e30]. It is thus critical to ensure that there are appropriate methods to measure the dynamics of genome-wide DNA methylation in neuronal cells/tissues. Weber M. et al. in 2005 first described a method known as methylated DNA immunoprecipitation (MeDIP)-chip, to assess genome-wide DNA methylation [31]. It consists of enriching methylated DNA fragments through an antibody against 5-methylcytosine (5 mC), and detecting the purified fraction of methylated DNA with high-throughput DNA methylation arrays (chips). MeDIP-chip (e.g., mouse and human promoter CpG arrays) can be used to map the dynamic alterations of genome-wide promoter CpG methylation in aging tissues [10]. Around the same time, Meissner et al. first reported a reduced representation bisulfite sequencing (RRBS) method to dissect the methylome of mammalian cells [32]. RRBS is based on the fact that CpG sites within the mammalian genome tend to cluster together as CpG islands (CGIs) that are usually located close to the promoters of known genes [33]. So, firstly cutting the genome into small fragments by a restriction enzyme that recognizes CpG and its flanking sequences, then most of the CGIs will be collected and sequenced with high coverage even with lower numbers of total sequencing reads (e.g., w50 million reads). RRBS has led to important findings regarding global methylation and demethylation processes during early developmental stages [34]. Lister et al. later described whole-genome bisulfite sequencing (WGBS) to map DNA methylations at single base resolution [35]. This is currently the gold standard

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for DNA methylome measurement and provides coverage for more than 90% of the approximately 28.7 million CpGs in the human genome [36]. However, it demands much higher sequencing reads, the minimum request for sequencing reads coverage is about 30X genome size. Those methods are not only good for tissue level study but also can be used for cell population interrogation of the DNA methylome. It is important to understand that the frontal brain region such as the hippocampus is not a unitary structure. Major types of hippocampus subregional neurons including cornu ammonis1 (CA1) pyramidal neurons, CA3 pyramidal neurons, and dentate gyrus (DG) granule neurons have been studied extensively, and are believed to play central roles for learning and memory and cognitive functions of the hippocampus. Hippocampal neurons are the main effectors of age-associated neurodegeneration. More specifically, CA1 and CA3 pyramidal neurons are more susceptible to neurodegenerative disorders such as Alzheimer disease, whereas granule neurons in DG are more vulnerable to age-related damage [7,37,38]. In addition, neurons from one type of population are possibly different from one to another, one is more stable and resistant to stressors while another is more vulnerable to the same stressors. Single cell transcriptome analysis has been achievable and has proven to be a powerful tool to understand the variation among the same type of cells [39]. The methods to examine the genome-wide DNA methylation at single level were also essentially desired. Guo et al. reported a methylome analysis method that enables single-cell at single-base resolution DNA methylation analysis based on reduced representation bisulfite sequencing (scRRBS) [40]. ScRRBS integrates all of the experimental processes in a single-tube reaction without including any purification steps prior to the bisulfite conversion step, since the multiple purification steps are the major problem for massive loss of DNA. This technique is sensitive and can detect the methylation status of up to 1.5 million CpG sites within the genome of an individual embryonic stem cell [41]. While Smallwood et al. described a single-cell bisulfite sequencing (scBS-seq) method, which can be applied to accurately measure DNA methylation at up to 48.4% of CpG sites [42]. In BS-seq protocols, bisulfite treatment is performed first then sequencing adaptors are ligated to fragmented DNA minimizing the DNA loss from single cell. In brief, these are all powerful tools for us to map DNA methylation at tissue, cell population and single cell level facilitating a better understanding of DNA methylation in neuronal gene regulation and thereby cognitive function.

2.2 DNA methylation and brain aging/cognitive dysfunction Studies showed that DNA methylation levels are particularly promising biomarkers of chronological aging (i.e. the calendar years that have passed since birth) and this implies a profound effect on DNA methylation levels in most human tissues and cell types [43]. In the central nervous system, DNA methylation is critical for proper postnatal neurodevelopment and undergoes age-dependent changes in the adult brain (Fig. 6.1; [27]). The overall decline in DNA methylation has been associated with cell and tissue aging including the brain for decades [1]. For example, DNA methylation in the PFC shows unique temporal patterns across life. The fastest changes occur during the prenatal period, slowing down markedly after birth and continuing to slow further with the aging process [44]. However, the effects of aging are complex, with some evidence pointing to age-related decreases in global DNA methylation, together with increased methylation at CpG islands across multiple brain regions in humans [44]. The enrichment of methylation at CpG sites tends to occur more frequently among functionally related gene transcripts,

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Methionine Homocysteine O2+ α-ketoglutarate succinate + CO 2 NH2 N O

SAM

SAH

NH2

-CH3 N H

Cytosine (C)

Dnmt

N O

NH2 CH3

N

H 5-methylcytosine (5mC)

OH

N Tet

O

N

H 5-hydroxymethylcytosine (5hmC)

Gene expression

Biological function

FIG. 6.1 DNA Methylation and Demethylation Play Important Roles in Neuronal Gene Expression and Brain Function. The cytosine methylation process relies on the activity of DNA methyltransferase (Dnmt) and demethylase (Tet), plus a supply of methyl groups. SAM: S-adenosylmethionine; SAH, S-adenosylhomocysteine. Credit: Xiangru Xu.

including gene classes that regulate DNA binding and transcription factors. This age-related aggregation of methylation might contribute to transcriptional abnormalities reported in the aged brain. Consistent with this possibility, altered methylation of activity regulated cytoskeleton associated protein (Arc) DNA in the CA1 and dentate gyrus of the hippocampus in aged rats is associated with decreased Arc transcription and spatial memory impairment [45]. Evidence suggesting that DNA methylation influences differential cognitive outcomes in aging derives from a targeted study examining methylation in the promoter regions of gamma-aminobutyric acid type a receptor alpha5 subunit (Gabra5), heat shock protein family a member 5 (Hspa5), and synapsin I (Syn1) previously implicated in age-related cognitive decline in the Long-Evans rat [46]. The overall results reveal an increase in the number of methylated sites across all three genes, but only in relation to chronological age and not cognitive status. The loss of Dnmt1 and Dnmt3a in the adult brain leads to cognitive deficits in mice [28]. Transient over-expression of Dnmt3a2, a Dnmt3a isoform, in mouse hippocampus restores age-associated cognitive deficits. Moreover, inhibition of hippocampal Dnmt3a2 expression by RNAi leads to cognitive behavioral deficits in young mice [28]. Mutant Tet1 animals exhibit abnormal hippocampal long-term depression and impaired memory extinction. In humans, mutations in Dnmt1 are associated with a form of neurodegenerative disease. These results suggest that impairment of DNA methylation plays a crucial role and is a fundamental mechanism that regulates mouse learning, memory and cognition [1].

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Both developmental programming and age-dependent alterations of 5hmC occur in the mammalian brain. In hippocampus and cerebellum, 5hmC patterns may be maintained in general across life, but may be further acquired with age at specified loci [27]. In the mouse hippocampus, for example, global 5hmC content increases during aging in the absence of 5 mC decrease, suggesting that 5hmC acts as an epigenetic marker and not simply as an intermediary in DNA demethylation [47]. Besides, 5hmC levels inversely correlate with the dosage of MeCP2, a protein encoded by a gene with mutations causes Rett syndrome [27]. These findings suggest that 5-hmCemediated epigenetic regulation is critical in neurodevelopment, aging and human diseases.

3. RNA methylation, brain aging and cognitive dysfunction 3.1 RNA methylation RNA methylation such as 5-methylcytidine (m5C) and N6-methyladenosine (m6A) is a rather new theme in the last few years, though m5C and m6A both have been discovered to widely exist across the whole spectrum of animal kingdoms by recently developed transcriptome-wide sequencing approaches. It is therefore not only created a new field of research e epitranscriptome, but also revealed essential roles/functions of RNA methylation in a wide range of fundamental cellular processes. Here, we mainly review and discuss the cutting-edge knowledge/techniques of m5C RNA methylations and its impact on brain aging and cognitive dysfunctions.

3.1.1 5-Methylcytidine (m5C) Post-transcriptional modifications of RNA add complexity to RNA-mediated functions by regulating how and when a primary RNA transcript is converted into a mature RNA. There are in total around 150 known RNA modifications [48], though our knowledge about their occurrences and functions in RNA is still far more limited. The existence of methylated bases in RNA including C5-methylcytidine (m5C) had been described 50 years ago [49]. But, until only very recently, m5C was thought to be mainly restricted to the stable and highly abundant transfer RNAs (tRNAs) and ribosome RNAs (rRNAs) [50]. The latest development of novel transcriptome-wide methods to map global m5C RNA methylomes has not only restored scientific interest in the field but also contributed to a better understanding of gene expression regulation at different levels. It has become evident that post-transcriptional methylation of cytosines regulates fundamental cellular processes that are essential for normal development. The importance of tightly controlled removal of m5C on RNA is further highlighted by the link of loss-of-function mutations in methylation and demethylation enzymes to severe human diseases. RNA m5C methyltransferases belong to a large and highly conserved group of proteins, yet their RNA substrate specificity is predicted to be different [51]. Among all RNA methyltransferases Dnmt2 is the best studied, mostly for its potential function in DNA methylation. Dnmt2 shares almost all sequence and structural features of DNA methyltransferases [52]. However, it turned out that Dnmt2 plays no critical role in influencing global DNA methylation. Dnmt2-deficient mouse embryonic stem (ES) cells do not display altered genomic methylation patterns and organisms expressing only Dnmt2 as the sole candidate DNA methyltransferase gene lack genomic methylation patterns [53,54]. Dnmt2 then was identified as one of the first cytosine-5 RNA methylases in a multicellular organism [55]. At least two more enzymes NOP2/Sun RNA Methyltransferase Family Member 2 (NSun2) and member 4 (NSun4) can generate 5-methylcytidine in RNA in mammals [56,57], yet their substrate specificities are unknown.

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3.2 RNA methylation and brain aging/cognitive dysfunction NSun2 was first described in the mammalian epidermis as a transcriptional target of the protooncogene c-Myc [56]. NSun2 is up-regulated in a wide range of cancers and knockdown of NSun2 in human squamous-cell-carcinoma xenografts decreased their growth [56e58]. Interestingly, genetic mutations in the NSUN2 gene in several human cohorts have been identified and primarily linked to autosomal-recessive intellectual disability and a Dubowitz-like syndrome [59e61]. The common symptoms of the disorder include growth and mental retardation [59e61]. Similar to the human syndrome, deletion of the NSun2 ortholog in Drosophila caused severe short-term-memory deficits [60], yet it is still unknown about whether and how loss of RNA methylation is the underlying cause for the symptoms of these complex diseases.

4. Interventions and drug development targeting DNA methylation in brain aging/cognitive dysfunction Epigenetics such as DNA/RNA methylation, unlike genetics, is not only inheritable but also reversible. Strategies aimed at reversing age-associated epigenetic alterations, therefore, may lead to the development of novel therapeutic interventions that can prevent brain aging/cognitive dysfunction or alleviate symptoms of devastating, age-associated neurodegenerative diseases.

4.1 Interventions Dietary restriction (DR), without malnutrition, appears to be a promising strategy to extend the life span and counteract detrimental age-related alterations in a fashion that is evolutionarily conserved from yeast to primates and humans [62], although studies in humans are very limited and with mixed results [63]. DR of caloric intake and enhanced levels of endogenous and exogenous antioxidants are approaches that are potentially able to mitigate age-related deterioration of the brain. The beneficial effects include, in mammals, the attenuation of age-associated cognitive impairment and neurodegeneration [64]. More specifically, synaptic plasticity was shown to be enhanced by DR, as evidenced by increased long-term potentiation [65]. Besides DR, rapamycin is the first drug intervention to reliably increase mammalian lifespan by 10% or more [66]. The link between aging and disease by rapamycin treatment has been carefully discussed [67]. Interestingly, rapamycin treatment suppresses brain aging in senescence-accelerated OXYS rats [68], and also produces an improvement in cognitive functions that normally decline with age in mice [69]. The results from our study in mouse brain unexpectedly demonstrated that both DR and rapamycin can restore, at least partially, the age-related alterations in histone methylation levels [53]. This allows us to suggest a novel and beneficial epigenetic mechanism for age-interventions. The overall alterations of histone modifications in brain suggest changes of histone modification related-enzymes by these interventions. In accordance with this, a recent study showed that, independent from genotype, DR prevents the age-related increase of HDAC2 in the hippocampus, particularly in the CA3 and CA1-2 subregions. Furthermore, HDAC2 correlates positively with 5 mC while these markers were shown to co-localize in the nucleus of hippocampal cells [60]. Interestingly, in mouse cerebellar Purkinje cells, aging is associated with an increase of 5 mC and 5hmC, and these age-related increases are mitigated by DR, and the ratio between 5 mC and 5hmC decreases

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with age and DR treatment, suggesting that DR has a stronger effect on DNA methylation than DNA hydroxymethylation [70]. As discussed above, DNA methylation is implicated in age-related changes in gene expression as well as in cognition and Dnmt3a is essential for memory formation and underlying changes in neuronal and synaptic plasticity. DR indeed attenuates age-related changes in Dnmt3a in mouse hippocampus [71]. These findings enforce the notion that aging is closely connected to marked epigenetic changes, affecting multiple brain regions, and that DR is an effective means to prevent or counteract deleterious age-related epigenetic alterations. Physical exercise improves the efficiency of the capillary system and increases oxygen supply to the brain, thus enhancing metabolic activity and oxygen intake in neurons, and increases neurotrophin levels and resistance to stress. Regular exercise and an active lifestyle during adulthood have been associated with reduced risk and protective effects for mild cognitive impairment. Recent studies have examined the epigenetic impact of exercise in the brain. For example, in a rodent study, epigenetic changes in the hippocampus and cerebral cortex have been correlated with an environmental enrichment that includes voluntary exercise, which increases synaptic integrity and neuroplasticity in the brain, while improving memory, learning and stress response [72]. This study clearly indicates that a lifestyle intervention can improve cognitive functions through epigenetic mechanisms. Another study revealed that regular physical exercise induces epigenetic modifications at the dentate gyrus, which may regulate gene expression responses involved in neuroplastic and cognitive responses to stressful events. These behavioral responses to exercise were found to correlate with changes in the levels of histone H3 acetylation at lysine 14(H3K14ac) and phosphorylation of histone H3 at Ser10 (H3S10p) [73]. Aside from histones, DNA methylation is significantly increased in the hypothalamus of rats by physical exercise. Physical exercise can also increase global DNA methylation in the hippocampus, cortex, and hypothalamus and decrease expression of the Dnmt1 gene in the hippocampus and hypothalamus of rats that undergo repeated restraint stress. These findings indicate that physical exercise affects DNA methylation of the hypothalamus and might modulate epigenetic responses evoked by repeated restraint stress in the hippocampus, cortex, and hypothalamus [74]. Although the experimental data that link physical exercise or DR and epigenetics are still limited, insight into the epigenetic mechanisms involved in the brain aging process and their modulation through lifestyle interventions such as DR and physical exercise might open new avenues for the development of preventive and therapeutic strategies to treat age-related neurodegenerative diseases.

4.2 Epigenetic drug development An utmost important phase is to explore the use of epigenetic drugs as potential therapeutics for age-related brain/cognitive diseases. Unlike genetic mutations or SNPs that cannot be reversed without gene therapy, epigenetic marks that accumulate in the brain during aging are reversible and can be modulated and possibly corrected through pharmacological approaches. Several DNA methylation inhibitors, including the cytidine analogs 5-azacytidine and zebularine and nucleoside analogs that sequester Dnmt after being incorporated into DNA, are approved or are in preclinical and clinical trials for the treatment of cancer. Interestingly, Dnmt inhibitors become powerful modulators of hippocampal learning and memory when administered directly into the brains of mice and rats [75].

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5. Perspectives and challenges As an emerging field, epigenetic investigations into brain aging/cognitive dysfunction have just started to attract attention from both academics and pharmaceutical industries. It has shown promising clues in dissecting the secrets to brain aging/cognitive dysfunction. More intriguingly, interventions and drugs targeting epigenetic factors such as DNA methylation are not only providing possible cures for age-related neurological symptoms, but also assisting a mechanistic understanding of the etiology and patho-physiology of brain aging and age-related cognitive function impairment. However, piecing together the role of DNA methylation in transcriptional regulation and its impact on age-related cognitive function remains challenging.

5.1 The complexity of transcriptional regulation by DNA methylation Learning and memory are two intimately linked cognitive processes that stem from interactions between genes and the environment (experience). These cognitive functions have also been associated with changes in gene expression, and a number of synaptic plasticity associated genes have been found to enhance or impair learning and memory. Dysregulation of these synaptic plasticity genes, such as brain-derived neurotrophic factor (Bdnf), cAMP response element binding (Creb) and activity regulated cytoskeletal-associated protein (Arc) have been strongly correlated with mammalian brain aging and cognitive decline. For instance, polymorphisms in the human BDNF gene have been associated with memory and hippocampal function [76]. Bdnf-deficient mice display premature age-associated decrements [77]. Hippocampus-specific deletion of Bdnf in adult mice impairs spatial memory and extinction of aversive memories [78]. Mice with Creb deficiency have a mild cognitive impairment, and exhibit a deficit in condition-dependent learning and memory tests [79]. Expression of Arc, a neuronal activity-relevant gene, decreases with age, and this decreased expression correlates with DNA hypermethylation of its promoter [46]. It is also known that upregulation of Dnmt3a2 in hippocampus can restore age-related cognitive function, though it is not known yet how precisely Dnmt3a2 contributes to cognitive function [28]. Higher levels of Dnmt3a2 presumably result in an increase of DNA methylation of Dnmt3a2 target genes. The expression of synaptic plasticity genes like Bdnf, c-Fos and Arc are increased significantly with over-expression of Dnmt3a2. This sounds counterintuitive on the basis of the traditional view that DNA methylation is associated with transcriptional repression. However, there are reports suggesting that exonic DNA methylation may serve as a transcriptional activator that triggers gene transcription [35]. This also could be caused by methylation-associated blocking of transcriptional repressor (TF) such as neuron restrictive silencer factor (Nrst/REST), which has been reported to be involved in transcriptional repression of Bdnf. However, BDNF expression will be released after NRST/REST being inhibited by the promoter methylation [80]. This is just one example, not to mention the impact of DNA methylation on other DNA elements such as enhancers, and non-coding RNAs including long non-coding RNAs and miRNAs, which may result in regulation of neuronal gene expression [81]. Taken together, it raises additional layers of complexity for understanding the role of DNA methylation in neuronal gene expression regulation. Deep sequencing methods such as BS-DNA-methyl-seq and RNA-seq at neuronal tissue and cell level will be an effective tool to better understand this complexity.

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5.2 Elucidating the functional significance of DNA methylation Animal models as well as cutting-edge genetic manipulation approaches should be employed to determine the biological significance of DNA methylation on learning, memory and cognitive function [82]. For example, a constitutive and an inducible forebrain neuron-specific Dnmt3a2 transgenic mouse line could be generated to test if higher level Dnmt3a2 in neurons can enhance/restore mouse learning, memory and cognitive functions. In contrary, a Dnmt3a2 conditional forebrain neuron-specific knockout mouse line will also be useful to measure if Dnmt3a2 is essential for maintaining learning and cognition. Lastly, genome-wide analysis of the DNA methylation landscape and transcriptome, in parallel, in neurons under various conditions including age and expression level of Dnmt3a2 via bisulfite sequencing and RNA-seq would allow for the identification of neuronal targets of Dnmt3a2.

5.3 Interplay of DNA methylation and histone modification It is critical to understand the coordination of DNA methylation and histone modification in the regulation of neuronal gene expression and learning, memory and cognition. The epigenetic processes associated with DNA methylation/demethylation and histone modifications do not always act independently, but could closely interact to form a complex and multilayered regulatory system to dynamically fine-tune gene expression. Dnmts cooperate with histone-modifying enzymes involved in adding and/or stripping histone markers in order to impose a repressive state on a gene region [11]. For instance, there is an interesting interplay between Dnmt3a-dependent DNA methylation and Polycombgroup (PcG)-dependent H3K27me3 marks. Dnmt3a activity at non-promoter regions correlate with increased expression of neurogenic genes, by interfering with PcG binding and H3K27me3-mediated gene repression. In contrast, Dnmt3a activity at promoter regions inhibits gene expression. Additionally, Dnmt inhibitors block changes in H3 acetylation associated with memory formation. Furthermore, deficits in memory and hippocampal synaptic plasticity induced by Dnmt inhibitors can be reversed by pretreatment with an HDAC inhibitor [55]. MeCP2 binding, preferentially to fully methylated DNA, is associated with both HDAC machinery and histone methyltransferases to alter specific histone modifications [83]. Thus, DNA methylation acts in concert with histones to regulate gene expression, through interference with transcription factor binding and chromatin compaction. It is also conceivable that histone modifications influence DNA methylation patterns, indicating a bidirectional relationship between histone and DNA modifications. DNA methylation patterns are established and maintained by specific combinations of chromatin modifications. Consistent with this hypothesis, elevated histone acetylation can trigger DNA demethylation and thereby gene expression in vitro [11]. Conversely, HDACs are known to interact with Dnmts and inhibit gene expression through the induction of DNA methylation [50], whereas transcription factors that recruit histone acetyltransferases can trigger demethylation of DNA. Likewise, HDAC inhibitors are capable of inducing DNA demethylation [83]. Taken together, these results reveal a complex relationship between histone modifications and DNA methylation.

5.4 Examine RNA methylation (m5C) and its role in brain aging and cognitive dysfunction Although the precise molecular and biological functions of RNA m5C methyltransferases are still poorly understood. A noticeably high number of NSun-proteins are associated with human disease

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syndromes that include growth retardation and neurological deficits. It will be critical to dig out the specific link between human diseases and RNA methylation, since it may be explained by (1) a direct role of 5-methylcytidine in rRNA and tRNA to regulate global protein translation, or (2) roles of 5-methylcytidine in functional mRNAs and subsequently proteins. Moreover, the methods to map the epitranscriptome were mostly adapted from DNA methylation approaches, the reproducibility and reliability are still unclear, and needs careful examination [50,51].

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