DNA methylation and hydroxymethylation dynamics in the aging brain and its impact on ischemic stroke

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DNA methylation and hydroxymethylation dynamics in the aging brain and its impact on ischemic stroke

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Neurochemistry International

Available online 11 June 2025, 106007

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https://doi.org/10.1016/j.neuint.2025.106007

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Highlights

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  5mC and 5hmC are crucial epigenetic modifications that drive context-specific gene regulation in brain.
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  Aging disrupts the balance of 5mC and 5hmC in several brain regions.
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  Aberrant 5mC and 5hmC patterns contribute to stroke pathophysiology.
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  TET activation drives 5hmC-dependent transcriptional induction of neuroprotective genes after stroke.
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  Targeting 5mC and 5hmC dependent epigenetic reprogramming is a promising strategy for stroke therapy.

Abstract

DNA methylation and hydroxymethylation patterns at the 5^th carbon of cytosine (5mC and 5hmC) in CpG dinucleotides tightly regulate gene transcription in normal physiology, aging, and associated diseases, including ischemic stroke. Resilience to ischemic brain injury depends on the interplay of diverse neural and non-neural cell types, whose gene expression and identity are predominantly regulated by brain-enriched epigenetic mechanisms, particularly the dynamics of 5mC and 5hmC in response to changing transcriptional demands under ischemic stress. In this review, we discussed the role of 5mC and 5hmC in aging and the pathophysiology of stroke. Given the high degree of inter-individual variability in stroke studies and its multifactorial etiology, we emphasize the need for personalized, temporally controlled, epigenome-based therapies to improve stroke outcomes.(Will your competent? doctor and hospital get further research going on this?

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Introduction

Epigenetic signatures in the mammalian genome, including DNA and histone modifications, control tissue- and context-specific gene expression, thereby regulating cellular differentiation and organism development (Ashe et al., 2021; Egger et al., 2004). In particular, methylation and hydroxymethylation marks on the cytosine at CpG (cytosine followed by guanine) dinucleotides tightly regulate transcription during various physiological and pathological contexts (Jin and Liu, 2018; Kriukienė et al., 2012). Methylation at the 5^th carbon position of cytosine forms 5-methylcytosine (5mC), which is a repressive, heritable epigenetic mark that negatively regulates gene expression by either restricting the binding of transcription factors or favoring a non-permissive chromatin architecture by interacting with chromatin remodelers, such as methyl-DNA-binding proteins (MBDs) (Boyes and Bird, 1991; Zhu et al., 2016). The 5mC will be oxidized to form 5-hydroxymethylcytosine (5hmC), which is a transient, yet stable, epigenetic mark associated with permissive chromatin state and enhanced gene transcription, achieved either by increasing chromatin accessibility to transcription machinery or inhibiting transcriptional repressor binding proteins, such as MBD1 or methyl cytosine binding proteins 1 and 2 (MeCP1 and 2) (Branco et al., 2012).

It is estimated that the mammalian genome contains ∼3 × 10⁷ residues of 5mC, mainly within CpG dinucleotides (Walsh and Bestor, 1999). 5mC plays a vital role in genomic stability by silencing transposable elements, allele-specific expression of imprinted genes, and X-chromosome inactivation (Schübeler, 2015). The formation of 5mC is catalyzed by a family of highly conserved enzymes known as DNA methyltransferases (DNMTs), which covalently transfer a methyl group from S-adenosyl methionine to the 5^th carbon of cytosine (Edwards et al., 2017). There are three classes of DNMTs: DNMT1, DNMT2, and DNMT3A/3B/3L. Of these, DNMT1 is primarily responsible for maintenance methylation, ensuring that methylation patterns are copied during DNA replication (Goyal et al., 2006). The exact biological functions of DNMT2 remain to be characterized, although some studies show that DNMT2 regulates centromere function and tRNA modifications (Goll et al., 2006; Jeltsch et al., 2017). In contrast, DNMT3A/3B/3L are involved in de novo DNA methylation, establishing new methylation patterns during gametogenesis, embryonic development, and cell differentiation (Okano et al., 1999). While the functional significance of differential DNA methylation patterns was thoroughly investigated in cancer pathophysiology, genome-wide changes in DNA methylome are also evident during aging and in the onset and progression of several age-related diseases (Kulis and Esteller, 2010; Noroozi et al., 2021).

Despite the chemical and genetic stability of 5mC, it can still be reverted to its unmethylated cytosine through two primary pathways: passive DNA demethylation, which occurs during DNA replication when methylation marks are not preserved, and active DNA demethylation, which involves enzymatic mechanisms that directly modify or remove the methyl group (Bhutani et al., 2011). During the active DNA demethylation process, the conversion of 5mC to 5hmC is mediated by the Fe(II)- and α-ketoglutarate-dependent DNA dioxygenase family of enzymes, the ten-eleven translocases (TETs): TET1, TET2, and TET3. These enzymes oxidize 5mC to 5hmC, which can be further oxidized to 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) (Tahiliani et al., 2009; Wu and Zhang, 2017). Among these oxidized cytosine derivatives, 5hmc serves as a stable and independent epigenetic mark, especially abundant in the mammalian brain (Jin et al., 2011). Dysregulated genome-wide hydroxy methylome patterns are associated with the development of several neuronal and non-neuronal diseases (Chen et al., 2016; Stöger et al., 2017). Given their influence on context and tissue-specific gene expression, 5mC and 5hmC are frequently referred to as the fifth and sixth bases of DNA (Kumar et al., 2018; Shi et al., 2017). These modifications regulate numerous biological processes, including learning, memory, aging, disease susceptibility, and overall health span (Moen et al., 2015). Therefore, precise control of 5mC and 5hmC dynamics is essential not only for tissue-specific transcriptional regulation but also for the adaptation to various forms of physiological and pathological stress throughout life.

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Section snippets

5mC and 5hmC imbalance during aging and stroke risk

Biologically, aging is characterized by cellular senescence associated with a progressive decline in tissue and organ function, resulting in a reduced capacity to respond to stress, disrupted homeostasis, increased susceptibility to different diseases, and ultimately, death (Guo et al., 2022). As organisms age, their epigenetic patterns, including global 5mC and 5hmC landscapes, undergo notable shifts, a process known as ‘epigenetic drift’, resulting in impaired transcriptional fidelity and

Differential 5mC patterns in post-stroke genome

Genome-wide association studies suggest that the risk of stroke has a substantial epigenetic component, especially the DNA methylation patterns across the genome. Aberrant DNA methylation is implicated in the onset of ischemic stroke, its severity, and associated neurological impairments (Choi et al., 2022). Hypomethylation of the long interspersed nucleotide element-1 (LINE-1) in DNA from peripheral blood leukocytes was shown to be strongly associated with ischemic stroke in elderly men (

Differential 5hmC patterns in post-stroke genome

While 5mC has long been the primary focus in epigenetic research, growing evidence now highlights 5hmC as a crucial epigenetic modification in CNS disorders (Khare et al., 2012; Tahiliani et al., 2009). Unlike 5mC, which can be measured using well-established techniques such as bisulfite sequencing, detecting 5hmC presents significant technical challenges due to its similarity in biochemical properties to 5mC (Huang et al., 2010). The standard bisulfite conversion method, commonly used in

Can the epigenome be targeted for stroke therapy in future?

The multifactorial nature of the pathological mechanisms underlying ischemic brain injury makes stroke an exceptionally challenging condition to treat. Stroke triggers a cascade of interconnected events, starting from the loss of tight junction proteins and endothelial cell integrity to disruption of the blood-brain barrier, excitotoxicity, oxidative stress, endoplasmic reticulum stress, inflammation, apoptosis, autophagy, mitochondrial damage, disrupted transcription and translation, altered

CRediT authorship contribution statement

Raghu Vemuganti: Writing – review & editing, Supervision, Funding acquisition, Conceptualization. Pallavi Joshi: Writing – review & editing, Writing – original draft, Conceptualization. Vijay Arruri: Writing – review & editing, Writing – original draft, Conceptualization

Conflict of Interest

The author(s) declared no potential conflicts of interest.

Funding Sources

These studies were supported in part by the Department of Neurological Surgery, University of Wisconsin, U.S. Department of Veterans Affairs (I01 BX005127, I01 BX004344, I01BX006062), National Institute of Health (R35NS132184, RO1 NS109459, RO1 NS099531 and RO1 NS101960) and American Heart Association (24POST1198978). Dr. Vemuganti is the recipient of a Research Career Scientist Award (IK6BX005690) from the US Department of Veterans Affairs.

Funding

These studies were supported in part by the Department of Neurological Surgery, University of Wisconsin, Madison, National Institute of Health (R35 NS132184), US Veterans Affairs (I01BX0062), and American Heart Association (24POST1198978). Dr. Vemuganti is the recipient of a VA Research Career Scientist award (IK6BX005690).

References at link.