Epigenetic Modifications and Their Potential Contribution to Traumatic Brain Injury Pathobiology and Outcome

Abstract

Epigenetic information is not permanently encoded in the DNA sequence, but rather consists of reversible, heritable modifications that regulate the gene expression profile of a cell. Epigenetic modifications can result in cellular changes that can be long lasting and include DNA methylation, histone methylation, histone acetylation, and RNA methylation. As epigenetic modifications are reversible, the enzymes that add (epigenetic writers), the proteins that decode (epigenetic readers), and the enzymes that remove (epigenetic erasers) these modifications can be targeted to alter cellular function and disease biology. While epigenetic modifications and their contributions are intense topics of current research in the context of a number of diseases, including cancer, inflammatory diseases, and Alzheimer disease, the study of epigenetics in the context of traumatic brain injury (TBI) is in its infancy. In this review, we will summarize the experimental and clinical findings demonstrating that TBI triggers epigenetic modifications, with a focus on changes in DNA methylation, histone methylation, and the translational utility of the universal methyl donor S-adenosylmethionine (SAM). Finally, we will review the evidence for using methyl donors as possible treatments for TBI-associated pathology and outcome.

Introduction

Identical twins do not always share the same heritable diseases, despite having the same primary genetic information.¹ Clearly, information other than the deoxyribonucleic acid (DNA) sequence can be passed from parent to offspring (or cell to daughter cell during division) that contributes to the identity, level, and timing of gene expression. The influence of epigenetic regulation of gene expression starts periconception and continues until death.²

Epigenetic information is not directly encoded into the DNA sequence itself, but rather consists of reversible, heritable modifications that regulate the gene expression profile of a cell. Mechanisms underlying epigenetic information include the transfer of chemical modifications to DNA, ribonucleic acid (RNA) (e.g., methylation), or histones (e.g., methylation and/or acetylation), as well as through the expression of non-coding RNAs (e.g., microRNA, piwi-interacting RNA, long non-encoding RNA).

While epigenetic mechanisms have been the subject of several in-depth reviews, little is known about the contribution of epigenetics to the pathophysiology of traumatic brain injury (TBI). In this review, we will discuss the evidence that TBI can trigger epigenetic modifications. We will focus on changes in methylation and its contributions to TBI pathophysiology and outcome and on possible therapeutic approaches using the universal methyl (-CH₃) group donor S-adenosylmethionine (SAM).

Epigenetics

Epigenetics is the study of information transfers that regulate gene expression but are not directly encoded in the DNA sequence. Although the concept of extrinsic factors that could modulate the expression of phenotypic traits was proposed almost 80 years ago, the biological basis for these modifications has only been explored recently.^3,4 As a result, the complexity and variety of cellular changes that can give rise to long-lasting, heritable alterations in gene expression is only just being appreciated. In 2016, the National Institutes of Health suggested the following definition for epigenetics: “Epigenetics refers to both heritable changes in gene activity and expression (in the progeny of cells or of individuals) and also stable, long-term alterations in the transcriptional potential of a cell that are not necessarily heritable.”⁵

Studies aiming at understanding the role of epigenetics have identified lasting modifications to gene expression that occur in a wide variety of illnesses, including respiratory disease, cardiovascular disease, autoimmune disease, neurodegenerative diseases, and virtually all cancers.^6–8 Epigenetics also plays a critical role in normal human health, such as cognition, reproduction, and aging.^9–11 In its simplest form, epigenetics involves the addition and/or removal of epigenetic “tags” that can either activate or impede cellular activity. To date, many types of epigenetic tags have been identified, including methylation, acetylation, phosphorylation, ubiquitination, and sumoylation. The study of epigenetics in the context of TBI is in its infancy,^12,13 so we will focus this review on methylation because it is the most studied of the epigenetic modifications.

DNA methylation

The DNA in mammalian cells exists in the form of nucleosomes in which negatively charged DNA wraps around octamers of positively charged histone proteins (Fig. 1). This interaction between the negatively charged DNA and the positively charged histones hinders recruitment of RNA polymerase and gene expression.¹⁴ The DNA methylation is the selective addition of a –CH₃ group in an enzymatic reaction catalyzed by a methyltransferase to the cytosine base within a cytosine-guanine dinucleotide repeat (CpG), forming a 5-methylcytosine residue (5mC). DNA methylation primarily leads to long-term repression of gene expression.¹⁵ There are a small number of studies, however, that have found that in some circumstances, DNA methylation can result in increased gene expression.^16,17

FIG. 1.

Epigenetic regulation of gene expression. Deoxyribonucleic acid (DNA) and histones are packaged into a compact structure called chromatin, which can exist in two forms: heterochromatin and euchromatin. Hypermethylation of DNA and histones causes chromatin to form a compact structure referred to as heterochromatin that is transcriptionally silent. Alterations in the methylation landscape loosens the chromatin, a state called euchromatin, which allows the DNA to be transcriptionally competent. Each nucleosome of chromatin consists of a histone octomer (dimers of H2a, H2b, H3, and H4) that is wound by ∼2 turns of double-stranded DNA. The histone tails (H4 tail shown) can be reversibly modified by epigenetic tags including methylation (arginine and lysine), acetylation (lysine), sumoylation, and phosphorylation that are added by “writers” (and removed by “erasers”). A group of proteins called “readers” are recruited by these tags where they can initiate downstream effects. Methylation of DNA in gene promotor regions can impede transcription factor binding and inhibit transcription. In addition to this mechanism, DNA methylation (in CpG islands) recruits readers that can facilitate the interaction of DNA methylation, histone modifications, and chromatin structure to affect gene expression. Enhancer binding proteins recognize specific enhancer sequences with high affinity and recruit coactivators that often have histone acetylation activity. This results in recruitment of transcription factor II and RNA polymerase 2 to the transcription initiation complex, and transcription of ribonucleic acid (RNA). The RNA can be also modified by methylation, a modification that can alter splicing, stability, and protein translation. For example, histone acetylation commonly enhances gene transcription. The resulting messenger RNA can also be modified by methylation, which can alter its splicing, stability, and protein translation.

DNA methylation plays an important role in many processes, including embryonic development, imprinting, X-chromosome inactivation, and regulation of gene expression. Recent studies have indicated that approximately 70% of all cytosines in CpG dinucleotides located within human gene promoters are methylated.^18,19 The majority of CpG methylation sites can be found in the promotor regions of housekeeping genes. CpG islands (long repeats of CpG dinucleotides) are also prevalent in housekeeping genes (e.g., ribosomal proteins, transcription factors, mitochondrial proteins, etc.) and are found in approximately 60% of all genes.^20,21 The methylation status of cytosine residues and CpG islands can be maintained and passed on to daughter cells through DNA replication. Thus, epigenetically regulated changes of gene expression are often maintained over several cell divisions.

Writers, readers and erasers

The group of enzymes that catalyze the attachment of epigenetic tags (e.g., methylation of DNA and histones) are collectively referred to as “writers.” For nucleotide methylation, three families of proteins with distinct 5mCpG-binding domains have been identified.²² These enzymes, known as DNA methyltransferases (DNMTs), catalyze the addition of methyl groups from the donor SAM to the cytosine residue of CpG dinucleotides.

DNMT1 is the most abundant mammalian DNA methyltransferase isoform, and it is predominately involved in maintaining the methylation state of DNA because it is capable of methylating both hemi-methylated and non-methylated DNA. After DNA replication, the newly synthesized DNA strand (without any methylation) is recognized by DNMT1 and methylated using the complementary hemi-methylated strand as the template to faithfully propagate the epigenetic information.²³

Methylated DNA acts as a recruitment signal for a group of proteins called methylation “readers” that bind to methylated DNA to cause local chromatin compaction, which can result in silencing of gene expression.^{19 24} A large group of readers have been identified that bind to 5mCpG (DNA methylation readers), N⁶-methyladenosine (RNA methylation readers), and methylated histones (histone methylation readers). Several in-depth reviews are available on this topic.^25–28

A third group of proteins, which are involved in the removal of epigenetic tags on DNA and histones, are referred to as epigenetic “erasers.” Removal of the methyl group on 5mC can occur by two different pathways: active enzymatic removal or passive non-enzymatic removal.²⁹ In passive 5mC removal, the DNA strands can lose methylation tags during successive DNA replications because of insufficient DNMT1 activity. Active removal of 5mC tags is performed by the ten-eleven translocation family of enzymes (Tet1-3). Knockout of Tet1 expression revealed that it plays a key role in removing primordial germ cell imprinting marks on paternal chromosomes in sperm.³⁰

Tet2 has been shown to be important for regulating 5mC and 5hmC levels to maintain normal neurogenesis and the adult neural stem cell niche.³¹ Similar to Tet1, Tet3 also plays an important role in epigenetic remodeling during development and is critical for removing paternal DNA methylation in fertilized eggs.³²

Histone methylases and demethylases

Methylation of histones on lysine (K) and arginine (R) amino acids is carried out by lysine methyl transferases and arginine methyl transferases, respectively. Lysine amino acids on histone H3 (K4, K9, K27, K36, and K79) and histone H4 (K20) are most often the targets for methylation. Histone methyl transferases can add up to three methyl groups on a single lysine (mono-, di-, and trimethylation). Addition of one, two, or three, methyl groups exerts different effects on gene transcription.³³

Arginine methyl transferases methylate the terminal nitrogen atom of arginine residues in histone proteins. Methylation of H4 arginine residues R4, R17, or R23 activates transcription.³⁴ Two families of histone demethylase enzymes have been identified: lysine-specific demethylase (LSD) and Jumonji C (JmjC). The LSD demethylates mono- and dimethylated lysine, while JmjC demethylates all three methylated (mono, di, and tri) forms of lysine.³⁵

RNA methylation

In addition to modifications of DNA and histones, RNA can also be methylated on the m6 position of adenosine residues (m6A) by methyltransferase-like enzyme (METTL) family members.³⁶ Because oocytes contain a large amount of RNAs, this modification can be passed on to progeny. The heterodimeric METTL3/METTL14 RNA methyltransferase complex is the dominant m6A writer expressed in mammals and is capable of methylating nuclear messenger RNAs (mRNAs) containing the short consensus sequence (G>A)m6AC(A/C/U).^37,38 m6A marks are recognized by various reader proteins to modulate RNA splicing, RNA stability, and RNA translation.³⁹ METTL16 is a highly conserved m6A writer and methylates adenosine residues in a subset of U6 snRNAs and structured mRNAs, including SAM synthetase, which harbor the longer consensus sequence UACm6AGAGAA.^40–44

SAM: the universal methyl donor

More than 100 methyl transferase enzymes, including methyl writers, utilize SAM as the methyl donor for DNA, histone, and RNA modification reactions,⁴⁵ making SAM the second most commonly used molecule after adenosine triphosphate (ATP). In addition to its role as an epigenetic marker important for mediating gene regulation, methylation regulates many important biological processes, such as axonal transport (via microtubule methylation) and plasma membrane integrity and signaling (via phospholipid methylation), that are critical for normal neuronal function.^46–49

SAM is generated from methionine by methionine adenosyl transferases as a part of the methionine cycle (Fig. 2). Once utilized as a methyl donor by one of the many SAM-dependent methyl transferases, SAM is converted to S-adenosyl homocysteine (SAH), which can act as a potent competitive inhibitor of transmethylation reactions. Intracellular SAH levels are controlled by S-adenosyl homocysteine hydroxylase that converts SAH to homocysteine, which can be further metabolized to yield cystathionine (a precursor for glutathione biosynthesis) or to regenerate methionine (Fig. 2). Alternatively, methionine can be regenerated from SAH via coupling with the folic acid cycle, in which 5-methyltetrahydrofolate serves as the methyl donor substrate in a reaction catalyzed by methionine synthase. Many nutrients, such as folate, vitamin B6, riboflavin, vitamin B12, and choline, can all influence methylation reactions.^50–53

FIG. 2.

Enzymatic pathways for S-adenosylmethionine (SAM) metabolism. SAM can be synthesized from dietary sources of methionine and folic acid (vitamin B9) through the methionine cycle (blue) and folate cycle (maroon), respectively. Various methylation enzymes use SAM as the methyl donor to methylate deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein. Once the methyl group is transferred, SAM is converted into S-adenosyl homocysteine (SAH), which further becomes homocysteine, and then methionine. SAM is also critical for the generation of homocysteine, a precursor for the amino acid cysteine and the antioxidant glutathione (green). Folic acid is converted to DHF (dihydrofolic acid) and then to THF (tetrahydrofolate) by the enzyme dihydrofolate reductase. The THF is subsequently converted to methionine via the sequential action of betaine homocysteine methyltransferase (BHMT), methyltetrahydrofolate reductase (MTF), 5-10-methylene-tetrahydrofolate reductase (MTHFR), and methionine synthase. In addition to producing methionine, homocysteine can be converted by cystathione beta-synthase to cystathione, which is converted to cysteine and alpha-ketoglutarate by the B6-dependent enzyme cystathionase. Cystenine is converted to the antioxidant glutathone by the rate limiting enzyme glutatmate-cysteine ligase (GCL).

Epigenetic contributions to experimental TBI pathology and outcome

A TBI affects nearly 1.8 million persons a year in the United States. Although the traumatic forces that result from the rapid acceleration/deceleration of the head are transmitted into the brain in less than 100 msec, the consequences of TBI can be lifelong. As such, TBI needs to be viewed as a chronic disease rather than a singular event.⁵⁴ Some of the long-term consequences of TBI include persistent cognitive impairments, cognitive slowing, pain, headache, sleep disturbances, and depression.

The persistence of these changes suggests that they may be caused, at least in part, by lasting alterations in cellular function such as epigenetic modifications. Because epigenetic modifications can be long-lasting yet are also reversible, examining their status in the context of TBI holds promise for developing therapies to lessen the long-term consequences of TBI. Further, because treatments that alter epigenetic pathways are likely to modulate the expression of groups of genes, epigenetic-based therapies may mimic a combination therapy that is more suitable to lessen the complex pathophysiology of TBI.

Experimental TBI alters nuclear genomic methylation

A summary of the experimental TBI studies performed in rodents that have examined overall DNA and histone methylation levels or the methylation status in promoter regions of methylation writers, erasers, and other selected genes after injury is presented in Table 1.

Table 1.

Summary of Epigenetic Methylation Studies Carried Out in Experimental and Clinical Traumatic Brain Injury

Experimental Studies
Injury type	Subject	Structure(s)	Reported change(s)	References
Controlled cortical impact	Rat	Hippocampus	Decreased H3 methylation Decreased H3 acetylation	13
Controlled cortical impact	Rat	Hippocampus	Reduced MBD1 expression	68
Controlled cortical impact	Rat pups	Hippocampus	Increased permissive DNA methylation and histone modifications	51
Controlled cortical impact	Mouse	Hippocampus	Increased permissive DNA methylation	85
Controlled cortical impact	Mouse	Contralateral cortex	Increased histone acetylation and methylation in response to ketogenic diet after injury	67
Controlled cortical impact	Mouse	Hippocampus	Increased histone 3 acetylation, induction of methyl-CpG-binding protein in response to fluoxetine treatment	68
Fluid percussion injury	Rat	Hippocampus, circulating leukocytes	Global methylation changes using bisulfite sequencing	55
Weight drop	Rat	Somatosensory cortex	Global cellular hypomethylation, predominately in microglia/macrophages	86
Weight drop	Rat	Hippocampus, sperm	Paternally transmitted alteration in DNA methylation	56
Repeated weight drop	Mouse	Hippocampus, mitochondria	Increased cytosine methylation of the TFAM promoter. Altered mitochondrial gene expression. Restoration of methylation using methionine treatment	61
Blast exposure	Rat	Hippocampus, prefrontal cortex	DNMT, TET and TDG expression changes and altered DNA methylation	52
Blast exposure	Rat	Frontal cortex	Differentially methylated neuronal and glial genes	53

Clinical Studies
Subjects	Study type	Samples	Reported change(s)	References
Children (GCS 13-15)	Prospective case-control	Blood	CpG methylation changes predictive of concussion	70
Adults (GCS ≤8)	Prospective, longitudinal,	CSF	High methylation of BDNF CpG sites was associated with better outcome	69
Adults (GCS 13-15; GCS ≤8)	Observational	Blood	Reduced circulating levels of the methyl donor methionine	74
Adults (GCS 13-15)	Prospective		Improved outcome	76

MBD, methyl CpG binding domain; DNA, deoxyribonucleic acid; CpG, cytosine-guanine dinucleotide; TFAM, mitochondrial transcription factor A; DNMT, DNA methyltransferase; TeT, ten-eleven translocation enzymes; TDG, thymine = DNA glycosylase; GCS, Glasgow Coma Scale; CSF, cerebrospinal fluid; BDNF, brain-derived neurotrophic factor.

Using a controlled cortical impact (CCI) model of moderate pediatric TBI in rats (PND17), Gao and associates¹³ (2006) reported that hippocampal CA3 histone H3 methylation is decreased as early as 6 h post-injury, a change that persisted for up to 72 h. These results suggested that epigenetic changes in the hippocampus might contribute to cognitive dysfunction after TBI, and targeting histone methylation might provide a new therapeutic avenue.

A study by Schober and colleagues⁵⁵ (2012) also in PND17 rats reported that experimental TBI acutely increased both histone methylation and acetylation and DNA methylation within promoter and exon splicing enhancer elements in the IGF-1 gene. These changes were associated with increased expression of IGF-1b mRNA, an alternatively spliced variant of IGF-1. Because IGF-1 plays important roles in brain growth, development, and neuronal repair, this study suggests that epigenetic modifications may contribute to both negative and positive changes after TBI.

More recently, Bailey and coworkers (2015)⁵⁶ examined DNA methylation and the expression of DNA methylation writers and erasers two weeks after blast overpressure TBI in adult (250 g) rats. In the prefrontal cortex, blast (23 psi) TBI caused a significant reduction in the mRNA expression level of the DNA writer DNMT3b, while a two-fold increase in the mRNA levels of the DNA eraser Tet2 was observed after both the 10 psi and 23 psi blast injuries.⁵⁶ The hippocampus appeared to be more vulnerable to epigenetic changes as the mRNA expression levels of DNMT1 (17 and 23 psi), DNMT3b (10 and 23 psi), Tet2 (10, 17, and 23 psi), Tet3 (10 psi), and TDG (17 and 23 psi) were all significantly enhanced in the hippocampus after blast injury.

These gene expression changes were associated with a significant negative correlation between global DNA methylation levels in hippocampal genomic DNA, as assessed by enzyme-linked immunoassay, and blast overpressure.⁵⁶ These results indicate that global hippocampal DNA is hypomethylated two weeks after a blast TBI, possibly contributing to long-term TBI pathophysiology.

Consistent with this suggestion, Haghighi and colleages⁵⁷ (2015) used bisulfite sequencing to examine DNA methylation changes in neurons and glia isolated from rat frontal cortex eight months after repeated blast overpressure TBI. The DNA methylation levels were found to be altered in 458 genes in cortical neurons and 379 genes in cortical astrocytes. Within neurons, genes exhibiting differential DNA methylation after blast injury were associated with cell death, cell survival, and development. A subset of 30 genes were further examined for changes in mRNA expression level. The authors found that 50% of these genes showed a significant negative correlation between the change in DNA methylation level and mRNA abundance.

One of the genes that was hypermethylated in cortical neurons, serotonin N-acetyltransferase (SNAT), also showed a 20% reduction in mRNA abundance. SNAT, which catalyzes the conversion of serotonin to melatonin, a hormone implicated in sleep and depression, as well as several other sleep-related genes (e.g., Per3, Nos1, Cacna1b, and Il1r1) that showed altered methylation levels after injury, may contribute to sleep disturbances observed after TBI.⁵⁸

In another study, Meng and associates⁵⁹ reported genome-wide DNA methylation changes, detected using bisulfite sequencing, in both hippocampal tissue and circulating leukocytes after a mild fluid percussion injury. This study identified 758 hypermethylated loci and 781 hypomethylated loci in hippocampal genomic DNA while 892 hypermethylated loci and 915 hypomethylated loci were found in leukocyte DNA after injury. Interestingly, 269 of these altered genomic loci were in common between the two tissues, suggesting there may be some overlap in the central and peripheral epigenetic responses to TBI. In addition, selected DNA methylation changes were associated with mRNA expression profiles, suggesting that some of the altered methylation islands could affect gene expression.

Together, these studies provide initial evidence to suggest that epigenetic modifications may contribute to the altered expression of specific genes after injury, and may influence TBI-associated pathology and outcome.

Experimental TBI-associated genomic methylation may be heritable

As stated above, a hallmark feature of epigenetic changes is that they are heritable and can be passed from one cell to a daughter cell, or from parent to offspring. Using high-fat diet as a means of altering DNA methylation, Hehar and coworkers⁶⁰ (2017) examined whether the methylation levels of genes associated with neurodevelopment, injury repair, high-fat diet, and aging (i.e., Bdnf, Lepr, Oxtr, Tert, Igf2, and Igf2r) are altered by paternal experience, and whether these changes could be passed along to offspring.

Interestingly, the authors found that the DNA methylation patterns of these six genes in male offspring (both sperm and brain tissue) were different between pups born to sires fed high-fat diets versus those on control diets. Thirty days after these pups received a mild TBI (mTBI), the promoter methylation patterns of these genes in their sperm were further modified, with the genes being affected related to the diet of the father. These observations suggest that paternal transmission of epigenetic information can influence the consequences of a TBI, and that these changes may have influences not only on the brain-injured individual but potentially on their offspring as well.

Experimental TBI alters mitochondrial DNA methylation

Although most mitochondrial proteins are derived from the cell's nuclear genomic DNA, mitochondrial DNA (mtDNA) encodes 13 proteins that comprise all the components of the electron transport chain (ETC), as well as two unique ribosomal RNAs and 22 mitochondrial transfer RNAs that are important for mitochondrial function.

The mtDNA gene expression is primarily regulated by four elements—the D-loop, the light-strand promoter (LSP), and two heavy-strand promoters, HSP1 and HSP2.^61–64 Unlike nuclear DNA promotor methylation, methylation at HSP1 can increase binding of the mitochondrial transcription factor A (TFAM), which regulates mtDNA replication and initiates transcription of mitochondrial-encoded RNAs.^65,66

Balasubramanian and associates⁶⁵ reported that repeated mTBI in rats acutely (48 h) and chronically (30 days) increased methylation of the promotor region of TFAM. While repeated mTBI increased methylation of the TFAM promotor, the promoter regions HSP1 and HSP2 were hypomethylated. This reduction in HSP1 and HSP2 promoter methylation was associated with both reduced mitochondrial biogenesis (as assessed by examining mtRNA) and reduced ATP levels.

Treatments aimed at altering methylation in experimental TBI

Studies aimed at altering methylation after injury have reported mixed results. There is a body of literature indicating that altering dietary components may be beneficial in some neurological diseases, and may, at least in part, act by influencing epigenetic markers.^67–70 Thau-Zuchman and coworkers⁷¹ (2021) found that placing adult TBI animals on a modified ketogenic diet after injury reduced TBI-associated pathologies such as contusion volume and neuroinflammation while it improved sensorimotor and spatial memory performance. These changes were associated with significantly increased histone methylation and acetylation (assessed on H3K9).

Although it remains unclear whether the histone alterations detected by Thau-Zuchman and colleagues⁷¹ are causative or correlative, the influence of diet and how it might affect pathology have promise as being a low risk but potentially effective intervention. In support of this, improvements in functional memory (Morris Water Maze) and smaller lesion volumes have been observed in brain-injured rodents that were administered vitamin B6, riboflavin, or choline.^72–74

DNA modifications, including methylation levels, may be altered in depression, and there is some evidence that antidepressant agents, including selective serotonin reuptake inhibitors (SSRIs), can affect the global level of DNA methylation.^75–79 In a TBI study by Wang and colleagues⁸⁰ (2011), the authors investigated the effects of SSRI administration after moderate-to-severe TBI. These authors found that fluoxetine treatment (4 weeks, initiated 3 days post-CCI) resulted in increased histone H3 acetylation in the hippocampus and increased expression of DNA methylation transcription factor (methyl-CpG-binding domain protein 1; MBD1) in the dentate gyrus. While these changes were associated with enhanced hippocampal neurogenesis, fluoxetine treatment did not improve locomotor or memory function after TBI.⁸⁰

Finally, Balasubramanian and associates⁶⁵ found that administration of the methyl donor methionine to rats receiving repeated mTBI restored the methylation levels of HSP1 and HSP2 in the hippocampus resulting in increased expression of mitochondrial proteins. Whether this restoration of mitochondrial protein expression, however, is associated with restored ATP levels or enhanced hippocampal function has not been explored.

Overall, these rodent models of TBI have shown changes in histone and DNA methylation that are strongly dependent on the specific brain subregion and on time post-injury. Generally, these results seem to indicate an increase in protective factors and a proinflammatory response aimed at repairing damage caused from TBI, particularly in neurogenic regions such as the hippocampus.

Human TBI pathobiology, outcome, and epigenetic methylation

Some of the common pathologies exhibited after human TBI include white matter damage, inflammation (both central and peripheral), and neuronal loss. These and other pathologies contribute to TBI outcomes, including cognitive impairments, pain, sleep disturbances, depression, and increased risk for development of neurodegenerative diseases. Studies aimed at investigating epigenetic contributions to human TBI pathologies and outcomes have not yet been undertaken.

Only a few recent studies have reported epigenetic DNA methylation changes, including the levels of methyl donor SAM in human TBI samples. Treble-Barna and coworkers⁸¹ (2021) found that higher DNA methylation associated with the BDNF gene positively correlated with improved outcome in severe human TBI as assessed using the Glasgow Outcome Scale, the Neurobehavioral Rating Scale-Revised, and the Disability Rating Scale. A recent human study applied artificial intelligence to identify numerous DNA methylation alterations after pediatric mild TBI.⁸² Most DNA loci were hypomethylated after injury, suggesting widespread gene activation and consistent with the results from the rodent experimental TBI studies discussed above.

TBI and methyl group donors: A possible therapeutic direction

As discussed above, SAM is the major methyl donor for almost all the epigenetic methylation reactions on DNA, RNA, and proteins. Thus, the availability of SAM can profoundly alter the landscape of epigenetic methylation. Since as early as 1997, studies have been examining the consequences of TBI on SAM biosynthesis and metabolism using both experimental models of TBI and in human samples.

For example, Henley and associates⁸³ found that s-adenosyl-L-methionine decarboxylase is acutely decreased in the injured motor cortex of brain-injured rats and speculated that this decrease may contribute to delayed pathological changes after TBI.⁸³ In a mouse model of TBI, cystathionine-β-synthase (CBS) levels have been shown to be decreased for up to 72 h after TBI⁸⁴; CBS catalyzes the condensation of serine with homocysteine to form cystathionine, the levels of which are enhanced by SAM.^84,85

More recently, using plasma samples collected from healthy volunteers and persons within 24 h of a TBI, the circulating levels of methionine, SAM, betaine, and 2-methylglycine were found to be decreased in subjects who had experienced a severe TBI.⁸⁶ Similar decreases in methionine and its metabolic products were observed in samples collected from subjects with mTBI, albeit to a lesser degree.

These data, along with a recent review of SAM metabolism and its alterations in disease states (including depressive disorders, pain, and neurodegeneration), suggest that SAM supplementation might be an effective strategy to manage the post-concussive sequelae commonly observed after TBI.⁸⁷ Consistent with this, in the only clinical trial performed to date, SAM administration to patients with concussion was found to be associated with a 77% reduction in clinical scores during recovery compared with a 49% reduction in the placebo group.⁸⁸

Conclusion

Overall, a substantial effort has been made to characterize and understand the epigenetic mechanisms that regulate gene expression and how changes in these mechanisms can affect various disease states. Only a limited number of studies have been performed, however, to examine the methylation changes that occur after TBI. Approaches to modulate components in the SAM pathway provide many interesting avenues for further research and for expanding our understanding of the brain's epigenetic response(s) to trauma.

Because initial studies have indicated, there is a significant reduction in the relative levels of SAM in the plasma of patients with TBI, SAM supplementation may be useful to manage some of the behavioral/cognitive problems observed in these patients. Evidence from other diseases has shown that altered epigenetic methylation may contribute to white matter damage,⁸⁹ inflammation,^90–92 and cognitive function,^93–96 suggesting that similar mechanisms may contribute to these and other TBI-associated pathologies. Findings from future epigenetic research holds substantial promise for improving outcomes for patients affected by TBI.

Authors' Contributions

LZ, RW, PS, NK, GH, HAC, ANM, JBR, and PKD all contributed to the review of relevant literature, writing, and/or editing of the article.

Funding Information

Research in authors' laboratories is supported by grants from the National Institutes of Health (R01NS118329, R01NS101686, R01NS121261, and R01NS109118).

Author Disclosure Statement

No competing financial interests exist.