Although the mechanisms are still largely unknown, foetus exposure to GDM and maternal diabetes has been shown to lead to lifelong metabolic complications because of metabolic imprinting. The risk of  Type II diabetes  developing in offspring is significantly higher in offspring where the mother was diagnosed with Type II diabetes before pregnancy rather than after. In addition, the age at which offspring are diagnosed with Type 2 diabetes is significantly younger in offspring exposed to maternal diabetes/GDM than those who are not. It is suggested that this is a result of  DNA methylation  during foetal development. [ 14 ]


Methylation , in the  chemical sciences , is the addition of a  methyl group  on a  substrate , or the substitution of an atom (or group) by a methyl group. Methylation is a form of  alkylation , with a methyl group replacing a  hydrogen  atom.  These terms are commonly used in  chemistry ,  biochemistry ,  soil science , and  biology .


In  biological systems , methylation is  catalyzed  by  enzymes ; such methylation can be involved in modification of  heavy metals , regulation of  gene expression , regulation of  protein function , and  RNA processing .  In vitro  methylation of tissue samples is also a way to reduce some  histological staining artifacts . The reverse of methylation is  demethylation .


In biological systems, methylation is accomplished by enzymes.  Methylation can modify heavy metals and can regulate gene expression, RNA processing, and protein function. It is a key process underlying  epigenetics . Sources of methyl groups include S-methylmethionine, methyl folate, methyl B12. [ 1 ]


Methanogenesis , the process that generates methane from CO 2 , involves a series of methylation reactions. These reactions are caused by a set of enzymes harbored by a family of anaerobic microbes. [ 2 ]


In reverse methanogenesis, methane is the methylating agent. [ 3 ]


A wide variety of  phenols  undergo O-methylation to give  anisole  derivatives.  This process, catalyzed by such enzymes as  caffeoyl-CoA O-methyltransferase , is a key reaction in the biosynthesis of  lignols ,  percursors  to  lignin , a major structural component of plants.


Plants produce flavonoids and isoflavones with methylations on hydroxyl groups, i.e.  methoxy bonds . This 5-O-methylation affects the flavonoid's water solubility. Examples are  5-O-methylgenistein ,  5-O-methylmyricetin , and  5-O-methylquercetin  (azaleatin).


Along with  ubiquitination  and  phosphorylation , methylation is a major biochemical process for modifying protein function.  The most prevalent protein methylations affect arginine and lysine residue of specific histones. Otherwise histidine, glutamate, asparagine, cysteine are susceptible to methylation.  Some of these products include  S -methylcysteine , two isomers of  N -methylhistidine, and two isomers of  N -methylarginine. [ 4 ]


Methionine synthase  regenerates  methionine  (Met) from  homocysteine  (Hcy). The overall reaction transforms  5-methyltetrahydrofolate  (N 5 -MeTHF) into  tetrahydrofolate  (THF) while transferring a methyl group to Hcy to form Met. Methionine Syntheses can be cobalamin-dependent and cobalamin-independent: Plants have both, animals depend on the methylcobalamin-dependent form.


In methylcobalamin-dependent forms of the enzyme, the reaction proceeds by two steps in a ping-pong reaction. The enzyme is initially primed into a reactive state by the transfer of a methyl group from N 5 -MeTHF to Co(I) in enzyme-bound  cobalamin  ((Cob), also known as vitamine B12)) ,
, forming methyl-cobalamin(Me-Cob) that now contains Me-Co(III) and activating the enzyme. Then, a Hcy that has coordinated to an enzyme-bound  zinc  to form a reactive thiolate reacts with the Me-Cob. The activated methyl group is transferred from Me-Cob to the Hcy thiolate, which regenerates Co(I) in Cob, and Met is released from the enzyme. [ 5 ]


Biomethylation is the pathway for converting some heavy elements into more mobile or more lethal derivatives that can enter the  food chain . The  biomethylation  of  arsenic  compounds starts with the formation of  methanearsonates .  Thus, trivalent inorganic arsenic compounds are methylated to give methanearsonate.  S-adenosylmethionine  is the methyl donor.  The methanearsonates are the precursors to dimethylarsonates, again by the cycle of  reduction  (to methylarsonous acid) followed by a second methylation. [ 6 ]  Related pathways are found in the  microbial methylation  of mercury to  methylmercury .


DNA methylation  is the conversion of the cytosine to  5-methylcytosine . The formation of Me-CpG is  catalyzed  by the enzyme  DNA methyltransferase . In vertebrates, DNA methylation typically occurs at  CpG sites  (cytosine-phosphate-guanine sites—that is, sites where a  cytosine  is directly followed by a  guanine  in the DNA sequence). In mammals, DNA methylation is common in body cells, [ 7 ]  and methylation of CpG sites seems to be the default. [ 8 ] [ 9 ]  Human DNA has about 80–90% of CpG sites methylated, but there are certain areas, known as  CpG islands , that are CG-rich (high cytosine and guanine content, made up of about 65% CG  residues ), wherein none is methylated. These are associated with the  promoters  of 56% of mammalian genes, including all  ubiquitously expressed genes . One to two percent of the human genome are CpG clusters, and there is an inverse relationship between CpG methylation and transcriptional activity. Methylation contributing to epigenetic inheritance can occur through either DNA methylation or protein methylation. Improper methylations of human genes can lead to disease development, [ 10 ] [ 11 ]  including cancer. [ 12 ] [ 13 ]


In  honey bees , DNA methylation is associated with alternative splicing and gene regulation based on functional genomic research published in 2013. [ 14 ]  In addition, DNA methylation is associated with expression changes in immune genes when honey bees were under lethal viral infection. [ 15 ]  Several review papers have been published on the topics of DNA methylation in social insects. [ 16 ] [ 17 ]


RNA methylation  occurs in different RNA species viz.  tRNA ,  rRNA ,  mRNA ,  tmRNA ,  snRNA ,  snoRNA ,  miRNA , and viral RNA. Different catalytic strategies are employed for RNA methylation by a variety of RNA-methyltransferases. RNA methylation is thought to have existed before DNA methylation in the early forms of life evolving on earth. [ 18 ]


N6-methyladenosine (m6A)  is the most common and abundant methylation modification in RNA molecules (mRNA) present in eukaryotes. 5-methylcytosine (5-mC) also commonly occurs in various RNA molecules. Recent data strongly suggest that m6A and 5-mC RNA methylation affects the regulation of various biological processes such as RNA stability and mRNA translation, [ 19 ]  and that abnormal RNA methylation contributes to etiology of human diseases. [ 20 ]


In social insects such as honey bees, RNA methylation is studied as a possible epigenetic mechanism underlying aggression via reciprocal crosses. [ 21 ]


Protein methylation  typically takes place on  arginine  or  lysine   amino acid  residues in the protein sequence. [ 22 ]   Arginine can be methylated once (monomethylated arginine) or twice, with either both methyl groups on one terminal nitrogen ( asymmetric dimethylarginine ) or one on both nitrogens (symmetric dimethylarginine), by  protein arginine methyltransferases  (PRMTs). Lysine can be methylated once, twice, or three times by  lysine methyltransferases . Protein methylation has been most studied in the  histones . The transfer of  methyl  groups from  S-adenosyl methionine  to histones is catalyzed by enzymes known as  histone methyltransferases . Histones that are methylated on certain residues can act  epigenetically  to repress or activate gene expression. [ 23 ] [ 24 ]  Protein methylation is one type of  post-translational modification .


Methyl metabolism is very ancient and can be found in all organisms on earth, from bacteria to humans, indicating the importance of methyl metabolism for physiology. [ 25 ]  Indeed, pharmacological inhibition of global methylation in species ranging from human, mouse, fish, fly, roundworm, plant, algae, and cyanobacteria causes the same effects on their biological rhythms, demonstrating conserved physiological roles of methylation during evolution. [ 26 ]


The term methylation in  organic chemistry  refers to the  alkylation  process used to describe the delivery of a  CH 3  group. [ 27 ]


Methylations are commonly performed using  electrophilic  methyl sources such as  iodomethane , [ 28 ]   dimethyl sulfate , [ 29 ] [ 30 ]   dimethyl carbonate , [ 31 ]  or  tetramethylammonium chloride . [ 32 ]  Less common but more powerful (and more dangerous) methylating reagents include  methyl triflate , [ 33 ]   diazomethane , [ 34 ]  and methyl fluorosulfonate ( magic methyl ). These reagents all react via S N 2  nucleophilic substitutions . For example, a  carboxylate  may be methylated on oxygen to give a methyl  ester ; an  alkoxide  salt  RO −  may be likewise methylated to give an  ether ,  ROCH 3 ; or a ketone  enolate  may be methylated on carbon to produce a new  ketone .


The  Purdie methylation  is a specific for the methylation at oxygen of  carbohydrates  using  iodomethane  and  silver oxide . [ 35 ]


The  Eschweiler–Clarke reaction  is a method for methylation of  amines . [ 36 ]  This method avoids the risk of  quaternization , which occurs when amines are methylated with methyl halides.


Diazomethane  and the safer analogue  trimethylsilyldiazomethane  methylate carboxylic acids, phenols, and even alcohols:


The method offers the advantage that the side products are easily removed from the product mixture. [ 37 ]


Methylation sometimes involve use of  nucleophilic  methyl reagents.  Strongly nucleophilic methylating agents include  methyllithium  ( CH 3 Li ) [ 38 ]  or  Grignard reagents  such as  methylmagnesium bromide  ( CH 3 MgX ). [ 39 ]  For example,  CH 3 Li  will add methyl groups to the  carbonyl  (C=O) of ketones and aldehyde.:


Milder methylating agents include  tetramethyltin ,  dimethylzinc , and  trimethylaluminium . [ 40 ]


The  missing heritability problem [ 1 ] [ 2 ] [ 3 ] [ 4 ] [ 5 ] [ 6 ]  arises from the difference between  heritability  estimates from genetic data and  heritability  estimates from twin and family data across many physical and mental traits, including diseases, behaviors, and other  phenotypes . This is a problem that has significant implications for medicine, since a person's  susceptibility to disease  may depend more on the combined effect of all the genes in the background than on the disease genes in the foreground, or the role of genes may have been severely overestimated.


The missing heritability problem was named as such in 2008 (after the " missing baryon problem " in  physics ). The  Human Genome Project  led to optimistic forecasts that the large genetic contributions to many traits and diseases (which were identified by quantitative genetics and behavioral genetics in particular) would soon be mapped and pinned down to specific genes and their genetic variants by methods such as  candidate-gene  studies which used small samples with limited genetic sequencing to focus on specific genes believed to be involved, examining  single-nucleotide polymorphisms  (SNPs). While many hits were found, they often failed to replicate in other studies.


The exponential fall in genome sequencing costs led to the use of  genome-wide association studies  (GWASes) which could simultaneously examine all candidate-genes in larger samples than the original finding, where the candidate-gene hits were found to almost always be false positives and only 2-6% replicate; [ 7 ] [ 8 ] [ 9 ] [ 10 ] [ 11 ] [ 12 ]  in the specific case of  intelligence  candidate-gene hits, only 1 candidate-gene hit replicated, [ 13 ]  the top 25 schizophrenia candidate-genes were no more associated with schizophrenia than chance, [ 14 ] [ 15 ]  and of 15 neuroimaging hits, none did. [ 16 ]  In 2012, the editorial board of  Behavior Genetics  noted, in setting more stringent requirements for candidate-gene publications, that "the literature on candidate gene associations is full of reports that have not stood up to rigorous replication...it now seems likely that many of the published findings of the last decade are wrong or misleading and have not contributed to real advances in knowledge". [ 17 ]  Other researchers have characterized the literature as having "yielded an infinitude of publications with very few consistent replications" and called for a phase out of candidate-gene studies in favor of  polygenic scores . [ 18 ]


Standard genetics methods have long estimated large heritabilities such as 80% for traits such as height or intelligence, yet none of the genes had been found despite sample sizes that, while small, should have been able to detect variants of reasonable effect size such as 1 inch or 5 IQ points. If genes have such strong cumulative effects - where were they? Several resolutions have been proposed, that the missing heritability is some combination of:


The  neurobiological effects of physical exercise  involve possible interrelated effects on brain structure, brain function, and  cognition . [ 1 ] [ 2 ] [ 3 ] [ 4 ]  Research in humans has demonstrated that consistent  aerobic exercise  (e.g., 30 minutes every day) may induce improvements in certain  cognitive functions ,  neuroplasticity  and  behavioral plasticity ; some of these long-term effects may include  increased  neuron growth , increased neurological activity (e.g.,  c-Fos  and  BDNF  signaling), improved stress coping, enhanced  cognitive control of behavior , improved  declarative ,  spatial , and  working  memory, and structural and functional improvements in brain structures and  pathways  associated with cognitive control and memory. [ 5 ] [ 6 ] [ 7 ]  The effects of exercise on cognition may affect  academic performance  in children and college students, improve adult productivity, preserve  cognitive function  in old age, preventing or treating certain  neurological disorders , and improving overall  quality of life . [ 8 ] [ 9 ] [ 10 ] [ 11 ]


In healthy adults, aerobic exercise has been shown to induce transient effects on cognition after a single exercise session and persistent effects on cognition following consistent exercise over the course of several months. [ 1 ] [ 7 ] [ 12 ]  People who regularly perform an aerobic exercise (e.g., running,  jogging , brisk walking, swimming, and cycling) have greater scores on  neuropsychological function and performance tests  that measure certain cognitive functions, such as  attentional control ,  inhibitory control ,  cognitive flexibility ,  working memory  updating and capacity,  declarative memory ,  spatial memory , and  information processing speed . [ 5 ] [ 7 ] [ 12 ] [ 13 ] [ 14 ]


Aerobic exercise has both short and long term effects on mood and emotional states by promoting  positive affect , inhibiting  negative affect , and decreasing the biological response to acute  psychological stress . [ 12 ]   Aerobic exercise may affect both self-esteem and overall well-being (including sleep patterns) with consistent, long term participation. [ 15 ]  Regular aerobic exercise may improve symptoms associated with  central nervous system disorders  and may be used as  adjunct therapy  for these disorders. There is some evidence of exercise treatment efficacy for  major depressive disorder  and  attention deficit hyperactivity disorder . [ 9 ] [ 16 ] [ 17 ] [ 18 ]  The  American Academy of Neurology 's  clinical practice guideline  for  mild cognitive impairment  indicates that clinicians should recommend regular exercise (two times per week) to individuals who have been diagnosed with these conditions. [ 19 ]


Some  preclinical  evidence and emerging clinical evidence supports the use of exercise as an adjunct therapy for the treatment and prevention of  drug addictions . [ 20 ] [ 21 ] [ 22 ] [ 23 ]


Reviews of clinical evidence also support the use of exercise as an adjunct therapy for certain  neurodegenerative disorders , particularly  Alzheimer's disease  and  Parkinson's disease . [ 24 ] [ 25 ]  Regular exercise may be associated with a lower risk of developing neurodegenerative disorders. [ 26 ]


Neuroplasticity  is the process by which  neurons  adapt to a disturbance over time, and most often occurs in response to repeated exposure to stimuli. [ 27 ]  Aerobic exercise increases the production of  neurotrophic factors [ note 1 ]  (e.g.,  BDNF ,  IGF-1 ,  VEGF ) which mediate improvements in cognitive functions and various forms of memory by promoting  blood vessel formation  in the brain,  adult neurogenesis , [ note 2 ]  and other forms of neuroplasticity. [ 2 ] [ 5 ] [ 29 ] [ 30 ]  Consistent aerobic exercise over a period of several months induces  clinically significant  improvements in  executive functions  and increased  gray matter  volume in nearly all regions of the brain, [ 31 ]  with the most marked increases occurring in brain regions that give rise to executive functions. [ 1 ] [ 5 ] [ 6 ]  The brain structures that show the greatest improvements in gray matter volume in response to aerobic exercise are the  prefrontal cortex ,  caudate nucleus , and  hippocampus ; [ 1 ] [ 5 ]  less significant increases in gray matter volume occur in the  anterior cingulate cortex ,  parietal cortex ,  cerebellum , and  nucleus accumbens . [ 5 ]  The prefrontal cortex, caudate nucleus, and anterior cingulate cortex are among the most significant brain structures in the  dopamine and norepinephrine systems  that give rise to cognitive control. [ 32 ]  Exercise-induced neurogenesis (i.e., the increases in gray matter volume) in the hippocampus is associated with measurable improvements in  spatial memory . [ 33 ] [ 34 ]  Higher  physical fitness  scores, as measured by  VO 2  max , are associated with better executive function, faster information processing speed, and greater  gray matter  volume of the hippocampus, caudate nucleus, and nucleus accumbens. [ 1 ]


Reviews of neuroimaging studies indicate that consistent aerobic exercise increases  gray matter  volume in nearly all regions of the brain, [ 31 ]  with more pronounced increases occurring in brain regions associated with memory processing, cognitive control,  motor function , and  reward ; [ 1 ] [ 5 ] [ 31 ]  the most prominent gains in gray matter volume are seen in the prefrontal cortex, caudate nucleus, and hippocampus, which support cognitive control and memory processing, among other cognitive functions. [ 1 ] [ 6 ]  Moreover, the left and right halves of the prefrontal cortex, the hippocampus, and the  cingulate cortex  appear to become more  functionally interconnected  in response to consistent aerobic exercise. [ 1 ]  Three reviews indicate that marked improvements in prefrontal and hippocampal gray matter volume occur in healthy adults that regularly engage in medium intensity exercise for several months. [ 1 ] [ 35 ]  Other regions of the brain that demonstrate moderate or less significant gains in gray matter volume during neuroimaging include the  anterior cingulate cortex ,  parietal cortex ,  cerebellum , and  nucleus accumbens . [ 5 ] [ 36 ]


Regular exercise has been shown to counter the shrinking of the hippocampus and memory impairment that naturally occurs in late adulthood. [ 5 ]  Sedentary adults over age 55 show a 1–2% decline in hippocampal volume annually. [ 37 ]  A neuroimaging study with a sample of 120 adults revealed that participating in regular aerobic exercise increased the volume of the left hippocampus by 2.12% and the right hippocampus by 1.97% over a one-year period. [ 37 ]  Subjects in the low intensity stretching group who had higher fitness levels at baseline showed less hippocampal volume loss, providing evidence for exercise being protective against age-related cognitive decline. [ 37 ]  In general, individuals that exercise more over a given period have greater hippocampal volumes and better memory function. [ 5 ]  Aerobic exercise has also been shown to induce growth in the  white matter  tracts in the anterior  corpus callosum , which normally shrink with age. [ 5 ] [ 35 ]


The various functions of the brain structures that show exercise-induced increases in gray matter volume include:


Concordant with the functional roles of the brain structures that exhibit increased gray matter volumes, regular exercise over a period of several months has been shown to persistently improve numerous executive functions and several forms of memory. [ 5 ] [ 6 ] [ 44 ] [ 45 ]  In particular, consistent aerobic exercise has been shown to improve  attentional control , [ note 3 ]   information processing speed ,  cognitive flexibility  (e.g.,  task switching ),  inhibitory control , [ note 4 ]   working memory  updating and capacity, [ note 5 ]   declarative memory , [ note 6 ]  and  spatial memory . [ 5 ] [ 6 ] [ 7 ] [ 44 ]  In healthy young and middle-aged adults, the  effect sizes  of improvements in cognitive function are largest for indices of executive functions and small to moderate for aspects of memory and information processing speed. [ 1 ] [ 7 ]  It may be that in older adults, individuals benefit cognitively by taking part in both aerobic and resistance type exercise of at least moderate intensity. [ 47 ]  Individuals who have a sedentary lifestyle tend to have impaired executive functions relative to other more physically active non-exercisers. [ 6 ]  A reciprocal relationship between exercise and executive functions has also been noted: improvements in executive control processes, such as attentional control and inhibitory control, increase an individual's tendency to exercise. [ 6 ]


One of the most significant effects of exercise on the brain is increased synthesis and expression of  BDNF , a  neuropeptide  and  hormone , resulting in increased signaling through its  receptor tyrosine kinase ,  tropomyosin receptor kinase B  (TrkB). [ 4 ] [ 50 ] [ 51 ]  Since BDNF is capable of crossing the  blood–brain barrier , higher peripheral BDNF synthesis also increases BDNF signaling in the brain. [ 30 ]  Exercise-induced increases in BDNF signaling are associated with improved cognitive function, improved mood, and improved memory. [ 29 ] [ 50 ]  Furthermore, research has provided a great deal of support for the role of BDNF in hippocampal neurogenesis, synaptic plasticity, and neural repair. [ 5 ] [ 50 ]  Engaging in moderate-high intensity aerobic exercise such as running, swimming, and cycling increases BDNF  biosynthesis  through  myokine  signaling, resulting in up to a threefold increase in  blood plasma  and BDNF levels; [ 4 ] [ 50 ] [ 51 ]  exercise intensity is positively correlated with the magnitude of increased BDNF biosynthesis and expression. [ 4 ] [ 50 ] [ 51 ]  A meta-analysis of studies involving the effect of exercise on BDNF levels found that consistent exercise modestly increases resting BDNF levels as well. [ 29 ]  This has important implications for exercise as a mechanism to reduce stress since stress is closely linked with decreased levels of BDNF in the hippocampus. In fact, studies suggest that BDNF contributes to the anxiety-reducing effects of antidepressants. The increase in BDNF levels caused by exercise helps reverse the stress-induced decrease in BDNF which mediates stress in the short term and buffers against stress-related diseases in the long term. [ 52 ]


IGF-1  is a  peptide  and  neurotrophic factor  that mediates some of the effects of  growth hormone ; [ 53 ]  IGF-1 elicits its physiological effects by binding to a specific  receptor tyrosine kinase , the  IGF-1 receptor , to control tissue growth and remodeling. [ 53 ]  In the brain, IGF-1 functions as a neurotrophic factor that, like  BDNF , plays a significant role in cognition, neurogenesis, and neuronal survival. [ 50 ] [ 54 ] [ 55 ]  Physical activity is associated with increased levels of IGF-1 in  blood serum , which is known to contribute to neuroplasticity in the brain due to its capacity to cross the  blood–brain barrier  and  blood–cerebrospinal fluid barrier ; [ 5 ] [ 50 ] [ 53 ] [ 54 ]  consequently, one review noted that IGF-1 is a key mediator of exercise-induced adult neurogenesis, while a second review characterized it as a factor which links "body fitness" with "brain fitness". [ 53 ] [ 54 ]  The amount of IGF-1 released into blood plasma during exercise is positively correlated with exercise intensity and duration. [ 56 ]


VEGF  is a neurotrophic and  angiogenic  (i.e., blood vessel growth-promoting)  signaling protein  that binds to two receptor tyrosine kinases,  VEGFR1  and  VEGFR2 , which are expressed in  neurons  and  glial cells  in the brain. [ 55 ]   Hypoxia , or inadequate cellular oxygen supply, strongly upregulates VEGF expression and VEGF exerts a neuroprotective effect in hypoxic neurons. [ 55 ]  Like  BDNF  and  IGF-1 , aerobic exercise has been shown to increase VEGF biosynthesis in peripheral tissue which subsequently crosses the blood–brain barrier and promotes neurogenesis and  blood vessel formation  in the  central nervous system . [ 30 ] [ 57 ]  Exercise-induced increases in VEGF signaling have been shown to improve cerebral blood volume and contribute to exercise-induced neurogenesis in the hippocampus. [ 5 ] [ 57 ]


A study using FNDC5  knock-out mice  as well as artificial elevation of circulating  irisin  levels showed that irisin confers beneficial cognitive effects of physical exercise and that it can serve an exercise mimetic in mice in which it could "improve both the cognitive deficit and neuropathology in  Alzheimer's disease  mouse models". The mediator and its regulatory system is therefore being investigated for potential interventions to improve – or further improve – cognitive function or alleviate Alzheimer's disease in humans. [ 58 ] [ 59 ] [ 60 ]  Experiments indicate irisin may be linked to regulation of BDNF and neurogenesis in mice. [ 61 ]


In addition to the persistent effects on cognition that result from several months of daily exercise, acute exercise (i.e., a single bout of exercise) has been shown to transiently improve a number of cognitive functions. [ 12 ] [ 62 ] [ 63 ]  Reviews and meta-analyses of research on the effects of acute exercise on cognition in healthy young and middle-aged adults have concluded that information processing speed and a number of executive functions – including attention, working memory, problem solving, cognitive flexibility, verbal fluency, decision making, and inhibitory control – all improve for a period of up to 2 hours post-exercise. [ 12 ] [ 62 ] [ 63 ]  A systematic review of studies conducted on children also suggested that some of the exercise-induced improvements in executive function are apparent after single bouts of exercise, while other aspects (e.g., attentional control) only improve following consistent exercise on a regular basis. [ 44 ]  Other research has suggested immediate performative enhancements during exercise, such as exercise-concurrent improvements in processing speed and accuracy during both visual attention and working memory tasks. [ 64 ] [ 65 ]


Continuous exercise can produce a transient state of  euphoria  – an emotional state involving the experience of  pleasure  and feelings of profound contentment, elation, and well-being – which is colloquially known as a " runner's high " in  distance running  or a "rower's high" in  rowing . [ 66 ] [ 67 ] [ 68 ] [ 69 ]


β-Phenylethylamine , commonly referred to as  phenethylamine , is a human  trace amine  and potent  catecholaminergic  and  glutamatergic   neuromodulator  that has similar  psychostimulant  and  euphoriant  effects and a similar  chemical structure  to  amphetamine . [ 73 ]  Thirty minutes of moderate to high intensity physical exercise has been shown to induce an enormous increase in urinary  β-phenylacetic acid , the primary metabolite of phenethylamine. [ 74 ] [ 75 ] [ 76 ]  Two reviews noted a study where the average 24 hour urinary  β-phenylacetic acid  concentration among participants following just 30 minutes of intense exercise increased by 77% relative to baseline concentrations in resting control subjects; [ 74 ] [ 75 ] [ 76 ]  the reviews suggest that phenethylamine synthesis sharply increases while an individual is exercising, during which time it is rapidly metabolized due to its short half-life of roughly 30 seconds. [ 74 ] [ 75 ] [ 76 ] [ 77 ]  In a resting state, phenethylamine is synthesized in  catecholamine  neurons from  L - phenylalanine  by  aromatic amino acid decarboxylase  (AADC) at approximately the same rate at which  dopamine  is produced. [ 77 ]


In light of this observation, the original paper and both reviews suggest that phenethylamine plays a prominent role in mediating the mood-enhancing  euphoric  effects of a runner's high, as both phenethylamine and amphetamine are potent euphoriants. [ 74 ] [ 75 ] [ 76 ]


β-Endorphin  (contracted from "endogenous morphine") is an endogenous  opioid   neuropeptide  that binds to  μ-opioid receptors , in turn producing  euphoria  and  pain relief . [ 78 ]  A  meta-analytic  review found that exercise significantly increases the secretion of  β-endorphin  and that this secretion is correlated with improved mood states. [ 78 ]  Moderate intensity exercise produces the greatest increase in  β-endorphin  synthesis, while higher and lower intensity forms of exercise are associated with smaller increases in  β-endorphin  synthesis. [ 78 ]  A review on  β-endorphin  and exercise noted that an individual's mood improves for the remainder of the day following physical exercise and that one's mood is positively correlated with overall daily physical activity level. [ 78 ]


However, humans studies showed that pharmacological blockade of endogenous endorphins does not inhibit a runner's high, while blockade of endocannabinoids may have such an effect. [ 79 ]


Anandamide  is an  endogenous cannabinoid  and  retrograde neurotransmitter  that binds to  cannabinoid receptors  (primarily  CB 1 ), in turn producing euphoria. [ 68 ] [ 80 ]  It has been shown that aerobic exercise causes an increase in plasma anandamide levels, where the magnitude of this increase is highest at moderate exercise intensity (i.e., exercising at ~70–80% maximum heart rate). [ 80 ]  Increases in plasma anandamide levels are associated with  psychoactive effects  because anandamide is able to cross the  blood–brain barrier  and act within the central nervous system. [ 80 ]  Thus, because anandamide is a euphoriant and aerobic exercise is associated with euphoric effects, it has been proposed that anandamide partly mediates the short-term mood-lifting effects of exercise (e.g., the euphoria of a runner's high) via exercise-induced increases in its synthesis. [ 68 ] [ 80 ]


The "stress hormone",  cortisol , is a  glucocorticoid  that binds to  glucocorticoid receptors . [ 81 ] [ 82 ] [ 83 ]   Psychological stress  induces the release of cortisol from the  adrenal gland  by activating the  hypothalamic–pituitary–adrenal axis  (HPA axis). [ 81 ] [ 82 ] [ 83 ]  Short-term increases in cortisol levels are associated with adaptive cognitive improvements, such as enhanced inhibitory control; [ 82 ] [ 83 ]  however, excessively high exposure or prolonged exposure to high levels of cortisol causes impairments in cognitive control and has  neurotoxic  effects in the human brain. [ 83 ]  For example, chronic psychological stress decreases  BDNF  expression, which has detrimental effects on hippocampal volume and can lead to depression. [ 81 ]


As a physical stressor, aerobic exercise stimulates cortisol secretion in an intensity-dependent manner; [ 82 ]  however, it does not result in long-term increases in cortisol production since this exercise-induced effect on cortisol is a response to transient negative  energy balance . [ note 7 ] [ 82 ]  Aerobic exercise increases  physical fitness  and lowers  neuroendocrine  (i.e.,  HPA axis ) reactivity and therefore reduces the biological response to psychological stress in humans (e.g., reduced cortisol release and attenuated  heart rate  response). [ 12 ] [ 84 ]  Exercise also reverses stress-induced decreases in  BDNF  expression and signaling in the brain, thereby acting as a buffer against stress-related diseases like depression. [ 81 ] [ 84 ]


Glutamate , one of the most common neurochemicals in the brain, is an  excitatory neurotransmitter  involved in many aspects of brain function, including learning and memory. [ 85 ]  Based upon animal models, exercise appears to normalize the excessive levels of glutamate neurotransmission into the  nucleus accumbens  that occurs in drug addiction. [ 21 ]  A review of the effects of exercise on neurocardiac function in preclinical models noted that exercise-induced neuroplasticity of the  rostral ventrolateral medulla  (RVLM) has an inhibitory effect on glutamatergic neurotransmission in this region, in turn reducing  sympathetic activity ; [ 86 ]  the review hypothesized that this neuroplasticity in the RVLM is a mechanism by which regular exercise prevents inactivity-related  cardiovascular disease . [ 86 ]


Exerkines  are putative "signalling moieties released in response to acute and/or chronic exercise, which exert their effects through  endocrine ,  paracrine  and/or  autocrine  pathways". [ 87 ]


Engaging in active physical pursuits has demonstrated positive effects on the mental health of children and adolescents, [ 88 ]  enhances their academic performance, [ 89 ]  boosts cognitive function, [ 90 ]  and diminishes the likelihood of obesity and cardiovascular diseases among this demographic. [ 91 ]  Establishing consistent exercise routines with regular frequency and duration is pivotal. [ 92 ] [ 93 ] [ 94 ]  Cultivating beneficial exercise habits and sustaining adequate physical activity may support the overall physical and mental well-being of young individuals. Therefore, identifying factors that either impede or encourage exercise behaviors could be a significant strategy in promoting the development of healthy exercise habits among children and adolescents.


A 2003 meta-analysis found a positive effect of exercise in children on perceptual skills, intelligence quotient, achievement, verbal tests, mathematic tests, and academic readiness. [ 95 ]  The correlation was strongest for the age ranges of 4–7 and 11–13 years. [ 95 ]


A 2010 meta-analysis of the effect of activity on children's executive function found that aerobic exercise may briefly aid children's executive function and also influence more lasting improvements to executive function. [ 96 ]  Other studies suggested that exercise is unrelated to academic performance, perhaps due to the parameters used to determine exactly what academic achievement is. [ 97 ]  This area of study has been a focus for education boards that make decisions on whether physical education should be implemented in the school curriculum, how much time should be dedicated to physical education, and its impact on other academic subjects. [ 95 ]


Another study found that sixth-graders who participated in vigorous physical activity at least three times a week had the highest scores compared to those who participated in moderate or no physical activity at all. Children who participated in vigorous physical activity scored three points higher, on average, on their academic test, which consisted of math, science, English, and world studies. [ 98 ]


Neuroimaging studies indicate that exercise may influence changes in brain structure and function. [ 97 ]  Some investigations have linked low levels of aerobic fitness in children with impaired executive function when older as adults, but lack of selective attention, response inhibition, and interference control may also explain this outcome. [ 99 ]


Clinical and preclinical evidence indicate that consistent aerobic exercise, especially endurance exercise (e.g.,  marathon running ), actually prevents the development of certain  drug addictions  and is an effective adjunct treatment for drug addiction, and psychostimulant addiction in particular. [ 20 ] [ 21 ] [ 22 ] [ 23 ]  Consistent aerobic exercise magnitude-dependently (i.e., by duration and intensity) may reduce drug addiction risk, which appears to occur through the reversal of drug-induced, addiction-related neuroplasticity. [ 21 ] [ 22 ]  Moreover, aerobic exercise decreases psychostimulant self-administration, reduces the  reinstatement  (i.e., relapse) of drug-seeking, and induces opposite effects on  striatal   dopamine receptor D 2  (DRD2) signaling (increased DRD2 density) to those induced by pathological stimulant use (decreased DRD2 density). [ 21 ] [ 22 ]  Consequently, consistent aerobic exercise may lead to better treatment outcomes when used as an adjunct treatment for drug addiction. [ 21 ] [ 23 ]  As of 2016 [update] , more clinical research is still needed to understand the mechanisms and confirm the efficacy of exercise in drug addiction treatment and prevention. [ 20 ]


Regular physical exercise, particularly aerobic exercise, is an effective  add-on treatment  for  ADHD  in children and adults, particularly when combined with stimulant medication (i.e.,  amphetamine  or  methylphenidate ), although the best intensity and type of aerobic exercise for improving symptoms are not currently known. [ 18 ] [ 100 ]  In particular, the long-term effects of regular aerobic exercise in ADHD individuals include better behavior and motor abilities, improved  executive functions  (including attention,  inhibitory control , and  planning , among other cognitive domains), faster information processing speed, and better memory. [ 18 ]  Parent-teacher ratings of behavioral and socio-emotional outcomes in response to regular aerobic exercise include: better overall function, reduced ADHD symptoms, better self-esteem, reduced levels of anxiety and depression, fewer somatic complaints, better academic and classroom behavior, and improved social behavior. [ 18 ]  Exercising while on stimulant medication augments the effect of stimulant medication on executive function. [ 18 ]  It is believed that these short-term effects of exercise are mediated by an increased abundance of  synaptic  dopamine and norepinephrine in the brain. [ 18 ]


Numerous  systematic reviews  and  meta-analyses  have indicated that exercise has a marked and persistent  antidepressant  effect in humans, [ 101 ] [ 16 ] [ 102 ] [ 17 ] [ 103 ]  an effect believed to be mediated through enhanced  BDNF  signaling in the brain. [ 17 ]  Several systematic reviews have analyzed the potential for physical exercise in the treatment of  depressive disorders . The 2013  Cochrane Collaboration  review on  physical exercise  for depression noted that, based upon limited evidence, it is more effective than a control intervention and comparable to psychological or antidepressant drug therapies. [ 103 ]  Three subsequent 2014 systematic reviews that included the Cochrane review in their analysis concluded with similar findings: one indicated that physical exercise is effective as an  adjunct treatment  (i.e., treatments that are used together) with antidepressant medication; [ 17 ]  the other two indicated that physical exercise has marked antidepressant effects and recommended the inclusion of physical activity as an adjunct treatment for mild–moderate depression and mental illness in general. [ 16 ] [ 102 ]  A 2016 meta-analysis concluded that physical exercise improves overall quality of life in individuals with depression relative to controls. One systematic review noted that  yoga  may be effective in alleviating symptoms of  prenatal depression . [ 104 ]  Another review asserted that evidence from  clinical trials  supports the efficacy of physical exercise as a treatment for depression over a 2–4 month period. [ 5 ]  These benefits have also been noted in  old age , with a review conducted in 2019 finding that exercise is an effective treatment for clinically diagnosed depression in older adults. [ 105 ]


A 2024 systematic review and network meta-analysis of 218  randomized controlled trials  involving over 14,000 participants found that various forms of exercise, including walking or  jogging , yoga,  resistance training , and mixed  aerobic activities , were associated with reductions in depressive symptoms. The review observed that the effects of exercise were comparable to those of  psychotherapy  and  pharmacotherapy , with more intensive exercise yielding greater benefits. Resistance training was identified as particularly effective for younger individuals, while yoga appeared to be more beneficial for older adults. While confidence in the findings was limited by methodological concerns in the included studies, the review noted that exercise produced significant improvements in symptoms across a wide range of participants and treatment contexts. [ 101 ]


Physical exercise plays a significant role in the prevention and management of  stroke . It is well established that physical activity decrease the risk of ischemic stroke and  intracerebral haemorrhage . [ 106 ] [ 107 ] [ 108 ]  Engaging in physical activity before experiencing a stroke has been found to have a positive impact on the severity and outcomes of stroke. [ 109 ]  Exercise has the potential to increase the expression of VEGF,  caveolin , and  angiopoietin  in the brain. These changes may promote  angiogenesis  and  neovascularization  that contribute to improved blood supply to the stroke affected areas of the brain. [ 110 ] [ 111 ] [ 112 ]  Exercise may affect the activation of  endothelial nitric oxide synthase  (eNOS) and subsequent production of  nitric oxide  (NO). [ 113 ] [ 114 ] [ 115 ]  The increase in NO production may lead to improved post-stroke cerebral blood flow, ensuring a sufficient oxygen and nutrient supply to the brain. Physical activity has been associated with increased expression and activation of  hypoxia-inducible factor 1 alpha  (HIF-1α),  heat shock proteins , and  brain-derived neurotrophic factor  (BDNF). [ 116 ] [ 117 ] [ 118 ]  These factors play crucial roles in promoting cellular survival, neuroprotection, and repair processes in the brain following a stroke. Exercise also inhibit  glutamate  and  caspase  activities, which are involved in neuronal death pathways. [ 119 ] [ 120 ] [ 121 ] [ 122 ]  Additionally, it may promote  neurogenesis  in the brain. These effects collectively contribute to the reduction of brain infarction and edema, leading to potential improvements in neurological and functional outcomes. The neuroprotective properties of physical activity in relation to haemorrhagic strokes are less studied. Pre-stroke physical activity has been associated with improved outcomes after intracerebral haemorrhages. [ 123 ]  Furthermore, physical activity may reduce the volume of intracerebral haemorrhages. [ 124 ] [ 125 ]   Being physically active after stroke also enhance the functional recovery. [ 126 ] [ 127 ] [ 128 ]


The  American Academy of Neurology 's January 2018 update of their  clinical practice guideline  for  mild cognitive impairment  states that clinicians should recommend regular exercise (two times per week) to individuals who have been diagnosed with this condition. [ 19 ]  This guidance is based upon a moderate amount of high-quality evidence which supports the efficacy of regular physical exercise (twice weekly over a 6-month period) for improving cognitive symptoms in individuals with mild cognitive impairment. [ 19 ]


Alzheimer's disease  is a cortical neurodegenerative disorder and the most prevalent form of  dementia , representing approximately 65% of all cases of dementia; it is characterized by impaired cognitive function, behavioral abnormalities, and a reduced capacity to perform  basic activities of daily life . [ 24 ]  Two reviews found evidence for possible positive effects of physical exercise on cognitive function, the rate of cognitive decline, and the ability to perform activities of daily living in individuals with Alzheimer's disease. [ 24 ]  A subsequent review found higher levels of physical activity may be associated with reduced risk of dementia and cognitive decline. [ 26 ]


Parkinson's disease  symptoms reflect various functional impairments and limitations, such as  postural instability ,  gait disturbance , immobility, and frequent falls. Some evidence suggests that physical exercise may lower the risk of Parkinson's disease. [ 129 ]  A 2017 study found that strength and endurance training in people with Parkinson's disease had positive effects lasting for several weeks. [ 130 ]  A 2023 Cochrane review on the effects of physical exercise in people with Parkinson's disease indicated that  aquatic exercise  might reduce severity of motor symptoms and improve quality of life. [ 131 ]  Furthermore,  endurance training ,  functional training , and multi-domain training (i.e., engaging in several types of exercise) may provide improvements. [ 131 ]


Neuroepigenetics  is the study of how  epigenetic  changes to  genes  affect the  nervous system .  These changes may effect underlying conditions such as  addiction ,  cognition , and  neurological development .


Neuroepigenetic mechanisms regulate gene expression in the neuron. Often, these changes take place due to recurring stimuli. Neuroepigenetic mechanisms involve proteins or protein pathways that regulate gene expression by adding, editing or reading epigenetic marks such as  methylation  or  acetylation . Some of these mechanisms include ATP-dependent  chromatin remodeling ,  LINE1 , and  prion  protein-based modifications. Other silencing mechanisms include the recruitment of specialized proteins that methylate DNA such that the  core promoter  element is inaccessible to  transcription factors  and  RNA polymerase . As a result, transcription is no longer possible. One such protein pathway is the  REST  co-repressor complex pathway. There are also several  non-coding RNAs  that regulate neural function at the epigenetic level. These mechanisms, along with neural  histone methylation , affect arrangement of  synapses , neuroplasticity, and play a key role in learning and memory.


DNA methyltransferases (DNMTs)  are involved in regulation of the electrophysiological landscape of the brain through methylation of  CpGs . Several studies have shown that inhibition or depletion of  DNMT1  activity during neural maturation leads to hypomethylation of the neurons by removing the cell's ability to maintain methylation marks in the chromatin. This gradual loss of methylation marks leads to changes in the expression of crucial developmental genes that may be dosage sensitive, leading to neural degeneration. This was observed in the mature neurons in the dorsal portion of the mouse  prosencephalon , where there was significantly greater amounts of neural degeneration and poor neural signaling in the absence of DNMT1. Despite poor survival rates amongst the DNMT1-depleted neurons, some of the cells persisted throughout the lifespan of the organism. The surviving cells reaffirmed that the loss of DNMT1 led to hypomethylation in the neural cell genome. These cells also exhibited poor neural functioning. In fact, a global loss of neural functioning was also observed in these model organisms, with the greatest amounts neural degeneration occurring in the prosencephalon. [ 1 ]


Other studies showed a trend for  DNMT3a  and  DNMT3b . However, these DNMT's add new methyl marks on unmethylated DNA, unlike DNMT1. Like DNMT1, the loss of DNMT3a and 3b resulted in neuromuscular degeneration two months after birth, as well as poor survival rates amongst the progeny of the mutant cells, even though DNMT3a does not regularly function to maintain methylation marks. This conundrum was addressed by other studies which recorded rare loci in mature neurons where DNMT3a acted as a maintenance DNMT. The  Gfap locus , which codes for the formation and regulation of the cytoskeleton of astrocytes, is one such locus where this activity is observed. The gene is regularly methylated to downregulate  glioma  related cancers. DNMT inhibition leads to decreased methylation and increased synaptic activity. [ 2 ]  Several studies show that the methylation-related increase or decrease in synaptic activity occurs due to the upregulation or downregulation of receptors at the neurological synapse. Such receptor regulation plays a major role in many important mechanisms, such as the 'fight or flight' response. The  glucocorticoid receptor  (GR) is the most studied of these receptors. During stressful circumstances, there is a signaling cascade that begins from the  pituitary gland  and terminates due to a  negative feedback loop  from the  adrenal gland . In this loop, the increase in the levels of the stress response hormone results in the increase of GR. Increase in GR results in the decrease of cellular response to the hormone levels. It has been shown that methylation of the I7  exon  within the GR locus leads to a lower level of basal GR expression in mice. These mice were more susceptible to high levels of stress as opposed to mice with lower levels of methylation at the I7 exon. Up-regulation or down-regulation of receptors through methylation leads to change in synaptic activity of the neuron.


CpG Islands  (CGIs) are regulatory elements that can influence gene expression by allowing or interfering with transcription initiation or enhancer activity. CGIs are generally interspersed with the promoter regions of the genes they affect and may also affect more than one promoter region. In addition they may also include enhancer elements and be separate from the transcription start site.  Hypermethylation at key CGIs can effectively silence expression of tumor suppressing genes and is common in gliomas.  Tumor suppressing genes  are those which inhibit a cell's progression towards cancer. These genes are commonly associated with important functions which regulate cell-cycle events. [ 3 ]  For example,  PI3K  and  p53  pathways are affected by CGI promoter hypermethylation, this includes the promoters of the genes CDKN2/p16, RB, PTEN, TP53 and p14ARF. Importantly,  glioblastomas  are known to have high frequency of methylation at CGIs/promoter sites. For example,  Epithelial Membrane Protein 3  (EMP3) is a gene which is involved in cell proliferation as well as cellular interactions. It is also thought to function as a tumor suppressor, and in glioblastomas is shown to be silenced via hypermethylation. Furthermore, introduction of the gene into EMP3-silenced neuroblasts results in reduced colony formation as well as suppressed tumor growth. [ 4 ]  In contrast, hypermethylation of promoter sites can also inhibit activity of oncogenes and prevent tumorigenesis. Such oncogenic pathways as the transformation growth factor (TGF)-beta signaling pathway stimulate cells to proliferate. In glioblastomas the overactivity of this pathway is associated with aggressive forms of tumor growth. Hypermethylation of  PDGF-B , the TGF-beta target, inhibits uncontrolled proliferation. [ 5 ]


Global reduction in methylation is implicated in  tumorigenesis . More specifically, wide spread CpG demethylation, contributing to global hypomethylation, is known to cause genomic instability leading to development of tumors. [ 3 ] [ 6 ]  An important effect of this DNA modification is its transcriptional activation of oncogenes. For example, expression of MAGEA1 enhanced by hypomethylation interferes with p53 function. [ 7 ]


Aberrant patterns of histone modifications can also take place at specific loci and ultimately manipulate gene activity. In terms of CGI promoter sites, methylation and loss of acetylation occurs frequently at H3K9. Furthermore, H3K9 dimethylation and trimethylation are repressive marks which, along with bivalent differentially methylated domains, are hypothesized to make tumor suppressing genes more susceptible to silencing. Abnormal presence or lack of methylation in glioblastomas are strongly linked to genes which regulate apoptosis, DNA repair, cell proliferation, and tumor suppression. One of the best known examples of genes affected by aberrant methylation that contributes to formation of glioblastomas is  MGMT , a gene involved in DNA repair which encodes the protein  O6-methylguanine-DNA methyltransferase . Methylation of the  MGMT  promoter is an important predictor of the effectiveness of alkylating agents to target glioblastomas. [ 8 ]  Hypermethylation of the  MGMT  promoter causes transcriptional silencing and is found in several cancer types including glioma, lymphoma, breast cancer, prostate cancer, and retinoblastoma. [ 9 ]


Neuroplasticity  refers to the ability of the brain to undergo synaptic rearrangement as a response to recurring stimuli. Neurotrophin proteins play a major role in synaptic rearrangement, amongst other factors. Depletion of  neurotrophin  BDNF or BDNF signaling is one of the main factors in developing diseases such as  Alzheimer's disease ,  Huntington's disease , and  depression . [ 10 ]  Neuroplasticity can also occur as a consequence of targeted epigenetic modifications such as methylation and acetylation. Exposure to certain recurring stimuli leads to demethylation of particular loci and  remethylation  in a pattern that leads to a response to that particular stimulus. Like the histone readers, erasers and writers also modify histones by removing and adding modifying marks respectively. An eraser, neuroLSD1, is a modified version of the original  Lysine Demethylase 1 (LSD1) that exists only in neurons and assists with neuronal maturation. Although both versions of LSD1 share the same target, their expression patterns are vastly different and neuroLSD1 is a truncated version of LSD1. NeuroLSD1 increases the expression of  immediate early genes (IEGs)  involved in cell maturation. Recurring stimuli lead to differential expression of neuroLSD1, leading to rearrangement of loci. The eraser is also thought to play a major role in the learning of many complex behaviors and is way through which genes interact with the environment.


Alzheimer's disease  (AD) is a neurodegenerative disease known to progressively affect memory and incite  cognitive  degradation. Epigenetic modifications both globally and on specific candidate genes are thought to contribute to the  etiology  of this disease.  Immunohistochemical  analysis of post-mortem brain tissues across several studies have revealed global decreases in both  5-methylcytosine  (5mC) and  5-hydroxymethylcytosine  (5hmC) in AD patients compared with controls. [ 11 ]  However, conflicting evidence has shown elevated levels of these epigenetic markers in the same tissues. Furthermore, these modifications appear to be affected early on in tissues associated with the  pathophysiology  of AD. [ 12 ]  The presence of 5mC at the promoters of genes is generally associated with gene silencing. 5hmC, which is the oxidized product of 5mC, via  ten-eleven-translocase  (TET), is thought to be associated with activation of gene expression, though the mechanisms underlying this activation are not fully understood. [ 13 ] [ 14 ]


Regardless of variations in results of methylomic analysis across studies, it is known that the presence of 5hmC increases with differentiation and aging of cells in the brain. Furthermore, genes which have a high prevalence of 5hmC are also implicated in the pathology of other age related neurodegenerative diseases, and are key regulators of ion transport, neuronal development, and cell death. [ 15 ] [ 16 ]   For example, over-expression of  5-Lipoxygenase  (5-LOX), an enzyme which generates pro-inflammatory mediators from arachidonic acid, in AD brains is associated with high prevalence of 5hmC at the 5-LOX gene promoter region. [ 17 ]


DNA modifications at different transcriptional sites have been shown to contribute to neurodegenerative diseases. These include harmful transcriptional alterations such as those found in motor neuron functionality associated with  Amyotrophic Lateral Sclerosis  (ALS). Degeneration of upper and lower motor neurons, which contributes to muscle atrophy in ALS patients, is linked to chromatin modifications among a group of key genes. One important site that is regulated by epigenetic events is the hexanucleotide repeat expansion in C9orf72 within the  chromosome 9p21 . Hypermethylation of the C9orf72 related CpG Islands is shown to be associated with repeat expansion in ALS affected tissues. [ 18 ]  Overall, silencing of the C9orf72 gene may result in  haploinsufficiency , and may therefore influence the presentation of disease. The activity of chromatin modifiers is also linked to prevalence of ALS.  DNMT3A is an important methylating agent and has been shown to be present throughout the central nervous systems of those with ALS. Furthermore, over-expression of this de novo methyl transferase is also implicated in cell death of motor-neuron  analogs . [ 19 ]


Mutations  in the  FUS gene , that encodes an RNA/DNA binding protein, are causally linked to ALS.  ALS patients with such mutations have increased levels of  DNA damage . [ 20 ] [ 21 ]   The protein encoded by the FUS gene is employed in the DNA damage response.  It is recruited to DNA double-strand breaks and catalyzes  recombinational repair  of such breaks.  In response to DNA damage, the FUS protein also interacts with  histone deacetylase  I, a protein employed in  epigenetic  alteration of  histones .  This interaction is necessary for efficient  DNA repair . [ 20 ]   These findings suggest that defects in epigenetic signalling and DNA repair contribute to the pathogenesis of ALS. [ 20 ]


A multitude of genetic and epigenetic changes in DNA profiles in brain cells are thought to be linked to  tumorgenesis . These alterations, along with changes in protein functions, are shown to induce uncontrolled cell proliferation, expansion, and  metastasis . While genetic events such as deletions,  translocations , and  amplification  give rise to activation of oncogenes and deactivation of tumor suppressing genes, epigenetic changes silence or up-regulate these same genes through key chromatin modifications.


Neurotoxicity  refers to damage made to the  central  or  peripheral nervous systems  due to chemical, biological, or physical exposure to  toxins . Neurotoxicity can occur at any age and its effects may be short-term or long-term, depending on the mechanism of action of the neurotoxin and degree of exposure.


Certain  metals  are considered essential due to their role in key biochemical and physiological pathways, while the remaining metals are characterized as being nonessential. Nonessential metals do not serve a purpose in any biological pathway and the presence and accumulation in the  brain  of most can lead to neurotoxicity.  These nonessential metals, when found inside the body compete with essential metals for binding sites, upset  antioxidant balance , and their accumulation in the brain can lead to harmful side effects, such as  depression  and  intellectual disability . [ 22 ]  An increase in nonessential heavy metal concentrations in air, water and food sources, and household products has increased the risk of chronic exposure.


Acetylation ,  methylation  and  histone  modification are some of the most common  epigenetic  markers. While these changes do not directly affect the  DNA sequence , they are able to alter the accessibility to genetic components, such as the  promoter  or  enhancer  regions, necessary for  gene expression . Studies have shown that long-term maternal exposure to  lead  (Pb) contributes to decreased methylation in areas of the fetal epigenome, for example the interspaced repetitive sequences (IRSs)  Alu1  and LINE-1. [ 23 ]  The hypomethylation of these IRSs has been linked to increased risk for  cancers  and  autoimmune diseases  later in life. [ 24 ]  Additionally, studies have found a relationship between chronic prenatal Pb exposure and neurological diseases, such as  Alzheimer's  and  schizophrenia , as well as developmental issues. Furthermore, the acetylation and methylation changes induced by overexposure to lead result in decreased  neurogenesis  and neuron  differentiation  ability, and consequently interfere with early  brain development . [ 25 ]


Overexposure to essential metals can also have detrimental consequences on the epigenome. For example, when  manganese , a metal normally used by the body as a  cofactor , is present at high concentrations in the blood it can negatively affect the central nervous system. Studies have shown that accumulation of manganese leads to dopaminergic cell death and consequently plays a role in the onset of  Parkinson's disease  (PD). A hallmark of Parkinson's disease is the accumulation of α-Synuclein in the brain. Increased exposure to manganese leads to the downregulation of protein kinase C delta (PKCδ) through decreased acetylation and results in the misfolding of the α-Synuclein protein that allows aggregation and triggers  apoptosis  of dopaminergic cells. [ 26 ] [ 27 ]


The field has only recently seen a growth in interest, as well as in research, due to technological advancements that facilitate better resolution of the minute modifications made to DNA. However, even with the significant advances in technology, studying the biology of neurological phenomena, such as cognition and addiction, comes with its own set of challenges. Biological study of cognitive processes, especially with humans, has many ethical caveats. Some procedures, such as brain biopsies of people with  Rett syndrome , usually call for a fresh tissue sample that can only be extricated from the brain of deceased individual. In such cases, the researchers have no control over the age of brain tissue sample, thereby limiting research options. In case of addiction to substances such as alcohol, researchers utilize mouse models to mirror the human version of the disease. However, the mouse models are administered greater volumes of ethanol than humans normally consume in order to obtain more prominent phenotypes. Therefore, while the model organism and the tissue samples provide an accurate approximation of the biology of neurological phenomena, these approaches do not provide a complete and precise picture of the exact processes underlying a  phenotype  or a disease.


Neuroepigenetics had also remained underdeveloped due to the controversy surrounding the classification of genetic modifications in matured neurons as  epigenetic  phenomena. This discussion arises due to the fact that neurons do not undergo  mitosis  after maturation, yet the conventional definition of epigenetic phenomena emphasizes heritable changes passed on from parent to offspring. However, various  histone  modifications are placed by epigenetic modifiers such as  DNA methyltransferases (DNMT)  in neurons and these marks regulate gene expression throughout the life span of the neuron. The modifications heavily influence gene expression and arrangement of synapses within the brain. Finally, although not inherited, most of these marks are maintained throughout the life of the cell once they are placed on chromatin.


In  chromatin , those proteins which remain after the  histones  have been removed, are classified as  non-histone proteins .  The non-histone proteins, are a large group of heterogeneous proteins that play a role in organization and compaction of the chromosome into higher order structures. They play vital roles in regulating processes like nucleosome remodeling, DNA replication, RNA synthesis and processing, nuclear transport, steroid hormone action and interphase/mitosis transition. [ 1 ]
 Scaffold proteins ,  DNA polymerase ,  Heterochromatin Protein 1  and  Polycomb  are common non-histone proteins.  This classification group also includes numerous other structural, regulatory, and motor proteins. Non-histone protein are acidic.


The  methylation  of non-histone proteins regulates responses to  DNA damage  including the modulation of  DNA repair  pathways in proliferating and post- mitotic   neuronal cells . [ 2 ]  Such modulation likely has implications for neuronal function. [ 2 ]


Hartwell, Leland; L. Hood; M. Goldberg; A. Reynolds; L. Silver; R. Veres (2004).  Genetics: From Genes to Genomes . New York, NY: McGraw-Hill. pp.  417– 422.  ISBN   0-07-246248-5 .


A  nucleosome  is the basic structural unit of  DNA  packaging in  eukaryotes . The structure of a nucleosome consists of a segment of DNA wound around eight  histone proteins [ 1 ]  and resembles thread wrapped around a spool. The nucleosome is the fundamental subunit of  chromatin . Each nucleosome is composed of a little less than two turns of DNA wrapped around a set of eight proteins called histones, which are known as a  histone octamer . Each histone octamer is composed of two copies each of the histone proteins  H2A ,  H2B ,  H3 , and  H4 .


DNA must be compacted into nucleosomes to fit within the  cell nucleus . [ 2 ]  In addition to nucleosome wrapping, eukaryotic  chromatin  is further compacted by being folded into a series of more complex structures, eventually forming a  chromosome . Each human cell contains about 30 million nucleosomes. [ 3 ]


Nucleosomes are thought to carry  epigenetically  inherited information in the form of  covalent modifications  of their core  histones . Nucleosome positions in the genome are not random, and it is important to know where each nucleosome is located because this determines the accessibility of the DNA to  regulatory proteins . [ 4 ]


Nucleosomes were first observed as particles in the electron microscope by Don and Ada Olins in 1974, [ 5 ]  and their existence and structure (as histone octamers surrounded by approximately 200 base pairs of DNA) were proposed by  Roger Kornberg . [ 6 ] [ 7 ]  The role of the nucleosome as a regulator of transcription was demonstrated by Lorch et al. in vitro [ 8 ]  in 1987 and by Han and Grunstein [ 9 ]  and Clark-Adams et al. [ 10 ]  in vivo in 1988.


The nucleosome core particle consists of approximately 146  base pairs  (bp) of  DNA [ 11 ]  wrapped in 1.67 left-handed  superhelical turns  around a  histone  octamer, consisting of 2 copies each of the core histones  H2A ,  H2B ,  H3 , and  H4 . [ 12 ]  Core particles are connected by stretches of  linker DNA , which can be up to about 80 bp long. Technically, a nucleosome is defined as the core particle plus one of these linker regions; however the word is often synonymous with the core particle. [ 13 ]  Genome-wide nucleosome positioning maps are now available for many model organisms and human cells. [ 14 ]


Linker histones such as  H1  and its isoforms are involved in chromatin compaction and sit at the base of the nucleosome near the DNA entry and exit binding to the linker region of the DNA. [ 15 ]  Non-condensed nucleosomes without the linker histone resemble "beads on a string of DNA" under an  electron microscope . [ 16 ]


In contrast to most eukaryotic cells, mature sperm cells largely use  protamines  to package their genomic DNA, most likely to achieve an even higher packaging ratio. [ 17 ]  Histone equivalents and a simplified chromatin structure have also been found in  Archaea , [ 18 ]  suggesting that eukaryotes are not the only organisms that use nucleosomes.


Pioneering structural studies in the 1980s by Aaron Klug's group provided the first evidence that an octamer of histone proteins wraps DNA around itself in about 1.7 turns of a left-handed superhelix. [ 19 ]  In 1997 the first near atomic resolution  crystal structure  of the nucleosome was solved by the Richmond group, showing the most important details of the particle. The human  alpha satellite   palindromic DNA  critical to achieving the 1997 nucleosome crystal structure was developed by the Bunick group at Oak Ridge National Laboratory in Tennessee. [ 20 ] [ 21 ] [ 22 ] [ 23 ] [ 24 ]  The structures of over 20 different nucleosome core particles have been solved to date, [ 25 ]  including those containing histone variants and histones from different species. The structure of the nucleosome core particle is remarkably conserved, and even a change of over 100 residues between frog and yeast histones results in electron density maps with an overall  root mean square deviation  of only 1.6Å. [ 26 ]


The nucleosome core particle (shown in the figure) consists of about 146  base pair  of DNA [ 11 ]  wrapped in 1.67 left-handed  superhelical turns  around the  histone octamer , consisting of 2 copies each of the core histones  H2A ,  H2B ,  H3 , and  H4 . Adjacent nucleosomes are joined by a stretch of free DNA termed  linker DNA  (which varies from 10 - 80 bp in length depending on species and tissue type [ 18 ] ).The whole structure generates a cylinder of diameter 11 nm and a height of 5.5 nm.


Nucleosome core particles are observed when chromatin in interphase is treated to cause the chromatin to unfold partially. The resulting image, via an electron microscope, is "beads on a string". The string is the DNA, while each bead in the nucleosome is a core particle. The nucleosome core particle is composed of DNA and histone proteins. [ 29 ]


Partial  DNAse  digestion of  chromatin  reveals its nucleosome structure. Because DNA portions of nucleosome core particles are less accessible for DNAse than linking sections, DNA gets digested into fragments of lengths equal to multiplicity of distance between nucleosomes (180, 360, 540 base pairs etc.). Hence a very characteristic  pattern similar to a ladder  is visible during  gel electrophoresis  of that DNA. [ 27 ]  Such digestion can occur also under natural conditions during  apoptosis  ("cell suicide" or programmed cell death), because  autodestruction of DNA  typically is its role. [ 30 ]


The core histone proteins contains a characteristic structural motif termed the "histone fold", which consists of three alpha-helices (α1-3) separated by two loops (L1-2). In solution, the histones form H2A-H2B heterodimers and H3-H4 heterotetramers. Histones dimerise about their long α2 helices in an anti-parallel orientation, and, in the case of H3 and H4, two such dimers form a 4-helix bundle stabilised by extensive H3-H3' interaction. The H2A/H2B dimer binds onto the H3/H4 tetramer due to interactions between H4 and H2B, which include the formation of a hydrophobic cluster. [ 12 ]
The histone octamer is formed by a central H3/H4 tetramer sandwiched between two H2A/H2B dimers. Due to the highly basic charge of all four core histones, the histone octamer is stable only in the presence of DNA or very high salt concentrations.


The nucleosome contains over 120 direct protein-DNA interactions and several hundred water-mediated ones. [ 31 ]  Direct protein - DNA interactions are not spread evenly about the octamer surface but rather located at discrete sites. These are due to the formation of two types of DNA binding sites within the octamer; the α1α1 site, which uses the α1 helix from two adjacent histones, and the L1L2 site formed by the L1 and L2 loops. Salt links and  hydrogen bonding  between both side-chain basic and hydroxyl groups and main-chain amides with the DNA backbone phosphates form the bulk of interactions with the DNA. This is important, given that the ubiquitous distribution of nucleosomes along genomes requires it to be a non-sequence-specific DNA-binding factor. Although nucleosomes tend to prefer some DNA sequences over others, [ 32 ]  they are capable of binding practically to any sequence, which is thought to be due to the flexibility in the formation of these water-mediated interactions. In addition, non-polar interactions are made between protein side-chains and the deoxyribose groups, and an arginine side-chain intercalates into the DNA minor groove at all 14 sites where it faces the octamer surface.
The distribution and strength of DNA-binding sites about the octamer surface distorts the DNA within the nucleosome core. The DNA is non-uniformly bent and also contains twist defects. The twist of free B-form DNA in solution is 10.5 bp per turn.  However, the overall twist of nucleosomal DNA is only 10.2 bp per turn, varying from a value of 9.4 to 10.9 bp per turn.


The histone tail extensions constitute up to 30% by mass of histones, but are not visible in the crystal structures of nucleosomes due to their high intrinsic flexibility, and have been thought to be largely unstructured. [ 33 ]  The N-terminal tails of histones H3 and H2B pass through a channel formed by the minor grooves of the two DNA strands, protruding from the DNA every 20 bp. The  N-terminal  tail of histone H4, on the other hand, has a region of highly basic amino acids (16–25), which, in the crystal structure, forms an interaction with the highly acidic surface region of a H2A-H2B dimer of another nucleosome, being potentially relevant for the higher-order structure of nucleosomes. This interaction is thought to occur under physiological conditions also, and suggests that  acetylation  of the H4 tail distorts the higher-order structure of chromatin. [ citation needed ]


The organization of the DNA that is achieved by the nucleosome cannot fully explain the packaging of DNA observed in the cell nucleus. Further compaction of  chromatin  into the cell nucleus is necessary, but it is not yet well understood.  The current understanding [ 25 ]  is that repeating nucleosomes with intervening "linker" DNA form a  10-nm-fiber , described as "beads on a string", and have a packing ratio of about five to ten. [ 18 ]  A chain of nucleosomes can be arranged in a  30 nm fiber , a compacted structure with a packing ratio of ~50 [ 18 ]  and whose formation is dependent on the presence of the  H1 histone .


A crystal structure of a tetranucleosome has been presented and used to build up a proposed structure of the 30 nm fiber as a two-start helix. [ 34 ]
There is still a certain amount of contention regarding this model, as it is incompatible with recent  electron microscopy  data. [ 35 ]   Beyond this, the structure of chromatin is poorly understood, but it is classically suggested that the 30 nm fiber is arranged into loops along a central protein scaffold to form transcriptionally active  euchromatin . Further compaction leads to transcriptionally inactive  heterochromatin .


Although the nucleosome is a very stable protein-DNA complex, it is not static and has been shown to undergo a number of different structural re-arrangements including nucleosome sliding and DNA site exposure. Depending on the context, nucleosomes can inhibit or facilitate transcription factor binding. Nucleosome positions are controlled by three major contributions: First, the intrinsic binding affinity of the histone octamer depends on the DNA sequence. Second, the nucleosome can be displaced or recruited by the competitive or  cooperative binding  of other protein factors. Third, the nucleosome may be actively translocated by ATP-dependent remodeling complexes. [ 36 ]


When incubated thermally, nucleosomes reconstituted onto the 5S DNA positioning sequence were able to reposition themselves translationally onto adjacent sequences. [ 37 ]  This repositioning does not require disruption of the histone octamer but is consistent with nucleosomes being able to "slide" along the DNA  in cis .  CTCF  binding sites act as nucleosome positioning anchors so that, when used to align various genomic signals, multiple flanking nucleosomes can be readily identified. [ 38 ]  Although nucleosomes are intrinsically mobile, eukaryotes have evolved a large family of ATP-dependent chromatin remodelling enzymes to alter chromatin structure, many of which do so via nucleosome sliding. Nucleosome sliding is one of the possible mechanism for large scale tissue specific expression of genes. The transcription start site for genes expressed in a particular tissue, are nucleosome depleted while, the same set of genes in other tissue where they are not expressed, are nucleosome bound. [ 39 ]


Nucleosomal DNA is in equilibrium between a wrapped and unwrapped state. DNA within the nucleosome remains fully wrapped for only 250 ms before it is unwrapped for 10-50 ms and then rapidly rewrapped, as measured using time-resolved  FRET . [ 40 ]  This implies that DNA does not need to be actively dissociated from the nucleosome but that there is a significant fraction of time during which it is fully accessible. Introducing a DNA-binding sequence within the nucleosome increases the accessibility of adjacent regions of DNA when bound. [ 41 ]


This propensity for DNA within the nucleosome to "breathe" has important functional consequences for all DNA-binding proteins that operate in a chromatin environment. [ 40 ]  In particular, the dynamic breathing of nucleosomes plays an important role in restricting the advancement of  RNA polymerase II  during transcription elongation. [ 42 ]


Promoters of active genes have nucleosome free regions (NFR). This allows for promoter DNA accessibility to various proteins, such as transcription factors. Nucleosome free region typically spans for 200 nucleotides in  S. cerevisiae [ 43 ]  Well-positioned nucleosomes form boundaries of NFR. These nucleosomes are called +1-nucleosome and −1-nucleosome and are located at canonical distances downstream and upstream, respectively, from transcription start site. [ 44 ]  +1-nucleosome and several downstream nucleosomes also tend to incorporate H2A.Z histone variant. [ 44 ]


Eukaryotic genomes are ubiquitously associated into chromatin; however, cells must spatially and temporally regulate specific loci independently of bulk chromatin. In order to achieve the high level of control required to co-ordinate nuclear processes such as DNA replication, repair, and transcription, cells have developed a variety of means to locally and specifically modulate chromatin structure and function. This can involve covalent modification of histones, the incorporation of histone variants, and non-covalent remodelling by ATP-dependent remodeling enzymes.


Since they were discovered in the mid-1960s, histone modifications have been predicted to affect transcription. [ 45 ]  The fact that most of the early post-translational modifications found were concentrated within the tail extensions that protrude from the nucleosome core lead to two main theories regarding the mechanism of histone modification. The first of the theories suggested that they may affect electrostatic interactions between the histone tails and DNA to "loosen" chromatin structure. Later it was proposed that combinations of these modifications may create binding epitopes with which to recruit other proteins. [ 46 ]  Recently, given that more modifications have been found in the structured regions of histones, it has been put forward that these modifications may affect histone-DNA [ 47 ]  and histone-histone [ 48 ]  interactions within the nucleosome core. Modifications (such as acetylation or phosphorylation) that lower the charge of the globular histone core are predicted to "loosen" core-DNA association; the strength of the effect depends on location of the modification within the core. [ 49 ]
Some modifications have been shown to be correlated with gene silencing; others seem to be correlated with gene activation. Common modifications include  acetylation ,  methylation , or  ubiquitination  of  lysine ;  methylation  of  arginine ; and  phosphorylation  of  serine . The information stored in this way is considered  epigenetic , since it is not encoded in the DNA but is still inherited to daughter cells. The maintenance of a repressed or activated status of a gene is often necessary for  cellular differentiation . [ 18 ]


Although histones are remarkably conserved throughout evolution, several variant forms have been identified. This diversification of histone function is restricted to H2A and H3, with H2B and H4 being mostly invariant. H2A can be replaced by  H2AZ  (which leads to reduced nucleosome stability) or  H2AX  (which is associated with DNA repair and  T cell  differentiation), whereas the  inactive X chromosomes  in mammals are enriched in macroH2A. H3 can be replaced by H3.3 (which correlates with activate genes and regulatory elements) and in  centromeres  H3 is replaced by  CENPA . [ 18 ]


A number of distinct reactions are associated with the term  ATP-dependent chromatin remodeling . Remodeling enzymes have been shown to slide nucleosomes along DNA, [ 50 ]  disrupt histone-DNA contacts to the extent of destabilizing the H2A/H2B dimer [ 51 ] [ 52 ]  and to generate negative superhelical torsion in DNA and chromatin. [ 53 ]  Recently, the Swr1 remodeling enzyme has been shown to introduce the variant histone H2A.Z into nucleosomes. [ 54 ]  At present, it is not clear if all of these represent distinct reactions or merely alternative outcomes of a common mechanism. What is shared between all, and indeed the hallmark of ATP-dependent chromatin remodeling, is that they all result in altered DNA accessibility.


Studies looking at gene activation  in vivo [ 55 ]  and, more astonishingly, remodeling  in vitro [ 56 ]  have revealed that chromatin remodeling events and transcription-factor binding are cyclical and periodic in nature. While the consequences of this for the reaction mechanism of chromatin remodeling are not known, the dynamic nature of the system may allow it to respond faster to external stimuli. A recent study indicates that nucleosome positions change significantly during mouse embryonic stem cell development, and these changes are related to binding of developmental transcription factors. [ 57 ]


Studies in 2007 have catalogued nucleosome positions in yeast and shown that nucleosomes are depleted in  promoter  regions and  origins of replication . [ 58 ] [ 59 ] [ 60 ]
About 80% of the yeast genome appears to be covered by nucleosomes [ 61 ]  and the pattern of nucleosome positioning clearly relates to DNA regions that regulate  transcription , regions that are transcribed and regions that initiate DNA replication. [ 62 ]  Most recently, a new study examined  dynamic changes  in nucleosome repositioning during a global transcriptional reprogramming event to elucidate the effects on nucleosome displacement during genome-wide transcriptional changes in yeast ( Saccharomyces cerevisiae ). [ 63 ]  The results suggested that nucleosomes that were localized to promoter regions are displaced in response to stress (like  heat shock ). In addition, the removal of nucleosomes usually corresponded to transcriptional activation and the replacement of nucleosomes usually corresponded to transcriptional repression, presumably because  transcription factor  binding sites became more or less accessible, respectively. In general, only one or two nucleosomes were repositioned at the promoter to effect these transcriptional changes. However, even in chromosomal regions that were not associated with transcriptional changes, nucleosome repositioning was observed, suggesting that the covering and uncovering of transcriptional DNA does not necessarily produce a transcriptional event. After transcription, the rDNA region has to protected from any damage, it suggested HMGB proteins play a major role in protecting the nucleosome free region. [ 64 ] [ 65 ]


DNA twist defects are when the addition of one or a few base pairs from one DNA segment are transferred to the next segment resulting in a change of the DNA twist. This will not only change the twist of the DNA but it will also change the length. [ 66 ]  This twist defect eventually moves around the nucleosome through the transferring of the base pair, this means DNA twists can cause nucleosome sliding. [ 67 ]  Nucleosome crystal structures have shown that superhelix location 2 and 5 on the nucleosome are commonly found to be where DNA twist defects occur as these are common remodeler binding sites. [ 68 ]  There are a variety of chromatin remodelers but all share the existence of an ATPase motor which facilitates chromatin sliding on DNA through the binding and hydrolysis of ATP. [ 69 ]  ATPase has an open and closed state. When the ATPase motor is changing from open and closed states, the DNA duplex changes geometry and exhibits base pair tilting. [ 68 ]  The initiation of the twist defects via the ATPase motor causes tension to accumulate around the remodeler site. The tension is released when the sliding of DNA has been completed throughout the nucleosome via the spread of two twist defects (one on each strand) in opposite directions. [ 69 ]


Nucleosomes can be assembled  in vitro  by either using purified native or recombinant histones. [ 70 ] [ 71 ]  One standard technique of loading the DNA around the histones involves the use of salt  dialysis . A reaction consisting of the histone octamers and a naked DNA template can be incubated together at a salt concentration of 2 M. By steadily decreasing the salt concentration, the DNA will equilibrate to a position where it is wrapped around the histone octamers, forming nucleosomes.  In appropriate conditions, this reconstitution process allows for the nucleosome positioning affinity of a given sequence to be mapped experimentally. [ 72 ]


A recent advance in the production of nucleosome core particles with enhanced stability involves site-specific  disulfide  crosslinks. [ 73 ]  Two different crosslinks can be introduced into the nucleosome core particle. A first one crosslinks the two copies of  H2A  via an introduced cysteine (N38C) resulting in  histone octamer  which is stable against H2A/H2B dimer loss during nucleosome reconstitution. A second crosslink can be introduced between the H3 N-terminal histone tail and the nucleosome DNA ends via an incorporated convertible nucleotide. [ 74 ]  The DNA-histone octamer crosslink stabilizes the nucleosome core particle against DNA dissociation at very low particle concentrations and at elevated salt concentrations.


Nucleosomes are the basic packing unit of genomic DNA built from histone proteins around which DNA is coiled. They serve as a scaffold for formation of higher order chromatin structure as well as for a layer of regulatory control of gene expression. Nucleosomes are quickly assembled onto newly synthesized DNA behind the replication fork.


Histones  H3  and  H4  from disassembled old nucleosomes are kept in the vicinity and randomly distributed on the newly synthesized DNA. [ 75 ]  They are assembled by the  chromatin assembly factor 1  (CAF-1) complex, which consists of three subunits (p150, p60, and p48). [ 76 ]  Newly synthesized H3 and H4 are assembled by the replication coupling assembly factor (RCAF). RCAF contains the subunit Asf1, which binds to newly synthesized H3 and H4 proteins. [ 77 ]   The old H3 and H4 proteins retain their chemical modifications which contributes to the passing down of the epigenetic signature. The newly synthesized H3 and H4 proteins are gradually acetylated at different lysine residues as part of the chromatin maturation process. [ 78 ]  It is also thought that the old H3 and H4 proteins in the new nucleosomes recruit histone modifying enzymes that mark the new histones, contributing to epigenetic memory.


In contrast to old H3 and H4, the old  H2A  and  H2B  histone proteins are released and degraded; therefore, newly assembled H2A and H2B proteins are incorporated into new nucleosomes. [ 79 ]  H2A and H2B are assembled into dimers which are then loaded onto nucleosomes by the nucleosome assembly protein-1 (NAP-1) which also assists with nucleosome sliding. [ 80 ]  The nucleosomes are also spaced by ATP-dependent nucleosome-remodeling complexes containing enzymes such as Isw1 Ino80, and Chd1, and subsequently assembled into higher order structure. [ 81 ] [ 82 ]


The crystal structure of the nucleosome core particle ( PDB :  1EQZ  [ 28 ] ) - different views showing details of histone folding and organization. Histones  H2A ,  H2B ,  H3 ,  H4  and  DNA  are coloured.


The  nucleosome repeat length , ( NRL ) is the average distance between the centers of neighboring  nucleosomes . NRL is an important physical  chromatin  property that determines its biological function. NRL can be determined genome-wide for the  chromatin  in a given cell type and state, or locally for a large enough genomic region containing several  nucleosomes . [ 1 ]


In  chromatin , neighbouring  nucleosomes  are separated by the  linker DNA  and in many cases also by the linker  histone H1 [ 2 ]  as well as non-histone proteins. Since the size of the nucleosome is typically fixed (146-147 base pairs), NRL is mostly determined by the size of the linker region between nucleosomes. Alternatively, partial DNA unwrapping from the  histone octamer  or partial disassembly of the  histone octamer  can decrease the effective nucleosome size and thus affect NRL.


Past studies going back to 1970s showed that, in general, NRL is different for different species and even for different cell types of the same organism. In addition, recent publications reported NRL variations for different genomic regions of the same cell type. [ 3 ]
 [ 4 ]  Recent works have compared the NRL around yeast transcription start sites (TSSs) in vivo and that for the reconstituted chromatin on the same  DNA  sequences in vitro. It was shown that ordered nucleosome positioning arises only in the presence of  ATP -dependent  chromatin remodeling . [ 5 ]  Furthermore, it was reported that the NRL determined around yeast TSSs is an invariant value universal for a given wild type yeast strain, although it can change when one of  chromatin remodelers  is missing. [ 6 ]  In general, NRL depends on the DNA sequence, concentrations of  histones  and non-histone  proteins , as well as long-range interactions between nucleosomes. [ 1 ]  NRL determines geometric properties of the nucleosome array, and therefore the higher-order packing of the DNA into the  chromatin fiber , [ 7 ]  which might affect  gene expression .


Nutriepigenomics  is the study of food  nutrients  and their effects on human health through  epigenetic  modifications.  There is now considerable evidence that nutritional imbalances during  gestation  and  lactation  are linked to  non-communicable diseases , such as  obesity ,  cardiovascular disease ,  diabetes ,  hypertension , and  cancer .  If metabolic disturbances occur during critical time windows of development, the resulting epigenetic alterations can lead to permanent changes in tissue and organ structure or function and predispose individuals to disease. [ 1 ]


Epigenetics  relates to heritable changes in  gene  function that occur independently of alterations in primary  DNA  sequence.  Two major epigenetic mechanisms implicated in nutriepigenomics are  DNA methylation  and  histone  modification.  DNA methylation in  gene promoter  regions usually results in  gene silencing  and influences gene expression.  While this form of gene silencing is extremely important in development and cellular differentiation, aberrant DNA methylation can be detrimental and has been linked to various disease processes, such as cancer. [ 2 ]   The methyl groups used in DNA methylation are often derived from dietary sources, such as folate and  choline , and explains why diet can have a significant impact on methylation patterns and gene expression. [ 3 ]   Gene silencing can also be reinforced through the recruitment of  histone deacetylases  to decrease transcriptional activation.  Conversely,  histone acetylation  induces transcriptional activation to increase gene expression.  Dietary components can influence these epigenetic events, thereby altering gene expression and disturbing functions such as appetite control, metabolic balance and fuel utilization. [ 1 ]


Various genetic sequences can be targeted for epigenetic modification.  A  transcriptome -wide analysis in mice found that a protein-restricted (PR) diet during gestation resulted in differential gene expression in approximately 1% of the fetal genes analyzed (235/22,690).  Specifically, increased expression was seen in genes involved in the  p53  pathway,  apoptosis , negative regulators of cell metabolism, and genes related to epigenetic control. [ 4 ]   Additional studies have investigated the effect of a PR-diet in rats and found changes in promoter methylation of both the  glucocorticoid receptor  and  peroxisome proliferator-activated receptor  (PPAR). [ 5 ] [ 6 ]  Altered expression of these receptors can result in elevated blood glucose levels and affect lipid and carbohydrate metabolism. [ 3 ]  Feeding a PR-diet to pregnant and/or lactating mice also increased expression of  glucokinase ,  acetyl-CoA carboxylase ,  PPARα , and  acyl-CoA oxidase . [ 7 ]   Changes in expression were reportedly due to epigenetic regulation of either the gene promoter itself, or promoters of transcription factors that regulate gene expression.  Additional genes that have been shown, either by  in vitro  or  in vivo  studies, to be regulated by epigenetic mechanisms include  leptin ,  SOCS3 ,  glucose transporter  (GLUT)-4,  POMC ,  11-β-hydroxysteroid dehydrogenase type 2  and  corticotrophin releasing hormone .  Epigenetic modification of these genes may lead to “metabolic programming” of the fetus and result in long-term changes in  metabolism  and energy homeostasis. [ 8 ]


The period of development in which the nutritional imbalance occurs is very important in determining which disease-related genes will be affected.  Different organs have critical developmental stages, and the time point at which they are compromised will predispose individuals to specific diseases. [ 9 ]   Epigenetic modifications that occur during development may not be expressed until later in life depending on the function of the gene. [ 3 ]  While the majority of studies implicate  prenatal  and  perinatal  periods as critical time windows, some research has shown that nutritional intake during  adulthood  can also affect the epigenome.


Developmental plasticity  is the process in which fetuses adapt to their environment.  Environmental cues, including dietary components, present in the  in utero  environment can induce significant changes in the expression of the genome through epigenetic modifications. [ 7 ]  Fetal developmental plastic responses can cause changes in lean body mass, endocrinology, blood flow and vascular loading, and lead to increased risk of various diseases in adulthood.


Fetal exposure to calcium, folate, magnesium, high or low protein, and zinc have all been associated with birth weight. [ 9 ]  Numerous studies have investigated the link between birth weight and risk of disease and have found that low birth weight is significantly associated with coronary heart disease, stroke and type-2 diabetes.  Most importantly, these associations occurred after adjusting for lifestyle factors, implying a genetic basis for onset of disease. [ 10 ]   Impaired  insulin  secretion is associated with low birth weight and can lead to insulin resistance as babies accumulate body fat. [ 11 ]  Studies using intrauterine growth retarded (IUGR) rats have found that growth inhibition can lead to decreased expression of Pdx1 transcription factor, which is essential for differentiation and function of pancreatic beta cells. [ 12 ]   Decreased histone acetylation at the proximal promoter of Pdx1 is responsible for reduced Pdx1 expression and subsequently results in a cascade of histone deacetylation and methylation events that can result in type-2 diabetes.


Obesity during pregnancy and high-fat maternal diets both show strong associations with obesity in offspring.  As the number of overweight reproductive-age women increases, the number of overweight children and infants also increases. [ 10 ]   It has been postulated that maternal obesity causes an accumulation of fat in fetal  adipose tissue  (adiposity) and predisposes babies for obesity in childhood and adulthood. [ 10 ]   Animal studies have shown that maternal overnutrition may impact brain development and cause disruptions to programming of the  hypothalamus .  Offspring that were exposed to a high-fat or high-caloric maternal diet had increased levels of insulin, glucose and leptin.  It is hypothesized that these elevations are due to disturbances in the complex neuronal network that includes the neuropeptide Y (NPY) and proopiomelanocortin (POMC) pathways. [ 8 ]   This altered neuronal signaling can consequently impact food-intake behavior and lead to diet-induced obesity in adulthood.  While epigenetic modifications are most likely involved in the development of obesity, the specific target genes have yet to be identified.  Genes involved in adipogenesis, such as fibroblast growth-factor-2, phosphatase and tensin homologue, cyclin-dependent kinase inhibitor 1A and oestrogen receptor-alpha, possess multiple  CpG islands  in their promoter sites and may act as epigenetic targets. [ 13 ]   Furthermore, it has been shown that prenatal exposure to a hypomethylating agent, such as  bisphenol A  (BPA), is associated with increased body weight and suggests modified DNA methylation as a mechanism for increasing susceptibility to obesity. [ 13 ]


It has long been realized that maternal folate intake during pregnancy is linked to fetal development and growth, and can reduce the risk of serious birth defects.  Folate is a source of  S-adenosyl methionine  (SAM), which is used to supply DNA methyltransferases with methyl groups.  Therefore, changes in folate supply have a substantial effect on DNA methylation patterns.  Low levels of folate are associated with an increased risk of preterm delivery, poor growth of the placenta and uterus, and intrauterine growth retardation. [ 3 ]   Several complex diseases, including cancer, cardiovascular diseases and  autism  have also been linked to maternal folate status.  Based on animal studies it has been hypothesized that reduced folate intake could increase the risk of  neural tube defects  by reducing the amount of methylayed DNA during cranial neural tube closure. [ 14 ]   Recently it was discovered that folate protection from congenital heart defects is linked to epigenetics and Wnt signaling.  Multiple environmental factors target the  Wnt signaling pathway  during embryogenesis and can cause misregulation of the pathway.   Folic acid  metabolism generates SAM, thereby altering the methylation states of histones H3K9, H3K4, and H3K27 and genetically altering Wnt signaling. [ 15 ]


Recently a  double-blind  placebo controlled trial of high dose  Folinic Acid  (Leucovorin Calcium) demonstrated efficacy at improving verbal communication in children with  autism . [ 16 ]


Another critical developmental time window is the perinatal period, the time period immediately before and after birth.   It has been shown that maternal diet in late pregnancy and an infant's diet in the beginning weeks can all have significant impacts on gene expression. Therefore, perinatal nutrition is both late-stage  in utero  nutrition and lactation.