Biological Understanding of Neurodevelopmental Disorders Based on Epigenetics, a New Genetic Concept in Education

Neurodevelopmental disorders, such as autism spectrum disorder, attention deficit hyperactive disorder, and learning disabilities, are heterogeneous conditions that are thought to have a multifactorial etiology including congenital genetic abnormalities and acquired environmental factors. Epigenetics is a biological mechanism that controls gene expression based on chemical modifications of DNA and chromosomal histone proteins. Environmental factors, such as severe mental stress, have been demonstrated to alter gene expression by changing epigenetic chemical modifications in the brain. Therefore, epigenetics is not only involved in congenital autism spectrum disorder-like conditions (e.g., Prader-Willi syndrome and Rett syndrome) but may also be involved in acquired attention deficit hyperactive disorder-like conditions (e.g., via child abuse and neglect). In this chapter, we introduce the basis of the epigenetic mechanism and the recent biological understanding of neurodevelopmental disorders based on epigenetics, which is a new genetic concept not only in medicine but also in education, which bridges internal brain mechanisms and external environmental factors.


Introduction
The number of children with autism spectrum disorder (ASD) is reportedly increasing by 10,000 cases per year in Japan [1], with similar increases observed in other countries, including the USA [2][3][4] and Korea [5]. These increases can be attributed, in part, to social factors, such as diagnostic substitution whereby children formerly diagnosed with mental retardation are now diagnosed as ASD. However, they cannot be explained fully by such diagnostic substitutions [6], and it is possible that biological changes in the brains of children may also play a role.
Thanks to advances in genomic DNA research, a number of genes associated with ASD have been identified. Mutations in genes encoding synaptic molecules, which facilitate communication between neuronal cells, have been identified in a subset of children with ASD [7,8]. However, the increase in ASD is unlikely to be simply a result of genetic factors because there is no reason to suspect that mutation Learning Disabilities 2 rates have suddenly increased in recent years. Rather, a more likely explanation is that environmental factors are involved.
Epigenetic mechanisms are one of the ways by which gene expression is controlled in higher vertebrates. These mechanisms are essential for normal development during embryogenesis [9] and for the differentiation of various types of cells including neural cells [10,11]. Therefore, it is important to gain an understanding of epigenetic mechanisms, which include chemical modifications of genetic components such as DNA, histone proteins, and microRNAs. Furthermore, the failure of epigenetic mechanisms results in neurodevelopmental disorders [12][13][14][15]. Actually, a number of congenital neurological and mental disorders are reportedly caused by epigenetic abnormalities [16][17][18][19][20][21][22].
Epigenetic modifications offer one mechanism by which environmental factors might lead to changes in population health [12]. This is partly supported by studies in twins showing that environmental factors contribute to the occurrence of autism [23][24][25]. These findings led us to propose the hypothesis that "various environmental factors can change the epigenetic status and alter the expression of a number of neuronal genes (namely synaptic genes), resulting in abnormal brain function (aberrant synaptic function) associated with some neurodevelopmental disorders." In this chapter, on the basis of such scientific evidence, we review the current understanding of congenital neurodevelopmental disorders caused by epigenetic abnormalities and also provide a basic description of acquired neurodevelopmental disorders caused by environment-induced epigenetic alterations. Finally, we discuss the future directions of medical and educational interventions for neurodevelopmental disorders (namely ASD).

Epigenetic abnormalities in "congenital" neurodevelopmental disorders
Epigenetic gene control is an essential mechanism for normal brain development. Abnormalities in the molecules associated with this process cause various congenital diseases. It is notable that defects in epigenetic phenomena and epigenetic molecules involved in gene regulation result in congenital neurological features and mental retardation. Here, we show four examples.

Genomic imprinting
Genes are believed to be expressed equally between the maternal and paternal chromosomes. However, an exceptional phenomenon, i.e., genomic imprinting, has been discovered that is the result of an epigenetic gene regulation mechanism. For an imprinted gene, one of the two parental alleles is active and the other is epigenetically inactivated (Figure 1A). Therefore, a defect in the active allele of the imprinted gene results in the loss of expression. This has been found in some neurodevelopmental diseases, including Angelman syndrome, which is characterized by severe mental retardation and epilepsy, and Prader-Willi syndrome, which is characterized by neurocognitive deficits, excessive daytime sleepiness, muscle hypotonia, short stature, small hands and feet, hypergonadism, hyperphagia and obesity that leads to type 2 diabetes [26].

X chromosome inactivation
The X chromosome has a large number of genes, whereas the Y chromosome has relatively few. Thus, females (XX) have more genes than males (XY). To minimize this sex imbalance, one of the two X chromosomes in females is inactivated by an epigenetic mechanism [27]. Improper X chromosome inactivation is thought to be an embryonic lethal condition [28,29].
When X chromosome inactivation does not occur in women with one normal X chromosome and a small X chromosome due to a large terminal deletion, an over-dosage effect of X-linked genes derived from the small X chromosome leads to severe neurodevelopmental delay [30]. This indicates that proper epigenetic gene regulation is essential for normal development ( Figure 1B).

DNA methyltransferases
DNA methylation is a fundamental step in epigenetic gene regulation that is regulated by DNA methyltransferases, which are enzymes that add a methyl group (CH 3 ) to CpG dinucleotides within human genomic DNA. A defect in a DNA methyltransferase causes ICF syndrome, which is characterized by immunodeficiency, centromere instability, facial anomalies, and mild mental retardation ( Figure 1C) [18][19][20].

Methyl-CpG-binding domain proteins
Methyl-CpG-binding domain proteins are also important molecules in the epigenetic control of gene expression. Abnormalities in the methyl-CpG-binding protein 2 (MECP2) gene cause Rett syndrome, which is characterized by seizures, ataxic gait, language dysfunction, and ASD-like behavior [21,22]. Therefore, abnormal MECP2 expression in the brain is considered to result in the neurological features of Rett syndrome. In fact, several studies have shown that MECP2 controls a subset of neuronal genes [29][30][31][32][33], suggesting that epigenetic dysregulation of neuronal genes may cause the neurological features of this disease ( Figure 1D).

Epigenetic abnormalities in "acquired" neurodevelopmental disorders
In neurodevelopmental disorders such as ASD, both environmental factors (e.g., environmental chemicals and infections) and genetic factors (e.g., defects in synaptic molecules) have historically been discussed [4,8]. However, the biological links between these two groups of factors have not been identified. Epigenetics may bridge these factors in normal and disease development [12].

Epigenetic bridge between genetic molecules and environmental factors
Besides intrinsic (congenital) epigenetic defects (described in Section 2), several lines of evidence suggest that extrinsic (environmental) factors, such as malnutrition [34,35], drugs [36][37][38][39][40], mental stress during the neonatal period [41], and neuronal stimulation [42], alter the epigenetic status, thereby affecting brain function. Therefore, it is intriguing to think that acquired neurodevelopmental disorders, including child abuse-and neglect-induced ADHD-like phenotypes, may be the result of epigenetic dysregulation caused by environmental factors (Figure 2).

Environmental factors that affect brain function via epigenetic mechanisms
Short-term mental stress after birth may alter gene expression in the brain and result in persistent abnormal behavior (Figure 2).
In rat pups from mothers exhibiting low levels of maternal care, an epigenetic DNA modification in the promoter of the glucocorticoid receptor gene was increased in the hippocampus, leading to the suppressed expression of this gene within the first week of life (Figure 3 right). Conversely, this promoter DNA modification was decreased in the brains of offspring who received high maternal care during the same period (Figure 3 left) [41].  This rat experiment provided a putative animal model for childhood neglect and maltreatment in humans. In fact, in a human study, postmortem analysis of the hippocampus of suicide victims with a history of childhood abuse revealed the hyper-modification of the neuron-specific promoter of the glucocorticoid receptor gene in combination with its decreased expression [43]. These findings suggest that the adverse effects of early-life stress on the DNA methylation program may last throughout life [44], and also indicate that neurodevelopmental problems may arise from epigenetic dysregulation caused by environmental factors in early life (Figure 3).
A similar epigenetic mechanism is also likely to be relevant in drug addiction. Gene expression in the dopaminergic and glutamatergic systems is mediated by epigenetic mechanisms, and cocaine and alcohol can alter the epigenetic state, which may be associated with permanent behavioral consequences [45,46].

Environment-induced epigenetic changes
The above findings were mainly obtained from animal studies, and there is little evidence from humans. However, the fact that epigenomic differences are larger in older monozygotic twins than in younger twins suggests that epigenetic status may be altered during aging by environmental factors in humans [47].
Likewise, the epigenomic patterns of monozygotic twins with discordant severity of Rett syndrome differ and they show differences in the expression of neuronal genes [25]. This indicates that environmental factors may alter the human epigenome and the resulting epigenomic differences may create phenotypic differences between twins.
Birth weight has decreased over the past 20 years, which is thought to be a result of the popularity of dieting among young women and of the recommendation by obstetricians to minimize pregnancy weight gain to reduce the risk of medical problems during pregnancy [48]. According to epidemiological studies of populations affected by famines in the Netherlands and China, offspring with low birth weight are expected to have an increased risk of not only metabolic disorders (e.g., obesity and diabetes mellitus) but also mental disorders [49][50][51]. Recent studies have demonstrated that malnutrition during the fetal period causes a hypomodification

Mental stress-induced DNA methylation changes. Maternal separation-stress during the first week of life induced hypermethylation in the glucocorticoid receptor gene promoter in the mouse hippocampus, and this environment-induced epigenetic changes persists life-time long with abnormal behavior.
of the peroxisome proliferator-activated receptor alpha (PPARa) gene in the rat liver [52]. Similar epigenetic changes have been identified in people who suffered malnutrition during a period of famine in the Netherlands [53]. The use of assisted reproductive technologies by women, which are now used widely due to increases in the age at which individuals wish to conceive, reportedly decreases the epigenetic modification of DNA at multiple maternally imprinted regions [54,55].

Medical interventions for epigenetics-associated neurodevelopmental disorders
The administration of folic acid to pregnant rats alters the DNA modification status of their offspring [56]. Furthermore, folic acid supplementation to pregnant rats under malnutrition conditions prevents the hypomodification of a hepatic gene in their offspring [57]. In addition to folic acid, various nutritional and other environmental factors, such as royal jelly [58], drugs for mental disorders [36,38,40], environmental chemicals [59,60], and external stimuli (electro-convulsive treatment for psychiatric diseases) [42], have also been demonstrated to alter the DNA or histone modification status of the brain.
As mentioned above, mental stress in the first week of life causes epigenetic abnormalities in the brains of mice. Conversely, several mouse studies have demonstrated that appropriate educational conditions may ameliorate the features of neurodevelopmental disorders. Environmental enrichment, consisting of larger-sized home cages with a variety of objects including running wheels, improves motor coordination and decreases anxiety-related behavior in female mice with an Mecp2 defect, a model of human Rett syndrome [61,62]. Environmental enrichment also improves locomotor activity with reduced ventricular volume, and restores the expression of synaptic proteins in the hypothalamus and syntaxin 1a and synaptotagmin expression in the cortex of the brain of these mice [63,64].
Children with congenital neurodevelopmental disorders caused by genetic defects are considered to be difficult to cure, because it is technically challenging to distribute gene products to the appropriate brain regions and at the appropriate time of development. However, it was recently demonstrated that Rett syndrome may be an exception, partly because MECP2 is not essential for brain structure, but rather encodes a "lubricant" that works at a relatively later period of brain development. As a consequence, the reintroduction of MECP2 into mice with a defect in Mecp2 after birth is sufficient to rescue Rett-like neurological symptoms [65,66]. Furthermore, the restoration of MECP2 function in astrocytes substantially improves locomotion, anxiety levels, and respiratory abnormalities in mice with a defect in Mecp2 [67]. These results suggest that the up-regulation of MECP2, possibly mediated by drug treatment, might help to improve the brain function of patients with Rett syndrome. Additionally, these results indicate that neurodevelopmental disorders caused by epigenetic abnormalities can be treated.

Evidence for epigenetic reversibility
Unlike DNA mutations, epigenetic modifications of DNA are reversible, since they are based on the attachment and detachment of chemical residues without any change to the DNA sequence. Therefore, environmental stress-induced epigenetic abnormalities are potentially reversible, and thus possibly treatable. Here, we show examples.
A mouse study demonstrated that chronic social defeat stress-induced epigenetic alterations can be reversed and brain-derived neurotrophic factor gene expression in the brain can be activated with a commonly used antidepressant (imipramine) by inducing histone acetylation via the down-regulation of histone deacetylases, which ameliorates depression-like behavior [36].
As mentioned above, malnutrition during the fetal period induces the chemical modification of PPARa in the peripheral blood of individuals who suffered malnutrition during a famine in the Netherlands [53] and in the liver of rats fed a proteinrestricted diet [52]. However, the protein-restricted diet-induced hypomethylation of PPARa in the offspring could be avoided by supplementation of the diet of maternal rats with folic acid (an essential substrate for methyl residues) [57].
Besides malnutrition, maternal smoking is known to have a negative impact on fetuses, e.g., stillbirth, low birth weight, and small for gestational age, and on offspring, e.g., sudden infant death syndrome, reduced lung function, bronchial asthma, and increased incidence of neurocognitive disorders, tobacco addiction, and obesity [68][69][70].
Recent cohort studies using cord blood samples originating from fetuses demonstrated that maternal smoking changes DNA methylation at several genes, including a CpG locus in the myosin IG (MYO1G) gene. Since MYO1G encodes a membrane protein of immune system-associated blood cells [71][72][73][74], epigenetic changes in DNA modification presumably down-regulate gene expression, which may be associated with a predisposition to bronchial asthma [73].
Whereas the epigenetic status of MYO1G is altered in individuals who smoke during pregnancy, this alteration is not found in individuals who stop smoking during pregnancy, suggesting that smoking-induced alterations in methylation can be reversed by smoking cessation or that they may be produced in a dose-dependent manner during pregnancy [72]. These findings further indicate that smoking cessation during pregnancy may be effective at preventing offspring from developing bronchial asthma (Figure 4). These findings also suggest that the epigenetic mechanism is reversible.

Epigenetic-based early educational intervention
In this chapter, we describe environmental stress-induced epigenetic alterations and their associated disorders. We also discuss the reversibility of the epigenetic mechanism to recover gene expression and potentially ameliorate disease conditions. As a number of molecules associated with epigenetic gene regulation have been identified, pharmacological companies are developing drugs to target these molecules with an aim to correct aberrant gene expression, especially for neurodevelopmental and psychiatric disorders [75].
Besides medical approach, "educational intervention" is another way taking advantage of use of epigenetic reversibility especially for children, because enriched nurturing environment that urged exercise and stimulated brain function ameliorated neurological features, which is demonstrated in a mouse model of Rett syndrome that is an autistic disorder caused by failure of epigenetic gene regulation as mentioned above [62][63][64]. Therefore, understanding of the epigenetic reversible concept is important for all staffs in a preschool and a nursery school, because they are the caregivers who will be able to urge development of children who had an adverse experience before and after birth, by offering appropriate nurture and education.

Conclusion
It was reported that the number of children with ASD is increasing in various countries including US and Japan. When we think of biological mechanism for this increase, one can imagine that some factors in recent society increased ASD via epigenetic mechanism based on chemical modification of DNA and histone proteins which control gene expression in the children's brain.
It has been known that abnormalities in epigenetic mechanisms lead to congenital neurodevelopmental disorders, such as Rett syndrome characterized by seizures, ataxic gait, language dysfunction, and ASD-like behavior.
Besides congenital epigenetic abnormalities, several lines of evidence suggest that environmental factors also alter the epigenetic status of brain-function associated genes. Therefore, it is intriguing to think that child abuse and neglect-induced ADHD-like phenotypes, which are thought to be increased in modern society, may be the result of epigenetic dysregulation caused by mental stress in early life.
Recent medical research demonstrated that some nutrients and drugs for mental illness reversed the epigenetic state and recover healthy physical and mental condition, and revealed that epigenetics is a reversible and thus treatable mechanism.
Besides such medical approach, "educational intervention" is another way taking advantage of use of epigenetic reversibility especially for children, because enriched nurturing environment that urged exercise and stimulated brain function ameliorated neurological features in mouse experiments. Therefore, epigenetics, described in this chapter, will be essential concept that contribute to future nurture and education.
In conclusion, epigenetics becomes a new genetic concept not only in medicine but also in education, which bridges internal brain mechanisms and external environmental factors.