Epigenetics and Neuroinflammation Associated With Neurodevelopmental Disorders: A Microglial Perspective

Neuroinflammation is a cause of neurodevelopmental disorders such as autism spectrum disorders, fetal alcohol syndrome, and cerebral palsy. Converging lines of evidence from basic and clinical sciences suggest that dysregulation of the epigenetic landscape, including DNA methylation and miRNA expression, is associated with neuroinflammation. Genetic and environmental factors can affect the interaction between epigenetics and neuroinflammation, which may cause neurodevelopmental disorders. In this minireview, we focus on neuroinflammation that might be mediated by epigenetic dysregulation in microglia, and compare studies using mammals and zebrafish.

Rodents have been successfully used to analyze the role of microglia in neuroinflammation associated with NDDs (Johnson and Kaffman, 2018). In mice, primitive microglia derived from yolk sac progenitors (erythromyeloid precursors) migrate into the brain around embryonic day (E) 9.5, where they differentiate into microglia, colonize various brain regions, and regulate neurodevelopment (Prinz et al., 2019;Stratoulias et al., 2019;Thion and Garel, 2020;Sharma et al., 2021). The entry of primitive macrophages and colonization of the brain are also conserved in zebrafish, an alternative animal model for various diseases, including NDDs Réu et al., 2017;Ferrero et al., 2018;Bian et al., 2020;Neely and Lyons, 2021). When the second wave of hematopoiesis occurs in mice, microglia progenitors expressing homeobox B8 are generated in the yolk sac, are present in the aorta-gonad-mesonephros (AGM) and fetal liver, and seed into the brain around E12.5 (De et al., 2018). In zebrafish, definitive hematopoiesis begins 15 days postfertilization in the ventral wall of the dorsal aorta, which is the analogous region of AGM in mammals, leading to the formation of adult microglia in the brain Ferrero et al., 2018). Embryonic microglia derived from primitive macrophages gradually disappear in zebrafish Ferrero et al., 2018;Sharma et al., 2021). In mice, microglia derived from both primitive and definitive hematopoiesis coexist in the adult brain . Despite these differences, the core microglial gene expression signature and microglial functions, such as immune surveillance, cellular debris cleaning, response to injury, and integration with neural circuits, are conserved between mammals and zebrafish (Mazzolini et al., 2020;Neely and Lyons, 2021).
In this minireview, we describe our current understanding of the interaction between epigenetics and neuroinflammation, focusing on microglia in relation to miRNA-124 and 153 (miR124 and miR153), methyl-CpG binding protein 2 (MECP2), and ubiquitin-like with PHD and ring finger domains 1 (UHRF1) (Figure 1). We also compare studies using mammals and zebrafish to provide a future direction for zebrafish-based research on the epigenetic regulation of microglia and neuroinflammation in NDDs (Table 1).

MIR124 AND MIR153
miRNAs play important roles in the regulation of neurodevelopment by modulating the expression of target genes via binding to the 3′-untranslated regions (Thomas et al., 2018). miRNAs are also involved in microglial function (Butovsky and Weiner, 2018;Cheray and Joseph, 2018;Guo et al., 2019;Qiu M. et al., 2021;Zhao et al., 2021). The expression of these miRNAs is epigenetically regulated by prenatal exposure (Knopik et al., 2019;Sushma et al., 2021). Prenatal cocaine exposure (PCE) dysregulates DNA methylation and the expression of miRNAs that are important for the neurodevelopment of offspring (Lambert and Bauer, 2012;Richardson et al., 2015;Vaillancourt et al., 2017). For example, PCE in mice can cause hypermethylation of insulin growth factor II (Igf2), leading to decreased expression of Igf2 in the hippocampus of offspring and impairment of cognitive function (Zhao et al., 2015). PCE also downregulates miR124 in microglia through promoter hypermethylation in mice (Guo et al., 2016). The cocaine-mediated downregulation of miR124 in rat primary microglia leads to increased expression of target genes, including Toll-like receptor 4 (TLR4) and signal transducer and activator of transcription 3, and aberrant activation of microglia (Periyasamy et al., 2018;Chivero et al., 2020). Inhibition of miR124 also activates microglia in zebrafish (Svahn et al., 2016). These studies suggest that the antiinflammatory role of miR124 in microglia is conserved between mammals and zebrafish.
Exposure to ethanol during development can have deleterious effects on various cell types, including neurons, oligodendrocytes, astrocytes, and microglia, depending on the dose and timing of The inhibition of miR124 activates zebrafish MG. Guo et al. (2016) The decrease of miR124 caused by cocaine exposure posttranscriptionally increases the expression of TLR4 and STAT3 in rat MG. The addition of miR153 mimic suppress the expression of Tnf in mouse MG.
The expression of miR153c is decreased in zebrafish embryo exposed to ethanol Qiu et al. (2021b) Knockdown of miR153c causes the phenotype similar to those of zebrafish exposed to ethanol exposure and the brain region (Wilhelm and Guizzetti, 2016;Wong et al., 2017;Stratoulias et al., 2019;Almeida et al., 2020;Kane and Drew, 2021;Lussier et al., 2021). The expression of miR153 is decreased in mouse fetal cerebral cortical-derived neural progenitor cells exposed to ethanol (Balaraman et al., 2012). In microglia located in the hypothalamus of a rat FAS model, the expression is increased (Chastain et al., 2019). TNF secreted from microglia exposed to ethanol can cause neuronal apoptosis and neuroinflammation (Boyadjieva and Sarkar, 2010;Shrivastava et al., 2017). The addition of an miR153 mimic to mouse microglia suppresses the production of TNF (Qiu T. et al., 2021). In zebrafish exposed to ethanol from 4 to 24 h postfertilization (hpf), the expression of miR153c, a zebrafish homolog of miR153, was decreased (Tal et al., 2012). Knockdown of miR153c causes phenotypes similar to those of zebrafish exposed to ethanol from 4 to 24 hpf (Tal et al., 2012). Supplementation with folic acid rescued developmental defects in zebrafish FAS models (Muralidharan et al., 2015;Jiang et al., 2020) and ameliorated the dysregulation of miRNA in a mouse FAS model (Wang et al., 2009). Folic acid also affects DNA methylation (Crider et al., 2012). Cocaine exposure decreases the expression of miR153 in a human neuroblastoma cell line (Cabana-Domínguez et al., 2018). These studies suggest that prenatal substance exposure may affect promoter methylation of miR153 and decrease its expression in both mammalian and zebrafish microglia, leading to neuroinflammation.

MECP2
Mutation in MECP2 is the most prevalent cause of Rett syndrome, a progressive NDD with ASD-like features (Amir et al., 1999;Fagiolini et al., 2020). MECP2 is a DNA methylation reader with two major domains: a methyl-binding domain and a transcriptional repressor domain (Fagiolini et al., 2020). MECP2 has a high affinity for methylated CpG (mCG), methylated CpA (mCA), and hydroxymethylated CpA (hmCA), but not for hydroxymethylated CpG (hmCG) (Ip et al., 2018;Connolly and Zhou, 2019;Lavery and Zoghbi, 2019;Tillotson and Bird, 2019). An integrative genome-wide analysis of the methylome and transcriptome using brains from patients with Rett syndrome, idiopathic ASD, and controls revealed that genes associated with the differentially-methylated regions in these NDDs compared with the controls showed significant enrichment in genes regulated during microglial development (Vogel Ciernia et al., 2020). Transcriptome analyses using mouse models have revealed that Mecp2 deficiency causes dysregulation of the microglial inflammatory response (Cronk et al., 2015;Zhao et al., 2017). Mecp2-null microglia also show increased uptake of glutamate, leading to an increase in mitochondrial reactive oxygen species and a decrease in mitochondrial ATP production in mice . These findings are consistent with other studies demonstrating the dysregulation of neuroinflammation and microglial/macrophage functions in Rett syndrome and ASD (Voineagu et al., 2011;Gupta et al., 2014;O'Driscoll et al., 2015;Parikshak et al., 2016;Schafer et al., 2016;Nance et al., 2017;Kahanovitch et al., 2019;Pecorelli et al., 2020;Marballi and Macdonald, 2021;Wittrahm et al., 2021). Furthermore, these findings suggest that dysregulation of microglia and peripheral immune cells may play pathogenic roles in NDDs and serve as therapeutic targets (Garay and Mcallister, 2010;Reemst et al., 2016;Kaur et al., 2017;Komada et al., 2017;Coomey et al., 2020). In a zebrafish model of Rett syndrome, a premature stop codon has been introduced before the methyl-binding domain of mecp2 (Pietri et al., 2013). This zebrafish Rett syndrome model shows increased expression of inflammatory cytokines, impaired locomotion, and decreased anxiety-like behavior, which may be associated with the phenotypes observed in patients with and rodent models of Rett syndrome (Pietri et al., 2013;Van der Vaart et al., 2017). Proteomic analysis using the zebrafish Rett model found that proteins associated with ATP generation and skeletal muscle are dysregulated, which may be associated with impaired motor behaviors in the model (Pietri et al., 2013;Cortelazzo et al., 2017). These findings suggest that MECP2 function is well conserved between zebrafish and mammals. It should be noted, however, that the total number of mpx-positive neutrophils, but not mpeg-positive microglia/macrophages in the body, is increased in the zebrafish model of Rett syndrome (Van der Vaart et al., 2017). Thus, the role of mecp2 in zebrafish microglia remains unclear.

UHRF1
The microbiome is involved in the development and maintenance of microglia (Stilling et al., 2014;Erny et al., 2015;Thion et al., 2018;Wang et al., 2018;Erny and Prinz, 2020;Davoli-Ferreira et al., 2021). The densities of microglia in the somatosensory cortex and striatum of germ-free (GF) mice were significantly higher than those of specific-pathogen-free (SPF) mice at E14.5 and E16.5 (Thion et al., 2018). In adults, microglia of GF mice show deficits in the signaling of type I interferon receptors and polarization towards specific phenotypes (Erny et al., 2015). The impairment of microglial maturation is also caused by temporal eradication of the host microbiota or limited microbiota complexity in SPF mice, whereas recolonization with a complex microbiota or supplementation with short-chain fatty acids (SCFA) restores microglial function in GF mice (Erny et al., 2015). SCFA, such as butyrate, propionate, and pyruvate, show inhibitory effects on HDAC activity, suggesting that the function of microglia may be epigenetically regulated by SCFA-producing microbes through the modulation of histone acetylation (Stilling et al., 2014;Fung et al., 2017). Consistent with this idea, genomewide analysis of chromatin accessibility revealed that there are differentially accessible regions between microglia in GF and SPF mice (Thion et al., 2018). Dysregulation of maternal microbiota caused by maternal infection and exposure to environmental factors during pregnancy can disrupt microglial function and fetal brain development, leading to NDDs (Davoli-Ferreira et al., 2021). The innate immunity regulated by commensal microbiota is conserved in zebrafish (Murdoch and Rawls, 2019).
UHRF1 is a RING E3 ubiquitin ligase that interacts with DNMT1 to copy pre-existing mCG to newly synthesized Frontiers in Cell and Developmental Biology | www.frontiersin.org May 2022 | Volume 10 | Article 852752 daughter strands during replication (Li et al., 2021). In mice, knockout of Uhrf1 decreases mCG at the Tnf promoter and increases the expression of Tnf in macrophages, which causes colitis, a type of inflammatory bowel disease (IBD) (Qi et al., 2019). In zebrafish, knockout of uhrf1 decreases mCG at the tnf promoter and increases the expression of tnf in intestinal epithelial cells, leading to IBD-like intestinal damage (Marjoram et al., 2015). Knockout of dnmt1 also increases the expression of tnf in intestinal epithelial cells (Marjoram et al., 2015). In both models, blocking TNF ameliorates IBD-like phenotypes (Marjoram et al., 2015;Qi et al., 2019). Intestinal damage activates peripheral immune cells, including T H 17 cells and macrophages, leading to breakdown of the blood-brain barrier and dysfunction of microglia (Fung et al., 2017;Wang et al., 2018;Abdel-Haq et al., 2019;Davoli-Ferreira et al., 2021). These findings suggest that zebrafish is a useful tool for analyzing the gut-microglia connection associated with epigenetics and NDDs.

DISCUSSION
In addition to the examples discussed above, several studies have demonstrated conserved functions of microglia associated with NDDs in mammals and zebrafish. Leukodystrophies are a group of NDDs characterized by white matter abnormalities (Van der Knaap and Bugiani, 2017). The clinical symptoms include cerebral palsy and cognitive decline (Van der Knaap and Bugiani, 2017). Microglial dysfunction plays an important role in the etiology of leukodystrophy (Garcia et al., 2020;Berdowski et al., 2021). Homozygous mutations in colony-stimulating factor 1 receptor (CSF1R) cause pediatric onset leukoencephalopathy (Oosterhof et al., 2019). Homozygous knockout of CSF1R homologs in rats and zebrafish causes a lack of microglia in the brain, which is consistent with the findings in humans (Oosterhof et al., 2018;Oosterhof et al., 2019;Patkar et al., 2021). Loss of function mutations in ribonuclease T2 (RNASET2) cause early onset leukoencephalopathy resembling congenital cytomegalovirus brain infection in humans (Henneke et al., 2009). Homozygous knockout of RNASET2 homologs in mice and zebrafish causes abnormal activation of microglia and increased expression of interferon-stimulated genes in the brains Kettwig et al., 2021;Rutherford et al., 2021). These results warrant further examination to reveal the epigenetic mechanisms underlying leukoencephalopathies using zebrafish models. Microglia can acquire a specific phenotype depending on the context , and epigenetics play an important role in the plasticity of microglia (Cheray and Joseph, 2018;Martins-Ferreira et al., 2021). For example, upon stimulation with lipopolysaccharide (LPS), the enhancer of zeste homolog 2, a component of polycomb repressive complex 2 (Prc2), which has histone methyltransferase activity, is increased in mouse microglia, leading to an increase in tri-methylation of histone H3 lysine 27 (H3K27) and pro-inflammatory gene expression through toll-like receptor-induced activation of nuclear factor κB (Nfkb1) (Arifuzzaman et al., 2017;Zhang et al., 2018). The Nfkb1 activation by LPS-TLR4 signaling increases the expression of tet methylcytosine dioxygenase 2 (TET2) and stimulates the expression of LPS-mediated pro-inflammatory cytokines in mouse microglia (Carrillo-Jimenez et al., 2019). TET2 catalyzes the oxidation of 5-methylcytosine (5mC) to 5hydroxymetylcytosine (5 hmC) (Macarthur and Dawlaty, 2021). 5mC is established de novo by two DNA methyltransferases, DNMT3A/B and maintained by DNMT1 (Wu and Zhang, 2014;Lavery and Zoghbi, 2019). Activation of TET2 and/or inhibition of DNMT3A/B decreases mCG levels, resulting in the detachment of MECP2 from genomic mCG sequences (Ip et al., 2018). DNMT3A haploinsufficiency in mice causes behavioral abnormalities and epigenomic dysregulation that overlap with Rett syndrome and ASD (Christian et al., 2020). Notably, the expression of Mecp2, Uhrf1, Tet2, Dnmt1, and Dnmt3a is also dysregulated in various rodent FAS models (Chen et al., 2013;Kim et al., 2013;Nagre et al., 2015;Varadinova and Boyadjieva, 2015;Veazey et al., 2017;Boschen et al., 2018;Alberry et al., 2021;Lussier et al., 2021). Epigenetic dysregulation caused by exposure to environmental chemicals during development may cause neuroinflammation and NDDs through polarization of microglia into pro-inflammatory phenotypes. Zebrafish are wellsuited for analyzing the epigenetic effects of developmental chemical exposure (Aluru, 2017;Cavalieri and Spinelli, 2017).
The phenotypes of microglia are also dependent on the region in which they colonize Thion and Garel, 2020). In mice, microglia located in the cerebellum show higher clearance activity than those located in the cerebral cortex or striatum (Ayata et al., 2018). In microglia located in the cerebellar cortex or striatum, PRC2 causes trimethylation of H3K27, resulting in the suppression of gene expression related to clearance activity (Ayata et al., 2018). Regional differences in microglial phenotypes have also been observed in zebrafish (Silva et al., 2021). Epigenetic regulation during development, as well as the ontogeny and function of microglia, is relatively well conserved between zebrafish and mammals (Balasubramanian et al., 2019), making zebrafish a suitable model for analyzing the association between epigenetics, neuroinflammation, and NDDs.

AUTHOR CONTRIBUTIONS
MK and YN planned and wrote the manuscript.

FUNDING
This work was supported in part by the Japan Society for the Promotion of Science KAKENHI (17K08500 to MK and 19K07318 to YN) and the Long-Range Research Initiative of the Japan Chemical Industrial Association (20-3-08 to YN).