Schizophrenia is a serious and debilitating neurodevelopmental disorder that typically occurs in early adulthood. Patients experience hallucinations, delusions, disorganization, and deterioration in cognitive and social functioning, such as amotivation and social withdrawal [1]. The average lifetime prevalence of schizophrenia is just under 1% [2]. Individuals from all ages with schizophrenia have high mortality and have their life expectancy reduced by approximately 20 years compared with the general population [3]. Alterations in cortical circuits have been reported to be involved in the pathogenesis of schizophrenia. The loss of dendritic spines in pyramidal cells results in reduced excitatory activity, while, at the same time, reduced excitatory input to GABAergic interneurons results in reduced inhibition in pyramidal cells. These changes are thought to lead to an abnormal functional network [4].

The heritability of schizophrenia is as high as 80% [5]. Genetic factors, including copy number variants [6] and de novo mutations [7], have been identified to be closely associated with the development of schizophrenia. Interestingly, studies of monozygotic twins have revealed the heterogeneity of schizophrenia, suggesting that non-genetic factors may be involved in the pathogenesis of schizophrenia. Environmental factors can induce enduring changes in gene expression through epigenetic mechanisms in neurodevelopment processes, aging, and neurodegenerative diseases [8]. DNA methylation, a critical epigenetic modification, contributes to alter gene expression without affecting the underlying genomic sequences; 5-methycytosine (5mC) and 5-hydroxylcytosine (5hmC) are two major forms of DNA methylation in mammals. DNA methyltransferases (DNMTs) transfer a methyl group to the 5-position of cytosine to form 5mC; the ten-eleven translocation (TET) dioxygenases catalyze the 5mC to 5hmC, 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) on DNA.

Altering the expression or activity of DNA methylation-related enzymes produce exacerbated phenotypes or therapeutic effects in different animal models. Cathrin Bayer et al. found that the depletion of DNMT1 increased the survival of mutant Huntington-transfected cells and accelerated perinuclear Huntington aggregation, suggesting that DNMT1-dependent degradation pathways mediate mutant Huntington-induced cytotoxicity [9]. Growing evidence suggests that epigenetic responses to environmental stimuli and plays an important role in schizophrenia [10]. By mapping thousands of 5mC sites in the prefrontal cortex of schizophrenia patients and healthy controls, differentially methylated regions (DMRs) at genes related to development and neurodifferentiation have been identified [11]. Disruptions of genome-wide 5mC have been observed in peripheral blood mononuclear cells (PBMCs) of schizophrenia patients, particularly in those experiencing their first episode of disease [12]. Despite inconsistent results from genome-wide association studies (GWAS), 108 replicable genomic loci have been identified in 2014 [6].

2. Altered 5mC Pattern in Schizophrenia

It is known that 5mC is the primary product of DNA methylation, which can influence various processes such as gene activity, individual development, and cancer progression ^[13][14][13,14]. DNMT1, DNMT3A, and DNMT3B possess catalytic activity and covalently add a methyl group from S-adenosylmethionine (SAM) onto the fifth carbon of the cytosine (C) pyrimidine ring, resulting in the formation of 5mC. While DNMT2 and DNMT3L contain conserved sequences of DNMT enzymes, they lack catalytic activity ^[15][16][15,16]. During late embryonic development and after maturation, genomic methylation patterns are established, and DNMT1 is responsible for maintaining the genomic methylation status. The DNMT1 is approximately one hundred times more active than DNMT3A and DNMT3B [17]. Abnormalities of 5mC have been widely reported in neurodegenerative diseases, such as Parkinson’s disease (PD) and Huntington’s disease (HD) ^[18][19][20][18,19,20]. Psychiatric disorders are characterized by impairment in cognition, emotion regulation, or behavior, and typically include bipolar disorder (BD) and schizophrenia. The pathogenesis of BD involves mitochondrial dysfunction, DNA oxidative damage, and DNA methylation changes. BD patients and their relatives exhibit genome-wide decreases in 5mC levels. After lithium treatment, 5mC levels remained unchanged in BD patients, whereas the levels were increased in their relatives, providing evidence of the significance of 5mC modification in BD pathology ^[21][22][23][21,22,23]. The overexpression of DNMT1 was considered to be one of the etiological factors of schizophrenia. Transcriptional dysregulation has been observed in embryonic stem cells overexpressing DNMT1. Approximately 50% of these genes have been implicated in schizophrenia, showing dysregulation independent of DNA methylation ^[24][25][24,25]. A novel GWAS in patients with schizophrenia identified schizophrenia-associated differential 5mC at 242 sites, of which mitotic arrest deficient 1-like 1 (MAD1L1) was robustly differentially methylated [26]. Alongside GWAS, a growing number of studies aim to identify the 5mC of candidate genes in patients with schizophrenia [27]. Specific 5mC differences have also been found in the gray and white matter of individuals with schizophrenia. These DMRs were identified within or near the Kruppel-like factor 9 (KLF9), sideroflexin 1 (SFXN1), Sprouty-related EVH1 domain-containing 2 (SPRED2), and AlS2 C-terminal-like (ALS2CL) genes [28]. Other key genes with dynamic 5mC were also involved in the pathogenesis of schizophrenia (Table 1). Reelin protein is a signaling molecule that plays a crucial role in the nervous system. In humans, the dysregulation of Reelin protein is associated with various neurological disorders, including BD, autism, depression, and schizophrenia ^[39][40][39,40]. It is encoded by the RELN gene and was originally discovered in recessive mutant mice. The abnormal expression of this protein can lead to manifestations such as gait disturbances, ataxia, and tremors [39]. Reelin protein regulates the adhesion and migration of nerve cells by activating receptors on the cell surface, thereby promoting the proper positioning and layering of cerebral cortex neurons [39]. In humans, the dysregulation of Reelin protein is associated with various neurological disorders, including schizophrenia, BD, autism, depression, and more ^[39][40][39,40]. Reelin was found to be altered in various animal models of schizophrenia, suggesting its key role in the pathogenesis of schizophrenia [41]. Reelin levels were significantly reduced in schizophrenia patients, with specific gender differences [42]. The reduction of Reelin in schizophrenia was attributed to a significant increase in 5mC levels at the RELN promoter, with unmethylated Reelin expressed 25-fold higher than methylated Reelin ^[29][30][29,30]. Therefore, 5mC is an important factor in regulating RELN expression, and exploring the precise mechanism of the methylation of RELN may help elucidate the pathogenesis of schizophrenia. Brain-derived neurotrophic factor (BDNF) regulates neurogenesis and synaptic plasticity as a biomarker for neuropsychiatric disorders [31]. such as bipolar disorder and major depressive disorder, and alterations in BDNF levels, the imbalance between pro-BDNF and m-BDNF, and defects in the BDNF signaling pathway have been observed [43]. When the BDNF Val66Met was knocked into mice, BDNF expression was significantly decreased, resulting in reduced neurogenesis and structural abnormalities in the hippocampus ^[44][45][44,45]. In patients with chronic schizophrenia, BDNF levels are closely related to memory, attention, information-processing speed, and cognitive impairment [46]. The 5mC of the BDNF gene could be regulated by environmental conditions and was believed to be involved in the pathogenesis of schizophrenia. Using bisulfite sequencing, no differences were found in BDNF 5mC status between schizophrenia patients and healthy controls, but there was a correlation between disease progression and 5mC [47]. An analysis of postmortem brains from schizophrenia patients revealed elevated levels of 5mC and 5hmC in the BDNF-regulatory regions in the frontal cortex and hippocampus, including the promoter regions, suggesting a strong association between BDNF methylation and schizophrenia [31]. Furthermore, schizophrenia patients with the same genotype and alleles exhibited different symptoms, which may be related to the differences in BDNF expression levels. Additionally, BDNF levels may differ in schizophrenia patients treated with different drugs [48]. The specific mechanisms underlying schizophrenia is still completely unclear, but classical theories believe that serotonin, glutamate, and dopamine networks are closely associated with the onset of schizophrenia [49]. Glutamate decarboxylase 1 (GAD1) is an important factor in the glutamate metabolism network; it plays a crucial role in conditions such as attention deficit/hyperactivity disorder (ADHD), heroin addiction, early infantile epilepsy, and developmental delay ^[50][51][52][50,51,52]. In patients with schizophrenia, 5mC levels in the promoter region of GAD1 were reduced, resulting in a significant increase in the overall expression of GAD1 ^[37][53][37,53]. The COMT gene encodes catechol-O-methyltransferase and is involved in the regulation of the dopamine metabolism network, it is involved in treatment-resistant depression, borderline personality disorder, post-traumatic stress disorder, and other mental disorders ^[54][55][56][54,55,56]. In patients with schizophrenia, the 5mC levels of the COMT gene are elevated, resulting in a significant increase in mRNA expression. The dysregulation of COMT expression has been demonstrated in the pathophysiology of schizophrenia ^[38][57][58][38,57,58] Dopamine transporter (DAT) is encoded by the Solute Carrier Family 6 Member 3 (SLC6A3) gene and is located on the neuronal cell membrane. It is responsible for the reuptake of dopamine from the synaptic cleft into neurons, thereby regulating the concentration and duration of the dopamine action in neurons. Therefore, it plays a role in emotional control, reward regulation, and other processes ^[59][60][59,60]. Moreover, 5mC levels in the DAT 5′-UTR of PD patients were different from healthy controls, and this methylation level may be related to different stages of the disease [61]. Cognitive impairment is one of the symptoms in schizophrenia patients. DAT availability in the striatum has been showed to be positively correlated with cognitive function in schizophrenia patients. Reduced DAT availability was primarily associated with increased 5mC levels ^[32][33][32,33]. These findings indicated that DNA methylation regulates key genes in the dopamine-network-involved pathological progress of schizophrenia. Dystrobrevin-binding protein 1 (DTNBP1) is also highly associated with schizophrenia [34]. DTNBP1 is considered to be one of the key genes in the glutamatergic system; it also plays a role in depression and temporal lobe epilepsy ^[62][63][62,63]. It can interact with BDNF to regulate activity-dependent BDNF secretion, neural development, and inhibitory circuit function. Abnormalities in the DTNBP1 pathway may lead to schizophrenia ^[64][65][64,65]. In rodents, the reduced expression of DTNBP1 resulted in abnormal neuronal growth and morphology. Additionally, DTNBP1 plays a role in schizophrenia by regulating neurotransmission through the modulation of N-methyl-D-aspartate receptors (NMDAR) and dopamine D2 receptors (DRD2) [34]. It has been reported that female schizophrenia patients have higher 5mC levels at the upstream CpG sites of DTNBP1. This result was consistent with the observed downregulation of DTNBP1 mRNA levels in schizophrenia patients. Furthermore, investigations of saliva and postmortem brain samples from schizophrenia patients have revealed increased levels of 5mC in the DTNBP1 promoter, suggesting the potential role for 5mC on DTNBP1 in schizophrenia ^[35][36][35,36]. To sum up, the above related 5mC changes are summarized in Figure 1.

3. Altered 5hmC Pattern in Schizophrenia

In as early as 1952, 5hmC was discovered in bacteriophage DNA [66] and was first detected in mammalian genomes in 1972 [67]. As of 2009, the TET family has been identified to convert 5mC to 5hmC both in vivo and in vitro ^[68][69][68,69]. The TET protein family includes three members, TET1, TET2, and TET3, which have a conserved double-stranded β-helix domain (DSBH) at their C-terminus, which contains α-ketoglutarate (α-KG)- and Fe(II)-binding sites. Additionally, there is a cysteine-rich domain (CD) that contains a high concentration of cysteine residues. Together, these two domains form the core catalytic domain, which mediates the oxidation reaction of 5mC to 5hmC [70]. The distribution of 5hmC in animal tissues is widespread but variable, displaying certain tissue specificity ^[71][72][73][71,72,73]. It has been found, through 5hmC profiling in mammals, that 5hmC is mainly enriched in the central nervous system (CNS), with lower levels in peripheral tissues, which is in sharp contrast to the global distribution of 5mC ^[73][74][73,74]. In the brain, 5hmC exhibits region-specific distribution, with a unique distribution pattern in the cerebellum ^[75][76][75,76]. This suggests that 5hmC may play an important role in tissue-specific functions. Furthermore, 5hmC levels increase during neuronal differentiation and are associated with genes critical for neuronal function during neurodevelopment [77]. In addition, 5hmC is involved in processes of cell development and differentiation, the regulation of chromatin structure, and bone marrow regeneration ^[78][79][80][78,79,80]. In the process of DNA methylation regulation, the epigenetic plasticity of the CNS affects the expression of neuroactivity-dependent genes, organism learning, and memory ^[81][82][81,82]. It is well-known that 5mC is considered a stable heritable modification and represents a static process in epigenetic regulation. In fact, the dynamic balance between 5mC and 5hmC is an important condition for organism homeostasis, and imbalances in their ratio are implicated in the pathogenesis of many neurodegenerative diseases ^{[78][79][83][84]}[78,79,83,84]. Abnormalities in 5hmC have been reported to be widespread in patients and animal models of AD and PD. At the same time, it was found that dynamic changes in 5hmC were positively correlated with aging ^[76][80][76,80]. Genome-wide DNA methylation changes may lead to genomic instability, and DNA methylation changes at promoter regions typically affect gene transcription. In schizophrenia, genome-wide 5hmC levels were elevated in male patients and reduced in female patients compared with healthy individuals [10], suggesting a potential association between the dysregulation of 5hmC levels and the development of schizophrenia. Many candidate genes in the peripheral blood or postmortem brain from schizophrenia patients have been identified with the differential methylation status associated with schizophrenia ^[11][85][11,85], including nitric oxide synthase 1 (NOS1), AKT serine/threonine kinase 1 (AKT1), DTNBP1, DNMT1, protein phosphatase 3 catalytic subunit gamma (PPP3CC), glutamic acid decarboxylase 67 (GAD67), and sex-determining region Y-box containing gene 10 (SOX10) ^[86][87][86,87]. These genes exhibit varying degrees of changes in 5mC or 5hmC (Figure 1 and Table 2). In the DNA demethylation pathway, apolipoprotein B mRNA-editing enzymes (APOBEC) are an important lipid carrier protein and participate in the conversion of 5hmC to 5fC and 5caC. TET1 expression was increased, while APOBEC3A and APOBEC3C expression was decreased in the frontal cortex of schizophrenia patients, suggesting that the disruption of the DNA methylation and demethylation pathways may contribute to elevated 5hmC levels in schizophrenia patients [87]. In addition, it has also been found that the increase in 5hmC occurring in the promoter region of the BDNF gene was associated with a reduced expression of BDNF in the frontal cortex of patients with schizophrenia [89]. Increased levels of 5mC and 5hmC in the BDNF promoter reduced the binding of GADD45b protein in the associated chromatin regulatory region and further reduced transcription levels in schizophrenia patients [89]. The dysregulation of methylation levels in schizophrenia-related genes plays a crucial role in the development of schizophrenia. However, research on 5hmC changes in schizophrenia remains limited. Further studies are needed to elucidate the detailed mechanisms by which 5hmC contributes to schizophrenia.