Choline metabolism is situated within the greater one-carbon metabolic pathway and is closely linked to the methionine cycle. Choline, methionine, and folate are key dietary nutrients that function as methyl donors, and their roles are closely interlinked since their metabolic pathways converge at the conversion of homocysteine to methionine. Research has shown that a disruption in one of these nutrients can trigger compensatory adjustments in the others (31). One of the few human studies investigating the metabolic relationship between choline and folate indicates that, in adults, choline serves as a methyl donor when folate levels are insufficient. This study further suggests that endogenous synthesis of choline alone does not sustain adequate levels of both choline and folate in plasma if they are under-consumed (32). In animal models, particularly pregnant mice, maternal diets rich in choline can partially mitigate the effects of folate deficiency, though not entirely (33), underscoring the unique and non-substitutable roles these nutrients play. Our manuscript will focus on the maternal choline dietary intake.

In the cell, choline can be phosphorylated by choline kinase (CK) to form phosphocholine, or it can be oxidized by choline dehydrogenase (CHDH) to form betaine aldehyde (34, 35). Phosphocholine (PC) is the precursor for the Kennedy pathway, where short-to-medium-chain phosphatidylcholines (PtdCho) are synthesized and incorporated into cell membranes (34, 36). Both lysophosphatidylcholine (LPC) and PC mediate the transport of docosahexaenoic acid (DHA) and eicosatetraenoic acid (EPA) across the blood–brain barrier via the Mfsd2a transporter (37, 38). These choline species not only support brain functionality and membrane integrity of neurons and glial cells but also facilitate the transport of DHA, which regulates the blood–brain barrier permeability (39).

Choline also serves as a precursor to sphingomyelin (SM), a crucial component of cell membranes. SM is synthesized by transferring a PC group from PtdCho to ceramide (40). During development, SM is particularly abundant due to its role in insulating neuronal axons, and it is one of the major choline compounds found in human milk (41). A recent study in humans revealed that higher levels of SM are significantly associated with improved verbal development during the first 2 years of life (42), likely due to enhanced axonal myelination.

Choline enters the brain through several transport modalities, including free choline transporters: CTL1, CTL2, and CHT1 (41, 42). CTL1 is a Na + independent choline transporter, which is present in astrocytes, cortical neurons, and the endothelial cells that constitute the blood–brain barrier (43, 44). In cases of CTL1 dysfunction, CTL2 provides additional support to maintain brain choline levels (45). Recently, a novel choline transporter, FLVCR2 (also known as SLC49A2), was identified in humans. It is highly expressed in endothelial cells at the blood–brain barrier and serves as one of the primary transporters of choline into the brain (46). However, more research is needed to fully understand the regulation of FLVCR2 in response to choline availability and its potential implications for neurodevelopment.

These brain transporters are crucial in the context of maternal nutrition, choline metabolism, and embryonic neurogenesis. Once choline is transported into cholinergic neurons, it is used to synthesize ACh via choline acetyltransferase and then stored in synaptic vesicles through the vesicular Ach transporter, ready for release. In mice, the development of the cholinergic neurons begins around embryonic day (E11.5) and continues postnatally until approximately postnatal day 30 (P30) (47). During this period, CHT1 expression increases in most regions of the brain (48). Interestingly, this critical window of development is where we have demonstrated that low choline can have long-lasting effects that persist into adulthood (49). This merits more in-depth research into the long-term consequences of prenatal low choline availability on cholinergic function in both childhood and adulthood.

In one-carbon metabolism, choline is converted into betaine, which then intersects with the methionine cycle as a methyl donor. Betaine acts as a substrate for betaine–homocysteine methyltransferase (BHMT), which remethylates homocysteine (Hcy) to form methionine (50). Methionine is subsequently used to generate S-adenosylmethionine (AdoMet), a key molecule for methylation reactions, including DNA and histone methylation. We demonstrated that during neurogenesis, low choline availability reduces methylation potential in embryonic brains (E17.5), leading to abnormal microRNA expression in neural progenitor cells (NPCs) (29). Specifically, we observed phenotypical changes in the cerebral cortex associated with the aberrant expression of miR-129p, miR-466 k, and miR-137 in NPCs under conditions of low choline availability (29). In addition, low choline intake induces CpG island hypomethylation, which results in decreased global histone methylation in hippocampal-derived murine NPCs (51). These epigenetic alterations were linked to changes in G9a histone methyltransferase activity and impacted the expression of calbindin 1, a protein involved in long-term potentiation and learning (51). In the developing cerebral cortex, the master transcription factor SRY-related HMG box 4 (SOX4) is reduced under low choline conditions. This reduction leads to decreased protein levels of its downstream target, enhancer of zeste homolog 2 (EZH2) (52). EZH2, a catalytic subunit of polycomb repressive complex 2 (PRC2), functions as a methyltransferase that tri-methylates lysine 27 of histone 3 (H3K27me3) (52).

Concurrent with the reduction in protein levels of SOX4 and EZH2, miR-129-5p has increased expression in the cerebral cortex with low choline availability (29, 52). miR-129-5p targets SOX4, leading to its reduced translation, suggesting a feedback loop in which increased miR-129-5p reduces SOX4 protein expression resulting in a lack of EZH2 transcription (52). Consequently, a reduction in total H3K27me3 was found in embryonic cerebral cortices with low choline availability compared to those with more choline availability, demonstrating the downstream effects of decreased EZH2 expression (52).

Rett syndrome is a complex neurodevelopmental disorder predominantly affecting female individuals, characterized by severe cognitive and physical impairments. Initially, children with RTT exhibit apparently typical development, followed by a progressive loss of fine and gross motor skills, deceleration of head growth, and repetitive hand movements. Diagnosis of RTT includes identifying abnormal breathing patterns, such as hyperventilation or period apnea (68, 69). In the early stages, some girls may be misdiagnosed with autism spectrum disorder until the hallmark motor skill deterioration becomes apparent, prompting a reevaluation of the clinical case.

Rett syndrome is an X-linked neurodevelopmental disorder linked to mutations in the MECP2 gene located on the X chromosome (70). This gene encodes methyl-CpG binding protein 2 (MeCP2), a critical protein for brain development, and is predominantly expressed in neurons after embryonic neurogenesis has concluded (71). MeCP2 contains a methyl-CpG-binding domain (MBD), which binds to chromosomes in a methylation-dependent manner (72–75). MeCP2 recruits other corepressors, through a transcriptional-repression domain (TRD), including the SIN3A histone deacetylase (HDAC) complex and NCoR/SMRT co-repressor complexes (72, 76). This process is essential for preserving chromatin structure and consequently gene expression (77, 78).

Methyl-CpG binding protein 2 is localized on chromatin and can bind to both the nucleosome complex and methylated DNA (79). DNA methylation not only can directly control MeCP2 expression but also can function. In a mouse model, locus-specific DNA methylation of the Mecp2 promoter led to autism-like behaviors (80). The localization of MeCP2 to chromocenters is suggested to be primarily determined by global DNA methylation patterns, independent of heterochromatin foci and phase separation (81). Although MeCP2 binding to DNA persists in the absence of DNA methylation, this binding is promiscuous and transient, suggesting that DNA methylation is the key factor in MeCP2 binding.

Low choline availability results in the alteration of global CpG island methylation in the developing brain (54, 82). Methylation of CpGs is carried out de novo by DNA methyltransferase 3A, with stabilization by DNA methyltransferase 3 L (DNMT3L) and 3B (DNMT 3A/3B). DNA methyltransferase 1 (DNMT1) maintains these marks through cell division, a process supported in part by DNMT3L (83). In the hippocampus, low choline availability resulted in CpG hypomethylation, leading to the increased expression of several genes including kinase-associated phosphatase, calretinin, and cyclin-dependent kinase inhibitor 2B. Conversely, in the cortex, 5-methylcytosine levels are elevated (82). This hypermethylation is linked to the increased expression of several DNMTs, including DNMT1, DNMT3a, and DNMT3L, which contributes to the aberrant expression of several genes including insulin-like growth factor 2, G9a, and calbindin 1 (51). MeCP2 preferentially binds to methyl-CG dinucleotides, predominantly CpG islands, which are adjacent to regions enriched in adenine (A) and thymine (T) bases (84).

In addition to its effects on CpG islands, choline also influences histone methylation. Low choline levels reduce methylation at the H3K27me3, as well as at H3K9me2 and H3K9me1. Methylation of H3K9 and H3K27 is critical for the formation of heterochromatin as it recruits and maintains heterochromatin protein 1α through PRC2-mediated cooperation. MeCP2 was found to bind with histones; however, binding was increased in the presence of H3K27me3 (85). It is suggested that H3K27me3 helps to recruit MeCP2 to facilitate gene repression as Mecp2 knockout mice have increased expression of genes that are regulated by H3K27me3 (85). Interestingly, our research, as mentioned above, shows that choline availability alters the methylation levels of H3K27me3; potentially, choline can play an indirect role in the recruitment of MeCP2 (81). Moreover, modified variants of MeCP2 that bind only to methyl-CpGs were found to be insufficient in maintaining neurological function (86). Taken together, these findings suggest that choline availability may influence MeCP2’s role in gene regulation through its effects on methylation and chromatin structure.

Despite the genetic origin of RTT, encouraging outcomes have been published (87), using an in vitro model of choline supplementation. In this model, the expression of the MeCP2 protein was knocked down with shRNA, leading to improvements in synaptic function and behavior. Furthermore, a study using a Rett syndrome mouse model (i.e., Mecp2-null) demonstrated that postnatal choline supplementation attenuated some behavioral deficits in the offspring (88). These promising findings underscore the need for further research in animals and humans to better understand the potential role of choline in alleviating RTT symptoms.

Fragile X syndrome is recognized as a leading cause of intellectual disability and autism spectrum disorder. Children with FXS often experience developmental delays and autism-like behaviors such as hand flapping, poor eye contact, and challenges with social interactions. In addition, behavioral alterations are common, including anxiety, delayed language milestones, and in some cases, seizures, strabismus, and obesity (89). These children meet the criteria for attention deficit hyperactivity disorder (ADHD) and autism. Typically, FXS is diagnosed around the age of three, often as a result of a follow-up assessment of recurrent developmental concerns observed during early childhood. In most cases, parents are referred to a clinical geneticist, where a blood test is conducted to detect mutation in the FMR1 gene, which confirms the diagnosis of FXS (90, 91).

Fragile X syndrome is caused by a reduction or complete absence of fragile X messenger ribonucleoprotein 1 (FMRP). FMRP is an RNA-binding protein that is involved in the regulation of many mRNAs within postsynaptic neurons, contributing to synaptic plasticity and brain structure. The most common cause of FXS is the expansion of the CGG trinucleotide repeat in the promoter region of the gene FMR1 (92). This leads to methylation of the FMR1 promoter region and subsequent transcriptional silencing. This altered epigenetic landscape occurs around the 11th week of gestation and is associated with H3 demethylation, which is mediated by the formation of DNA–RNA duplexes between the CGG repeat region of the FMR1 promoter and its mRNA counterpart (93). Consequently, this leads to the silencing of the FMR1 gene by FMR1 mRNA (93).

Individuals with more than 200 CGG (full mutation) experience complete transcriptional silencing of FMR1, while individuals with 55–200 CGG repeats (premutation) exhibit increased transcription of FMR1 (94). In addition, some individuals can have a mosaicism, where certain cells show methylation of the FMR1 promoter, while others are unmethylated, leading to varying levels of FMRP expression (94). The degree of methylation of the FMR1 promoter is correlated with a spectrum of behavioral deficits in which the severity of the disease is related to the extent of altered FMR1 gene expression and subsequent FMRP protein levels (95).

Fragile X messenger ribonucleoprotein 1 protein is predominantly located in the cytoplasm, associated with free ribosomes within dendritic spines. In neurons, it is involved in RNA stability, splicing, and RNA interference (96). Preclinical models of FXS replicate some of the phenotypes observed in humans, contributing to our understanding of the disease and revealing potential pathways for treatment (97).

Fragile X syndrome is characterized by the disruption of cholinergic function, which contributes to the behavioral and cognitive impairments observed in the disorder. A recent study using proton magnetic resonance spectroscopy explored choline concentration in the brains of male individuals with FXS. The findings revealed a significant reduction in the choline concentration and choline/creatine ratio (98). This suggests that increasing choline availability might partially restore cholinergic function. A recent study demonstrated that a combined intervention of donepezil and choline improved language abilities in children with autism (99). Interestingly, donepezil is also a recognized treatment for FXS. This evidence establishes a foundation for investigating the potential benefits of choline supplementation in combination with pharmaceutical treatment for FXS.

In a forebrain organoid model of FXS, dysregulated neurogenesis was observed, characterized by a reduced number of actively proliferating Ki67+ NPCs (100). As previously discussed, prenatal choline availability regulates NPC proliferation in the developing cortex (49). This raises the question of whether choline supplementation could enhance the reduced NPC proliferation observed in FXS. In addition, in a knockout model of FXS, a phenotype of dysregulated neuronal maturation was noted. This aligns with findings where Fmr1 is identified as a significant regulatory target of miR-129-5p, which controls neuronal migration during cortical neurogenesis. Choline regulates miR-129-5p in the cerebral cortex, and in utero inhibition of miR-129-5p was able to rescue the neuronal migration defects caused by low choline (29). Further investigation into how choline influences cortical development in different FXS models would be valuable.

Down syndrome results from a trisomy of chromosome 21 (Hsa21), leading to the abnormal function of multiple bodily systems, including musculoskeletal, neurological, and cardiovascular systems (16). Apart from the facial phenotype, DS is characterized by impaired language and memory capacities. Understanding the pathology of DS and its impact on these systems is complex, primarily due to the triplication of over 200 protein-coding genes. Advances in human genome sequencing have enabled the identification of specific genes and their epigenetic regulation on Hsa21, shedding light on the potential consequences of this genetic alteration. For example, it is now well-established that a subset of individuals with DS are at a higher risk for early-onset Alzheimer’s disease, attributed to a dose-dependent increase in amyloidogenic fragments (101, 102). Effective management of DS requires a holistic approach, addressing psychiatric care to manage behavioral changes, as well as nutritional and metabolic health (103).

In the brains of individuals with DS, a reduction in cortical volume and a decrease in overall number of neurons have been observed (104). This finding aligns with studies in mouse models that show a reduction in the pool of NPCs in the cerebral cortex, suggesting that impaired neurogenesis may contribute to the brain phenotype observed in DS. Not only is gene expression altered, but methylation and epigenetic marks are also altered in DS (105). Interestingly, some dysregulated genes, such DYRK1A, play a role in this process by phosphorylating downstream proteins such as p21, which regulates the G1/G0-S cell phase transition (106). This dysregulation impacts NPC differentiation, promoting a shift toward glycogenesis, which may explain the altered proliferation and reduced number of neurons observed in DS (107).

Altered methylation patterns on HSA21 are reported in individuals with DS (108). This may be linked to a reduction in the methylation potential, as reduced levels of AdoMet have been observed in DS (109). This alteration in one-carbon metabolism, including reduced methionine levels and increased cystathionine, may contribute to the underlying causes of altered DNA methylation patterns in DS.

Environmental conditions, such as nutritional and choline status during critical periods of development, particularly during pregnancy and early childhood, may play a critical role in DS. Findings from animal studies have shown that prenatal choline supplementation can improve attention and reduce cognitive and affective dysfunctions in the Ts65Dn mouse model of DS (110). In addition, maternal choline supplementation has been shown to enhance hippocampal neurogenesis in the offspring of the Ts65Dn mouse model, compared to the non-supplemented groups. The improvement in hippocampal neurogenesis was directly correlated with coordination and performance, suggesting that an improvement in neurogenesis causes better cognitive special performance (111).

The relevance of maternal choline intake extends beyond early development as studies in adult wild-type rats have demonstrated its positive impact on neurogenic responses, indicating long-lasting effects on adult neurogenesis (112). It is likely that the mechanism by which choline supplementation exerts its effects in Ts65dn mice is also through enhanced neurogenesis. Choline, via its metabolite betaine, influences methylation potential and, therefore, regulates gene expression. Specifically, in the Ts65Dn mouse, maternal choline supplementation produced long-term effects on gene expression in pyramidal neurons. Interestingly, GABAergic genes were upregulated in response to choline, which could explain the observed improvements in learning and memory (113).

Moreover, a case–control study revealed that in humans, genetic variants of the BHMT gene (rs3733890) increase the risk of DS, particularly in populations with folate impairment (114). Although larger studies are needed, these findings highlight the importance of considering genetic factors, as well as the potential of choline and its metabolites, in the context of DS risk.