Impaired folate 1-carbon metabolism causes formate-preventable hydrocephalus in glycine decarboxylase–deficient mice

Ventriculomegaly and hydrocephalus are associated with loss of function of glycine decarboxylase (Gldc) in mice and in humans suffering from non-ketotic hyperglycinemia (NKH), a neurometabolic disorder characterized by accumulation of excess glycine. Here, we showed that ventriculomegaly in Gldc-deficient mice is preceded by stenosis of the Sylvian aqueduct and malformation or absence of the subcommissural organ and pineal gland. Gldc functions in the glycine cleavage system, a mitochondrial component of folate metabolism, whose malfunction results in accumulation of glycine and diminished supply of glycine-derived 1-carbon units to the folate cycle. We showed that inadequate 1-carbon supply, as opposed to excess glycine, is the cause of hydrocephalus associated with loss of function of the glycine cleavage system. Maternal supplementation with formate prevented both ventriculomegaly, as assessed at prenatal stages, and postnatal development of hydrocephalus in Gldc-deficient mice. Furthermore, ventriculomegaly was rescued by genetic ablation of 5,10-methylene tetrahydrofolate reductase (Mthfr), which results in retention of 1-carbon groups in the folate cycle at the expense of transfer to the methylation cycle. In conclusion, a defect in folate metabolism can lead to prenatal aqueduct stenosis and resultant hydrocephalus. These defects are preventable by maternal supplementation with formate, which acts as a 1-carbon donor.


Introduction
Hydrocephalus results from abnormal cerebrospinal fluid (CSF) hydrodynamics (overproduction, diminished drainage, or impaired flow) leading to progressive enlargement of the cerebral ventricular system and subsequent pathology (1). Hydrocephalus can be acquired (following injury, infection, tumor formation, or trauma) or arise as a congenital condition, in isolation or associated with structural abnormalities such as Dandy-Walker or Chiari II malformations (2,3). Although common (affecting 0.5-1 per 1,000 live births), the molecular pathophysiology of isolated congenital hydrocephalus is known in only a relatively small proportion of cases, owing to heterogeneity and multifactorial etiology (4)(5)(6).
A potential causal effect of impaired function of the glycine cleavage system (GCS) is highlighted by occurrence of hydrocephalus in association with non-ketotic hyperglycinemia (NKH), a life-limiting autosomal recessive neurometabolic disorder characterized by accumulation of glycine in body fluids and tissues (7,8). NKH results from mutation of GCS-encoding genes, with the majority of patients carrying mutations in GLDC (glycine decar-boxylase) (9,10). Hydrocephalus arises in approximately 8% of NKH patients and enlarged ventricles are commonly found on imaging (15 of 41 patients in one clinical survey) (8,11).
A direct link between GLDC loss of function and hydrocephalus was confirmed by analysis of mice carrying hypomorphic (Gldc GT1 ) or null (Gldc GT2 ) alleles of the murine homolog, Gldc. A proportion of Gldc mutants die perinatally, owing to neural tube defects (NTDs) resulting from failed neural tube closure (12,13). However, among Gldc-deficient mice that survive postnatally, hydrocephalus becomes evident by 5-7 weeks of age in 20%-25% of homozygotes (Gldc GT1/GT1 ), with a characteristic domed head, distorted cranium, and severely enlarged lateral ventricles (12). These mice also show signs of NKH, including elevated glycine concentration in body fluids and tissues and premature lethality (12).
The physiological (communicating vs. noncommunicating) and metabolic mechanisms underlying GLDC-related hydrocephalus have not been determined. The GCS is a mitochondrial enzyme complex that mediates decarboxylation of glycine, Ventriculomegaly and hydrocephalus are associated with loss of function of glycine decarboxylase (Gldc) in mice and in humans suffering from non-ketotic hyperglycinemia (NKH), a neurometabolic disorder characterized by accumulation of excess glycine. Here, we showed that ventriculomegaly in Gldc-deficient mice is preceded by stenosis of the Sylvian aqueduct and malformation or absence of the subcommissural organ and pineal gland. Gldc functions in the glycine cleavage system, a mitochondrial component of folate metabolism, whose malfunction results in accumulation of glycine and diminished supply of glycine-derived 1-carbon units to the folate cycle. We showed that inadequate 1-carbon supply, as opposed to excess glycine, is the cause of hydrocephalus associated with loss of function of the glycine cleavage system. Maternal supplementation with formate prevented both ventriculomegaly, as assessed at prenatal stages, and postnatal development of hydrocephalus in Gldc-deficient mice. Furthermore, ventriculomegaly was rescued by genetic ablation of 5,10-methylene tetrahydrofolate reductase (Mthfr), which results in retention of 1-carbon groups in the folate cycle at the expense of transfer to the methylation cycle. In conclusion, a defect in folate metabolism can lead to prenatal aqueduct stenosis and resultant hydrocephalus. These defects are preventable by maternal supplementation with formate, which acts as a 1-carbon donor.

Results and Discussion
In our previous ultrasound analysis of litters at E18.5, Gldc-deficient (Gldc GT1/GT2 ) fetuses that exhibited enlarged lateral ventricles did not differ from wild types in volume of the fourth ventricle or cerebellum (14). Similarly, we found that among Gldc GT1/GT1 fetuses with obvious dilatation of the lateral and third ventricles (7 of 13 Gldc GT1/GT1 ), more posterior structures including the fourth ventricle appeared comparable to wild types ( Figure 1).
Specific expansion of the ventricular system rostral to the aqueduct of Silvius indicated the possibility of mechanical obstruction. Consistent with this hypothesis, after injection into the lateral ventricles, aqueous dye circulated to the posterior aqueduct and fourth with concomitant transfer of a 1-carbon group to tetrahydrofolate (THF), generating 5,10-methylene THF in folate 1-carbon metabolism (FOCM) (9). Hence, GCS loss of function not only causes accumulation of glycine, but also prevents transfer of glycinederived 1-carbon units to FOCM (13). Excess glycine is thought to lead to neurological features of NKH, such as epilepsy (8,9), whereas NTDs result from impaired FOCM (13). Potential mechanisms underlying the development of ventriculomegaly and hydrocephalus could therefore include consequences of FOCM suppression or glycine accumulation in CSF or neural tissues. For example, it is proposed that altered osmolality of CSF (a potential effect of excess glycine) could result in net movement of water into the ventricles, leading to abnormal CSF hydrodynamics (16). Alternatively, FOCM is required for provision of 1-carbon groups for key cellular processes including nucleotide biosynthesis and methylation reactions, whose disturbance could plausibly lead to agenesis is associated with development of both communicating and noncommunicating forms of hydrocephalus in mice, as seen in genetic mutants for Msx1, Pax6, and Rfx3 or overexpression of Sox3 (17,18). A hypoplastic pineal gland is also observed with postnatal hydrocephalus in Lhx9-null mice (19).
Gldc is expressed throughout the neuroepithelium at E9.5 (12). At developmental stages when ventriculomegaly arose, we found widespread expression of Gldc in the brain at E16.5, including the pineal gland, SCO, and pituitary gland (Figure 2, Q, T, and R). Immunostaining confirmed the presence of Gldc protein in these structures at E18.5 (Figure 2, S, U, and V).
Hydrocephalus has been associated with denudation of the ependymal cell layer lining the ventricles and subsequent occlusion of the aqueduct (17,20). Lack or abnormal function of ventricles in wild-type and Gldc GT1/+ neonatal mice but not in some Gldc GT1/GT1 littermates (Figure 1, P-U and Supplemental Figure 1; supplemental material available online with this article; https:// doi.org/10.1172/JCI132360DS1). Among wild-type and unaffected Gldc GT1/GT1 fetuses at E18.5, the aqueduct lumen was continuous and could be followed in serial sections from the third to fourth ventricles ( Figure 2). In contrast, the aqueduct was very narrow or completely occluded in all Gldc GT1/GT1 fetuses that displayed ventriculomegaly (Figure 2, M-P, and Supplemental Figure 2). At E18.5, the subcommissural organ (SCO) and pineal gland are visible in the roof of the aqueduct in wild-type and unaffected Gldc-deficient fetuses (Figure 2, A-L). However, in all the affected Gldc-deficient fetuses the SCO was absent and the pineal gland was absent or malformed ( Figure 1M and Figure 2, M-P). SCO We found that exogenous supply of 1-carbon units by maternal formate supplementation normalized the folate profile in Gldcdeficient embryos and prevented NTDs, despite the tissue glycine concentration remaining elevated (12,13). Based on this strategy, pregnant dams were supplemented with formate prior to collection of litters for evaluation of ventriculomegaly ( Figure 3A). Histological examination divided Gldc GT1/GT1 fetuses into 2 main categories: a group that exhibited severely dilated lateral ventricles with absent SCO and pineal gland ( Figure 1M) and another with normal appearance, indistinguishable from wild type ( Figure 1H and Supplemental Figure 4). A few fetuses displayed an intermediate phenotype in which the SCO or pineal gland was detectable but appeared abnormal, accompanied by mild ventricle dilation (Supplemental Figure 4). Formate supplementation led to significant normalization of development ( Figure 3B), with none of the treated Gldc GT1/GT1 fetuses displaying severe ventriculomegaly or absence of the SCO and pineal gland. Similarly, the ependymal cell layer was intact in all formate-treated fetuses examined (n = 6 unaffected and 1 intermediate; Figure 3, D-I).
Having found that formate supplementation could prevent fetal ventriculomegaly, we asked whether this prenatal treatment rescued postnatal onset of hydrocephalus. Gldc GT1/+ dams were supplemented with formate for the first 15 days of pregnancy and offspring were monitored for development of abnormalities until 6-7 weeks of age. Notably, none of the Gldc GT1/GT1 offspring (n = 35) of formate-supplemented mice developed hydrocephalus, unlike ependymal motile cilia may also result in hydrocephalus without aqueduct stenosis, possibly owing to impaired CSF flow (17,20). We found that the ependymal cell layer was intact in the ventricular system of wild-type (n = 5) and unaffected Gldc GT1/GT1 (n = 4) fetuses at E18.5 (Figure 2, W, X, Z, AA, CC, and DD, and Supplemental Figure 2). Abnormalities were not observed in the ependymal cell layer of the lateral ventricles in Gldc GT1/GT1 fetuses with ventriculomegaly (Supplemental Figure 2, G-L), but this layer appeared disrupted in the dorsal region of the third ventricle in most (4 out of 5; Figure 2, Y, BB, and EE) but not all (Supplemental Figure 3) fetuses. The fetal onset of ventriculomegaly, prior to maturation of multiciliated ependyma (in the first postnatal week in mice), and largely intact ependyma suggest that disruption of this cell layer is unlikely to be causal. Together, the presence of enlarged lateral and third ventricles, unaffected fourth ventricle, impaired dye distribution, and late-fetal onset of ventriculomegaly implicate aqueduct stenosis as the structural abnormality causing a noncommunicating form of hydrocephalus in Gldc-deficient fetuses.
We next investigated the metabolic basis of hydrocephalus in Gldc-deficient mice. Loss of function of GCS causes accumulation of glycine, with significantly elevated levels in Gldc-deficient embryos by E11.5 (12). Metabolic labeling shows that the contribution of glycine-derived 1-carbon units to FOCM is also ablated in Gldc-deficient embryos, with consequent alterations in the relative abundance of folates (12,13). duct stenosis. Given the key role of the folate cycle in providing precursors for thymidylate and purine synthesis we speculate that defects result from impaired nucleotide biosynthesis in the neuroepithelium. The next step in this research will be to further refine the developmental stage and tissue site(s) in which folate cycle disruption subsequently leads to malformations of the SCO, pineal gland, and aqueduct. In animal models, a potential link between folate status and hydrocephalus was noted in dietary studies examining nutritional effects of folate and vitamin B 12 (21,22). Some neonatal offspring of rats fed synthetic diets lacking folic acid and vitamin B 12 exhibited enlarged ventricles attributed to possible blockage of the cerebral aqueduct (21). However, speculation remained over the relative importance of folic acid and vitamin B 12 and whether FOCM was impaired in these models (22).
Abnormal folate transport to the brain has been proposed to contribute to aqueduct obstruction in the hydrocephalic Texas (H-Tx) rat (23,24). Interestingly, however, hydrocephalus is not commonly described among neurological signs associated with the very low levels of folate in CSF that occur in hereditary folate malabsorption, caused by mutations in SLC46A1 (proton-coupled folate transporter; PCFT) or cerebral folate deficiency, caused by mutations in FOLR1 (folate receptor α; FRα) (25)(26)(27). On the other hand, hydrocephalus can arise in patients with severe MTHFR nonsupplemented Gldc GT1/GT1 in which hydrocephalus arose in 30% of mice ( Figure 3C), similar to previous studies (12,14).
Prevention of ventriculomegaly/hydrocephalus by prenatal formate supplementation implicates insufficient supply of 1-carbon groups to FOCM as the underlying causative mechanism. The folate cycle intermediates, 5,10-methylene tetrahydrofolate (THF) and 10-formyl THF, act as 1-carbon donors in biosynthesis of thymidylate and purines. Alternatively, 1-carbon groups can be transferred to the methionine cycle via donation of a methyl group from 5-methyl THF to homocysteine ( Figure 4A). To ask whether ventriculomegaly derives from suppression of the folate cycle or inadequate supply of 1-carbon groups to the methionine cycle we bred Gldc-deficient fetuses that lack 5,10-methylene tetrahydrofolate reductase (Mthfr). Compound null Gldc GT2/GT2 Mthfr -/embryos cannot generate 5-methyl THF and 1-carbon groups are therefore retained in the folate cycle (13).
Analysis of litters produced by intercross of compound heterozygous, Gldc GT2/+ Mthfr +/mice revealed venticulomegaly in Gldc GT2/GT2 fetuses that were wild type or heterozygous at the Mthfr locus (Figure 4). In contrast, all Gldc/Mthfr double-knockout fetuses lacked ventriculomegaly, demonstrating a significant protective effect of the Mthfr null mutation. This preventive effect highlights the folate cycle, rather than methionine cycle, as the subset of FOCM reactions that are critical in prevention of aque- YJP's present address is: RIKEN Center for Brain Science, Wako, Saitama, Japan. deficiency or remethylation disorders (including cblC disease) (28). MTHFR-related hydrocephalus may be of the communicating form (29), and it is not clear to what extent aqueduct stenosis also contributes. Nevertheless, impaired methionine synthase activity (as in cblC patients) could lead to accumulation of 5-methyl THF and thereby deplete 1-carbon units from the folate cycle (methyl trap). This suggests the potential for a shared biochemical mechanism underlying hydrocephalus caused by methionine synthase or Gldc deficiency.
Here, we found that impaired enzymatic activity within mitochondrial FOCM, as opposed to diminished exogenous supply or a methyl trap, can be a direct cause of aqueduct stenosis, the most common known cause of congenital hydrocephalus in humans. The presence of hydrocephalus and/or enlarged ventricles in NKH patients and in Gldc-deficient mice suggests that the requirements for GCS activity are shared. Mutations in GLDC may also predispose to failed neurulation in humans as in mice, missense variants having been identified in some patients with NTDs (30)(31)(32), including functional mutations found in both NTDs and NKH (30,31).
Further evidence for a molecular link between congenital hydrocephalus and NTDs has come from identification of putative causal mutations in TRIM71, SMARCC1, and PTCH1 in patients with communicating and obstructive forms of hydrocephalus (6). Loss of function of each of these genes in mice causes cranial NTDs (33)(34)(35). Although the association of hydrocephalus with spina bifida is usually considered a secondary manifestation, one could speculate that they may be independent malformations with a shared genetic origin in some individuals. Hence, although NTDs and hydrocephalus arise at different developmental stages, the potential for prevention of both defects by formate supplementation suggests that there may be a related cellular mechanism, serving as a starting point for further research toward understanding the causation, pathogenesis, and primary prevention of these related conditions.

Methods
Further details are provided in the supplemental material.