Medical Hypotheses 134 (2020) 109429

Contents lists available at ScienceDirect

Medical Hypotheses
journal homepage: www.elsevier.com/locate/mehy

Why is folate effective in preventing neural tube closure defects? T

⁎
Kohji Sato
Department of Organ & Tissue Anatomy, Hamamatsu University School of Medicine, 1-20-1 Handayama, Higashiku, Hamamatsu, Shizuoka 431-3192, Japan

A R T I C LE I N FO A B S T R A C T

Keywords: Neural tube defects (NTDs) originate from a failure of the embryonic neural tube to close. The pathogenesis of
Neural tube defects NTDs is largely unknown. Fortunately, adequate maternal folate application is known to reduce the risk of
Folate human NTDs. However, why folate reduces NTDs is largely unknown.
Glycine cleavage system The main cause for NTDs is the disturbance of the cell growth in the neuroepithelium. Of course, rapid cell
Nucleic acids synthesis
growth needs enough synthesis of nuclei acids. Interestingly, folate is used as a source for the synthesis of nucleic
Neuroepithelium
acids. Furthermore, glycine cleavage system (GCS) is essential for the synthesis of nucleic acids from folate, and
very strongly expressed in neuroepithelial cells, suggesting that these highly proliferating cells need enough
synthesis of nuclei acids and high amounts of folate. Taken together, I speculate the following hypothesis; (1)
The closure of the neural tube requires rapid growth of neuroepithelial cells. (2) High rates of nuclei acids
synthesis are needed for the rapid growth. (3) GCS, which is requisite in nucleic acid synthesis from folate, is
expressed very strongly and functions robustly in neuroepithelial cells. (4) Pregnant women require 5–10-fold
higher amounts of folate compared to non-pregnant women. (5) So, folate-deficient situations are easy to occur
in neuroepithelial cells, resulting in NTDs. (6) Thus, folate is effective to prevent NTDs.

Introduction carbon metabolism comprises a network of enzymatic reactions re-

The process of neurulation occurs during early embryogenesis,
starting with the formation of the neural plate from specialized ecto-
dermal cells and the neural plate develops bilateral neural folds, which
elevate and fuse at the midline to create the neural tube [1]. Neural
tube defects (NTDs), such as spina bifida and anencephaly, originate
from a failure of the embryonic neural tube to close [2] with a world-
wide incidence ranging from 1.0 to 10.0 per 1000 births [3]. The pa-
thogenesis of NTD is genetically complex and diverse, and the patho-
genetic mechanisms are largely unknown [2]. Fortunately, adequate
maternal folate application is known to reduce the risk of human NTDs
[1]. However, why folate reduces NTDs is largely unknown. Thus, in
this manuscript, I try to find the missing link between them.
Hypothesis
NTDs originate from a failure of the embryonic neural tube to close.
The main cause is the disturbance of the cell growth in the neuroe-
pithelium of the neural plate and neural fold. Of course, rapid cell
growth needs enough synthesis of nuclei acids. Interestingly, folate is
very deeply involved in the synthesis of nucleic acids. Folate one-
quired for synthesis of purines and thymidylate for DNA synthesis [4].
Furthermore, glycine cleavage system (GCS), a key enzyme complex in
folate one-carbon metabolism and very important for DNA synthesis, is
very strongly expressed and functions in the neuroepithelium [5],
suggesting that these highly proliferating cells need enough synthesis of
nuclei acids and high amounts of folate. On the other hand, adequate
folate intake is particularly important for pregnant women, in whom
folate requirements are 5–10-fold higher compared to non-pregnant
women [6]. So, folate-deficient situations are easy to occur in neuroe-
pithelial cells of the neural plate and neural fold, resulting in NTDs.
Taken together, I speculate the following hypothesis (Figs. 1 and 2).
(1) The closure of the neural tube requires rapid growth of neuroe-
pithelial cells.
(2) High rates of nuclei acids synthesis are needed for the rapid growth.
(3) GCS, which is requisite in nucleic acid synthesis from folate, is
expressed very strongly and functions robustly in neuroepithelial
cells.
(4) Pregnant women require 5–10-fold higher amounts of folate com-
pared to non-pregnant women.
(5) So, folate-deficient situations are easy to occur in neuroepithelial

⁎
Address: Department of Anatomy & Neuroscience, Hamamatsu University School of Medicine, 1-20-1 Handayama, Higashiku, Hamamatsu, Shizuoka 431-3192,
Japan.
E-mail address: ksato@hama-med.ac.jp.

https://doi.org/10.1016/j.mehy.2019.109429
Received 24 June 2019; Received in revised form 3 October 2019; Accepted 10 October 2019
0306-9877/ © 2019 Elsevier Ltd. All rights reserved.
K. Sato
Fig. 1. Basic concept.
cells, resulting in NTDs.
(6) Thus, folate is effective to prevent NTDs.
Evaluation of the hypothesis
GCS is highly expressed in neuroepithelium
GCS is the main system to degrade glycine in mammalians. The
defect of this pathway causes nonketotic-hyperglycinemia (NKH),
which is an autosomal recessive disorder associated with very severe
clinical symptoms [7] and also grave brain malformations including
NTDs [8–10]. These severe symptoms and grave brain malformations
are not normally observed in the other amino acid metabolic disorders,
suggesting that GCS and glycine should have unknown pivotal roles in
brain development.
The distribution of GCS has been investigated in the developing
2
Medical Hypotheses 134 (2020) 109429
Fig. 2. Cartoon for the theory. The neural plate develops bilateral neural folds
(arrows in A). The neural folds elevate and make the neural groove (arrows in
B). The neural groove fuses at the midline to create the neural tube (C). NTDs
originate from a failure of the embryonic neural tube to close. In every stage,
neuroepithelium expresses abundant GCS (red areas), and GCS functions to
make enough nucleic acids for cell growth from abundant up-taken folate (blue
circles). Thus, folate-deficient situations are easy to occur in neuroepithelial
cells, resulting in NTDs. (For interpretation of the references to colour in this
figure legend, the reader is referred to the web version of this article.)
brain [5]. Interestingly, GCS is very strongly expressed and functions
robustly in neuroepithelial cells during the neural tube formation [5].
As shown in Fig. 2, the neural plate develops bilateral neural folds
(arrows in Fig. 2A), and the neural folds elevate and make the neural
groove (arrows in Fig. 2B), and finally the neural groove fuses at the
midline to create the neural tube (Fig. 2C). NTDs originate from a
failure of the embryonic neural tube to close. In every stage, neuroe-
pithelium expresses abundant GCS (red areas in Fig. 2), further sup-
porting pivotal roles of GCS in the neural tube formation.
Glycine cleavage system is requisite in nucleic acid synthesis from folate in
the neuroepithelium
GCS is indispensable in supplying proliferating cells with 5,10-me-
thylenetetrahydrofolate as a one-carbon donor, which is essential for
the synthesis of nucleic acids in cell proliferation [11]. Because neu-
roepithelial cells proliferate very rapidly, a large amount of 5,10-me-
thylenetetrahydrofolate should be supplied via GCS in these cells. As
shown in Fig. 3, in the neuroepithelium, up-taken folate is converted to
K. Sato
Fig. 3. GCS in the synthesis of nucleic acids from folate. In the neuroepithe-
lium, folate is up-taken from the blood, and folate is converted to tetra-
hydrofolate (THF). GCS breaks down glycine and generates 5,10-methylane-
THF from THF. And, 5,10-methylane-THF is used to make nucleic acids.
tetrahydrofolate (THF), and GCS generates 5,10-methylane-THF from
THF. And, 5,10-methylane-THF is used to make nucleic acids [12].
Interestingly, the neuroepithelium also expresses very high levels of
folate-binding proteins, which are needed to take up folate as a source
of 5,10-methylenetetrahydrofolate [13,14]. In addition, it has been
demonstrated that folate deficiency affects proliferation of neural stem
cells [15] and the association between neural tube defects and in-
adequate folate intake has already been established [11,16]. Thus, I
speculate that embryonic neuroepithelial cells transport folate for de
novo synthesis of sufficient nucleic acids for proliferation, and that GCS
may be indispensable for this process. Therefore, I think that the lack of
folate greatly impairs proliferation of neural stem cells, leading to
NTDs. Actually, GCS-encoding genes represent candidates for involve-
ment in NTDs [12]. In addition, loss-of-function mutation in GCS genes
predisposes to NTDs in mice and humans [12], supporting my theory.
Additional possibilities about the involvement of folate-depletion in the
pathogenesis of NTDs
Besides the functions in nucleic acid synthesis, folate has many
other roles in various biological events. Thus, folate-depletion may also
cause NTDs via the disturbance of many other biological events in the
development of the neuroepithelium. For example, maternal folate
deficiency has been shown to affect DNA repair and DNA methylation
[17], synthesis and transport of fatty acids [18] and choline metabolism
[19]. Since these events are also very important in the development of
the neuroepithelium, the disturbance of these events may impair pro-
liferation of neural stem cells, leading to NTDs.
Furthermore, cell–cell adhesion molecules, such as cadherins, are
reported to be very important for the complete closure of neural tube
[20]. For example, during neural tube formation, E-cadherin and N-
cadherin play pivotal roles [21]. Interestingly, neural tube formation is
impaired in N-cadherin knockout mice [22]. In addition, Su et al. have
reported that folate deficiency causes the down-regulation of E-cad-
herin [23]. Taken together, there is a possibility that folate-depletion
induces aberrant functions of cell–cell adhesion molecules, such as
cadherins, leading to NTDs.
In addition, it has been reported that the number of apoptotic cells
was almost doubled in the septum and hippocampus of offspring of
folate-depleted mothers [24], suggesting that the increase of apoptosis
might be also involved in the pathogenesis of NTDs.
Folate is effective in preventing neural tube defects
The importance of an adequate periconceptional maternal folate
status to prevent fetal neural tube defects has been well demonstrated
and resulted in the recommendation for women to use folate supple-
ments during the periconceptual period [25]. For example,
3
Medical Hypotheses 134 (2020) 109429
supplementary maternal peri-conceptional intake of folate has been
demonstrated to reduce the occurrence of human NTD by as much as
70% [26] and has led to government policies of fortification of various
foods with folate in some countries [27]. Pregnant women require
5–10-fold higher amounts of folate compared to non-pregnant women
[6]. So, folate-deficient situations are easy to occur in neuroepithelial
cells, where large amounts of folate are needed to make nuclei acids,
resulting in NTDs. Thus, folate is effective to prevent NTDs.
Consequences of the hypothesis and discussion
Neural tube defects (NTDs) originate from a failure of the em-
bryonic neural tube to close. In this report, I speculate the following
hypothesis; (1) The closure of the neural tube requires rapid growth of
neuroepithelial cells. (2) High rates of nuclei acids synthesis are needed
for the rapid growth. (3) GCS, which is requisite in nucleic acid
synthesis from folate, is expressed very strongly and functions robustly
in neuroepithelial cells. (4) Pregnant women require 5–10-fold higher
amounts of folate compared to non-pregnant women. (5) So, folate-
deficient situations are easy to occur in neuroepithelial cells, resulting
in NTDs. (6) Thus, folate is effective to prevent NTDs. Although sup-
plementary maternal peri-conceptional intake of folate has led to gov-
ernment policies of fortification of various foods with folate in some
countries [27]. Further promotion of mandatory folate fortification is
needed to prevent NTDs.
Grant Sponsor
The Ministry of Education, Science and Culture of Japan;
Shintenkai.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgement
The author would like to thank Ms Chiemi Kawamoto for her ex-
cellent technical help.
References
[1] Blom HJ, Shaw GM, den Heijer M, Finnell RH. Neural tube defects and folate: case
far from closed. Nat Rev Neurosci 2006;7:724–31.
[2] Juriloff DM, Harris MJ. Insights into the etiology of mammalian neural tube closure
defects from developmental, genetic and evolutionary. Studies J Dev Biol
2018;6:E22.
[3] Au KS, Ashley-Koch A, Northrup H. Epidemiologic and genetic aspects of spina
bifida and other neural tube defects. Dev Disabil Res Rev 2010;16:6–15.
[4] Beaudin AE, Stover PJ. Insights into metabolic mechanisms underlying folate-re-
sponsive neural tube defects: a minireview. Birth Defects Res A Clin Mol Teratol
2009;85:274–84.
[5] Ichinohe A, Kure S, Mikawa S, Ueki T, Kojima K, Fujiwara K, et al. Glycine cleavage
system in neurogenic regions. Eur J Neurosci 2004;19:2365–70.
[6] Tamura T, Picciano MF. Folate and human reproduction. Am J Clin Nutr
2006;83:993–1016.
[7] Hoover-Fong JE, Shah S, Van Hove JL, Applegarth D, Toone J, Hamosh A. Natural
history of nonketotic hyperglycinemia in 65 patients. Neurology 2004;63:1847–53.
[8] Press GA, Barshop BA, Haas RH, Nyhan WL, Glass RF, Hesselink JR. Abnormalities
of the brain in nonketotic hyperglycinemia: MR manifestations. AJNR Am J
Neuroradiol 1989;10:315–21.
[9] Dobyns WB. Agenesis of the corpus callosum and gyral malformations are frequent
manifestations of nonketotic hyperglycinemia. Neurology 1989;39:817–20.
[10] Nissenkorn A, Michelson M, Ben-Zeev B, Lerman-Sagie T. Inborn errors of meta-
bolism: a cause of abnormal brain development. Neurology 2001;56:1265–72.
[11] Fleming A, Copp AJ. Embryonic folate metabolism and mouse neural tube defects.
Science 1998;280:2107–9.
[12] Narisawa A, Komatsuzaki S, Kikuchi A, Niihori T, Aoki Y, Fujiwara K, et al.
Mutations in genes encoding the glycine cleavage system predispose to neural tube
defects in mice and humans. Hum Mol Genet 2012;21:1496–503.
K. Sato
[13] Finnell RH, Wlodarczyk BC, Craig JC, Piedrahita JA, Bennett GD. Strain-dependent
alterations in the expression of folate pathway genes following teratogenic exposure
to valproic acid in a mouse model. Am J Med Genet 1997;70:303–11.
[14] Piedrahita JA, Oetama B, Bennett GD, et al. Mice lacking the folic acid-binding
protein Folbp1 are defective in early embryonic development. Nat Genet
1999;23:228–32.
[15] Mattson MP, Shea TB. Folate and homocysteine metabolism in neural plasticity and
neurodegenerative disorders. Trends Neurosci 2003;26:137–46.
[16] Whitehead AS, Gallagher P, Mills JL, Kirke PN, Burke H, Molloy AM, et al. A genetic
defect in 5,10 methylenetetrahydrofolate reductase in neural tube defects. QJM
1995;88:763–6.
[17] Langie SA, Achterfeldt S, Gorniak JP, Halley-Hogg KJ, Oxley D, van Schooten FJ,
et al. Maternal folate depletion and high-fat feeding from weaning affects DNA
methylation and DNA repair in brain of adult offspring. FASEB J 2013;27:3323–34.
[18] da Silva RP, Kelly KB, Al Rajabi A, Jacobs RL. Novel insights on interactions be-
tween folate and lipid metabolism. BioFactors 2014;40:277–83.
[19] Jadavji NM, Deng L, Malysheva O, Caudill MA, Rozen R. MTHFR deficiency or
reduced intake of folate or choline in pregnant mice results in impaired short-term
memory and increased apoptosis in the hippocampus of wild-type offspring.
Neuroscience 2015;300:1–9.
Medical Hypotheses 134 (2020) 109429
[20] Nishimura T, Honda H, Takeichi M. Planar cell polarity links axes of spatial dy-
namics in neural-tube closure. Cell 2012;149:1084–97.
[21] Hatta K, Takeichi M. Expression of N-cadherin adhesion molecules associated with
early morphogenetic events in chick development. Nature 1986;320:447–9.
[22] Radice GL, Rayburn H, Matsunami H, Knudsen KA, Takeichi M, Hynes RO.
Developmental defects in mouse embryos lacking N-cadherin. Dev Biol
1997;181:64–78.
[23] Su YH, Huang WC, Huang TH, Huang YJ, Sue YK, Huynh TT, et al. Folate deficient
tumor microenvironment promotes epithelial-to-mesenchymal transition and
cancer stem-like phenotypes. Oncotarget 2016;7:33246–56.
[24] Craciunescu CN, Brown EC, Mar MH, Albright CD, Nadeau MR, Zeisel SH. Folic acid
deficiency during late gestation decreases progenitor cell proliferation and in-
creases apoptosis in fetal mouse brain. J Nutr 2004;134:162–6.
[25] Naninck EFG, Stijger PC, Brouwer-Brolsma EM. The importance of maternal folate
status for brain development and function of offspring. Adv Nutr 2019;10:502–19.
[26] MRC Vitamin Study Research Group. Prevention of neural tube defects: results of
the Medical Research Council Vitamin Study. MRC Vitamin Study Research Group
Lancet 1991;338:131–7.
[27] Garrett GS, Bailey LB. A public health approach for preventing neural tube defects:
folic acid fortification and beyond. Ann N Y Acad Sci 2018;1414:47–58.