DISCUSSION
Previous studies have indicated that CIC interaction with ATXN1
contributes to the pathogenesis of spinocerebellar ataxia type 1
(Rousseaux et al., 2018). Disruption of CIC-ATXN1 complex results in
hyperactivity, impaired learning and memory, and abnormal maturation and
maintenance of upper-layer cortical neurons in Emx1-cre mice (Cao et
al., 2021). Forebrain–specific deletion of CIC in Foxg1-cre mice caused
abnormal increases in oligodendrocyte progenitor and immature
oligodendrocytes populations (Yang et al., 2017). Brain-specific
deletion of CIC compromised neuroblasts transition to immature neurons
in mouse hippocampus and compromises normal neuronal differentiation
(Hwang et al., 2020). While CIC deletion clearly impacts numerous
aspects of neurodevelopment, ours is the first evidence indicating a
potential relationship between CIC and neural tube development.
In the present study, we performed and support the first genetic
association analysis between CIC variants and an increased risk for
NTDs. We also explored the possible underlying mechanisms by which CIC
could contribute to NTDs in humans. We initially focused on the folate
receptors, as they are membrane proteins that mediate cellular uptake of
folates. FOLR1 is important for neural tube closure during early
embryogenesis, as inactivation of this gene leads to embryoic death and
NTDs in mice which can be rescued by folate supplementation (Piedrahita
et al., 1999). In humans, homozygous biallelic LoF FOLR1 mutations lead
to extremely low 5-methyltetrohydrofolate level in the cerebrospinal
fluid (CFD) but have not been observed to result in NTDs (Steinfeld et
al., 2009). However, it has been reported that increased level of
maternal serum FOLR1 autoantibody during pregnancy is associated with
the occurrence of fetal NTDs (Rothenberg et al., 2004). Although
evidence is lacking to show that isolated FOLR1 gene variants are
associated with human NTDs, Saitsu (2017) found that altered spatial and
temporal Folr1 expression patterns in mice are associated with anterior
neural tube closure, and this expression pattern is conserved between
human and mice engineered to express a lacZ reporter transgene.
Additionally, there have been studies of human folate transport genes
that described 12 novel variants in FOLR1, FOLR2, and FOLR3 (Findley et
al., 2017) found in NTD cases. This included four large insertion
deletion variants in FOLR3 as well as a single stop gain variant. While
far from being conclusive evidence, it is suggestive that FOLR1
abnormalities might be involved in a subset of human NTDs. According to
our previous study of CIC and CFD, CIC could regulate FOLR1 expression
through binding to its promoter region. In this study, we confirmed that
CIC variants can decrease FOLR1 expression levels in human cell lines.
While we previously found that CIC binding motifs lie within the human
FOLR1 promoter region, they do not exist in the mouse Folr1promoter region.
Convergent extension (CE) is a crucial process during neural tube
closure by which the neural plate undergoes narrowing along its
mediolateral axis and extends along anteroposterior axis (Tada &
Heisenberg, 2012). The progression of convergent extension is driven by
planar polarized cell intercalation, which in turn is reported to be
driven by subcellular processes including extension of mediolaterally
directed cellular protrusions and shrinkage of mediolaterally oriented
cell-cell junctions (Butler & Wallingford, 2018; Blankenship, Backovic,
Sanny, Weitz & Zallen, 2006). The PCP signaling pathway is the most
well-characterized regulator of cell intercalation (Butler &
Wallingford, 2017). Several genes in the PCP pathway have been
associated with NTDs (Humphries, Narang & Mlodzik, 2020). Over 300
genes are known to be causative of NTDs in mice, and many of them
participate in the PCP pathway (Wang etal., 2018). Core PCP genes are
highly conserved from invertebrates to mammals, among which Van
Gogh (Vang), Vangl1/2 in mammals, is the critical regulator for normal
extension conversion. Other PCP genes include FZD3/FZD6,
CELSR1/CELSR2/CELSR3, DVL1/DVL2/DVL3,
PRICKLE1/PRICKLE2/PRICKLE3/PRICKLE4 and ANKRD6. Vangl2 was the first
genetically mapped PCP gene through studying Loop-tail mutant mice.
Homozygous Vangl2 mutants cause craniorachischisis, the most severe type
of NTD (Kibar et al., 2001), while Vangl1 and Vangl2 compound
heterozygous mice also exhibit a craniorachischisis phenotype (Torban et
al., 2008). Besides Vangl2, inactivation of Dvl1 and Dvl2, Celsr1, and
Fzd3 or both Fzd3 and Fzd6 also lead to severe NTD phenotypes, primarily
craniorachischisis and exencephaly in mice (Kibar et al., 2001; Wang et
al., 2006; Ybot-Gonzalez et al., 2007; Curtin et al., 2003). In addition
to these core PCP genes, some noncore PCP genes also exhibit severe NTD
in mice when gene targeted, such as protein tyrosine kinase 7 (PTK7),
scribbled PCP protein, the gene responsible for the circle tail mouse
phenotype, Scrib, and dishevelled binding antagonist of beta-catenin 1
(Dact-1) (Mohd-Zin, Marwan, Abou Chaar, Ahmad-Annuar & Abdul-Aziz,
2017). In humans, PCP genes have been examined in case–control
association studies or directly sequenced in mutation screens (Juriloff
& Harris, 2012). Vangl2 variants in human NTD samples were reported by
Lei et al.(2010) and Kibar et al.(2011). In the present study, we found
that all eight CIC variants lead to decreased Vangl2 protein levels in
human cell lines, and the CIC LoF variant also limited Vangl2 protein
levels in a murine cell line. We suspect this contributes to the
observed NTD phenotype.
Acting downstream of Vangl2, RhoA plays a crucial role
in regulating cell shape and movement (Oishi, Makita, Sato & Iiri,
2012). In this study, protein levels of Vangl2 and RhoA showed similar
trends for different CIC variants among groups in both human and mouse
cell lines. Our in vitro assays showed that CIC variants
diminished both FOLR1 expression level and PCP signals, both of which
could increase the likelihood of human NTDs. However, the relationship
between inhibiting Folr1 and associated PCP pathway gene expression and
the underlying mechanisms by which CIC affects Vangl2, remain unclear.
Balashova, Visina and Borodinsky (2017) found that FOLR1 is enriched in
the apical surface of the neural plate in Xenopus laevis ,
colocalized with C-cadherin and β-catenin, and that FOLR1 is necessary
for neural plate cell apical constriction during Xenopus neural tube
formation. The seemingly contradictory evidence emphasizes the
importance of continued experimentation on this system.
In this study, we identified a novel gene contributing to human NTDs. We
demonstrated the possible underlying mechanisms. Future studies are
needed to further elucidate these cellular and molecular mechanisms,
which will help us to better understand the genetic etiology of NTDs and
develop effective strategies to prevent these severe birth defects.