Impaired autophagy and excessive apoptosis disrupt cellular homeostasis and contribute to neural tube defects (NTDs), which are a group of fatal and disabling birth defects caused by the failure of neural tube closure during early embryonic development. However, the regulatory mechanisms underlying NTDs and outcomes remain elusive. Here, we report the role of the transcription factor nuclear factor I-C (NFIC) in maintaining cellular homeostasis in NTDs. We demonstrated that abnormally elevated levels of NFIC in a mouse model of NTDs can interact with the miR-200b promoter, leading to the activation of the transcription of miR-200b, which plays a critical role in NTD formation, as reported in our previous study. Furthermore, miR-200b represses autophagy and triggers apoptosis by directly targeting the autophagy-related gene Ambra1 (Autophagy/Beclin1 regulator 1). Notably, miR-200b inhibitors mitigate the unexpected effects of NFIC on autophagy and apoptosis. Collectively, these results indicate that the NFIC-miR-200b-Ambra1 axis, which integrates transcription- and epigenome-regulated miRNAs and an autophagy regulator, disrupts cellular homeostasis during the closure of the neural tube, and may provide new insight into NTD pathogenesis.

Congenital heart defects (CHDs) are the most common group of major birth defects. Presently there are no clinically used biomarkers for prenatally detecting CHDs. Here, we performed a comprehensive maternal serum proteomics assessment, combined with immunoassays, for the discovery of non-invasive biomarkers for prenatal diagnosis of CHDs. A total of 370 women were included in this study. An isobaric tagging for relative and absolute quantification (iTRAQ) proteomic approach was used first to compare protein profiles in pooled serum collected from women who had CHD-possessing or normal fetuses, and 47 proteins displayed significant differential expressions. Targeted verifications were performed on 11 proteins using multiple reaction monitoring mass spectrometry (MRM-MS), and the resultant candidate biomarkers were then further validated using ELISA analysis. Finally, we identified a biomarker panel composed of 4 cytoskeletal proteins capable of differentiating CHD-pregnancies from normal ones [with an area under the receiver operating characteristic curve (AUC) of 0.938, P < 0.0001]. The discovery of cytoskeletal protein changes in maternal serum not only could help us in prenatal diagnosis of CHDs, but also may shed new light on CHD embryogenesis studies.

Neural tube defects (NTDs) are complex abnormalities associated with gene-environment interactions. The underlying cause has not been determined. Spinal cord tissues from cases with NTDs and healthy controls were collected. Methylation patterns between cases and normal individuals were compared using 450K Infinium Methylation BeadChip Illumina. DNA methylation analysis by pyrosequencing (PyroMark Q96) and mRNA and protein expression were analyzed using real-time quantitative PCR and Western blotting, respectively. Next-generation and Sanger sequencing were used to determine genetic variants in the target genes. Spinal cord tissues from cases with NTDs had more hypomethylated than hypermethylated genes. Further evaluation showed that the exon 1 region of TRIM4 was hypomethylated, and TRIM4 mRNA and protein levels were significantly increased in NTDs compared to controls. A rare missense variant (rs76665876) in TRIM4 was found in 3 of the 14 NTD cases but was not related to TRIM4 expression. TRIM4 mRNA levels were significantly increased in cases with hypomethylation and without the rs76665876 variant. These findings suggest that spinal cord tissues in cases with NTDs had a different genome-wide methylation pattern compared to controls. Abnormal methylation patterns in TRIM4 in immunity pathways might be involved in NTD pathogenesis. Genetic variants in TRIM4 genes only slightly contribute to the etiology of human NTDs.

Aims: Failure of neural tube closure resulting from excessive apoptosis leads to neural tube defects (NTDs). NADPH oxidase 4 (NOX4) is a critical mediator of cell growth and death, yet its role in NTDs has never been characterized. NOX4 is a potential target of miR‐322, and we have previously demonstrated that miR‐322 was involved in high glucose‐induced NTDs. In this study, we investigated the effect of NOX4 on the embryonic neuroepithelium in NTDs and reveal a new regulatory mechanism for miR‐322 that disrupts neurulation by ameliorating cell apoptosis.Methods: All‐trans‐retinoic acid (ATRA)‐induced mouse model was utilized to study NTDs. RNA pull‐down and dual‐luciferase reporter assays were used to confirm the interaction between NOX4 and miR‐322. In mouse neural stem cells and whole‐embryo culture, Western blot and TUNEL were carried out to investigate the effects of miR‐322 and NOX4 on neuroepithelium apoptosis in NTD formation.Results: NOX4, as a novel target of miR‐322, was upregulated in ATRA‐induced mouse model of NTDs. In mouse neural stem cells, the expression of NOX4 was inhibited by miR‐322; still further, NOX4‐triggered apoptosis was also suppressed by miR‐322. Moreover, in whole‐embryo culture, injection of the miR‐322 mimic into the amniotic cavity attenuated cell apoptosis in NTD formation by silencing NOX4.Conclusion: miR‐322/NOX4 plays a crucial role in apoptosis‐induced NTD formation, which may provide a new understanding of the mechanism of embryonic NTDs and a basis for potential therapeutic target against NTDs.

Metallic elements play a pivotal role in maternal and fetal health. Metals can cross the placental barrier and be absorbed by fetuses, where they may affect closure of the neural tube during embryonic development. Neural tube defects (NTDs), which result from aberrant closure of the neural tube three to four weeks post-conception, have a multifactorial and complex etiology that combines genetic variants and environmental exposure. Recent advances in population-level association studies have investigated the link between maternal environmental exposure and NTDs, particularly the influence of metals on the incidence of NTDs. Herein, we present a broad and qualitative review of current literature on the association between maternal and prenatal metal exposure via the maternal peripheral blood, amniotic fluid, placenta, umbilical cord, and maternal hair, and the risk of developing NTDs. Specifically, we identify the various aggravating or attenuating effects of metallic exposure on the risk of NTD formation. This review provides novel insights into the association between environmental metals and NTDs and has important applications for NTD prevention and mitigating environmental exposure to metals.

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