Diabetes-induced Neural Tube Defects Research Articles

High glucose-increased miR-200c contributes to cellular senescence and DNA damage in neural stem cells.

Maternal diabetes increases the risk for neural tube defects (NTDs). It is unclear if miRNAs, senescence, and DNA damage are involved in this process. In this study, we used neural stem cells as an in vitro proxy of embryonic neuroepithelium to investigate whether high glucose triggers neural stem cell senescence and DNA damage by upregulating miR-200c, which may be responsible for NTDs. C17.2 neural stem cells were cultured with normal glucose (5 mM) or high glucose (≥16.7 mM) at different doses and time points for detecting miR-200c levels, markers of senescence and DNA damage. Neural stem cells were exposed to antioxidant SOD1 mimetic Tempol and high glucose for 48 h to test roles of oxidative stress on the miR-200c, senescence, and DNA damage levels. An miR-200c mimic and an inhibitor were transfected into neural stem cells to increase or decrease miR-200c activities. High glucose upregulated miR-200c in neural stem cells. A time course study of the effect of high glucose revealed that miR-200c initially increased at 12 h and reached its zenith at 18 h. Tempol reduced miR-200c levels caused by high glucose. High glucose induced markers of senescence and DNA damage in neural stem cells. Tempol abolished high glucose-induced markers of senescence and DNA damage. The miR-200c inhibitor suppressed high glucose-induced markers of senescence and DNA damage. Treatment with miR-200c mimic imitates high glucose-induced markers of senescence and DNA damage. We show that high glucose increases miR-200c, which contributes to cellular senescence and DNA damage in neural stem cells and provides a potential pathway for maternal diabetes-induced neural tube defects.

Synergistic Effects of Folic Acid and n-3 Polyunsaturated Fatty Acid in Preventing Neural Tube Defects in Diabetic Mice.

The present study aimed to investigate whether a combination of folic acid (FA) and n-3 polyunsaturated fatty acids (PUFA) has a better preventive effect on maternal diabetes-induced neural tube defects (NTD) than FA alone. The experiment included five groups of pregnant mice: healthy control (HC), diabetes mellitus control (DMC), diabetes + n-3 PUFA (DMn-3), diabetes + FA (DMFA), and diabetes + FA + n-3 PUFA (DMFA + n-3). The incidence of NTD in DMFA + n-3 (1.04%) was significantly lower than that in DMFA (8.57%) and DMn-3 (7.82%). The incidence of NTD in DMFA and DMn-3 was significantly lower than that in DMC (19.41%). DMFA + n-3 had a lower apoptosis of neuroepithelial cells, a lower expression of P53 and Bax, and a higher expression of Pax3 and Bcl-2, compared with DMFA and DMn-3. Combination of FA and n-3 PUFA attenuated diabetes-induced hypermethylation of Pax3, overexpression and overactivity of Dnmt3b, abnormal expression of genes involved in one-carbon metabolism and elevation of homocysteine, and these improving effects were better than FA or n-3 PUFA alone. In conclusion, the combination of FA and n-3 PUFA has a synergistic effect on preventing maternal diabetes-induced NTD.

Functional cargos of exosomes derived from Flk-1+ vascular progenitors enable neurulation and ameliorate embryonic anomalies in diabetic pregnancy

Various types of progenitors initiate individual organ formation and their crosstalk orchestrates morphogenesis for the entire embryo. Here we show that progenitor exosomal communication across embryonic organs occurs in normal development and is altered in embryos of diabetic pregnancy. Endoderm fibroblast growth factor 2 (FGF2) stimulates mesoderm Flk-1+ vascular progenitors to produce exosomes containing the anti-stress protein Survivin. These exosomes act on neural stem cells of the neuroepithelium to facilitate neurulation by inhibiting cellular stress and apoptosis. Maternal diabetes causes Flk-1+ progenitor dysfunction by suppressing FGF2 through DNA hypermethylation. Restoring endoderm FGF2 prevents diabetes-induced survivin reduction in Flk-1+ progenitor exosomes. Transgenic Survivin expression in Flk-1+ progenitors or in utero delivery of survivin-enriched exosomes restores cellular homeostasis and prevents diabetes-induced neural tube defects (NTDs), whereas inhibiting exosome production induces NTDs. Thus, functional inter-organ communication via Flk-1 exosomes is vital for neurulation and its disruption leads to embryonic anomalies.

Restoring BMP4 expression in vascular endothelial progenitors ameliorates maternal diabetes-induced apoptosis and neural tube defects

During mouse embryonic development, vasculogenesis initially occurs in the yolk sac, preceding neurulation. Our previous studies have demonstrated that maternal diabetes induces embryonic vasculopathy at early embryonic developmental stage by suppressing the expression of vascular growth factors including BMP4 (bone morphogenetic protein 4). This study aimed to determine whether restoring diabetes-inhibited BMP4 expression in Flk-1+ progenitors effectively prevented maternal diabetes-induced embryonic vasculopathy and NTDs. Transgenic (Tg) BMP4 expression in the vascular endothelial growth factor receptor 2 (Flk-1)-positive (Flk-1+) progenitors was achieved by crossing a Floxed BMP4 Tg mouse line with the Flk-1-Cre mouse line. Non-BMP4 Tg and BMP4 Tg embryos were harvested at E8.5 to assess the expression of BMP4, markers of endoplasmic reticulum stress, and expression of the Id genes, direct targets of BMP4; and the presence of cleaved caspase 3 and 8, apoptosis, and Smad signaling. BMP4 Tg overexpression neutralized its down-regulation by maternal diabetes in E8.5 embryos. Maternal diabetes-induced Flk-1+ progenitor apoptosis, impairment of blood island formation, and reduction of Flk-1+ progenitor number and blood vessel density, which were reversed by BMP4 Tg expression. BMP4 Tg expression in Flk-1+ progenitors blocked maternal diabetes-induced vasculopathy in early stage embryos (E7.5-E8.5) and consequently led to amelioration of maternal diabetes-induced neural tube defects (NTDs) at E10.5. BMP4 Tg expression inhibited maternal diabetes-induced endoplasmic reticulum stress and caspase cascade activation in the developing neuroepithelium, and reduced neuroepithelial cell apoptosis. BMP4 Tg expression re-activated Smad1/5/8 phosphorylation and reversed maternal diabetes-suppressed Smad4 expression. BMP4 Tg expression restored Id1 and Smad6 expression inhibited by maternal diabetes. In vitro, recombinant BMP4 protein blocked high glucose-induced Flk-1+ progenitor apoptosis and NTDs. These data demonstrate that BMP4 down-regulation in Flk-1+ progenitors are responsible for diabetes-induced yolk sac vasculopathy, and that restoring BMP4 expression prevents vasculopathy and rescues neuroepithelial cells from cellular organelle stress, leading to NTD reduction.

Identification of histone malonylation in the human fetal brain and implications for diabetes-induced neural tube defects.

BackgroundNeural tube defects (NTDs) are severe congenital malformations. Diabetes during pregnancy is a risk factor for NTDs, but its mechanism remains elusive. Emerging evidence suggests that protein malonylation is involved in diabetes. Here, we report the correlation between histone lysine malonylation in diabetes‐induced NTDs.MethodsNano‐HPLC/MS/MS was used to screen the histone malonylation profile in human embryonic brain tissue. Then, the histone malonylation level was compared between the brains of normal control mice and mice with diabetes‐induced NTDs. Finally, the histone malonylation level was compared under high glucose exposure in an E9 neuroepithelial cell line (NE4C).ResultsA total of 30 histone malonylation sites were identified in human embryonic brain tissue, including 18 novel sites. Furthermore, we found an increased histone malonylation level in brain tissues from mice with diabetes‐induced NTDs. Finally, both the histone malonylation modified sites and the modified levels were proved to be increased in the NE4C treated with high glucose.ConclusionOur results present a comprehensive map of histone malonylation in the human fetal brain. Furthermore, we provide experimental evidence supporting a relationship between histone malonylation and NTDs caused by high glucose‐induced diabetes. These findings offer new insights into the pathological role of histone modifications in human NTDs.

1327-P: Maternal Diabetes Suppresses Neural Tube Closure Gene Shroom3 through DNA Hypermethylation and Thus Disrupts Planar Cell Polarity

Neural Tube Defects (NTDs) are among the most frequent and severe malformations associated with maternal diabetes. DNA hypermethylation inhibits gene expression. Our recent studies have revealed a group of neural tube closure-related genes are downregulated by maternal diabetes. Planar cell polarity (PCP) is conserved in metazoans and is essential for development, deficiencies of PCP genes result in NTDs. Shroom3 encodes a PDZ-domain-containing protein that regulates cell shape in epithelium. Shroom3 interacts with PCP genes, loss of function of Shroom3 leads to NTDs. We hypothesize that DNA hypermethylation induced by maternal diabetes is responsible for the downregulation of Shroom3, which disrupts the PCP pathway leading to NTD formation. We found global DNA methylation levels were increased about 3 times and all 3 DNA methyltransferase levels were increased in the embryos from diabetic dams compared to the nondiabetic control group. Conditional double knockout of Dnmt3a/Dnmt3b gene in the developing neuroepithelium ameliorated maternal diabetes-induced NTDs, while overexpression Dnmt3b in the neuroepithelium induced NTDs in a dose dependent manner. Expression levels of PCP genes and Shroom3 were reduced by maternal diabetes and Dnmt3b transgenic overexpression. Similarly, PCP protein subcellular expression patterns were also disrupted. DNA methyl-sequencing revealed maternal diabetes-induced hypermethylation in the Shromm3 gene promoter. De-methylation rescued Shroom3 promoter activity suppressed by high glucose. Our study reveals that diabetes-induced DNA hypermethylation inhibits Shroom3 gene expression and Shroom3 deficiency mediates the teratogenic effect of maternal diabetes by disrupting PCP and resulting in NTDs. Since de-methylation can rescue Shroom3 activity, our findings suggest that targeting DNA hypermethylation could be therapeutically effective in treating maternal diabetes-induced NTDs. Disclosure C. Xu: None. P. Yang: None. Funding National Institutes of Health (R01DK083243, R01DK101972, R01HL131737, R01HL134368, R01DK103024)

679: The newly determined role of miR17 and its target, Txnip, in the diabetes-induced Congenital Malformations

The mechanism underlying diabetic embryopathy is elusive. MicroRNAs (miRNAs) may play an important role in such process. We previously found that miR-17 expression was suppressed in C17.2 neural stem cell line, by high glucose and in mouse early embryo at day 8.5 (E8.5) from diabetic pregnancy. Here we aim to investigate the role of miR-17 and its putative target gene, Txnip, in maternal diabetes-induced neural tube defects (NTDs). We generated a miR-17 transgenic (Tg) mice in which miR-17 was driven by nestin promoter along with a separate GFP gene. miR-17 Tg nondiabetic male mice mated with nondiabetic and streptozotocin (STZ)-induced type 1 diabetic Wild-Type (WT) female mice. To further determine the role of the miR-17 target gene, Txnip (Thioredoxin Interacting Protein), in the induction of maternal diabetes-induced NTDs, Txnipf/w; nestin-cre male mice crossed with nondiabetic or diabetic Txnipf/f (f: floxed condition allele) female mice. The Txnip gene was deleted specifically in the neuroepithelium in Txnipf/f; nestin-cre embryos. Embryos are collected for NTD analysis at E10.5. 3 out of 37 (8.1%) miR-17 Tg embryos exhibited NTDs under maternal diabetic conditions, whereas 22 out of 42 (52.4%) WT embryos had NTDs. The results indicate that miR-17 overexpression in the neuroepithelium significantly reduced the maternal diabetes-induced NTD rate. Comparing to the NTD rate of 19.5% (8 out of 41 embryos) in WT embryos, embryos with conditional Txnip gene deletion (homozygous) had a significantly decreased NTD rate of 6.7% (1 out of 15 embryos). However, the NTDs rate from Txnip gene heterozygous deletion embryos was 21.1% (4 out of 19) which was comparable to that from WT embryos in diabetic pregnancy. Our study demonstrates that miR-17 is suppressed by maternal diabetes and it protects embryos from NTDs in diabetic pregnancy, and that the antioxidant negative regulator and the pro-apoptotic gene, Txnip, is a miR-17 target gene in diabetic embryopathy. The miR-17-Txnip circuit is critically involved in the induction of maternal diabetes-induced NTDs.

1693-P: Rapamycin Reduces Maternal Diabetes-Induced Neural Tube Defects by Suppressing Premature Senescence

Maternal diabetes significantly increases the risk of structural birth defects. Senescence occurs after neural tube closure. We aimed to determine whether maternal diabetes induces premature senescence in the developing neuroepithelium. Maternal diabetes induced robust senescence associated β-galactosidase (SAβG) activity in the apical side of the developing neuroepithelium at embryonic day 8.5 (E8.5). The major senescence mediator p21 was induced preceding the SAβG signal at the same region. p21 gene deleted embryos of diabetic dams had a significantly lower neural tube defects (NTDs) incidence (3.6%) compared to wild type litter mates (25.9%). The transcriptional factor Forkhead transcription factor 3a (FoxO3a) is activated in diabetic embryopathy, and Foxo3a knockout reduces maternal diabetes-induced NTDs. Our data showed Foxo3a deletion diminished the signals of SAβG, and abrogated diabetes-increased abundance of p21 and DNA damage response proteins. MicroRNAs have been identified as novel inducers of cellular senescence. Two FoxO3a binding sites were found in the promote region of miR-200c. In vitro study confirmed both FoxO3a and high glucose stimulated miR-200 promoter activity. miR-200c null embryos had a lower NTD rate (4.3%) compared with that of WT embryos (29.2%) from diabetic dams. miR-200c repressed the expression of ZEB1 and ZEB2, which are transcription repressors for p21. In addition to the revelation of the gene regulation pathway in maternal diabetes-induced premature senescence, we found intraperitoneal injection of the senescence inhibitor Rapamycin in diabetic dams during E5.5-E9.5 diminished SAβG signal, and reduced the NTD rate from 24.0% to 4.5%) in diabetic pregnancy. Thus, our study revealed a novel FoxO3a-miR-200c-p21-mediated premature senescence pathway, and provided the mechanistic basis for targeting premature senescence in preventing diabetes-induced NTDs. Disclosure C. Xu: None. P. Yang: None. Funding American Diabetes Association (1-13-BS-220 to P.Y.); National Institutes of Health

1692-P: miR17 Overexpression or Deleting Its Target Gene Txnip Ameliorates Neural Tube Defect Formation in Diabetic Embryopathy

microRNAs (miR) play important roles in the induction of diabetic embryopathy. Our previous studies have demonstrated that high glucose in vitro and maternal diabetes in vivo suppressed miR-17 expression. To determine whether miR17 down-regulation is involved in maternal diabetes-induced neural tube defects (NTDs) formation, we created a miR-17 transgenic (Tg) mouse line in which miR-17 specifically overexpresses in the developing neuroepithelium. miR-17 Tg male mice crossed with nondiabetic and diabetic (streptozotocin-induced type 1 diabetes) wild type (WT) female mice to generate WT and Tg embryos. Under nondiabetic conditions, there was no NTDs in both WT and Tg embryos. The NTD rate of WT embryos was 52.4% (22 out of 42 embryos) in diabetic pregnancy, whereas miR-17 overexpression in the neuroepithelium significantly reduced the NTD rate to 8.1% (3 out of 37 embryos). Txnip up-regulation is implicated in the pathogenesis of diabetes and diabetic complications. Txnip is a predicted miR-17 target genes, and miR-17 overexpression abolished maternal diabetes-increased Txnip expression. To investigate whether Txnip participates in the pathogenesis of diabetic embryopathy, the Txnip gene was deleted specifically in the neuroepithelium using the Cre-flox (f) approach by crossing Txnipf/w; nestin-cre male mice with nondiabetic or diabetic Txnipf/f female mice. Comparing to the NTD rate of 19.4% (7 out of 36 embryos) in WT embryos, embryos with conditional Txnip deletion had an NTD rate of 8.3% (1 out of 12 embryos). Our findings indicated that the miR-17-Txnip circuit is critically involved in the induction of diabetic embryopathy. Disclosure P. Yang: None. P. Yang: None. Funding American Diabetes Association (1-13-BS-220 to P.Y.); National Institutes of Health

The increased activity of a transcription factor inhibits autophagy in diabetic embryopathy

Maternal diabetes induces neural tube defects and stimulates the activity of the forkhead box O3 (Fox)O3a in the embryonic neuroepithelium. We previously demonstrated that deleting the FOXO3a gene ameliorates maternal diabetes-induced neural tube defects. Macroautophagy (hereafter referred to as "autophagy") is essential for neurulation. Rescuing autophagy suppressed by maternal diabetes in the developing neuroepithelium inhibits neural tube defect formation in diabetic pregnancy. This evidence suggests a possible link between FoxO3a and impaired autophagy in diabetic embryopathy. We aimed to determine whether maternal diabetes suppresses autophagy through FoxO3a, and if the transcriptional activity of FoxO3a is required for the induction of diabetic embryopathy. We used a well-established type 1 diabetic embryopathy mouse model, in which diabetes was induced by streptozotocin, for our invivo studies. To determine if FoxO3a mediates the inhibitory effect of maternal diabetes on autophagy in the developing neuroepithelium, we induced diabetic embryopathy in FOXO3a gene knockout mice and FoxO3a dominant negative transgenic mice. Embryos were harvested at embryonic day 8.5 to determine FoxO3a and autophagy activity and at embryonic day 10.5 for the presence of neural tube defects. We also examined the expression of autophagy-related genes. C17.2 neural stem cells were used for invitro examination of the potential effects of FoxO3a on autophagy. Deletion of the FOXO3a gene restored the autophagy markers, lipidation of microtubule-associated protein 1A/1B-light chain 3I to light chain 3II, in neurulation stage embryos. Maternal diabetes decreased light chain 3I-positive puncta number in the neuroepithelium, which was restored by deleting FoxO3a. Maternal diabetes also decreased the expression of positive regulators of autophagy (Unc-51 like autophagy activating kinase 1, Coiled-coil myosin-like BCL2-interacting protein, and autophagy-related gene 5) and the negative regulator of autophagy, p62. FOXO3a gene deletion abrogated the dysregulation of autophagy genes. Invitro data showed that the constitutively active form of FoxO3a mimicked high glucose in repressing autophagy. In cells cultured under high-glucose conditions, overexpression of the dominant negative FoxO3a mutant blocked autophagy impairment. Dominant negative FoxO3a overexpression in the developing neuroepithelium restored autophagy and significantly reduced maternal diabetes-induced apoptosis and neural tube defects. Our study revealed that diabetes-induced FoxO3a activation inhibited autophagy in the embryonic neuroepithelium. We also observed that FoxO3a transcriptional activity mediated the teratogenic effect of maternal diabetes because dominant negative FoxO3a prevents maternal diabetes-induced autophagy impairment and neural tube defect formation. Our findings suggest that autophagy activators could be therapeutically effective in treating maternal diabetes-induced neural tube defects.

Survivin from Flk1+ Endothelial Progenitor Cells in the Developing Embryo Ameliorates Maternal Diabetes–Induced Neural Tube Defects through Exosome

Neural tube defects (NTDs) are among the most common and severe structural birth defects and maternal diabetes induces NTDs. During mouse embryonic development, blood vessel formation initially occurs in the yolk sac, preceding neurulation which takes place from embryonic day 8 (E8) to E10.5. Our previous studies have demonstrated that maternal diabetes induces embryonic vasculopathy at early embryonic developmental stage. We hypothesize that early vasculopathy accounts for late failed neural tube closure in diabetic pregnancy. To test this hypothesis, we created the Flk1 promoter driven survivin (the cell cycle-regulated apoptosis inhibitor) transgenic mice. Maternal diabetes induced the loss of Flk1+ progenitors, impaired blood island formation and reduced blood vessel densities. Expression of survivin in the Flk1+ progenitors ameliorated diabetes-induced NTDs. Under nondiabetic conditions, survivin overexpression did not affect embryonic development. Overexpression of survivin significantly reduced diabetes-induced NTDs. Flk1+ progenitors produced survivin-containing exosomes. Immunocytochemistry staining indicated that survivin-containing exosomes were transported and incorporated into cells of the developing neuroepithelium, leading to blockage of neuroepithelial cell apoptosis induced by maternal diabetes. Furthermore, we isolated exosomes from Flk1+ progenitors derived from E7.5 embryos. In utero delivery of the survivin-containing exosomes by microinjection into the amniotic cavity of diabetic mice at E9 significantly inhibited maternal diabetes-induced neuroepithelial cell apoptosis and reduced NTDs. Taken together, these data revealed that the cross-talk between the extra-embryonic structures and the developing neuroepithelium through the survivin-containing exosome plays a critical role for the proper neural tube closure. Disclosure S. Cao: None. W. Shen: None. P. Yang: None.

Trehalose prevents sciatic nerve damage to and apoptosis of Schwann cells of streptozotocin-induced diabetic C57BL/6J mice

Type 1 diabetes (T1DM) affects approximately 1 in 500 children. Diabetic peripheral neuropathy (DPN) is the most common form of peripheral neuropathy in diabetes and is a significant risk factor for serious pathological change. It is difficult and costly to treat DPN and although there have been several pivotal trials. The development of new drugs to treat DPN remains a high priority. Trehalose is a naturally occurring disaccharide, which is indicated to prevent maternal type 1 diabetes-induced neural tube defects. Thus, the primary aim of this study is to determine whether trehalose ameliorates DPN-induced sciatic nerve injury in TIDM. To establish a T1DM mouse model, wild-type (WT) male C57BL/6 J mice were injected with streptozotocin (STZ). WT mice, T1DM mice, and mice fed with trehalose were assayed for myelin-related gene expression and with behavioral tests. To mimic high glucose in vivo, Schwann cells were cultured under high glucose conditions with or without trehalose. In addition, oxidative damage, apoptosis, and mitochondrial translocation of the pro-apoptotic B-cell lymphoma-2 (Bcl-2) family members were assessed in Schwann cells. Results showed that treatment by trehalose prevented DPN and preserved diabetes-decreased expression of myelin-related genes in T1DM mice. Furthermore, trehalose abolished diabetes-suppressed regeneration of the sciatic nerve. More importantly, trehalose suppressed high glucose-induced oxidative damage and apoptosis in Schwann cells. In summary, trehalose ameliorates DPN-induced sciatic nerve injury in T1DM by preventing apoptosis, which makes it a promising candidate for the treatment of DPN.

Modulation of nuclear factor-κB signaling and reduction of neural tube defects by quercetin-3-glucoside in embryos of diabetic mice

Diabetes mellitus in early pregnancy increases the risk of birth defects in infants. Maternal hyperglycemia stimulates the expression of nitric oxide synthase 2, which can be regulated by transcription factors of the nuclear factor-κB family. Increases in reactive nitrogen species generate intracellular stress conditions, including nitrosative, oxidative, and endoplasmic reticulum stresses, and trigger programmed cell death (or apoptosis) in the neural folds, resulting in neural tube defects in the embryo. Inhibiting nitric oxide synthase 2 can reduce neural tube defects; however, the underlying mechanisms require further delineation. Targeting nitric oxide synthase 2 and associated nitrosative stress using naturally occurring phytochemicals is a potential approach to preventing birth defects in diabetic pregnancies. This study aims to investigate the effect of quercetin-3-glucoside, a naturally occurring polyphenol flavonoid, in reducing maternal diabetes-induced neural tube defects in an animal model, and to delineate the molecular mechanisms underlying quercetin-3-glucoside action in regulating nitric oxide synthase 2 expression. Female mice (C57BL/6) were induced to develop diabetes using streptozotocin before pregnancy. Diabetic pregnant mice were administered quercetin-3-glucoside (100 mg/kg) daily via gavage feeding, introduction of drug to the stomach directly via a feeding needle, during neurulation from embryonic day 6.5-9.5. After treatment at embryonic day 10.5, embryos were collected and examined for the presence of neural tube defects and apoptosis in the neural tube. Expression of nitric oxide synthase 2 and superoxide dismutase 1 (an antioxidative enzyme) was quantified using Western blot assay. Nitrosative, oxidative, and endoplasmic reticulum stress conditions were assessed using specific biomarkers. Expression and posttranslational modification of factors in the nuclear factor-κB system were investigated. Treatment with quercetin-3-glucoside (suspended in water) significantly decreased neural tube defect rate and apoptosis in the embryos of diabetic mice, compared with those in the water-treated diabetic group (3.1% vs. 24.7%; P < .001). Quercetin-3-glucoside decreased the expression of nitric oxide synthase 2 and nitrosative stress (P < .05). It also increased the levels of superoxide dismutase 1 (P < .05), further increasing the antioxidative capacity of the cells. Quercetin-3-glucoside treatment also alleviated of endoplasmic reticulum stress in the embryos of diabetic mice (P < .05). Quercetin-3-glucoside reduced the levels of p65 (P < .05), a member of the nuclear factor-κB transcription factor family, but augmented the levels of the inhibitor of κBα (P < .05), which suppresses p65 nuclear translocation. In association with these changes, the levels of inhibitor of κB kinase-α and inhibitor of κBα phosphorylation were elevated (P< .05). Quercetin-3-glucoside reduces the neural tube defects rate in the embryos of diabetic dams. Quercetin-3-glucoside suppresses nitric oxide synthase 2 and increases superoxide dismutase 1 expression, leading to alleviation of nitrosative, oxidative, and endoplasmic reticulum stress conditions. Quercetin-3-glucoside may regulate the expression of nitric oxide synthase 2 via modulating the nuclear factor-κB transcription regulation system. Quercetin-3-glucoside, a naturally occurring polyphenol that has high bioavailability and low toxicity, is a promising candidate agent to prevent birth defects in diabetic pregnancies.

Oxidative stress-induced miR-27a targets the redox gene nuclear factor erythroid 2-related factor 2 in diabetic embryopathy

Maternal diabetes induces neural tube defects, and oxidative stress is a causal factor for maternal diabetes-induced neural tube defects. The redox gene nuclear factor erythroid 2-related factor 2 is the master regulator of the cellular antioxidant system. In this study, we aimed to determine whether maternal diabetes inhibits nuclear factor erythroid 2-related factor 2 expression and nuclear factor erythroid 2-related factor 2-controlled antioxidant genes through the redox-sensitive miR-27a. We used a well-established type 1 diabetic embryopathy mouse model induced by streptozotocin for our invivo studies. Embryos at embryonic day 8.5 were harvested for analysis of nuclear factor erythroid 2-related factor 2, nuclear factor erythroid 2-related factor 2-controlled antioxidant genes, and miR-27a expression. To determine if mitigating oxidative stress inhibits the increase of miR-27a and the decrease of nuclear factor erythroid 2-related factor 2 expression, we induced diabetic embryopathy in superoxide dismutase 2 (mitochondrial-associated antioxidant gene)-overexpressing mice. This model exhibits reduced mitochondria reactive oxygen species even in the presence of hyperglycemia. To investigate the causal relationship between miR-27a and nuclear factor erythroid 2-related factor 2 invitro, we examined C17.2 neural stem cells under normal and high-glucose conditions. We observed that the messenger RNA and protein levels of nuclear factor erythroid 2-related factor 2 were significantly decreased in embryos on embryonic day 8.5 from diabetic dams compared to thosefrom nondiabetic dams. High-glucose also significantly decreased nuclear factor erythroid 2-related factor 2 expression in a dose- and time-dependent manner in cultured neural stem cells. Our data revealed that miR-27a was up-regulated in embryos on embryonic day 8.5 exposed to diabetes, and that high glucose increased miR-27a levels in a dose- and time-dependent manner in cultured neural stem cells. In addition, we found that a miR-27a inhibitor abrogated the inhibitory effect of high glucose on nuclear factor erythroid 2-related factor 2 expression, and a miR-27a mimic suppressed nuclear factor erythroid 2-related factor 2 expression in cultured neural stem cells. Furthermore, our data indicated that the nuclear factor erythroid 2-related factor 2-controlled antioxidant enzymes glutamate-cysteine ligase catalytic subunit, glutamate-cysteine ligase modifier subunit, and glutathione S-transferase A1 were down-regulated by maternal diabetes in embryos on embryonic day 8.5 and high glucose in cultured neural stem cells. Inhibiting miR-27a restored expression of glutamate-cysteine ligase catalytic subunit, glutamate-cysteine ligase modifier subunit, and glutathione S-transferase A1. Overexpressing superoxide dismutase 2 reversed the maternal diabetes-induced increase of miR-27a and suppression of nuclear factor erythroid 2-related factor 2 and nuclear factor erythroid 2-related factor 2-controlled antioxidant enzymes. Our study demonstrates that maternal diabetes-induced oxidative stress increases miR-27a, which, in turn, suppresses nuclear factor erythroid 2-related factor 2 and its responsive antioxidant enzymes, resulting in diabetic embryopathy.

Endoplasmic Reticulum Stress-Induced CHOP Inhibits PGC-1α and Causes Mitochondrial Dysfunction in Diabetic Embryopathy.

Endoplasmic reticulum (ER) stress has been implicated in the development of maternal diabetes-induced neural tube defects (NTDs). ER stress-induced C/EBP homologous protein (CHOP) plays an important role in the pro-apoptotic execution pathways. However, the molecular mechanism underlying ER stress- and CHOP-induced neuroepithelium cell apoptosis in diabetic embryopathy is still unclear. Deletion of the Chop gene significantly reduced maternal diabetes-induced NTDs. CHOP deficiency abrogated maternal diabetes-induced mitochondrial dysfunction and neuroepithelium cell apoptosis. Further analysis demonstrated that CHOP repressed the expression of peroxisome-proliferator-activated receptor-γ coactivator-1α (PGC-1α), an essential regulator for mitochondrial biogenesis and function. Both CHOP deficiency in vivo and knockdown in vitro restore high glucose-suppressed PGC-1α expression. In contrast, CHOP overexpression mimicked inhibition of PGC-1α by high glucose. In response to the ER stress inducer tunicamycin, PGC-1α expression was decreased, whereas the ER stress inhibitor 4-phenylbutyric acid blocked high glucose-suppressed PGC-1α expression. Moreover, maternal diabetes in vivo and high glucose in vitro promoted the interaction between CHOP and the PGC-1α transcriptional regulator CCAAT/enhancer binding protein-β (C/EBPβ), and reduced C/EBPβ binding to the PGC-1α promoter leading to markedly decrease in PGC-1α expression. Together, our findings support the hypothesis that maternal diabetes-induced ER stress increases CHOP expression which represses PGC-1α through suppressing the C/EBPβ transcriptional activity, subsequently induces mitochondrial dysfunction and ultimately results in NTDs.

Formation of neurodegenerative aggresome and death-inducing signaling complex in maternal diabetes-induced neural tube defects

Diabetes mellitus in early pregnancy increases the risk in infants of birth defects, such as neural tube defects (NTDs), known as diabetic embryopathy. NTDs are associated with hyperglycemia-induced protein misfolding and Caspase-8-induced programmed cell death. The present study shows that misfolded proteins are ubiquitinylated, suggesting that ubiquitin-proteasomal degradation is impaired. Misfolded proteins form aggregates containing ubiquitin-binding protein p62, suggesting that autophagic-lysosomal clearance is insufficient. Additionally, these aggregates contain the neurodegenerative disease-associated proteins α-Synuclein, Parkin, and Huntingtin (Htt). Aggregation of Htt may lead to formation of a death-inducing signaling complex of Hip1, Hippi, and Caspase-8. Treatment with chemical chaperones, such as sodium 4-phenylbutyrate (PBA), reduces protein aggregation in neural stem cells in vitro and in embryos in vivo. Furthermore, treatment with PBA in vivo decreases NTD rate in the embryos of diabetic mice, as well as Caspase-8 activation and cell death. Enhancing protein folding could be a potential interventional approach to preventing embryonic malformations in diabetic pregnancies.

Superoxide dismutase 2 overexpression alleviates maternal diabetes-induced neural tube defects, restores mitochondrial function and suppresses cellular stress in diabetic embryopathy

Pregestational diabetes disrupts neurulation leading to neural tube defects (NTDs). Oxidative stress resulting from reactive oxygen species (ROS) plays a central role in the induction of NTD formation in diabetic pregnancies. We aimed to determine whether mitochondrial dysfunction increases ROS production leading to oxidative stress and diabetic embryopathy. Overexpression of the mitochondrion-specific antioxidant enzyme superoxide dismutase 2 (SOD2) in a transgenic (Tg) mouse model significantly reduced maternal diabetes-induced NTDs. SOD2 overexpression abrogated maternal diabetes-induced mitochondrial dysfunction by inhibiting mitochondrial translocation of the pro-apoptotic Bcl-2 family members, reducing the number of defective mitochondria in neuroepithelial cells, and decreasing mitochondrial membrane potential. Furthermore, SOD2 overexpression blocked maternal diabetes-increased ROS production by diminishing dihydroethidium staining signals in the developing neuroepithelium, and reducing the levels of nitrotyrosine-modified proteins and lipid hydroperoxide level in neurulation stage embryos. SOD2 overexpression also abolished maternal diabetes-induced endoplasmic reticulum stress. Finally, caspase-dependent neuroepithelial cell apoptosis enhanced by oxidative stress was significantly reduced by SOD2 overexpression. Thus, our findings support the hypothesis that mitochondrial dysfunction in the developing neuroepithelium enhances ROS production, which leads to oxidative stress and endoplasmic reticulum (ER) stress. SOD2 overexpression blocks maternal diabetes-induced oxidative stress and ER stress, and reduces the incidence of NTDs in embryos exposed to maternal diabetes.

High glucose-induced oxidative stress represses sirtuin deacetylase expression and increases histone acetylation leading to neural tube defects.

Aberrant epigenetic modifications are implicated in maternal diabetes-induced neural tube defects (NTDs). Because cellular stress plays a causal role in diabetic embryopathy, we investigated the possible role of the stress-resistant sirtuin (SIRT) family histone deacetylases. Among the seven sirtuins (SIRT1-7), pre-gestational maternal diabetes in vivo or high glucose in vitro significantly reduced the expression of SIRT 2 and SIRT6 in the embryo or neural stem cells, respectively. The down-regulation of SIRT2 and SIRT6 was reversed by superoxide dismutase 1 (SOD1) over-expression in the in vivo mouse model of diabetic embryopathy and the SOD mimetic, tempol and cell permeable SOD, PEGSOD in neural stem cell cultures. 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), a superoxide generating agent, mimicked high glucose-suppressed SIRT2 and SIRT6 expression. The acetylation of histone 3 at lysine residues 56 (H3K56), H3K14, H3K9, and H3K27, putative substrates of SIRT2 and SIRT6, was increased by maternal diabetes in vivo or high glucose in vitro, and these increases were blocked by SOD1 over-expression or tempol treatment. SIRT2 or SIRT6 over-expression abrogated high glucose-suppressed SIRT2 or SIRT6 expression, and prevented the increase in acetylation of their histone substrates. The potent sirtuin activator (SRT1720) blocked high glucose-increased histone acetylation and NTD formation, whereas the combination of a pharmacological SIRT2 inhibitor and a pan SIRT inhibitor mimicked the effect of high glucose on increased histone acetylation and NTD induction. Thus, diabetes in vivo or high glucose in vitro suppresses SIRT2 and SIRT6 expression through oxidative stress, and sirtuin down-regulation-induced histone acetylation may be involved in diabetes-induced NTDs. The mechanism underlying pre-gestational diabetes-induced neural tube defects (NTDs) is still elusive. Our study unravels a new epigenetic mechanism in which maternal diabetes-induced oxidative stress represses sirtuin deacetylase 2 (SIRT2) and 6 (SIRT6) expression leading to histone acetylation and gene expression. SIRT down-regulation mediates the teratogenicity of diabetes leading to (NTD) formation. The study provides a mechanistic basis for the development of natural antioxidants and SIRT activators as therapeutics for diabetic embryopathy.

The green tea polyphenol EGCG alleviates maternal diabetes–induced neural tube defects by inhibiting DNA hypermethylation

Maternal diabetes increases the risk of neural tube defects in offspring. Our previous study demonstrated that the green tea polyphenol, Epigallocatechin gallate, inhibits high glucose-induced neural tube defects in cultured embryos. However, the therapeutic effect of Epigallocatechin gallate on maternal diabetes-induced neural tube defects is still unclear. We aimed to examine whether Epigallocatechin gallate treatment can reduce maternal diabetes-induced DNA methylation and neural tube defects. Nondiabetic and diabetic pregnant mice at embryonic day 5.5 were given drinking water with or without 1 or 10 μM Epigallocatechin gallate. At embryonic day 8.75, embryos were dissected from the visceral yolk sac for the measurement of the levels and activity of DNA methyltransferases, the levels of global DNA methylation, and methylation in the CpG islands of neural tube closure essential gene promoters. embryonic day 10.5 embryos were examined for neural tube defect incidence. Epigallocatechin gallate treatment did not affect embryonic development because embryos from nondiabetic dams treated with Epigallocatechin gallate did not exhibit any neural tube defects. Treatment with 1 μM Epigallocatechin gallate did not reduce maternal diabetes-induced neural tube defects significantly. Embryos from diabetic dams treated with 10 μM Epigallocatechin gallate had a significantly lower neural tube defect incidence compared with that of embryos without Epigallocatechin gallate treatment. Epigallocatechin gallate reduced neural tube defect rates from 29.5% to 2%, an incidence that is comparable with that of embryos from nondiabetic dams. Ten micromoles of Epigallocatechin gallate treatment blocked maternal diabetes-increased DNA methyltransferases 3a and 3b expression and their activities, leading to the suppression of global DNA hypermethylation. Additionally, 10 μM Epigallocatechin gallate abrogated maternal diabetes-increased DNA methylation in the CpG islands of neural tube closure essential genes, including Grhl3, Pax3, and Tulp3. Epigallocatechin gallate reduces maternal diabetes-induced neural tube defects formation and blocks the enhanced expression and activity of DNA methyltransferases, leading to the suppression of DNA hypermethylation and the restoration of neural tube closure essential gene expression. These observations suggest that Epigallocatechin gallate supplements could mitigate the teratogenic effects of hyperglycemia on the developing embryo and prevent diabetes-induced neural tube defects.

Dominant negative FADD dissipates the proapoptotic signalosome of the unfolded protein response in diabetic embryopathy.

Endoplasmic reticulum (ER) stress and caspase 8-dependent apoptosis are two interlinked causal events in maternal diabetes-induced neural tube defects (NTDs). The inositol-requiring enzyme 1α (IRE1α) signalosome mediates the proapoptotic effect of ER stress. Diabetes increases tumor necrosis factor receptor type 1R-associated death domain (TRADD) expression. Here, we revealed two new unfolded protein response (UPR) regulators, TRADD and Fas-associated protein with death domain (FADD). TRADD interacted with both the IRE1α-TRAF2-ASK1 complex and FADD. In vivo overexpression of a FADD dominant negative (FADD-DN) mutant lacking the death effector domain disrupted diabetes-induced IRE1α signalosome and suppressed ER stress and caspase 8-dependent apoptosis, leading to NTD prevention. FADD-DN abrogated ER stress markers and blocked the JNK1/2-ASK1 pathway. Diabetes-induced mitochondrial translocation of proapoptotic Bcl-2 members mitochondrial dysfunction and caspase cleavage were also alleviated by FADD-DN. In vitro TRADD overexpression triggered UPR and ER stress before manifestation of caspase 3 and caspase 8 cleavage and apoptosis. FADD-DN overexpression repressed high glucose- or TRADD overexpression-induced IRE1α phosphorylation, its downstream proapoptotic kinase activation and endonuclease activities, and apoptosis. FADD-DN also attenuated tunicamycin-induced UPR and ER stress. These findings suggest that TRADD participates in the IRE1α signalosome and induces UPR and ER stress and that the association between TRADD and FADD is essential for diabetes- or high glucose-induced UPR and ER stress.