Inpp5e Regulated the Cilium-Related Genes Contributing to the Neural Tube Defects Under 5-Fluorouracil Exposure.

Primary cilia are crucial for neurogenesis, and cilium-related genes are involved in the closure of neural tubes. Inositol polyphosphate-5-phosphatase (Inpp5e) was enriched in primary cilia and closely related to the occurrence of neural tube defects (NTDs). However, the role of Inpp5e in the development of NTDs is not well-known. To investigate whether Inpp5e gene is associated with the neural tube closure, we established a mouse model of NTDs by 5-fluorouracil (5-FU) exposure at gestational day 7.5 (GD7.5). The Inpp5e knockdown (Inpp5e-/-) mouse embryonic stem cells (mESCs) were produced by CRISPR/Cas9 system. The expressions of Inpp5e and other cilium-related genes including intraflagellar transport 80 (Ift80), McKusick-Kaufman syndrome (Mkks), and Kirsten rat sarcoma viral oncogene homolog (Kras) were determined, utilizing quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), western blot, PCR array, and immunofluorescence staining. The result showed that the incidence of NTDs was 37.10% (23 NTDs/62 total embryos) and significantly higher than that in the control group (P < 0.001). The neuroepithelial cells of neural tubes were obviously disarranged in NTD embryos. The mRNA and protein levels of Inpp5e, Ift80, Mkks, and Kras were significantly decreased in NTD embryonic brain tissues, compared to the control (P < 0.05). Knockdown of the Inpp5e (Inpp5e-/-) reduced the expressions of Ift80, Mkks, and Kras in mESCs. Furthermore, the levels of α-tubulin were significantly reduced in NTD embryonic neural tissue and Inpp5e-/- mESCs. These results suggested that maternal 5-FU exposure inhibited the expression of Inpp5e, which resulted in the downregulation of cilium-related genes (Ift80, Mkks, and Kras), leading to the impairment of primary cilium development, and ultimately disrupted the neural tube closure.

The role and mechanism of AZD5363 anti-leukemia activity in T-cell acute lymphoblastic leukemia.

BACKGROUND: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive and heterogeneous hematologic malignancy. Chemotherapy resistance and refractory relapses are the most important challenges in T-ALL. PI3K/Akt/mTOR pathway has been implicated in regulating cell survival, T-ALL development and resistance to chemotherapy. We explored the effects of AZD5363 (a potent pan-Akt inhibitor) alone and in combination with autophagy inhibitor hydroxycholoroquine sulfate (HCQ) in cultured CCRF-CEM, Jurkat and PF382 cells and a T-ALL xenograft mouse model. METHODS: A xenograft mouse model was used to investigate the effect of AZD5363 on T-ALL progression. MTT assay, flow cytometry, siRNA, transmission electron microscopy and western blotting were performed in cultured CCRF-CEM, Jurkat and PF382 cells. The interaction between AZD5363 and HCQ was explored by molecular docking. RESULTS: AZD5363 delayed T-ALL progression and increased the expression of cleaved caspase-3 and LC3B-II in mice. AZD5363 decreased cells viability by arresting cell cycle in the G1 phase and inducing apoptosis, and, significantly increased the number of autophagosomes (p < 0.01). The increased expression of cleaved caspase-3 and LC3B-II, and phosphorylation of Akt and mTOR were significantly, inhibited by AZD5363. HCQ blocked AZD5363-induced autophagy and enhanced AZD5363-induced cell death (p < 0.01). CONCLUSIONS: AZD5363 suppressed T-ALL progression and its anti-leukemia activity was enhanced by HCQ in T-ALL cells, which might provide a potential therapeutic strategy for human T-ALL.

Down-Regulation of Inpp5e Associated With Abnormal Ciliogenesis During Embryonic Neurodevelopment Under Inositol Deficiency.

The inositol polyphosphate-5-phosphatase E (Inpp5e) gene is located on chromosome 9q34.3. The enzyme it encodes mainly hydrolyzes the 5-phosphate groups of phosphatidylinositol (3,4,5)-trisphosphate (PtdIns (3,4,5) P3) and phosphatidylinositol (4,5)-bisphosphate (PtdIns (4,5)P2), which are closely related to ciliogenesis and embryonic neurodevelopment, through mechanisms that are largely unknown. Here we studied the role of Inpp5e gene in ciliogenesis during embryonic neurodevelopment using inositol-deficiency neural tube defects (NTDs) mouse and cell models. Confocal microscopy and scanning electron microscope were used to examine the number and the length of primary cilia. The dynamic changes of Inpp5e expression in embryonic murine brain tissues were observed during Embryonic Day 10.5-13.5 (E 10.5-13.5). Immunohistochemistry, western blot, polymerase chain reaction (PCR) arrays were applied to detect the expression of Inpp5e and cilia-related genes of the embryonic brain tissues in inositol deficiency NTDs mouse. Real-time quantitative PCR (RT-qPCR) was used to validate the candidate genes in cell models. The levels of inositol and PtdIns(3,4) P2 were measured using gas chromatography-mass spectrometry (GC-MS) and enzyme linked immunosorbent assay (ELISA), respectively. Our results showed that the expression levels of Inpp5e gradually decreased in the forebrain tissues of the control embryos, but no stable trend was observed in the inositol deficiency NTDs embryos. Inpp5e expression in inositol deficiency NTDs embryos was significantly decreased compared with the control tissues. The expression levels of Inpp5e gene and the PtdIns (3,4) P2 levels were also significantly decreased in the inositol deficient cell model. A reduced number and length of primary cilia were observed in NIH3T3 cells when inositol deficient. Three important cilia-related genes (Ift80, Mkks, Smo) were down-regulated significantly in the inositol-deficient NTDs mouse and cell models, and Smo was highly involved in NTDs. In summary, these findings suggested that down-regulation of Inpp5e might be associated with abnormal ciliogenesis during embryonic neurodevelopment, under conditions of inositol deficiency.

Genetic Polymorphisms in DNA Repair Gene APE1/Ref-1 and the Risk of Neural Tube Defects in a High-Risk Area of China.

Apurinic/apyrimidinic endonuclease 1/redox-factor 1 (APE1/Ref-1) gene encodes a multifunctional protein involved in the DNA base excision repair (BER) pathway, which initiates repair of apurinic/apyrimidinic (AP) sites in DNA by catalyzing hydrolytic incision of the phosphodiester backbone. APE1/Ref-1 polymorphisms are related to the occurrence of neural tube defects (NTDs), but the association between APE1/Ref-1 polymorphisms and NTDs is not reported in Chinese Han population. The aim of the present study was to evaluate the association of APE1/Ref-1 polymorphism and the risk of NTD occurrence for Han population in a high-risk area of China. APE1/Ref-1 genotypes were determined by iPLEX Gold SNP genotyping. AP sites and folate level of brain tissues were measured. The results showed that three polymorphisms (rs3136817, rs77794916, and rs1760944) of APE1/Ref-1 were statistically associated with NTD subtypes. Allele C of rs3136817, allele T of rs77794916, and allele G of rs1760944 were associated with an increased risk for encephalocele (OR = 2.52, 95% CI [1.25-5.07], P < 0.01; OR = 1.80, 95% CI [1.04-3.12], P = 0.04; and OR = 1.96, 95% CI [1.12-3.45], P = 0.02), compared with those harboring the alleles T, C, and T, respectively. The folate level in NTDs was lower than that in controls. DNA AP sites in the encephalocele were significantly higher than the control (P < 0.01). The three polymorphisms of APE1/Ref-1 were significantly related to NTD occurrence, which indicated that APE1/Ref-1 might be a potential genetic risk factor for encephalocele in a high-risk area of NTDs in China.

Neural tube defects: role of lithium carbonate exposure in embryonic neural development in a murine model.

BACKGROUND: Lithium carbonate (Li2CO3) is widely used in the treatment of clinical-affective psychosis. Exposure to Li2CO3 during pregnancy increases the risk of neural tube defects (NTDs) in offspring, which are severe birth defects of the central nervous system. The mechanism of Li2CO3-induced NTDs remains unclear. METHODS: C57BL/6 mice were injected with different doses of Li2CO3 intraperitoneally on gestational day 7.5 (GD7.5), and embryos collected at GD11.5 and GD13.5. The mechanisms of Li2CO3 exposure-induced NTDs were determined utilizing immunohistochemistry, western blotting, EdU imaging, enzymatic method, gas chromatography-mass spectrometry (GC-MS), ELISA and HE staining. RESULTS: The NTDs incidence was 33.7% following Li2CO3 exposure. Neuroepithelial cell proliferation and phosphohistone H3 level were significantly increased in NTDs embryos, compared with control group (P < 0.01), while the expressing levels of p53 and caspase-3 were significantly decreased. IMPase and GSK-3ß activity was inhibited in Li2CO3-treated maternal and embryonic neural tissues (P < 0.01 and P < 0.05, respectively), along with decreased levels of inositol and metabolites, compared with control groups (P < 0.01). CONCLUSIONS: Lithium-induced NTDs model in C57BL/6 mice was established. Enhanced cell proliferation and decreased apoptosis following lithium exposure were closely associated with the impairment of inositol biosynthesis, which may contribute to lithium-induced NTDs. IMPACT: Impairment of inositol biosynthesis has an important role in lithium exposure-induced NTDs in mice model. Lithium-induced NTDs model on C57BL/6 mice was established. Based on this NTDs model, lithium-induced impairment of inositol biosynthesis resulted in the imbalance between cell proliferation and apoptosis, which may contribute to lithium-induced NTDs. Providing evidence to further understand the molecular mechanisms of lithium-induced NTDs and enhancing its primary prevention.

The Effects of Inositol Metabolism in Pregnant Women on Offspring in the North and South of China.

BACKGROUND Inositol is an essential nutrient for cell growth, survival and embryonic development. Myo-inositol is the predominant form in natural. To investigate the correlation between inositol metabolism and embryonic development, we assessed the metabolic characteristics of myo-inositol, phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3) of pregnant women in the North China (Yangquan and Weihai) and South China (Nanchang and Haikou) China. MATERIAL AND METHODS All data were collected by face-to-face interview during pregnant women health visits using a questionnaire. Plasma levels of myo-inositol, PI(4,5)P2 and PI(3,4,5)P3 from 89 randomly collected pregnant women were detected by gas chromatography-mass spectrometry and enzyme linked immunosorbent assay. RESULTS A total of 400 pregnant women were included in this survey. The plasma levels of myo-inositol and PI(4,5)P2 in the North China group of pregnant women were significantly higher than that in the South China group (P<0.01). The birth weight of fetuses in the North China group was heavier than that in the South China group (P<0.01). The birth length of fetuses in Yangquan was the longest among the 4 cities (P<0.01). The incidence rate of birth defects was 3.05% in the North China group, and 0.0% in the South China group. In bivariate linear correlation analysis, the body weight correlated with myo-inositol (r=0.5044, P<0.0001), PI(4,5)P2 (r=0.5950, P<0.0001) and PI(3,4,5)P3 (r=0.4710, P<0.0001), the body length was correlated with PI(4,5)P2 (r=0.3114, P=0.0035) and PI(3,4,5)P3 (r=0.2638, P<0.0130). CONCLUSIONS The plasma levels of myo-inositol and PI(4,5)P2 in pregnant women had significant difference between the North and the South of China, which might be correlated with fetal development and birth defects.

Relationship Between INPP5E Gene Expression and Embryonic Neural Development in a Mouse Model of Neural Tube Defect.

BACKGROUND The INPP5E gene encodes for the inositol polyphosphate-5-phosphatase (INPP5E) 72 kDa protein that regulates the phosphoinositide signaling pathway and other cellular activities, but the functional role of this gene in embryonic neurodevelopment and neural tube defect (NTD) remains unclear. The aim of this study was to use a mouse model of NTD to investigate the expression levels of the INPP5E gene during neural development and the occurrence of NTD. MATERIAL AND METHODS In an established NTD mouse model, stereoscopy was used to look for morphological defects. Transcription and expression levels of the INPP5E gene in neural tissues were detected using real-time fluorescence quantitative polymerase chain reaction (PCR) and Western blotting in the NTD mouse embryos and compared with control mouse embryos. RESULTS The expression levels of the INPP5E gene decreased as embryonic development progressed in the neural tissue of control mice embryos, but showed no obvious trend in the neural tissues of the NTD mouse embryos. The expression levels of the INPP5E gene in NTD mouse embryos were significantly lower compared with control embryos, at the time of neural tube closure (gestational day 11.5). CONCLUSIONS The INPP5E gene regulates the process of embryonic neural development. Abnormal levels of expression of the INPP5E gene may contribute to NTDs. Increased knowledge of the expression pattern of the INPP5E gene may lead to an advanced understanding of the molecular mechanism of embryonic neurodevelopment and identify more specific directions to explore potential treatments for NTDs associated with abnormalities in INPP5E gene expression levels.