The effect of inhibiting glycinamide ribonucleotide formyl transferase on the development of neural tube in mice.

Lin Xu,Xinli Tian,Ying Guo,Zhiqiang Zhu,Bo Niu,Ge Chang,Li Wang,Jianhua Wang

doi:10.1186/s12986-016-0114-x

Abstract

BackgroundFolate deficiency is closely related to the development of neural tube defects (NTDs). However, the exact mechanism is not completely understood. This study aims to induce murine NTDs by inhibiting one of the folate metabolic pathways, de novo purine synthesis and preliminarily investigate the potential mechanisms. The key enzyme, glycinamide ribonucleotide formyl transferase (GARFT) was inhibited by a specific inhibitor, lometrexol (DDATHF) in the pregnant mice.MethodsPregnant mice were intraperitoneally injected with various doses of DDATHF on gestational day 7.5 and embryos were examined for the presence of NTDs on gestational day 11.5. GARFT activity and levels of ATP, GTP, dATP and dGTP were detected in embryonic brain tissue. Proliferation and apoptosis was analyzed by real-time quantitative polymerase chain reaction (RT-qPCR), immunohistochemical assay and western blotting.Results40 mg kg−1 body weight (b/w) of DDATHF caused the highest incidence of NTDs (30.8 %) and therefore was selected as the optimal dose to establish murine NTDs. The GARFT activity and levels of ATP, GTP, dATP and dGTP in embryonic brain tissue were significantly decreased after DDATHF treatment. Furthermore, Levels of proliferation-related genes (Pcna, Foxg1 and Ptch1) were downregulated and apoptosis-related genes (Bax, Casp8 and Casp9) were upregulated. Expression of phosphohistone H3 was significantly decreased while expression of cleaved caspase-3 was greatly increased.ConclusionsResults indicate that DDATHF induced murine NTDs by disturbing purine metabolism and further led to abnormal proliferation and apoptosis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12986-016-0114-x) contains supplementary material, which is available to authorized users.

Highlights

Folate deficiency is closely related to the development of neural tube defects (NTDs)
This study aims to investigate whether the murine model of Neural tube defects (NTDs) can be induced by impairment of purine biosynthesis via inhibition of glycinamide ribonucleotide formyl transferase (GARFT) using a specific inhibitor, DDATHF, and to further explore the potential mechanisms
Murine NTDs induced by DDATHF Embryos were isolated and observed under a dissecting microscope on gestational day 11.5

Summary

Introduction

Folate deficiency is closely related to the development of neural tube defects (NTDs). This study aims to induce murine NTDs by inhibiting one of the folate metabolic pathways, de novo purine synthesis and preliminarily investigate the potential mechanisms. The key enzyme, glycinamide ribonucleotide formyl transferase (GARFT) was inhibited by a specific inhibitor, lometrexol (DDATHF) in the pregnant mice. Murine NTDs have been successfully induced by inhibiting the key enzyme, DHFR, in folate metabolic pathway [7]. We suppose that folate deficiency refers to folate and folate related metabolic disorder, which may lead to NTDs. Obviously, the inhibition of DHFR results in dysmetabolism of THF and one carbon units. Defects in any of these processes may lead to the occurrence of NTDs. Glycinamide ribonucleotide formyl transferase (GARFT) is a key enzyme in the process of de novo purine biosynthesis. The inhibition of this enzyme may impair the biosynthesis of purine nucleotide [9]

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