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. 2015 Mar 1;399(1):129-138.
doi: 10.1016/j.ydbio.2014.12.025. Epub 2014 Dec 31.

A requirement for Gch1 and tetrahydrobiopterin in embryonic development

Gillian Douglas  1 , Ashley B Hale  2 , Mark J Crabtree  2 , Brent J Ryan  3 , Alex Hansler  4 , Katrin Watschinger  5 , Steven S Gross  4 , Craig A Lygate  2 , Nicholas J Alp  2 , Keith M Channon  2
Affiliations

A requirement for Gch1 and tetrahydrobiopterin in embryonic development

Gillian Douglas et al. Dev Biol. .

Abstract

Introduction: GTP cyclohydrolase I (GTPCH) catalyses the first and rate-limiting reaction in the synthesis of the enzymatic cofactor, tetrahydrobiopterin (BH4). Loss of function mutations in the GCH1 gene lead to congenital neurological diseases such as DOPA-responsive dystonia and hyperphenylalaninemia VSports手机版. However, little is known about how GTPCH and BH4 affects embryonic development in utero, and in particular whether metabolic replacement or supplementation in pregnancy is sufficient to rescue genetic GTPCH deficiency in the developing embryo. .

Methods and results: Gch1 deficient mice were generated by the insertion of loxP sites flanking exons 2-3 of the Gch1 gene. Gch1(fl/fl) mice were bred with Sox2cre mice to generate mice with global Gch1 deficiency V体育安卓版. Genetic ablation of Gch1 caused embryonic lethality by E13. 5. Despite loss of Gch1 mRNA and GTPCH enzymatic activity, whole embryo BH4 levels were maintained until E11. 5, indicating sufficient maternal transfer of BH4 to reach this stage of development. After E11. 5, Gch1(-/-) embryos were deficient in BH4, but an unbiased metabolomic screen indicated that the lethality was not due to a gross disturbance in metabolic profile. Embryonic lethality in Gch1(-/-) embryos was not caused by structural abnormalities, but was associated with significant bradycardia at E11. 5. Embryonic lethality was not rescued by maternal supplementation of BH4, but was partially rescued, up to E15. 5, by maternal supplementation of BH4 and l-DOPA. .

Conclusion: These findings demonstrate a requirement for Gch1 in embryonic development and have important implications for the understanding of pathogenesis and treatment of genetic BH4 deficiencies, as well as the identification of new potential roles for BH4. V体育ios版.

Keywords: Development; Embryo; GCH; Heart; Tetrahydrobiopterin VSports最新版本. .

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Figures

Fig. 1
Fig. 1
A, Schematic showing the targeting of the murine Gch1 locus with loxP sites flanking the exons encoding the active site of the GTPCH protein. B, Genomic DNA was probed for the presence of loxP sites, a predicted 1056 bp band was observed in GCHfl/fl mice. Sox2 Cre mediated excision results in the presence of a knockout allele, a predicted 1332 bp product was observed in Gch1−/− mice, Gch1+/− mice had both a knockout and wild type band. C, Gch1 mRNA levels were significantly reduced (*=P<0.05, n=6 per group) in heart tissue from adult Gch1+/− mice compared with Gch1+/+ littermates. D, GTPCH activity was significantly reduced (*=P<0.05, n=6 per group) in heart tissue from adult Gch1+/− mice compared with Gch1+/+ littermates. E, BH4 levels were significantly reduced (*=P<0.05, n=6 per group) in heart tissue from adult Gch1+/− mice compared with Gch1+/+ littermates. F, Percentage embryo survival of each genotype from E9.5 to birth, at E11.5 and 12.5 live Gch1−/− are significantly under-represented and completely absent after E12.5 (P<0.05, n=35–10 litters per group; number of alive embryos for each group is written in the bar).
Fig. 2
Fig. 2
A, Whole embryo mRNA expression of Gch1 in Gch1+/+, Gch1+/− and Gch1−/− embryos Gch1 expression increased with development (*=P<0.05 of 4–6 embryos) in Gch1+/+ and Gch1+/− embryos whereas in Gch1−/− embryos Gch1 was not detected at any time point. B, Whole embryo Gch1 activity in E11.5 embryos, activity was significantly reduced in Gch1+/− and further reduced in Gch1-/- embryos (*=P<0.05, n=8–6 per group). C, BH4 levels in E9.5 embryos, D, E10.5 embryos and E, E11.5 embryos, no difference in embryonic BH4 levels between genotypes at either E9.5 or E10.5. At E11.5 there was a significant reduction in BH4 levels in Gch1−/− embryos (P<0.05, n=7–8 per group). F, l-DOPA levels in E11.5 embryos were significantly reduced in Gch1−/− embryos compared with Gch1+/+ and Gch1+/− embryos (*=P<0.05, n=8–6 per group). G, Phenylalanine levels were not different between any groups at E11.5. H, Whole embryos tyrosine levels were not different between Gch1+/+, Gch1+/− and Gch1−/− embryos at E11.5. I, Hierarchical clustering of an unbiased metabolomics screen of 590 compounds and J, 38 detected compounds from a targeted screen of 150 common metabolites in E11.5 Gch1+/+ (blue) and Gch1−/− (red) embryos. No significant difference were observed between groups and unsupervised clustering did not result in separation of embryos by genotype (P>0.05, n=8–9 per group).
Fig. 3
Fig. 3
A, Representative photographs of E11.5 Gch1+/+, Gch1+/− and Gch1−/− embryos. B, Embryos crow to rump length at E11.5, no differences between groups was observed. C, Placenta weights of Gch1+/+, Gch1+/− and Gch1−/− embryos at E10.5 and E11.5, no significant difference was observed between groups. D, Representative images of the heart, outflow tracks and dorsal aorta from E11.5 Gch1+/+ and Gch1−/− embryos. E, Representative images of whole mount PECAM stained E9.5 and E10.5 Gch1+/+ and Gch1−/− embryos.
Fig. 4
Fig. 4
Gch1-/- embryos were significantly bradycardic at E11.5 A, In utero heart rate at E10.5 in Gch1+/+, Gch1+/− and Gch1−/−, embryos no significant differences were observed between groups (n=6–9 per group from 6 independent litters). B, In utero heart rates at E11.5, Gch1−/− embryos had a significant reduction in heart rate compared with Gch1+/+ and Gch1+/− littermates (*=P<0.05, n=5–9 per group of 10 independent litters).
Fig. 5
Fig. 5
A, l-DOPA levels in Gch1+/+ E11.5 embryos from l-DOPA treated dams (drinking water 1 mg/ml in 0.25% ascorbic acid, E7.5-E11.5) a significant increase in embryonic l-DOPA levels was observed in treated dams (*=P<0.05). B, BH4 levels in Gch1+/+ E11.5 after sepiapterin treatment (2×5 mg/kg, SC, E7.5-E11.5) a significant increase in BH4 levels was observed 1 and 6 h after treatment (*=P<0.05, n=3–4 per group of 3 independent litters). C, BH4 levels in Gch1+/+ E11.5 embryos 14 h after SC injection with either sepiapterin (2×5 mg/Kg SC, E7.5-E11) or saline, the significant decrease in BH4 levels observed in Gch1−/− embryos was abolished 14 h after maternal sepiapterin treatment (*=P< 0.05, n=4–9 per group of 4 independent litters). D, Embryonic survival after maternal supplementation with either sepiapterin (2×5 mg/kg/day) or sepiapterin with l-DOPA (drinking water, 1 mg/ml in 0.25% ascorbic acid) from E7.5-E13.5, maternal treatment with sepiapterin and l-DOPA but not saline or sepiapterin alone resulted in a significant increase in alive Gch1−/− embryos at E13.5 (*=P<0.05, from 10–16 independent litters; total number of embryos alive+dead for each group written in the bar). E, Embryonic survival after maternal supplementation with sepiapterin (2×5 mg/kg/day) and l-DOPA (drinking water 1 mg/ml in 0.25% ascorbic acid) or saline from E7.5-E15.5, in both groups Gch1−/− were significantly under represented (*=P<0.05 from 8–10 independent litters; total number of embryos alive+dead for each group written in the bar).
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References

    1. Bendall J.K., Douglas G., McNeill E., Channon K., Crabtree M.J. Tetrahydrobiopterin in cardiovascular health and disease. Antioxid. Redox Signal. 2013 - PMC - PubMed
    1. Brüggemann N., Spiegler J., Hellenbroich Y. BEneficial prenatal levodopa therapy in autosomal recessive guanosine triphosphate cyclohydrolase 1 deficiency. Arch. Neurol. 2012;69:1071–1075. - VSports注册入口 - PubMed
    1. Cosentino F., Barker J.E., Brand M.P., Heales S.J., Werner E.R., Tippins J.R., West N., Channon K.M., Volpe M., Luscher T.F. Reactive oxygen species mediate endothelium-dependent relaxations in tetrahydrobiopterin-deficient mice. Arterioscler Thromb. Vasc. Biol. 2001;21:496–502. - VSports手机版 - PubMed
    1. Costigan M., Latremoliere A., Woolf C.J. Analgesia by inhibiting tetrahydrobiopterin synthesis. Curr. Opin. Pharmacol. 2012;12:92–99. - "VSports注册入口" PMC - PubMed
    1. Cotton R.G.H. A model for hyperphenylalaninaemia due to tetrahydrobiopterin deficiency. J. Inherit. Metab. Dis. 1986;9:4–14. - PubMed

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