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1.
FASEB J ; 38(11): e23738, 2024 Jun 15.
Artículo en Inglés | MEDLINE | ID: mdl-38855924

RESUMEN

Maternal nutrition contributes to gene-environment interactions that influence susceptibility to common congenital anomalies such as neural tube defects (NTDs). Supplemental myo-inositol (MI) can prevent NTDs in some mouse models and shows potential for prevention of human NTDs. We investigated effects of maternal MI intake on embryonic MI status and metabolism in curly tail mice, which are genetically predisposed to NTDs that are inositol-responsive but folic acid resistant. Dietary MI deficiency caused diminished MI in maternal plasma and embryos, showing that de novo synthesis is insufficient to maintain MI levels in either adult or embryonic mice. Under normal maternal dietary conditions, curly tail embryos that developed cranial NTDs had significantly lower MI content than unaffected embryos, revealing an association between diminished MI status and failure of cranial neurulation. Expression of inositol-3-phosphate synthase 1, required for inositol biosynthesis, was less abundant in the cranial neural tube than at other axial levels. Supplemental MI or d-chiro-inositol (DCI) have previously been found to prevent NTDs in curly tail embryos. Here, we investigated the metabolic effects of MI and DCI treatments by mass spectrometry-based metabolome analysis. Among inositol-responsive metabolites, we noted a disproportionate effect on nucleotides, especially purines. We also found altered proportions of 5-methyltetrahydrolate and tetrahydrofolate in MI-treated embryos suggesting altered folate metabolism. Treatment with nucleotides or the one-carbon donor formate has also been found to prevent NTDs in curly tail embryos. Together, these findings suggest that the protective effect of inositol may be mediated through the enhanced supply of nucleotides during neural tube closure.


Asunto(s)
Inositol , Defectos del Tubo Neural , Inositol/metabolismo , Inositol/farmacología , Defectos del Tubo Neural/metabolismo , Defectos del Tubo Neural/prevención & control , Animales , Femenino , Ratones , Embarazo , Embrión de Mamíferos/metabolismo , Fenómenos Fisiologicos Nutricionales Maternos , Metaboloma , Ácido Fólico/metabolismo
2.
Mol Genet Metab ; 142(3): 108496, 2024 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-38761651

RESUMEN

Non-Ketotic Hyperglycinemia (NKH) is a rare inborn error of metabolism caused by impaired function of the glycine cleavage system (GCS) and characterised by accumulation of glycine in body fluids and tissues. NKH is an autosomal recessive condition and the majority of affected individuals carry mutations in GLDC (glycine decarboxylase). Current treatments for NKH have limited effect and are not curative. As a monogenic condition with known genetic causation, NKH is potentially amenable to gene therapy. An AAV9-based expression vector was designed to target sites of GCS activity. Using a ubiquitous promoter to drive expression of a GFP reporter, transduction of liver and brain was confirmed following intra-venous and/or intra-cerebroventricular administration to neonatal mice. Using the same capsid and promoter with transgenes to express mouse or human GLDC, vectors were then tested in GLDC-deficient mice that provide a model of NKH. GLDC-deficient mice exhibited elevated plasma glycine concentration and accumulation of glycine in liver and brain tissues as previously observed. Moreover, the folate profile indicated suppression of folate one­carbon metabolism (FOCM) in brain tissue, as found at embryonic stages, and reduced abundance of FOCM metabolites including betaine and choline. Neonatal administration of vector achieved reinstatement of GLDC mRNA and protein expression in GLDC-deficient mice. Treated GLDC-deficient mice showed significant lowering of plasma glycine, confirming functionality of vector expressed protein. AAV9-GLDC treatment also led to lowering of brain tissue glycine, and normalisation of the folate profile indicating restoration of glycine-derived one­carbon supply. These findings support the hypothesis that AAV-mediated gene therapy may offer potential in treatment of NKH.


Asunto(s)
Encéfalo , Dependovirus , Modelos Animales de Enfermedad , Terapia Genética , Vectores Genéticos , Glicina-Deshidrogenasa (Descarboxilante) , Glicina , Hiperglicinemia no Cetósica , Hígado , Animales , Hiperglicinemia no Cetósica/genética , Hiperglicinemia no Cetósica/metabolismo , Hiperglicinemia no Cetósica/terapia , Glicina-Deshidrogenasa (Descarboxilante)/genética , Glicina-Deshidrogenasa (Descarboxilante)/metabolismo , Dependovirus/genética , Ratones , Humanos , Vectores Genéticos/genética , Glicina/metabolismo , Hígado/metabolismo , Encéfalo/metabolismo , Biomarcadores/metabolismo , Ácido Fólico/metabolismo
3.
Mol Cell Proteomics ; 23(3): 100718, 2024 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-38224738

RESUMEN

A functional role has been ascribed to the human dihydrofolate reductase 2 (DHFR2) gene based on the enzymatic activity of recombinant versions of the predicted translated protein. However, the in vivo function is still unclear. The high amino acid sequence identity (92%) between DHFR2 and its parental homolog, DHFR, makes analysis of the endogenous protein challenging. This paper describes a targeted mass spectrometry proteomics approach in several human cell lines and tissue types to identify DHFR2-specific peptides as evidence of its translation. We show definitive evidence that the DHFR2 activity in the mitochondria is in fact mediated by DHFR, and not DHFR2. Analysis of Ribo-seq data and an experimental assessment of ribosome association using a sucrose cushion showed that the two main Ensembl annotated mRNA isoforms of DHFR2, 201 and 202, are differentially associated with the ribosome. This indicates a functional role at both the RNA and protein level. However, we were unable to detect DHFR2 protein at a detectable level in most cell types examined despite various RNA isoforms of DHFR2 being relatively abundant. We did detect a DHFR2-specific peptide in embryonic heart, indicating that the protein may have a specific role during embryogenesis. We propose that the main functionality of the DHFR2 gene in adult cells is likely to arise at the RNA level.


Asunto(s)
ARN , Tetrahidrofolato Deshidrogenasa , Humanos , Línea Celular , Péptidos/metabolismo , Biosíntesis de Proteínas , Ribosomas/metabolismo , ARN/metabolismo , ARN Mensajero/metabolismo , Tetrahidrofolato Deshidrogenasa/genética , Tetrahidrofolato Deshidrogenasa/metabolismo
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