PhD Scientific Days 2023

Molecular Sciences - Posters L

The Epigenetic Role of Ascorbate in GLUT10/L-Gulonolactone Oxidase Double Knockout Mice

Text of the abstract

Introduction: Arterial tortuosity syndrome (ATS) is a recessiveley inherited, rare connective tissue disorder, mainly characterized by tortuosity and aneurysm formation of the major arteries. ATS is caused by loss-of-function mutation in the gene coding for glucose transporter GLUT10, which is responsible for the transportation of dehydroascorbic acid from cytosol to endomembranes. Ascorbate (Vitamin C) is a well-known antioxidant since its discovery, however, its role in epigenetic regulation as a cofactor for DNA and histone demethylases has only been recently described. We hypothesized that altered nuclear ascorbate concentration might be present in animal models of ATS, which can lead to a modified epigenetic pattern. Earlier, a GLUT10 knockout mouse model was developed to model ATS on animals, however, these mice didn’t show any phenotypical changes, normal redox homeostasis and intracellular distribution of ascorbate was detected.

Aims: Therefore, our aim is to generate double knockout mice – that besides GLUT10, also lack L-gulonolactone oxidase, the enzyme catalyzing the final step of ascorbate biosynthesis– and investigate whether they show an altered subcellular distribution of ascorbate and epigenetic pattern compared to wild type mice.

Methods: We determined the ascorbate levels in liver and brain homogenates and subcellular fractions by RP-HPLC. The amount and distribution of ascorbate was further studied with electron microscopy. We examined the protein expression of ascorbate-related epigenetic enzymes (DNMT1, TET1, Ezh2 and STAT3) by Western blot analysis. The expression of these proteins was also examined by immunofluorescent microscopy in primary hepatocytes isolated from the mice.

Results: Double knockout mice showed a decreased level of ascorbate in both liver and brain compared to wild type mice. Electron microscopy results showed a diminished ascorbate accumulation in double knockout mice. In case of DNMT1, we detected no changes in the protein expression. TET1 and Ezh2 protein expression and STAT3 phosphorylation were higher in double knockout mice.

Conclusions: Our results suggest that decreased ascorbate levels in double knockout mice might lead to altered epigenetic patterns, which are possibly also present in ATS patients; and might help to gain a deeper understanding of this disorder.

Funding: Semmelweis 250+ Kiválósági PhD Ösztöndíj