Epigenetic alterations in creatine transporter deficiency: a new marker for dodecyl creatine ester therapeutic efficacy monitoring

Abstract

Creatine transporter deficiency (CTD) is an X-linked disease caused by mutations in the Slc6a8 gene. The impaired creatine uptake in the brain leads to developmental delays with intellectual disability. We hypothesized that deficient creatine uptake in CTD cerebral cells impact methylation balance leading to alterations of genes and proteins expression by epigenetic mechanism. In this study, we determined the status of nucleic acid methylation in both Slc6a8 knockout mouse model and brain organoids derived from CTD patients' cells. We also investigated the effect of dodecyl creatine ester (DCE), a promising prodrug that increases brain creatine content in the mouse model of CTD. The level of nucleic acid methylation was significantly reduced compared to healthy controls in both in vivo and in vitro CTD models. This hypo-methylation tended to be regulated by DCE treatment in vivo. These results suggest that increased brain creatine after DCE treatment restores normal levels of DNA methylation, unveiling the potential of using DNA methylation as a marker to monitor the drug efficacy.

Keywords: cerebral organoids; creatine transporter deficiency; dodecyl creatine ester; epigenetic; methylation.

FIGURE 1

DNA methylation level in brain tissue of wild type (WT) and CrT KO mice with and without DCE treatment. N = 3–4 mice per group (WT = wild type; veh = CrT KO mice treated with the vehicle; DCE 2% = CrT KO mice treated with the dodecyl creatine ester formulation 2% w/w, 0.48 mg/day intranasally for 30 days). Data express as relative quantification to controls and analyzed using Kruskal–Wallis test with Dunn’s multiple comparisons test. *P ≤ 0.05.

FIGURE 2

DNA and RNA methylation level in CTD brain organoids (CTD 1–4, 2–3 and 3–7) vs. healthy brain organoids. (A) 5-mC in DNA (n = 6). (B) m6A in mRNA (n = 6–14). Data express as relative quantification to controls, and analyzed using Kruskal–Wallis test with Dunn’s multiple comparisons test. *P < 0.05; **P < 0.01; ***P < 0.001.

FIGURE 3

Schematic representation of the link between creatine intracerebral synthesis and DNA methylation in CTD patient brain. SAM, S-adenosylmethionine; AGAT, glycine amidinotransferase; GAMT, guanidinoacetate methyltransferase; DNMT, DNA methyltransferase; METTL, methyltransferase-like; GAA, guanidinoacetic acid.