Methylenetetrahydrofolate Reductase Deficiency Reduces Brain Microglia in Zebrafish During Embryonic Development and Is Not Corrected by Folic Acid

Abstract

ObjectivesNeuronal development and function is dependent on the interaction between the central nervous system and immune system. Microglia are resident macrophages of the brain critical for regulating neuronal activity during embryonic development. 5-methyltetrahydrofolate (5MTHF), the bioactive folate form, is essential for fetal brain development and immune function. Common variants in methylenetetrahydrofolate reductase (MTHFR), required for conversion of folic acid (FA) to 5-MTHF, limits its production. High dose FA supplementation is recommended but high FA may have the converse effect of reducing MTHFR activity. The objective of this study was to determine the effects of mthfr deficiency and its interaction with FA during embryonic development on microglia in a zebrafish model. MethodsThe mthfr gene in zebrafish was disrupted using two CRISPR mutagenesis methods. A set of 4 guide RNAs (gRNAs) + cas9 protein or cas9 alone (control) were injected to assay F0 zebrafish, or 2 gRNAs + cas9 mRNA were used to induce a germline mutation. To visualize macrophages at 4 days post fertilization (dpf) in live zebrafish, the transgenic mpeg1: mcherry line was used. In a subset of embryos, FA was added at 0, 50, 75, or 100- μM from 0–4dpf. At 4dpf, live neutral red staining for microglia was performed and the number in the optic tectum was quantified. 5MTHF, s-adenosylmethionine (SAM) and s-adenosylhomocysteine (SAH) were assayed in whole zebrafish at 5dpf. ResultsIn vivo imaging revealed a reduction in macrophage number (∼30%, P < 0.001) in the head region of mthfr disrupted zebrafish, but not in the periphery. mthfr zebrafish also had less microglia compared to controls (15%, P < 0.001). These changes were associated with lower 5MTHF (90%, P < 0.0001) and SAM: SAH (∼50%, P < 0.001) at 5dpf indicative of lower methylation potential. Exposure with FA did not correct the phenotype and at 100μM FA, control zebrafish also showed a decrease in microglia similar to mthfr zebrafish, confirming inhibitory effects of the high FA dose. Conclusionsmthfr deficiency reduces microglia in zebrafish but supplementation with FA does not prevent and may exacerbate the negative effects. The 5MTHF form of folate may be a better alternative to FA for brain health in patients with underlying genetic conditions. Funding SourcesSupported by CIHR-INMD.