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. 2023 Jan 23;22(2):e13772. doi: 10.1111/acel.13772

FIGURE 6.

FIGURE 6

5‐methyl tetrahydrofolate (5‐mTHF) supplementation supports mismatch repair signalling, and prevents neuronal cell senescence and brain function decline induced by chronic ethanol exposure. (a) Representative Western blots show that CIE treatment for 72 h in primary neurons results in reduced activities of SETD2, as reflected by reduced levels of H3K36me3. Quantification of the relative band intensities between H3K36me3 over total histone H3 levels was shown (N = 6, ***p < 0.0001, two‐tailed unpaired t‐test). (b) Quantification of 5‐mTHF in primary neurons subjected to 72 h of CIE treatment (N = 9, ***p < 0.0001, two‐tailed unpaired t‐test). (c) Representative Western blots showing reduced activities of SETD2 in primary neurons subjected to CIE treatment for 72 h was prevented by co‐administration of 100 nM 5‐mTHF but not the same quantity of folate. Quantification of the relative band intensities between H3K36me3 over total histone H3 levels was shown (N = 6, ***p < 0.0001, ns = non‐significant, one‐way ANOVA). (d) Representative Western blots showing the effect mediated by 100 nM 5‐mTHF on H3K36me3 was dependent on active SETD2 enzyme. Quantification of the relative band intensities between H3K36me3 over total histone H3 levels was shown (N = 6, ***p < 0.0001, ns = non‐significant, one‐way ANOVA). (e) Schematic diagram showing the possible repair pathway choices for ICL lesions in neurons subjected to CIE and 100 nM 5‐mTHF supplementation. (f and g) Representative immunofluorescent staining images of markers in (f) different repair ICL repair pathways, (g) cell cycle reengagement and cellular senescence in primary neurons subjected to the exposure of alcohol for 72 h in the CIE treatment paradigm. Quantifications of relative cell populations were shown below (N = 10, ***p < 0.0001, ns = non‐significant, one‐way ANOVA, scale bar: 100 μm). (h) Quantification of 5‐mTHF in brain cortex tissues subjected to the entire 2BC‐DID paradigm (N = 9, ***p < 0.0001, two‐tailed unpaired t‐test). (i) Representative Western blots showing the reduction in activities of SETD2 in the brain cortex subjected to the entire 2BC‐DID was prevented by intra‐nasal co‐administration of 100 ng/day of 5‐mTHF but not the same quantity of folate (N = 6, ***p < 0.0001, ns = non‐significant, one‐way ANOVA). (j) Representative SA‐β‐gal and immunofluorescent staining images indicating cellular senescence and neuronal cell cycle re‐entry phenomenon in the prefrontal cortex regions were alleviated upon intra‐nasal co‐administration of 100 ng/day 5‐mTHF (N = 10, ***p < 0.0001, own‐way ANOVA, scale bar: 200 μm). (k) Representative swimming patterns of mice revealed that the lasting impact of chronic binge‐like drinking was alleviated upon intra‐nasal co‐administration of 100 ng/day 5‐mTHF. The latency to the target of the probe trial in the MWM test was quantified (N = 10, ***p < 0.0001, **p < 0.001, ns = non‐significant, one‐way ANOVA). (l) Representative walking patterns of mice revealed that the lasting impact of chronic binge‐like drinking was alleviated upon intra‐nasal co‐administration of 100 ng/day 5‐mTHF. The percentage of alternation was recorded and calculated (N = 10, ***p < 0.0001, **p < 0.001, *p < 0.01, ns = non‐significant, one‐way ANOVA). Values represent the mean ± SEM.