Fig. 4: Gdh localization and influence of Gdh on Tet enzymes, gene expression and metabolism during neuronal stimulation.
From: Redirected nuclear glutamate dehydrogenase supplies Tet3 with α-ketoglutarate in neurons
a Comparison of hmdC and fdC levels in HEK293T (n = 5 biologically independent samples) expressing GFP-Tet3 + Gdh-FLAG or GFP-Tet3 + Gdh-FLAG-NES for 24 h. A two-sided paired t-test was performed. b ICC in HEK239T showing FLAG and GFP signal after ectopic co-expression of GFP-Tet3 + Gdh-FLAG or GFP + Gdh-FLAG from different promoters. Scale bar is 50 µm. c ICC in HEK293T showing FLAG, Gdh, and CoxIV after expressing FLAG3-Tet3 and Gdh from a bicistronic vector compared to untransfected cells. Merged images: FLAG (magenta), Gdh (cyan), CoxIV (red) and Hoechst (yellow). Scale bar is 5 µm. d Hippocampal hmdC levels upon neuronal stimulation with 25 mM KCl for 6 h with and without 20 µM Gdh inhibitor R162 (n = 7 biologically independent animals) or 40 µM PDC inhibitor UK5099 (n = 6 biologically independent animals). Connected dots represent matched hmdC values from one individual mouse. RM one-way ANOVA combined with Tukey’s multiple comparisons test was performed. e αKG level and αKG/succinate ratio in hippocampus (n = 5 biologically independent animals) after depolarization with and without R162. Ordinary one-way ANOVA combined with Tukey’s multiple comparisons test was performed. A.U. arbitrary units. f Relative gene expression levels of Npas4 and Bdnf (n = 7 biologically independent animals) in hippocampus after depolarization with and without R162 normalized to expression levels in unstimulated control. An unpaired two-sided t-test with Welch’s correction was performed. g PLA of Tet3 + NPC and Gdh + NPC in the hippocampus. Images show Z-stacks. Scale bar is 5 µm. a, d–f p (a, f) or p(adj) (d, e) <0.05 (*), <0.01 (**), <0.001 (***); exact p values and statistical details are provided in the Statistics and Reproducibility sub-section within the “Methods” section. e, f Boxes display interquartile range, median is shown as center line, mean as + , whiskers represent the minimum and the maximum. Source data are provided as a Source Data file.
