Journal: Nature Communications

Article Title: Dysregulation of REV-ERBα impairs GABAergic function and promotes epileptic seizures in preclinical models

doi: 10.1038/s41467-021-21477-w

Figure Lengend Snippet: a mRNA expression of REV-ERBα in hippocampus and cortex from TLE patients ( n = 10 biologically independent samples) and controls ( n = 5 biologically independent samples). Two-sided t test p values: p < 0.0001 (hippocampus); p = 0.003 (cortex). b Protein expression of REV-ERBα in hippocampus and temporal cortex from TLE patients and controls. TLE and control samples were pooled for western blotting analysis. Each western blot is representative of three independent experiments. Western blot strips (a target protein and a loading control) were cut from one gel. Two-sided t test p values: p = 0.0013 (hippocampus); p = 0.0013 (cortex). c – e REV-ERBα colocalizes with neuronal marker NeuN, astrocytic marker GFAP, and microglial marker Iba1 in human epileptic and control tissues. Similar results were obtained in three independent experiments. Scale bar = 20 μm. f Heatmap of clock gene transcripts in hippocampus and cortex from kainic acid (KA)-treated and control mice. g Rev-erbα protein levels in hippocampus and cortex from KA-treated mice ( n = 6 biologically independent samples) and controls ( n = 6 biologically independent samples). Western blot strips (a target protein and a loading control) were cut from one gel. P values (hippocampus, from left to right): p = <0.0001, 0.0015, 0.0050, 0.0002, <0.0001, and 0.0001 (two-way ANOVA and Bonferroni post hoc test). P values (cortex, from left to right): p = 0.0017, 0.0405, 0.0247, 0.0023, 0.0027, and 0.0016 (two-way ANOVA and Bonferroni post hoc test). h Seizure stages of wild-type (WT) mice ( n = 10 per group) treated with KA (20 mg/kg, i.p.) at each of six circadian time points (ZT2, ZT6, ZT10, ZT14, ZT18, and ZT22). i Mortality rates of WT mice treated with KA (20 mg/kg, i.p.) at each of six circadian time points (ZT2, ZT6, ZT10, ZT14, ZT18, and ZT22). Three independent experiments (each with ten mice) were performed to determine the mortality rate. In a , b and g – i , data are presented as mean ± SEM. * represents a p value of <0.05. TLE temporal lobe epilepsy, Rel relative, ZT zeitgeber time.

Article Snippet: ANOVA (one-way or two-way) with Bonferroni post hoc test or Kruskal–Wallis test was used for multiple comparisons (GraphPad Prism, San Diego, CA).

Techniques: Expressing, Control, Western Blot, Marker

Journal: Nature Communications

Article Title: Dysregulation of REV-ERBα impairs GABAergic function and promotes epileptic seizures in preclinical models

doi: 10.1038/s41467-021-21477-w

Figure Lengend Snippet: a Seizure stages of Rev-erbα −/− (KO) mice ( n = 8 biologically independent samples) and wild-type (WT) mice ( n = 8 biologically independent samples) injected with kainic acid (KA, 20 mg/kg) at ZT6. P < 0.0001 (two-sided Kruskal–Wallis test). b Seizure parameters (severity, onset, and duration) of Rev-erbα −/− mice ( n = 8 biologically independent samples) and WT mice ( n = 8 biologically independent samples) injected with KA (20 mg/kg) at ZT6. Two-sided t test p values: p < 0.0001 (seizure severity); p < 0.0001 (onset); p < 0.0001 (seizure duration). c Representative EEG tracings in mice before (3 h) and after (3 h) KA injection (20 mg/kg). d FJB staining of hippocampus sampled at 24 h after injection of KA (20 mg/kg) to Rev-erbα −/− and WT mice. e TUNEL staining of hippocampus sampled at 24 h after injection of KA (20 mg/kg). f NeuN and GFAP staining of hippocampus at 24 h after KA injection (20 mg/kg). g Seizure stages of Rev-erbα −/− mice ( n = 8 biologically independent samples) and WT mice ( n = 8 biologically independent samples) injected with KA (20 mg/kg) at ZT6 and ZT18. Two-sided Kruskal–Wallis test p values: p < 0.0001 (ZT6); p = 0.1213 (ZT18). h Seizure severity of Rev-erbα −/− mice ( n = 8 biologically independent samples) and WT mice ( n = 8 biologically independent samples) injected with KA (20 mg/kg) at ZT6 and ZT18. P < 0.0001 (ZT6); p = 0.0893 (ZT18; two-way ANOVA and Bonferroni post hoc test). i Mortality rates of Rev-erbα −/− and WT mice injected with KA (20 mg/kg) at ZT6 and ZT18. Three independent experiments (each with ten mice) for each group were performed to determine the mortality rate. P = 0.0002 (ZT6); p = 0.2302 (ZT18; two-way ANOVA and Bonferroni p ost hoc test). Scale bar = 50 μm. In a , b and g – i , data are presented as mean ± SEM. For d – f , similar results were obtained in three independent experiments. * represents a p value of <0.05. FJB Fluoro-Jade-B, n.s no significant, ZT zeitgeber time.

Article Snippet: ANOVA (one-way or two-way) with Bonferroni post hoc test or Kruskal–Wallis test was used for multiple comparisons (GraphPad Prism, San Diego, CA).

Techniques: Injection, Staining, TUNEL Assay

Journal: Nature Communications

Article Title: Dysregulation of REV-ERBα impairs GABAergic function and promotes epileptic seizures in preclinical models

doi: 10.1038/s41467-021-21477-w

Figure Lengend Snippet: a Effects of SR8278 pretreatment (25 mg/kg, i.p.) on seizure stages of wild-type (WT) mice induced by kainic acid (KA, 20 mg/kg, i.p.) at ZT6. P < 0.0001 (two-sided Kruskal–Wallis test). b Effects of SR8278 pretreatment on seizure parameters (severity, onset, and duration) of KA-induced acute seizure mice. Two-sided t test p values: p < 0.0001 (seizure severity); p < 0.0001 (onset); p < 0.0001 (seizure duration). c Representative EEG tracings in mice (pretreated with SR8278 or vehicle) before (3 h) and after (3 h) KA injection (20 mg/kg, i.p.). d FJB staining of hippocampus at 24 h after KA (20 mg/kg, i.p.) administration to WT mice pretreated with SR8278 (25 mg/kg, i.p.) or vehicle. Similar results were obtained in three independent experiments. e TUNEL staining of hippocampus at 24 h after KA (20 mg/kg, i.p.) administration to WT mice pretreated with SR8278 (25 mg/kg, i.p.) or vehicle. Similar results were obtained in three independent experiments. f Hippocampus NeuN and GFAP staining in mice (pretreated with SR8278 or vehicle) at 24 h after treatment with KA (20 mg/kg, i.p.). Similar results were obtained in three independent experiments. g Effects of SR8278 pretreatment (25 mg/kg, i.p.) on seizure stages of Rev-erbα −/− and WT mice induced by KA (20 mg/kg, i.p.) at ZT6. Two-sided Kruskal–Wallis test p values: p < 0.0001 (ZT6); p = 0.1027 (ZT18). h Effects of SR8278 pretreatment (25 mg/kg, i.p.) on seizure severity of Rev-erbα −/− and WT mice induced by KA (20 mg/kg, i.p.) at ZT6. P < 0.0001 (WT, seizure severity); p = 0.0893 (KO, seizure severity); p < 0.0001 (WT, onset); p = 0.0635 (KO, onset); p < 0.0001 (WT, seizure duration); p = 0.0643 (KO, seizure duration; two-way ANOVA followed by Bonferroni post hoc test). Scale bar = 50 μm. In a , b , g and h , data are presented as mean ± SEM. n = 6 mice per group. * represents a p value of <0.05. FJB Fluoro-Jade-B, n.s. no significant.

Article Snippet: ANOVA (one-way or two-way) with Bonferroni post hoc test or Kruskal–Wallis test was used for multiple comparisons (GraphPad Prism, San Diego, CA).

Techniques: Injection, Staining, TUNEL Assay

Journal: Nature Communications

Article Title: Dysregulation of REV-ERBα impairs GABAergic function and promotes epileptic seizures in preclinical models

doi: 10.1038/s41467-021-21477-w

Figure Lengend Snippet: a mRNA expressions of Slc6a1 and Slc6a11 in the hippocampus of Rev-erbα −/− (KO) and wild-type (WT) mice. n = 6 mice per group. Data are presented as the fold change in gene expression normalized to Cyclophilin b and relative to ZT2 of WT mice. b mRNA expressions of Slc6a1 and Slc6a11 in the cortex of Rev-erbα −/− and WT mice. n = 6 mice per group. c Protein levels of Slc6a1, Slc6a11, and E4bp4 in the hippocampus of Rev-erbα −/− and WT mice. n = 6 mice per group. Western blot strips (a target protein and a loading control) were cut from one gel. d Protein levels of Slc6a1, Slc6a11, and E4bp4 in the cortex of Rev-erbα −/− and WT mice. n = 6 mice per group. Western blot strips (a target protein and a loading control) were cut from one gel. e Effects of Rev-erbα overexpression or knockdown on mRNA expressions of Slc6a1 and Slc6a11 in Neuro-2a cells ( n = 3 biologically independent samples). Two-sided t test p values (from left to right): 0.0008, 0.0004, 0.0149, and 0.0062. f Effects of Rev-erbα overexpression or knockdown on Slc6a1 and Slc6a11 mRNA expressions in primary hippocampus neurons ( n = 3 biologically independent samples). Two-sided t test p values (from left to right): 0.0006, 0.0005, 0.0012, and 0.0004. g Effects of Rev-erbα overexpression or knockdown on mRNA expressions of Slc6a1 and Slc6a11 in primary cortex neurons ( n = 3 biologically independent samples). Two-sided t test p values (from left to right): 0.0022, 0.0046, 0.0387, and 0.0266. h SR8278 dose-dependently represses mRNA expressions of Slc6a1 and Slc6a11 in primary hippocampus and cortex neurons ( n = 3 biologically independent samples). P values (from left to right): 0.0065, 0.0016, 0.0024, 0.0016, 0.0341, 0.0065, 0.0019, 0.0004, 0.0499, 0.0193, 0.0089, 0.0046, 0.1162, 0.0118, 0.0033, and 0.0031 (one-way ANOVA and Bonferroni post hoc test). All data are presented as mean ± SEM. Statistics for a – d were performed with two-way ANOVA and Bonferroni post hoc test. * Represents a p value of <0.05. ZT zeitgeber time, Rel relative.

Article Snippet: ANOVA (one-way or two-way) with Bonferroni post hoc test or Kruskal–Wallis test was used for multiple comparisons (GraphPad Prism, San Diego, CA).

Techniques: Gene Expression, Western Blot, Control, Over Expression, Knockdown

Journal: Nature Communications

Article Title: Dysregulation of REV-ERBα impairs GABAergic function and promotes epileptic seizures in preclinical models

doi: 10.1038/s41467-021-21477-w

Figure Lengend Snippet: a mRNA expressions of Slc6a1 and Slc6a11 in hippocampus and cortex of E4bp4 −/− and wild-type (WT) mice. n = 6 mice per group. * p < 0.05 (two-way ANOVA and Bonferroni post hoc test). b Protein levels of Slc6a1 and Slc6a11 in hippocampus and cortex of E4bp4 −/− and WT mice. n = 6 mice per group. Western blot strips (a target protein and a loading control) were cut from one gel. c Effects of E4bp4 overexpression or knockdown on mRNA expressions of Slc6a1 and Slc6a11 in Neuro-2a cells ( n = 3 biologically independent samples). Two-sided t test p values: 0.0001, 0.0007, 0.0001, and 0.0001. d Effects of E4bp4 overexpression or knockdown on mRNA expressions of Slc6a1 and Slc6a11 in primary hippocampus neurons ( n = 3 biologically independent samples). Two-sided t test p values: 0.0014, 0.0018, 0.0004, and 0.0002. e Effects of E4bp4 overexpression or knockdown on mRNA expressions of Slc6a1 and Slc6a11 in primary cortex neurons ( n = 3 biologically independent samples). Two-sided t test p values: 0.0004, 0.0003, 0.001, and 0.0001. f Effects of E4bp4 on Slc6a1 and Slc6a11 promoter activities ( n = 3 biologically independent samples). P values: 0.0002, 0.0001, 0.0033, and 0.0001 (one-way ANOVA and Bonferroni post hoc test). g ChIP assays showing recruitment of E4bp4 protein to Slc6a1 and Slc6a11 promoters ( n = 3 biologically independent samples). Two-sided t test p values: 0.0001, 0.4384, 0.0001, and 0.2098. h Knockdown of E4bp4 attenuates the activation effects of Rev-erbα on Slc6a1 and Slc6a11 promoter activities ( n = 3 biologically independent samples). Two-sided t test p values: 0.0011 and 0.0003. i Seizure stages of E4bp4 −/− and WT mice after injection of kainic acid at ZT6. n = 6 mice per group. P = 0.0008 (two-sided Kruskal–Wallis test). j Seizure parameters (severity, onset, and duration) of E4bp4 −/− and WT mice after injection of kainic acid at ZT6. n = 6 mice per group. Two-sided t test p values: <0.0001, <0.0001, and <0.0001. All data are mean ± SEM. * Represents a p value of <0.05. Rel relative, Mut mutation.

Article Snippet: ANOVA (one-way or two-way) with Bonferroni post hoc test or Kruskal–Wallis test was used for multiple comparisons (GraphPad Prism, San Diego, CA).

Techniques: Western Blot, Control, Over Expression, Knockdown, Activation Assay, Injection, Mutagenesis