Journal: Molecular Psychiatry

Article Title: Transcriptomic dysregulation and autistic-like behaviors in Kmt2c haploinsufficient mice rescued by an LSD1 inhibitor

doi: 10.1038/s41380-024-02479-8

Figure Lengend Snippet: a Schematic diagram of the mutation introduced in Kmt2c +/fs mutant mouse line. The 19 bp deletion was introduced by the CRISPR/Cas9 system. b Western blot analysis of KMT2C using anti-KMT2C antibody. Proteins of the nucleic fraction were extracted from the whole brain samples derived from murine embryos at E15.5. The red arrowhead indicates the full-length KMT2C protein. c Measurement of the body weights. The Kmt2c +/fs mice show significantly lower body weights ( Kmt2c +/+ , N = 47; Kmt2c +/fs , N = 41, 12 weeks old). *** P < 0.001. d Measurement of the brain weights. The weights of the whole brains of Kmt2c +/fs mice are significantly higher ( Kmt2c +/+ , N = 29; Kmt2c +/fs , N = 28, 11 − 13 weeks old). *** P < 0.001. e Three-chamber social interaction test. The wildtype Kmt2c +/+ mice stay significantly longer in the chamber with a stranger mouse (S) than in the empty chamber (E), whereas Kmt2c +/fs mice do not show a significant difference ( Kmt2c +/+ , N = 30; Kmt2c +/fs , N = 30, 16 − 21 weeks old). * P < 0.05. f Y-maze test. The alteration rates of Kmt2c +/fs mice are significantly lower. ( Kmt2c +/+ , N = 30; Kmt2c +/fs , N = 30, 16 − 21 weeks old). * P < 0.05. g Schematic diagram of the Barnes maze test. After the training session of the Barnes maze to learn the goal among the twelve holes (upper image, the goal is schematically shown as a black circle. Results are shown in supplementary fig. ), the behaviors of mice immediately after the removal of the goal box were evaluated (probe test, lower image, the removed goal [the previous correct hole] is shown as the dashed circle). h Stay time near the previous correct hole in the probe test in the Barnes maze test. The Kmt2c +/fs mice stay significantly longer near the previous correct hole (dashed circle in lower image of ( g )) in the first minute of the probe test in the Barnes maze test ( Kmt2c +/+ , N = 15; Kmt2c +/fs , N = 15, 14 − 22 weeks old). * P < 0.05. i Schematic diagram of the serial reversal test in the IntelliCage analysis. In this test, one of the four corners (correct corner) is accessible to drinking water, and the correct corner is changed to the diagonally opposite corner every day. The diagonal line of the change of the correct corner is also switched every four days (i.e., days 1, 5, and 9 are the days of a rule change). j , k Rates of the visits to the previously correct corner in the IntelliCage serial reversal test on each day ( j ) and on the days of the rule change ( k ). The arrows in ( j ) indicate the days of the rule changes. On the days of the rule change (i.e., day 1, 5, and 9), the Kmt2c +/fs mice significantly more frequently visit the incorrect corner which had been the correct corner one day before the day of the diagonal line change during the first 15 min of the test session ( k ) ( Kmt2c +/+ , N = 6; Kmt2c +/fs , N = 6, 40 − 41 weeks old). Two-way repeated measures ANOVA was used for the statistical comparison in panel ( k ). Genotype, P = 0.004; Day, P = 0.027; Interaction, P = 0.549. In these analyses except for ( k ), Welch’s t-test was used for the statistical comparison. The data are presented as mean ± standard error of the mean (SEM). We used male mice.

Article Snippet: The social behavior of animals was measured in Crawley’s social interaction test chamber (O’HARA & CO., LTD., Tokyo, Japan).

Techniques: Mutagenesis, CRISPR, Western Blot, Derivative Assay, Comparison

Journal: Molecular Psychiatry

Article Title: Transcriptomic dysregulation and autistic-like behaviors in Kmt2c haploinsufficient mice rescued by an LSD1 inhibitor

doi: 10.1038/s41380-024-02479-8

Figure Lengend Snippet: a Overlap of the target genes of KMT2C and SETD1A. There is a significant overlap between the protein-coding genes whose promotor-TSS regions are bound by KMT2C and those bound by SETD1A. A hypergeometric test was used for statistical comparison. b The structural formula of vafidemstat drawn by ChemSketch. c Schematic representation of the experimental schedule. Administration of vafidemstat was started 4 weeks before the behavioral tests consisting of the open field, Y-maze, three-chamber social interaction, and prepulse inhibition tests. The brain samples were dissected after the behavioral test battery. Vafidemstat was administered via drinking water with 0.96 mg/kg/day. d Effect of vafidemstat on sociality in the three-chamber social interaction test. Though Kmt2c +/fs mice treated with vehicle (DMSO) did not exhibit significantly longer stay time near the stranger mice ( P = 0.586, red bars), DMSO-treated Kmt2c +/+ and vafidemstat-treated Kmt2c +/fs mice showed significant difference ( P = 0.0190 for DMSO-treated Kmt2c +/+ mice [blue bars] and P = 0.0314 for vafidemstat-treated Kmt2c +/fs mice [green bars]). (N = 15 in each condition). Welch’s t-test was used for statistical comparison between the stay time in the empty (E) and stranger (S) chambers. * P < 0.05. The data are presented as mean ± standard error of the mean (SEM). e , f Volcano plot of differential gene expression analysis comparing DMSO-treated Kmt2c +/fs and DMSO-treated Kmt2c +/+ mice (“untreated Kmt2c mutant-wildtype analysis” in the main text). A total of 210 upregulated and 217 downregulated DEGs (uncorrected P < 0.05) were identified by RNA-seq of bulk forebrain samples ( Kmt2c +/+ , N = 3; Kmt2c +/fs , N = 2, 22 weeks old). The red and blue circles in ( e ) represent the upregulated and downregulated DEGs, respectively. In ( f ), the green circles indicate the DEGs in untreated Kmt2c mutant-wildtype analysis whose expression levels were changed in the opposite direction (reversed), i.e., normalized toward the status of wildtype mice, in a comparison between vafidemstat-treated Kmt2c +/fs and DMSO-treated Kmt2c +/fs mice (“vafidemstat effect analysis“ in the main text). The other DEGs are indicated by the red and blue circles as in ( e ). In ( e , f ) the gray dotted horizontal lines indicate uncorrected P < 0.05 and the gray circles indicate non-DEGs. g Mosaic plot of the 210 upregulated and 217 downregulated DEGs in untreated Kmt2c mutant-wildtype analysis according to the direction of their expression changes in the vafidemstat effect analysis. As shown in the green areas of the figure, 198 of the 210 upregulated DEGs were decreased ( P = 1.44 × 10 −44 ) and 179 of the 217 downregulated DEGs were increased ( P = 4.47 × 10 −23 ) in the vafidemstat effect analysis. Binomial tests were used for statistical comparison. h Scatter plot of the fold changes of each gene in the untreated Kmt2c mutant-wildtype analysis (the X-axis) and the fold changes in the vafidemstat effect analysis (the Y-axis). Pearson’s correlation coefficient (r) and the corresponding P value are shown in the upper right of the panel. The black line and the (very narrow) gray area surrounding the line represent the regression line and its 95% confidence interval, respectively. i Scatter plot of the fold changes of significant DEGs in Kmt2c +/fs mice identified in Fig. (the X-axis) and the fold changes of these genes observed in the untreated Kmt2c mutant-wildtype analysis (red circles) or those observed in a comparison between vafidemstat-treated Kmt2c +/fs and DMSO-treated Kmt2c +/+ mice (i.e., an analysis of treated mutant and untreated wildtype mice, green circles) (the Y-axis). The red and green lines indicate the corresponding regression lines. The gray areas surrounding the regression line represent the 95% confidence intervals. Pearson’s correlation coefficients (r) and the corresponding P values for each analysis, indicated by red or green, are shown. We observed that the correlation coefficient observed for the fold changes in the untreated Kmt2c mutant-wildtype analysis (0.319, red) is significantly different from that observed for the fold changes in the analysis of treated mutant and untreated wildtype mice (0.195, green) ( P = 2.82 × 10 −8 , Steiger’s method ).

Article Snippet: The social behavior of animals was measured in Crawley’s social interaction test chamber (O’HARA & CO., LTD., Tokyo, Japan).

Techniques: Comparison, Inhibition, Battery, Gene Expression, Mutagenesis, RNA Sequencing, Expressing