Journal: International Journal of Molecular Sciences

Article Title: Haploinsufficiency of a Circadian Clock Gene Bmal1 ( Arntl or Mop3 ) Causes Brain-Wide mTOR Hyperactivation and Autism-like Behavioral Phenotypes in Mice

doi: 10.3390/ijms23116317

Figure Lengend Snippet: Heterozygous Bmal1 mutation leads to decreased Bmal1 expression and increased mTOR activation in the mouse brain . ( A ) Haploinsufficiency of Bmal1 expression and brain-wide mTOR hyperactivation in Bmal1 +/− mice. Top: Representative Western blots indicating decreased Bmal1 protein levels and increased phospho-S6 levels in the cerebellum (left) and forebrain (right) of the Bmal1 +/− mice. β-actin was used as a loading control. Bottom: Bar graphs indicate the quantification of Bmal1 protein levels (left) (cerebellum: t (6) = 5.279, p = 0.0019; forebrain: t (6) = 9.553, p < 0.0001, Student’s t -test) and phospho-S6 protein levels (right) (cerebellum: t (6) = 2.455, p = 0.0495; forebrain: t (6) = 2.619, p = 0.0396, Student’s t -test). n = 4 mice/group. ( B ) Confocal microscopic images of mouse cerebellar sections immunolabeled for phospho-S6 (p-S6, green) and Calbindin-D (28k) (red). Cell nuclei were counterstained by DAP1 (blue). Scale bar = 20 μm. ( C ) mTOR hyperactivation in the cerebellum of Bmal1 +/− mice. Left: Representative bright field microscopic images of sagittal mouse cerebellar sections immunolabeled for phospho-S6 as an indicator of mTOR activities. Scale bar = 500 μm. Right: Quantification of the phospho-S6 levels in different cerebellar lobules ( F (1224)genotype = 138.941, p < 0.0001, two-way ANOVA and lobule II: t = 2.934, p = 0.0296; lobule III: t = 4.369, p = 0.0002; lobule IV/V: t = 5.505, p < 0.0001; lobule VI: t = 3.994, p = 0.0007; lobule VII: t = 4.932, p < 0.0001; lobule VIII: t = 5.557, p < 0.0001; lobule IX: t = 3.042, p = 0.0210; lobule X: t = 3.006, p = 0.0236, Bonferroni’s post hoc tests). N = 15 sections from 3 mice of each genotype. Note that the p -S6 levels were increased in all cerebellar lobules in the Bmal1 +/− mice as compared to the WT mice. ( A , C ) Data are shown as individual values and/or mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: Membranes were blocked in 10% skim milk (Fisher scientific, Fair Lawn, NJ, USA) and then incubated (overnight, 4 °C) in PBST (PBS with 1% Triton X-100) containing 5% BSA and a primary antibody for Bmal1 (1:1000, NB100-2288, Novus Biologicals, Littleton, CO, USA), p-S6 (Ser240/244) (1:1000, CST2215, Cell Signaling Tech, Danvers, MA, USA), S6 (1:2000, SC-74459, Santa Cruz Biotechnology, Inc., Dallas, TX, USA), or β-actin (1:5000, A5441, Sigma-Aldrich, Inc., St. Louis, MO, USA).

Techniques: Mutagenesis, Expressing, Activation Assay, Western Blot, Control, Immunolabeling

Journal: International Journal of Molecular Sciences

Article Title: Haploinsufficiency of a Circadian Clock Gene Bmal1 ( Arntl or Mop3 ) Causes Brain-Wide mTOR Hyperactivation and Autism-like Behavioral Phenotypes in Mice

doi: 10.3390/ijms23116317

Figure Lengend Snippet: Impaired social communication by ultrasonic vocalizations (USVs) in Bmal1 +/− and Bmal1 −/− pups. ( A ) Typical sonograms of USVs from a WT and a Bmal1 −/− pup at P7. ( B ) Bar graphs indicating the number of calls and mean call duration of USVs over a 5-min period in pups at P7 and P14. Note that the number of calls was decreased at P14 compared to at P7 in the WT but not the Bmal1 −/− and Bmal1 +/− pups ( F (1, 38) = 0.02423, p = 0.8771, two-way ANOVA; WT, P7 vs. P14: p = 0.1181; Bmal1 −/− , P7 vs. P14: p = 0.3054; Bmal1 +/− , P7 vs. P14: p = 0.7323, Fisher’s LSD). The number of USVs was increased at P7 in the Bmal1 −/− mice and at P14 in the Bmal1 +/− and Bmal1 −/− mice as compared to the WT mice ( F (2, 38) = 11.66, p = 0.0001, two-way ANOVA; P7: WT vs. Bmal1 +/− , p = 0.1501; WT vs. Bmal1 −/− , p = 0.0428; P14: WT vs. Bmal1 +/− , p = 0.0012; WT vs. Bmal1 −/− , p < 0.0001, Fisher’s LSD). n = 7–9 pups/group. When exhibiting ultrasonic vocalizations, Bmal1 +/− mice exhibited longer call durations as compared to the WT mice at P7 ( F (2, 42) = 7.587, p = 0.0015; P7: Bmal1 +/− vs. WT, p = 0.0250). Bmal1 −/− mice exhibited longer call durations as compared to Bmal1 +/− and WT at P14 and Bmal1 −/− at P7 ( Bmal1 −/− vs. Bmal1 +/− , p < 0.0001; Bmal1 −/− vs. WT, p < 0.0001; Bmal1 −/− P14 vs. P7, p = 0.0006; Fisher’s LSD). n = 7–9 pups/group. Data are shown as the mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

Article Snippet: Membranes were blocked in 10% skim milk (Fisher scientific, Fair Lawn, NJ, USA) and then incubated (overnight, 4 °C) in PBST (PBS with 1% Triton X-100) containing 5% BSA and a primary antibody for Bmal1 (1:1000, NB100-2288, Novus Biologicals, Littleton, CO, USA), p-S6 (Ser240/244) (1:1000, CST2215, Cell Signaling Tech, Danvers, MA, USA), S6 (1:2000, SC-74459, Santa Cruz Biotechnology, Inc., Dallas, TX, USA), or β-actin (1:5000, A5441, Sigma-Aldrich, Inc., St. Louis, MO, USA).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Haploinsufficiency of a Circadian Clock Gene Bmal1 ( Arntl or Mop3 ) Causes Brain-Wide mTOR Hyperactivation and Autism-like Behavioral Phenotypes in Mice

doi: 10.3390/ijms23116317

Figure Lengend Snippet: Impaired sociability and preference for social novelty in Bmal1 +/− mice as determined by the three-chamber sociability tests. ( A ) The Bmal1 +/− mice exhibited impaired sociability. Left: Representative images of heat maps (top) and track plots (bottom) of a WT and a Bmal1 +/− mouse in the three-chamber sociability test. S1: stranger 1, C: center, and E: empty. The heat map indicates time spent at the location, and the track plot indicates the total distance traveled. Right: Bar graphs indicating time spent in individual chambers ( F (2, 45) chamber × genotype = 15.21, p < 0.0001, two-way ANOVA), time spent sniffing each wire cage ( F (1, 30) chamber × genotype = 43.75, p < 0.0001, two-way ANOVA), and the number of entries to each chamber ( F (1, 30) chamber × genotype =0.4676, p = 0.4994, two-way ANOVA). Note that the WT mice spent more time in the S1 than E chamber, while Bmal1 +/− spent similar time in the S1 and E chambers ( F (2,45) chamber = 50.37, p < 0.0001, two-way ANOVA and WT: S1 vs. E, p < 0.0001, Bmal1 +/− : S1 vs. E, p > 0.9999, Bonferroni’s post hoc comparisons). WT mice also spent more time sniffing S1 than E, while Bmal1 +/− spent similar times sniffing the S1 and E cages ( F (1,30) chamber = 93.54, p < 0.0001, two-way ANOVA and WT: S1 vs. E, p < 0.0001, Bmal1 +/− : S1 vs. E, p = 0.1109, Bonferroni’s post hoc comparisons). No difference was detected in the number of entries to the S1 and E chambers between WT and Bmal1 +/− ( F (1,30) chamber = 4.445, p = 0.0435, two-way ANOVA and WT: S1 vs. E, p = 0.0752, Bmal1 +/− : S1 vs. E, p = 0.7209, Bonferroni’s post hoc comparisons). n = 7–10 mice/group. ( B ) The Bmal1 +/− mice exhibited an impaired preference for social novelty. Left: Representative images of heat maps (top) and track plots (bottom) of a WT and a Bmal1 +/− mouse in the three-chamber social novelty test. S1: stranger 1 and S1: stranger 2 (the novel stranger). Right: Bar graphs indicating time spent in individual chambers ( F (2,45) chamber × genotype = 21.97, p < 0.0001, two-way ANOVA), time sniffing wire cages ( F (1, 30) chamber × genotype = 4.928, p = 0.0341, two-way ANOVA), and the number of entries to the S1 and S2 chambers ( F (1, 30) chamber × genotype =1.416, p = 0.2434, two-way ANOVA). Note that the WT mice spent a longer time in the S2 chamber than S1, while the Bmal1 +/− mice spent more time in the S1 chamber ( F (2,45) chamber = 52.01, p < 0.0001, two-way ANOVA and WT: S1 vs. S2, p < 0.0001, Bmal1 +/− : S1 vs. S2, p = 0.0019, Bonferroni’s post hoc comparisons). WT mice also spent more time sniffing S2 than S1, while Bmal1 +/− spent similar time sniffing the S1 and S2 cages ( F (1,30) chamber = 1.190, p = 0.2841, two-way ANOVA; WT: S1 vs. S2, p = 0.0298, Bmal1 +/− : S1 vs. S2, p = 0.7163, Bonferroni’s post hoc comparisons). No difference was detected in the number of entries to the S1 and E chambers for WT or Bmal1 +/− ( F (1,30) chamber = 0.5472, p = 0.4652, two-way ANOVA and WT: S1 vs. S2, p > 0.9999, Bmal1 +/− : S1 vs. S2, p = 0.4362, Bonferroni’s post hoc comparisons). n = 7–10 mice/group. Data are shown as individual values, as well as the mean ± SEM. * p < 0.05, ** p < 0.01, and **** p < 0.0001. n.s., not significant.

Article Snippet: Membranes were blocked in 10% skim milk (Fisher scientific, Fair Lawn, NJ, USA) and then incubated (overnight, 4 °C) in PBST (PBS with 1% Triton X-100) containing 5% BSA and a primary antibody for Bmal1 (1:1000, NB100-2288, Novus Biologicals, Littleton, CO, USA), p-S6 (Ser240/244) (1:1000, CST2215, Cell Signaling Tech, Danvers, MA, USA), S6 (1:2000, SC-74459, Santa Cruz Biotechnology, Inc., Dallas, TX, USA), or β-actin (1:5000, A5441, Sigma-Aldrich, Inc., St. Louis, MO, USA).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Haploinsufficiency of a Circadian Clock Gene Bmal1 ( Arntl or Mop3 ) Causes Brain-Wide mTOR Hyperactivation and Autism-like Behavioral Phenotypes in Mice

doi: 10.3390/ijms23116317

Figure Lengend Snippet: Excessive repetitive behaviors in Bmal1 +/− mice. ( A ) The marble burying test. Left: Representative images of marble burying by a WT and a Bmal1 +/− mouse. Right: A bar graph showing the number of marbles buried. Note that the Bmal1 +/− mice buried more marbles as compared to the WT mice ( t (12) = 4.902, p = 0.0004, Student’s t -test). n = 7 mice/group. ( B ) Mouse grooming behavior analysis. Left: Spontaneous grooming test. Bar graphs indicating grooming bouts ( t (12) = 2.426, p = 0.0320, Student’s t -test) and grooming time ( t (12) = 1.577, p = 0.1408, Student’s t -test). n = 7 mice/group. Note that the Bmal1 +/− mice showed increased grooming bouts but a similar total grooming time as compared to the WT mice. Right: Induced grooming test. A gentle water puff was given to mice to induce grooming behavior. Bar graphs indicating grooming bouts ( t (12) = 3.025, p = 0.0106, Student’s t -test) and total grooming time ( t (12) = 2.466, p = 0.0297, Student’s t -test). n = 7 mice/group. Note that the Bmal1 +/− mice showed increased grooming bouts as well as total grooming time as compared to the WT mice. Data represented as individual values, as well as the mean ± SEM. *** p < 0.001 and * p < 0.05, n.s., not significant.

Article Snippet: Membranes were blocked in 10% skim milk (Fisher scientific, Fair Lawn, NJ, USA) and then incubated (overnight, 4 °C) in PBST (PBS with 1% Triton X-100) containing 5% BSA and a primary antibody for Bmal1 (1:1000, NB100-2288, Novus Biologicals, Littleton, CO, USA), p-S6 (Ser240/244) (1:1000, CST2215, Cell Signaling Tech, Danvers, MA, USA), S6 (1:2000, SC-74459, Santa Cruz Biotechnology, Inc., Dallas, TX, USA), or β-actin (1:5000, A5441, Sigma-Aldrich, Inc., St. Louis, MO, USA).

Techniques: Gentle

Journal: International Journal of Molecular Sciences

Article Title: Haploinsufficiency of a Circadian Clock Gene Bmal1 ( Arntl or Mop3 ) Causes Brain-Wide mTOR Hyperactivation and Autism-like Behavioral Phenotypes in Mice

doi: 10.3390/ijms23116317

Figure Lengend Snippet: Increased anxiety-like behavior and deficits in motor coordination but intact novel object recognition memory in Bmal1 +/− mice. ( A ) The open field test. Left: Representative heat maps (top) and track plots (bottom) from a WT and a Bmal1 +/− mouse. The heat map indicates time spent at the location, and the track plot indicates the distance traveled during the test. Right: Bar graphs indicate time in the center and outside zones, distances traveled in the center and outside zones, and the total distances traveled. Note that the Bmal1 +/− mice spent less time in the center zone and more time in the outer zone as compared to the WT mice. ( F (1,24) genotype = 2.566 × 10 −29 , p > 0.999, two-way ANOVA and center, t = 3.081; p = 0.0102; outside, t = 3.081; p = 0.0102, Bonferroni’s post hoc comparison). The Bmal1 +/− mice also traveled a longer distance in the outer zone but a similar distance in the center as compared to WT (F (1,24) genotype = 6.938, p = 0.0145, two-way ANOVA and center t = 0.1799; p >0.9999; outside, t = 3.905; p = 0.0013, Bonferroni’s post hoc comparison). Bmal1 +/− also showed a significant increase in the total distance traveled as compared to WT mice ( t (12) = 2.362, p = 0.0359, Student’s t -test). n = 7 mice/group. ( B ) The rotarod test. Mice were pretrained to stay on the rotating rod (4 rpm) for at least 1 min before entering the trails. Left: A diagram of the rotarod testing equipment. Middle: A bar graph indicates that the number of pretrials was not different between the WT and Bmal1 +/− mice ( t (12) = 1.769, p= 0.1024, Student’s t -test). Right: Line graphs indicate the speed at falling and latency to fall. Note that the performances were improved over the eight trials in both the WT and the Bmal1 +/− mice ( F (7,96) trial = 24.63, p < 0.0001, two-way ANOVA). However, Bmal1 +/− mice fell at lower speeds as compared to the WT mice (F (1,96) trial = 58.36, p < 0.0001, two-way ANOVA; trial1: t = 3.068, p = 0.0224; trial 6: t = 2.934, p = 0.0334; trial 7: t = 4.135, p = 0.0006; trial 8: t = 3.601, p = 0.0040, Bonferroni’s post hoc comparisons) and showed significantly lower latencies to fall (F (1,96) trial = 66.45, p < 0.0001, two-way ANOVA and trial1: t = 3.279, p = 0.0116; trial 6: t = 2.933, p = 0.0335; trial 7: t = 4.107, p = 0.0007; trial 8: t = 3.882, p = 0.0015, Bonferroni’s post hoc comparisons) as compared to the WT. n = 7 mice/group. ( C ) The novel object recognition test. Left: Representative heat maps indicate the time spent at the location from a WT and a Bmal1 +/− mouse. Right: Bar graphs indicate the time spent in investigating novel or familiar objects and the discrimination indices. The discrimination index was calculated as (Time novel − Time familiar )/(Time novel+familiar) . Note that both the WT and the Bmal1 +/− mice spent more time exploring the novel object than the familiar object (F (1,24) objects = 61.51, p < 0.0001, two-way ANOVA and WT, t = 6.935, p < 0.0001; Bmal1 +/− , t = 4.157, p = 0.0007, Bonferroni’s post hoc comparisons).The discrimination index was not significantly different between the WT and Bmal1 +/− mice ( t (12) = 1.643, p = 0.1263, Student’s t -test). n = 7 mice/group. Data are shown as individual values and/or the mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. n.s., not significant.

Article Snippet: Membranes were blocked in 10% skim milk (Fisher scientific, Fair Lawn, NJ, USA) and then incubated (overnight, 4 °C) in PBST (PBS with 1% Triton X-100) containing 5% BSA and a primary antibody for Bmal1 (1:1000, NB100-2288, Novus Biologicals, Littleton, CO, USA), p-S6 (Ser240/244) (1:1000, CST2215, Cell Signaling Tech, Danvers, MA, USA), S6 (1:2000, SC-74459, Santa Cruz Biotechnology, Inc., Dallas, TX, USA), or β-actin (1:5000, A5441, Sigma-Aldrich, Inc., St. Louis, MO, USA).

Techniques: Comparison