Research Article

Hypnotic effect of thalidomide is independent of teratogenic ubiquitin/proteasome pathway

Yuki Hirose, Tomohiro Kitazono, Maiko Sezaki, Manabu Abe, Kenji Sakimura, Hiromasa Funato, Hiroshi Handa, Kaspar E. Vogt, and Masashi Yanagisawa
PNAS September 15, 2020 117 (37) 23106-23112; first published August 26, 2020; https://doi.org/10.1073/pnas.1917701117

  1. Contributed by Masashi Yanagisawa, July 19, 2020 (sent for review October 10, 2019; reviewed by Ying-Hui Fu and Toru Takumi)

Article Figures & SI

Figures

  • Fig. 1.
    Fig. 1.

    Hypnotic effect of thalidomide in C57BL/6N mice. C57BL/6N mice were treated with vehicle (0.5% methylcellulose) or thalidomide suspension at dark onset. The EEG/EMG signals were recorded for 24 h after treatment. n = 8. Data are mean ± SEM. (A) Total time of each state for 24 h after the treatments. *, †,‡ indicate significant difference between object and vehicle, thalidomide 100 mg/kg, and thalidomide 200 mg/kg, respectively. Upper bars indicate significant differences among vehicle and thalidomide 100, 200, and 400 mg/kg (P < 0.05). (B) The variation of each state per 3 h. (C) FFT spectrum analysis of EEG during each state in the 3 h after drug administration. (D) Effect of thalidomide (100 µM) on excitatory synaptic transmission. (Top) Sample traces from one experiment under baseline conditions (black) and after application of thalidomide (red). (Bottom) Summary of the effects on the amplitude (Left) and IEI (Right) of sEPSCs. (E) The effect of thalidomide (100 µM) on inhibitory synaptic transmission. (Top) Sample traces under baseline conditions (black) and after application of thalidomide (red). (Bottom) Summary of the effects of thalidomide on the amplitude (Left) and IEI (Right) of sIPSCs.

  • Fig. 2.
    Fig. 2.

    Crbn YW/AA KI mice. (A) Generation of Crbn YW/AA KI mice. Direct sequencing data at the mutated portion of exon 11 are shown. (B) HEK293A cells stably expressing mouse FH-CRBN, FH-CRBNWT, or FH-CRBNYW/AA were incubated with the indicated concentrations of thalidomide and MG132. Cell lysates were immunoprecipitated with anti-FLAG antibody and immunoblotted. (C) Densitometry quantification of immunoblots in arbitrary units (a.u.). (Left) FH-CRBNWT autoubiquitination is inhibited by thalidomide in a dose-dependent manner (n = 5; F = 4.22, P = 0.03, one-way repeated-measures ANOVA; *0 µM vs. 100 µM thalidomide, n = 5; P = 0.03, Bonferroni-corrected, paired Student's t test). (Right) FH-CRBNYW/AA autoubiquitination is not affected by thalidomide (n = 5; F = 0.83, P > 0.5, one-way repeated-measures ANOVA).

  • Fig. 3.
    Fig. 3.

    Hypnotic effect of thalidomide in Crbn YW/AA KI mice. Crbn YW/AA KI mice were treated with vehicle or thalidomide suspension. The EEG/EMG signals were recorded for 24 h after treatment. m/m, n = 4; m/+, n = 5; +/+, n = 4. Data are mean ± SEM. (A) Total time of each state in the 12-h dark period after treatment in each genotype. (B) The variation of each state per 3 h. (C) Depression of excitatory synaptic transmission by thalidomide. Sample plot of the amplitude of EPSCs as a function of time–thalidomide was added as indicated. (Inset) Average synaptic response before (black) and after (red) the addition of thalidomide. (D) Summary. (Left) Effect of thalidomide on the amplitude of evoked excitatory transmission in m/m and +/+ mice. (Middle) Pooled data for all mice; amplitude of evoked EPSCs before (black) and after (red) application of thalidomide. (Right) Paired pulse ratio (PPR) before (black) and after (red) the addition of thalidomide.

  • Fig. 4.
    Fig. 4.

    Effect of thalidomide on hypothalamic FOS expression. Immunostaining against FOS after thalidomide (200 mg/kg i.p.). (A) Coronal section of mouse brain with DAB labeling of FOS-expressing neurons. (B and C) Enlarged area marked in A after vehicle treatment (B) and after thalidomide treatment (C) (Scale bars: 1 mm in A; 200 μm in B and C.) Blue circle marks the supraoptic nucleus. (D) Quantification of the effect of thalidomide on FOS expression (mean ± SEM) in different brain regions. Data are mean ± SEM. SON, supraoptic nucleus; PVHm, paraventricular hypothalamic nucleus, magnocellular division; PVHp, paraventricular hypothalamic nucleus, parvicellular division; VLPO, ventrolateral preoptic nucleus; SCN, suprachiasmatic nuclei; MRN, median raphe nucleus; DR, dorsal raphe nucleus; TMN, tuberomammillary nucleus. (E) Double immunofluorescence staining in SON against FOS (red) and neurophysin 1 (oxytocin [OT]) (green) (Scale bar: 100 μm.) (F) Double-immunofluorescence staining in SON against FOS (red) and copeptin (vasopressin [VP]) (green) (Scale bar: 100 μm.) (G) Fraction of FOS-OT and FOS-VP double labeled neurons after vehicle and thalidomide treatment (OT, n = 8, T = −1.66, P = 0.149; VP, n = 8, T = −5.29, P = 0.002; unpaired Student’s t test. Interaction treatment*cell type (OT/VP), n = 16, F = 22, P = 0.001, two-way ANOVA.

Tables

  • Table 1.

    Delta (1 to 6 Hz) and theta (7 to 11 Hz) power as a percentage of total EEG power for increasing doses of thalidomide in WT mice

    DoseDelta power, %Theta power, %
    Vehicle67.4 ± 1.822.6 ± 1.3
    Thal 100 mg/kg73.7 ± 1.3*17.8 ± 0.8*
    Thal 200 mg/kg76.1 ± 1.5*15.7 ± 1.2*
    Thal 400 mg/kg77.6 ± 1.3*14.8 ± 0.9*

    • Data are mean ± SEM.

    • ↵* Indicates significant differences from vehicle (n = 8; delta power, F = 9.09, P < 0.001; theta power, F = 10.52, P < 0.001, one-way ANOVA, post hoc comparison to vehicle condition using Tukey’s correction).

  • Table 2.

    Power in different frequency bands for increasing doses of thalidomide for WT (m/m), YW/AA KI heterozygous (m/+), and YW/AA KI homozygous (+/+) mice

    Dosem/mm/++/+
    Delta power, %Theta power, %Delta power, %Theta power, %Delta power, %Theta power, %
    Vehicle68.6 ± 2.921.5 ± 2.065.5 ± 1.324.5 ± 1.067.9 ± 3.322.7 ± 2.4
    Thal 100 mg/kg73.8 ± 3.117.7 ± 2.272.3 ± 0.819.3 ± 0.672.2 ± 2.119.3 ± 1.5
    Thal 200 mg/kg75.2 ± 2.916.8 ± 2.173.4 ± 0.918.4 ± 0.673.1 ± 1.918.6 ± 1.4
    Thal 400 mg/kg78.0 ± 2.514.7 ± 1.675.6 ± 1.116.7 ± 0.873.6 ± 0.918.2 ± 0.6

    • Delta (1 to 6 Hz) and theta (7 to 11 Hz) power as a percentage of total EEG power (m/m, n = 4; m/+, n = 5; +/+, n = 4). Data are mean ± SEM.

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Hypnotic effect of thalidomide is independent of teratogenic ubiquitin/proteasome pathway
Yuki Hirose, Tomohiro Kitazono, Maiko Sezaki, Manabu Abe, Kenji Sakimura, Hiromasa Funato, Hiroshi Handa, Kaspar E. Vogt, Masashi Yanagisawa
Proceedings of the National Academy of Sciences Sep 2020, 117 (37) 23106-23112; DOI: 10.1073/pnas.1917701117
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