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

Abstract

Thalidomide exerts its teratogenic and immunomodulatory effects by binding to cereblon (CRBN) and thereby inhibiting/modifying the CRBN-mediated ubiquitination pathway consisting of the Cullin4-DDB1-ROC1 E3 ligase complex. The mechanism of thalidomide's classical hypnotic effect remains largely unexplored, however. Here we examined whether CRBN is involved in the hypnotic effect of thalidomide by generating mice harboring a thalidomide-resistant mutant allele of Crbn (Crbn YW/AA knock-in mice). Thalidomide increased non-REM sleep time in Crbn YW/AA knock-in homozygotes and heterozygotes to a similar degree as seen in wild-type littermates. Thalidomide similarly depressed excitatory synaptic transmission in the cortical slices obtained from wild-type and Crbn YW/AA homozygous knock-in mice without affecting GABAergic inhibition. Thalidomide induced Fos expression in vasopressin-containing neurons of the supraoptic nucleus and reduced Fos expression in the tuberomammillary nuclei. Thus, thalidomide's hypnotic effect seems to share some downstream mechanisms with general anesthetics and GABA_A-activating sedatives but does not involve the teratogenic CRBN-mediated ubiquitin/proteasome pathway.

Keywords: cereblon; electroencephalography/electromyography (EEG/EMG); mouse; sleep; supraoptic nucleus.

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.

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-CRBN^WT, or FH-CRBN^YW/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-CRBN^WT 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-CRBN^YW/AA autoubiquitination is not affected by thalidomide (n = 5; F = 0.83, P > 0.5, one-way repeated-measures ANOVA).

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.

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.