BMAL1 knockout macaque monkeys display reduced sleep and psychiatric disorders

Abstract

Circadian disruption is a risk factor for metabolic, psychiatric and age-related disorders, and non-human primate models could help to develop therapeutic treatments. Here, we report the generation of BMAL1 knockout cynomolgus monkeys for circadian-related disorders by CRISPR/Cas9 editing of monkey embryos. These monkeys showed higher nocturnal locomotion and reduced sleep, which was further exacerbated by a constant light regimen. Physiological circadian disruption was reflected by the markedly dampened and arrhythmic blood hormonal levels. Furthermore, BMAL1-deficient monkeys exhibited anxiety and depression, consistent with their stably elevated blood cortisol, and defective sensory processing in auditory oddball tests found in schizophrenia patients. Ablation of BMAL1 up-regulated transcriptional programs toward inflammatory and stress responses, with transcription networks associated with human sleep deprivation, major depressive disorders, and aging. Thus, BMAL1 knockout monkeys are potentially useful for studying the physiological consequences of circadian disturbance, and for developing therapies for circadian and psychiatric disorders.

Keywords: BMAL1; aging; circadian rhythms; macaque monkey; psychosis; sleep disruption.

Figure 1.

Generation of BMAL1 knockout cynomolgus monkeys. (A) Designated PAS.3 domain and the respective sequences in BMAL1 exon 13 that were targeted in cynomolgus monkeys. Red, sgRNA1 and 3 sequences. (B) Summary of all BMAL1-edited monkeys in this study. (C) Sequence alterations in the five CRISPR/Cas9-edited mutant monkeys, examined via PCR amplification of BMAL1 exon 13, followed by sequence analysis. (D) Images of male A5 (BMAL1-WT), male A6 (BMAL-KO), female A7 (BMAL1-WT), and female A3 (BMAL1-KO) cynomolgus monkeys at 4 months of age. (E) Expression of WT BMAL1 transcript over zeitgeber time (ZT) 2, 8, 14 and 20 for BMAL1-WT versus BMAL1-KO monkeys, shown by average blood mRNA levels. (F) Immunoblots of BMAL1 and PER2 levels in major tissues collected from stillbirths A1 and A9. GAPDH was used as the reference. *P < 0.05, **P < 0.01 and ***P < 0.001; Student's t-test.

Figure 2.

Dysregulated circadian locomotor activities and hormonal levels in BMAL1-deficient monkeys. (A) Continuous 14-d locomotor activity recorded in BMAL1-WT and BMAL1-KO monkeys. Example actograms of A5, A6, A7 and A3 are shown under L/D conditions. (B and C) Activity amplitude and chi-squared periodograms of the 14-d actograms. (D) Levels of melatonin, testosterone, DHEA and cortisol were assayed for monkey plasma samples obtained from temporal collections at 6-h intervals over 48 h. Results shown are from the male cohort. (E) Female hormonal assays in the same manner as in (D).

Figure 3.

Altered sleep states and diurnal EEG oscillations in BMAL1-KO monkeys. (A) Telemetric EEG recoding in male BMAL1-WT (A5) and BMAL1-KO (A6) monkeys under 3-d L/D cycles (upper panels), or 3-d L/L cycles (lower panels). Stage scores for the proportions of awake, NREM and REM states are shown. (B) Pie charts presented the averaged proportions of the three stages over the 3-d recording period in male monkeys. (C) Telemetric EEG recoding in female BMAL1-WT (A7) and BMAL1-KO (A3) monkeys under 3-d L/D cycles (upper panels) and 3-d L/L cycles (lower panels). (D) Pie charts are presented as in (B) for female monkeys.

Figure 4.

Depression-related behavior and sensory processing impairment in BMAL1-KO monkeys. (A) Activity tracks (marked red) obtained by 20-min videotape recordings for all BMAL1-edited and WT monkeys. (B) Summary of locomotor activities, including total distance, moving velocity, stationary time, and (C) time spent on-ground versus time spent off-ground were analyzed. Black, blue and red indicate BMAL1-WT, BMAL1 mosaic and BMAL1-KO monkeys, respectively. Data also include two WT age-matched male monkeys, in addition to the eight monkeys listed in Fig. 1B. *P < 0.05 and **P < 0.01; Student's t-test. (D) Topographic voltage maps of BMAL1 WT and KO monkeys. Asterisk indicates Fz electrode position. (E) Heat maps of voltage amplitudes induced by regular or deviant sound trials. ERPs at Fz electrode were analyzed. (F) Average ERP summarized from (E). (G) MMN of BMAL1 WT and KO monkeys, as indicated by the differences between regular and deviant in dashed lines. (H) MMN of BMAL1-WT versus BMAL-KO monkeys summarized from (G). Significant differences are indicated with star labels on top (P < 0.05, Student's t-test), and SEMs are shown as shaded areas.

Figure 5.

Blood transcriptome analysis of BMAL1-KO monkeys. (A) Representative examples of the expression levels of circadian controlled genes over time points ZT2, ZT8, ZT14 and ZT20. Results from male BMAL1-WT (A5) and BMAL1-KO (A6) monkeys are shown. (B) Heat maps of genes that were up- and down-regulated in their expression over time points ZT2, ZT8, ZT14 and ZT20 for monkeys A5 and A6. Results of greater than twofold changes are shown. (C) GO terms for up-regulated genes shown in the transcriptome analysis in (B). (D and E) STRING assembly for functional network in A5 (D, upper panel) and A6 (E, lower panel) at time point ZT2. The result shows that the network assembly became distinctly different between WT and BMAL1-KO monkeys (BMAL1 is labeled as ‘ARNTL' in the network). (F) Over-representing STRING network assembly of up-regulated targets in BMAL1-KO A6. Targets related to sleep deprivation (GSE39445) and major depressive disorder (GSE76826) are shown in black and blue, respectively, and those related to both are shown in red.