Timing of expression of the core clock gene Bmal1 influences its effects on aging and survival

Abstract

The absence of Bmal1, a core clock gene, results in a loss of circadian rhythms, an acceleration of aging, and a shortened life span in mice. To address the importance of circadian rhythms in the aging process, we generated conditional Bmal1 knockout mice that lacked the BMAL1 protein during adult life and found that wild-type circadian variations in wheel-running activity, heart rate, and blood pressure were abolished. Ocular abnormalities and brain astrogliosis were conserved irrespective of the timing of Bmal1 deletion. However, life span, fertility, body weight, blood glucose levels, and age-dependent arthropathy, which are altered in standard Bmal1 knockout mice, remained unaltered, whereas atherosclerosis and hair growth improved, in the conditional adult-life Bmal1 knockout mice, despite abolition of clock function. Hepatic RNA-Seq revealed that expression of oscillatory genes was dampened in the adult-life Bmal1 knockout mice, whereas overall gene expression was largely unchanged. Thus, many phenotypes in conventional Bmal1 knockout mice, hitherto attributed to disruption of circadian rhythms, reflect the loss of properties of BMAL1 that are independent of its role in the clock. These findings prompt reevaluation of the systemic consequences of disruption of the molecular clock.

Fig. 1. Loss of circadian rhythms in iKO mice

(A) Representative double-plotted actograms of wheel-running activity of 3-month-old Bmal1^f/f and Bmal1^f/f-EsrCre mice (red dots, TAM treatment). Similar results were obtained in N=8 to 9 mice/group. (B) The counts of wheel revolutions per hour from control mice (Ctrls) and iKO mice under conditions of constant darkness (DD) (N=8 to 9, Student’s t-test; ns, no significant difference). (C) Representative double-plotted actograms of wheel-running activity from 18-month-old Ctrl and iKO mice under DD. Similar results were obtained in N=6 to 7 mice/group. (D) The counts of wheel revolutions from 18-month-old Ctrls and iKOs under DD (N=6 to 7, Student’s t-test; ns, no significant difference). (E) Representative radiotelemetry results of locomotor activity, systolic blood pressure (SBP), and heart rate (HR) in Bmal1^f/f and Bmal1^f/f-EsrCre mice (red inverted triangles, TAM treatment). Similar results were obtained in N=3 mice/group. (F) Hepatic mRNA levels of canonical clock genes and clock-controlled gene Dbp, were determined by qRT-PCR [N=4/genotype/time point; x-axis, circadian time (CT); y-axis, relative mRNA levels; 2-way ANOVA; *, P<0.05; **, P<0.01; ***, P<0.001].

Fig. 2. General status of iKO mice

(A) Life span of Ctrls and iKOs with medians of 745 days and 765 days, respectively (log-rank test; P=0.9852). (B) Body weight of Ctrls and iKOs for both genders (Student’s t-test; ns, no significant difference). (C) The ratio of organ to body weight in 5-month-old and 11-month-old old Ctrls and iKOs (multiple t-tests with Holm-Sidak correction; **, P<0.01). (D) Fertility analysis in both male and female Ctrl, iKO, and untreated Cre+ mice (χ2 test; ns, no significant difference; **, P<0.01; ***, P<0.001). (E) Blood glucose concentrations of Ctrls and iKOs that underwent GTT and ITT (N=6 to 7, 2-way ANOVA; no significant difference between Ctrls and iKOs at any time point).

Fig. 3. Hair cycling and arthropathy

(A) 6-week-old Bmal1^f/f and Bmal1^f/f-EsrCre mice were treated with tamoxifen. Meanwhile, dorsal hair was shaved. Pictures were taken 7 weeks after hair shaving. Mice with obvious hair regrowth are highlighted with orange boxes. (B to C) Contingency bar graphs show frequency distribution of hair regrowth in both cKO and iKO strains. The hair regrowth was observed 7 w (B) or 12 w (C) after hair shaving. (D) Hair regrowth assay in 6-month-old and 1.5-year-old mice. The hair regrowth was observed 7 w after hair shaving. (E) Representative photographs of alizarin red–stained ribcages and hind limbs from both cKO and iKO strains. Blue circles indicate calcification.

Fig. 4. HFD-induced atherosclerosis

Both (A) cKO and (B) iKO mice and their littermate controls were in a LDLR^−/− background and fed with a HFD (42% kcal from fat) from 12 w of age. 16 w after HFD treatment, mice were euthanized and whole aortas were dissected and stained with oil red O. Plaque areas (shown in red) were quantified with Image-Pro. The percentage of plaque area relative to the entire area was calculated (Student’s t-test; *, P<0.05; **, P<0.01; ***, P<0.001).

Fig. 5. Ocular abnormalities and astrogliosis in iKO mice

Representative gross images (left) and H&E-stained sections of eyes (middle and right) from (A) Ctrl and (B–D) iKOs. (A) Unremarkable globe from a Ctrl mouse. AC, anterior chamber; Co, cornea; Ir, iris; Le, lens; Re, retina. (B) Pathologic changes in a male mouse eye. Grossly, there is a leukoplakic plaque on the cornea (left, arrow). Histologically, the cornea appears thickened with keratinization of the epithelium (right, arrow) and chronic inflammation and neovascularization of the stroma (right, arrowhead). (C) In the contralateral eye of the same mouse, the corneal surface appears irregular with a flattened chamber. Histologically, the cornea appears thickened. The retina is adherent to the lens. The corneal epithelium is attenuated (right, arrow) with chronic inflammation and neovascularization in the stroma. A subcapsular anterior cataract is present (right, arrowhead). (D) A female mouse eye shows an irregular corneal surface with leukoplakia (left, arrow) and corneal neovascularization (right, arrow). (E) Immunofluorescent staining of DAPI (all nuclei), NeuN (neuronal nuclei), BMAL1 and GFAP (activated astrocytes) in the motor cortex from cKO, iKO, and nKO mouse strains. Representative images show loss of Bmal1 immunoreactivity in all KOs, which is accompanied by severe astrogliosis (GFAP panels). Similar results were obtained in N=5 mice/group. Scale bar: 150 μm.

Fig. 6. Hepatic transcriptome

RNA samples from iKOs and their littermates under DD condition were used for RNA-Seq. (A) Out of the 37,681 transcripts, 5,457 exhibited circadian variability in Ctrl mice. By contrast, only 1 gene in iKOs exhibited a circadian pattern. Heat map rendering of the temporal gene expression pattern of these 5,457 circadian genes is shown with the average gene expression level measured for each time point. (B) Erh is the only hepatic gene that shows a circadian expression pattern in iKOs, and the q-value (JTK_CYCLE analysis) and fold change (ratio of peak/trough) are shown [x-axis, circadian time; y-axis, fragments per kilobase of transcript per million mapped reads (FPKM)]. (C) A Venn diagram (sizes not to scale) depicting the number of differentially expressed genes in cKO and iKO strains. (D) MGI Mammalian Phenotype Level 3 enrichment analysis for cKO differentially expressed genes. The top 17 (adjusted P value<0.05) phenotypes related to these genes are given. Overlap, the number of appearing gene/the number of background genes. (E) Mup3, Serpina3k, and Apoa1 are the only differentially expressed genes in the iKO strain.