Early aging and age-related pathologies in mice deficient in BMAL1, the core componentof the circadian clock

Abstract

Mice deficient in the circadian transcription factor BMAL1 (brain and muscle ARNT-like protein) have impaired circadian behavior and demonstrate loss of rhythmicity in the expression of target genes. Here we report that Bmal1(-/-) mice have reduced lifespans and display various symptoms of premature aging including sarcopenia, cataracts, less subcutaneous fat, organ shrinkage, and others. The early aging phenotype correlates with increased levels of reactive oxygen species in some tissues of the Bmal1(-/- )animals. These findings, together with data on CLOCK/BMAL1-dependent control of stress responses, may provide a mechanistic explanation for the early onset of age-related pathologies in the absence of BMAL1.

Figure 1.

Premature aging and early death in Bmal1 KO mice. (A) Kaplan-Meyer survival curve. (Closed circles) Wild-type (WT) mice; (open circles) KO mice. Wild-type animals older than 1 yr were sacrificed. (B) Age-dependent changes in body weight of wild-type (WT) and KO males. (Closed circles) Wild-type mice; (open circles) KO mice. (C) Gross appearance of 1-yr-old wild-type (WT) (right) and KO (left) male mice. (D) Weight of major organs from the 40-wk-old mice. (Black bars) Wild type (WT); (gray bars) KO. All organs from KO animals, with the exception of liver, are significantly smaller than wild type (p < 0.05). (E) The diameter of muscle fibers is reduced in Bmal1^−/− animals. Hematoxylin and eosin (H&E) staining of cross-sections of femoral quadriceps from 10- and 40-wk-old animals of both genotypes. (F) WBC differentials in young (10-wk-old) and aged (40-wk-old) wild-type (WT) and KO animals. Each stack in the bar represents cell type percentage. (Neutr) Neutrophils; (Lymph) lymphocytes; (Mono) monocytes; (Eos) eosinophils; (Baso) basophils; (LUC) large unstained cells. In aged KO mice, the percentage of lymphocytes is decreased and the percentage of monocytes/neutrophils is increased.

Figure 2.

Age-related pathologies of the skin in KO mice. (A) Dorsal hair regrowth 1 mo after shaving was observed in wild-type (WT) (left) but not KO (right) mice. (B) H&E staining of skin cross-sections from 10-wk-old wild-type (WT) and KO mice does not reveal any morphological differences between genotypes. (SAT) Subcutaneous adipose tissue. (C) H&E staining of skin cross-sections shows a dramatic reduction in SAT in 40-wk-old KO mice.

Figure 3.

Age-related eye pathologies in Bmal1^−/− mice. H&E staining of cross-sections of eyes from 30-wk-old wild-type (WT) and KO mice. (Co) Cornea; (Ir) iris; (Le) lens; (Re) retina. (A) Pathological changes of the cornea and lens of KO mice. (B) Magnification of the lens posterior zone (blue rectangle in A). The arrowheads point to the nuclei of epithelial cells infiltrating the posterior zone of the KO lens. (C) Magnification of the indicated zone of cornea (red rectangle in A). Acute cornea inflammation in the KO mice is characterized by neovascularization (yellow arrowheads), lymphoid cell infiltration (blue arrowheads), and ulcer (red arrowhead).

Figure 4.

BMAL1 is involved in regulation of ROS levels. (A) ROS levels indicated by relative DCF fluorescence in tissue extracts from 30-wk-old wild-type (WT) and KO mice. (B,C) Age-dependent increases in the level of ROS in kidney (B) and spleen (C) of KO mice. At least four animals of each age group and genotype were used. (*) p < 0.05. (D) BMAL1 suppression by siRNA. Western blot of lysates from L929 cells infected with a control lentivirus (V) or three different siBmal1 lentiviral constructs (539, 660, and 1686) probed with a BMAL1-specific antibody. (E,F) BMAL1 suppression by siBmal-1686 results in down-regulation of Per1 gene expression (E) and up-regulation of ROS levels (F).