The circadian system in cystic fibrosis mice is regulated by histone deacetylase 6

Abstract

Disordered sleep experienced by people with cystic fibrosis (CF) suggest a possible disruption in circadian regulation being associated with the loss of cystic fibrosis transmembrane conductance regulator (Cftr) function. To test this hypothesis, circadian regulation was assessed in an F508del/F508del CF mouse model. CF mice exhibited significant alterations in both timing of locomotor activity and in mean activity per hour in both light-dark (LD) and dark-dark (DD) photoperiods compared with wild-type (WT) controls. It was also noted that in DD periodicity increased in CF mice, whereas shortening in WT mice as is expected. CF mice also exhibited altered timing of circadian gene expression and a reduction of melatonin production at all time points. Mechanistically, the role of microtubules in regulating these outcomes was explored. Mice lacking expression of tubulin polymerization promoting protein (Tppp) effectively mimicked CF mouse phenotypes with each measured outcome. Depleting expression of the microtubule regulatory protein histone deacetylase 6 (Hdac6) from CF mice (CF/Hdac6) resulted in the reversal of each phenotype to WT profiles. These data demonstrate an innate disruption of circadian regulation in CF mice and identify a novel microtubule-related mechanism leading to this disruption that can be targeted for therapeutic intervention.

Keywords: HDAC6; circadian; cystic fibrosis; melatonin; microtubule.

Graphical abstract

Figure 1.

Targeted deletion of Hdac6 restores circadian phenotypes in cystic fibrosis mice. A: representative actograms of WT, CF, Tppp^−/−, and CF/Hdac6^−/− mice during 12/12 LD photoperiod (top) and DD photoperiod (bottom). Actogram data are double plotted (48 h/line) with light schedule indicated by the black and white bars at the top of each actogram (white represents lights on, black representing lights off). B: characteristics of locomotor activity in WT (n = 8), CF (n = 8), Tppp^−/− (n = 8), and CF/Hdac6^−/− (n = 8) mice during 12/12 LD photoperiod and DD photoperiod. Values are displayed as means ± SE. *Statistical significance at the 0.05 level compared with the WT value. **Significance at the 0.01 level compared with the WT value. C: mean activity per hour measured in counts in WT (n = 8), CF (n = 8), Tppp^−/− (n = 8), and CF/Hdac6^−/− (n = 8) mice during 12/12 LD photoperiod and DD photoperiod. Values are displayed as means ± SE. *Statistical significance at the 0.05 level. CF, cystic fibrosis; DD, dark-dark; Hdac6, histone deacetylase 6; LD, light-dark; Tppp, tubulin polymerization promoting protein; WT, wild type.

Figure 2.

Deletion of Tppp causes clock gene expression changes that mimic CF, which are reverted to WT patterns by deletion of Hdac6. Serial measurements of Clock (A), Bmal1 (B), Per1 (C), Per2 (D), Cry1 (E), Cry2 (F) in SCN tissue of CF (red, n = 8), Tppp^−/− (yellow, n = 8), and CF/Hdac6 (green, n = 8) animals. All measurements are compared with WT (blue, n = 8) at four time points: ZT 6, ZT 12, ZT 18, and ZT 0. All values are displayed as fold change of the experimental group relative to WT (the WT value for each transcript is represented by 1.00 on the y-axis). Yellow * represent statistical significance of the Tppp^−/− group compared with WT. Red * represent statistical significance of the CF group compared with WT. Error bars represent means ± SE. CF, cystic fibrosis; Hdac6, histone deacetylase 6; Tppp, tubulin polymerization promoting protein; WT, wild type.

Figure 3.

Microtubule dysfunction and CF independently result in decreased serum melatonin, which is corrected in CF by restoration of microtubule function. Serum melatonin concentration; WT (black) vs. CF (gray) vs. Tppp^−/− (dotted) vs. CF/Hdac6 (dashed) at four timepoints (n = 3/group/timepoint). Mean concentrations are displayed in nanograms per milliliter of serum. Black * represent statistical significance of the Tppp^−/− group compared with WT. Gray * represent statistical significance of the CF group compared with WT. Error bars represent means ± SE. CF, cystic fibrosis; Hdac6, histone deacetylase 6; Tppp, tubulin polymerization promoting protein; WT, wild type.

Figure 4.

Cystic fibrosis transmembrane conductance regulator (Cftr) mRNA is expressed in suprachiasmatic nucleus tissue. Box and whisker plot showing fold change of Cftr expression in CF (n = 4), Tppp^−/− (n = 4), and CF/Hdac6^−/− (n = 4) animals relative to WT (n = 4). All values are displayed as fold change of the experimental group relative to WT, which is represented as 1.00 on the y-axis. CF, cystic fibrosis; Hdac6, histone deacetylase 6; Tppp, tubulin polymerization promoting protein; WT, wild type.

Figure 5.

CFTR function in WT, Tppp^−/−, and F508del (CF) mouse nasal epithelial (MNE) cells. Representative trace of short-circuit current (I_sc/cm²) in MNE treated with amiloride (100 µM), forskolin (10 µM), and CFTR^inh172 (10 µM) as indicated by dotted lines. CF mice were also treated with genistein (30 µM). Changes in I_sc (DIsc/cm²) induced by each compound. Data represent n = 3–6 replicates with means ± SE shown. CF, cystic fibrosis; Tppp, tubulin polymerization promoting protein; WT, wild type.