Circadian alterations during early stages of Alzheimer's disease are associated with aberrant cycles of DNA methylation in BMAL1

Abstract

Introduction: Circadian alterations are prevalent in Alzheimer's disease (AD) and may contribute to cognitive impairment, behavioral symptoms, and neurodegeneration. Epigenetic mechanisms regulate the circadian clock, and changes in DNA methylation have been reported in AD brains, but the pathways that mediate circadian deregulation in AD are incompletely understood. We hypothesized that aberrant DNA methylation may affect circadian rhythms in AD.

Methods: We investigated DNA methylation, transcription, and expression of BMAL1, a positive regulator of the circadian clock, in cultured fibroblasts and brain samples from two independent cohorts of aging and AD.

Results: DNA methylation modulated rhythmic expression of clock genes in cultured fibroblasts. Moreover, rhythmic methylation of BMAL1 was altered in AD brains and fibroblasts and correlated with transcription cycles.

Discussion: Our results indicate that cycles of DNA methylation contribute to the regulation of BMAL1 rhythms in the brain. Hence, aberrant epigenetic patterns may be linked to circadian alterations in AD.

Keywords: Alzheimer's disease; BMAL1; Brain; Circadian clock; Circadian rhythms; DNA methylation; Epigenetics; Fibroblasts; Methylation cycles; Neurodegeneration.

Figure 1. Altered circadian rhythms in AD patients’ fibroblasts associate with aberrant BMAL1 transcription

A. Clinical and demographic characterization of cell line donors. (¹Age at skin biopsy. ²Calculated in years from clinical diagnosis to biopsy date). B. Lowess curves representing BMAL1 transcript abundance at different circadian times. Symbols represent duplicate determinations per cell line tested. C. Immunohistochemical detection of BMAL1 comparing protein abundance in control and AD fibroblasts at the times when BMAL1 transcripts reached peak and nadir values in control cells (indicated by arrows in Figure 1B). Imaged at 60 ×. Scale bar = 50 µm. D. Quantification of pixel intensity of BMAL1 immunoreactivity. ** p<0.01 and ***p<0.001 in comparison to control cells at peak time; or nadir time ## p<0.01 as per unpaired non-parametric Mann-Whitney t test. E–F. Rhythmic Per2::luc expression was measured from transduced control and AD-derived fibroblasts to determine circadian parameters. E. Representative data for Per2::luc rhythms obtained from a control subject and a patient with diagnosis of probable AD. F. Mean circadian rhythm parameters in AD case-control group analyses. * p<0.05 probable AD (n=13) vs. control fibroblasts (n=7) as determined by unpaired, non-parametric Mann-Whitney t test.

Figure 2. Pharmacological modulation of DNA methylation induces changes in circadian rhythms in cultured fibroblasts

Rhythmic Per2::luc expression in NIH 3T3 mouse fibroblasts treated with 5-Aza-dC; SAM or vehicle (NT). A–B. Concentration-dependent changes in circadian rhythms were observed in response to both compounds. C–E. Reduced global methylation shortens period; increases amplitude and advances phase. F–H. Increased methylation levels lengthen period; decreases amplitude and delays phase. I–J. Both treatments significantly changed global DNA methylation (I) and expression level of Bmal1 transcripts (J). K. Circadian rhythms are modulated by changes in methylation in human fibroblasts treated with 50 M 5-Aza-dC. Error bars represent S.E.M. * p<0.05; ** p<0.01 and *** p<0.001 in comparison to vehicle treated cells as per One Way ANOVA (C–I) or paired parametric t test (J–K).

Figure 3. Rhythmic oscillation of DNA methylation correlate with BMAL1 transcription in AD brains

A. Schematic representation of BMAL1 genomic region screened. Vertical bars indicate the location of probes in the Illumina 450k array. Blue * denotes probes showing significant rhythms with 24 h period. Red bars depict 5’ UTR probes that showed altered methylation rhythms in AD brains. Gray bar indicates the position of CG island Chr11:13298796–13300735 in ChrBuild37. B. Comparison of methylation rhythms at 5’UTR CpGs between control, early and late AD cases. Panels show non-linear fit of methylation values to sinusoidal curves. Blue * p<0.05; red ** p<0.01 in comparison to controls at the same time (one way ANOVA, with Tukey’s posthoc test). C. Representation of fit-sinusoidal curves for the combined 5’UTR sites from (B). D. Locally weighted scatterplot smoothing (Lowess) curves representing BMAL1 transcript abundance as a function of time of death as 4 h bins (T.O.D.). Symbols represent mean values ± S.E.M. E–F. Analysis of cg13286116 methylation and BMAL1 transcript abundance. Lower panel shows significant correlation (Pearson’s R) for control cases, which is lost in AD brains. G. Immunohistochemical detection of BMAL1 protein in frontal cortex samples at different T.O.D. ACTIN immunoreactivity is showed as loading control. H. Densitometric analysis of BMAL1 protein comparing abundance during AM vs. PM hours (as indicated in G). Data represents normalized values of BMAL1/ACTIN ratios.

Figure 4. Validation of rhythmic DNA methylation, BMAL1 transcription correlations and acrophase alterations in the ROS/MAP cohorts

A–B. Individual CpG site analysis on 396 ROS/MAP participants adjusting for age at death and sex. Lines represents best-fit cosine curve. Selected panels showing comparison of fitted cosinor curves per group for probes located at BMAL1 promoter (A) and 5’ UTR (B) regions. Top p value refers to rhythmicity in the group. Bottom p value refers to rhythmicity differences in comparison to the control group. C. Selected scatterplots showing association of the combined signal of all BMAL1 isoforms detected by RNAseq with methylation levels at individual CpGs. Pearson’s R coefficients and p values for the correlation are indicated. D–F. Distribution of methylation acrophase times for all 22 BMAL1 probes detected (D); or for probes located at BMAL1 promoter (E) or 5’UTR regions (F) in controls, early and late AD brains.

Figure 5. Differences in BMAL1 methylation between AD patients and controls are also evident in fibroblast cells

Bisulfite sequencing was used to compare the levels of methylation at site cg13286116 between control subjects and AD patients’ cells at the times when BMAL1 transcription is close to nadir (C.T.30) and peak (C.T.40) in control fibroblasts. A. Panel B from Fig. 1 is provided as a reference of BMAL1 transcript levels at the time points tested. B. The graph shows the percentage of sequenced clones that showed fully methylated cg13286116 at each time point. * p<0.05 in comparison to control cells, ## p<0.01 in comparison to Probable AD cells at C.T.30 as per unpaired Mann-Whitney t test.