Circadian Rhythm Dysfunction Accelerates Disease Progression in a Mouse Model With Amyotrophic Lateral Sclerosis

Abstract

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease caused by interactions between environmental factors and genetic susceptibility. Circadian rhythm dysfunction (CRD) is a significant contributor to neurodegenerative conditions such as Alzheimer's disease and Parkinson's disease. However, whether CRD contributes to the progression of ALS remains little known. We performed behavioral and physiological tests on SOD1G93A ALS model mice with and without artificially induced CRD, and on wild-type controls; we also analyzed spinal cord samples histologically for differences between groups. We found that CRD accelerated the disease onset and progression of ALS in model mice, as demonstrated by aggravated functional deficits and weight loss, as well as increased motor neuron loss, activated gliosis, and nuclear factor κB-mediated inflammation in the spinal cord. We also found an increasing abundance of enteric cyanobacteria in the ALS model mice shortly after disease onset that was further enhanced by CRD. Our study provides initial evidence on the CRD as a risk factor for ALS, and intestinal cyanobacteria may be involved.

Keywords: NF-κB; amyotrophic lateral sclerosis; circadian rhythm dysfunction; cyanobacteria; inflammation.

Figure 1

Circadian rhythm dysfunction (CRD) accelerated disease onset and progression in SOD1G93A mice. (A) A schematic showing the experimental procedures. (B,C) Survival curves: disease onsets and disease progression in the ALS + CRD and the ALS group. (D) Body weights loss of four groups starting from the day 50 to the day 119. N = 4–6 animals per group, *p < 0.05. Error bars represent SEM.

Figure 2

Circadian rhythm dysfunction (CRD) aggravated motor neuron (MN) loss and activated glial cells in SOD1G93A mice. (A) MNs [choline acetyltransferase (ChAT)-positive cells, red] and astrocytes [glial fibrillary acidic protein (GFAP)-positive cells, green] expression in four groups for three stages. (B) MNs (ChAT-positive cells, red) and microcytes (Iba1-positive cells, green) expression in four groups for three stages. Scale bars represent 100 µm. (C) Statistical evaluation of surviving MNs radio by counting the number of ChAT-positive cells. **p < 0.01; * p < 0.05 (one-way ANOVA, n = 4–6 animals per group). (D,E) GFAP and Iba1 expression was quantified at three stages by western blotting (one-way ANOVA, n = 2–3 animals per group). (F) Expression of GFAP and Iba1 in the spinal cord. **p < 0.01; *p < 0.05. Error bars represent SEM.

Figure 3

Circadian rhythm dysfunction (CRD) aggravated nuclear factor κB-mediated inflammation in SOD1G93A mice. (A) Expression of p65, p-p65, and p-IKK in the spinal cord. (B,C) Expression of p-IKK and normalized p-p65 was quantified at three stages by western blotting (one-way ANOVA, n = 2–3 animals per group). **p < 0.01; *p < 0.05. Error bars represent SEM.

Figure 4

Circadian rhythm dysfunction (CRD) increased enteric cyanobacteria abundance in SOD1G93A mice. (A) Intestinal bacterial abundance profiles at the level of phylum among the four groups at three time points using 16S high throughput sequencing. Flora were presented in the form of averages. Sample capacity of each group was 4–8. (B) Differences and distances of each sample between the amyotrophic lateral sclerosis (ALS) + CRD group and the ALS group at day 60 and 90, respectively, reflected by Principle Component Analysis (PCA). (C) Comparisons of relative abundances of cyanobacteria among the four groups at different disease stages. N = 4–6 per group per time point, *p < 0.05. Error bars represent SEM.