Cell-Autonomous Regulation of Astrocyte Activation by the Circadian Clock Protein BMAL1

Abstract

Circadian clock dysfunction is a common symptom of aging and neurodegenerative diseases, though its impact on brain health is poorly understood. Astrocyte activation occurs in response to diverse insults and plays a critical role in brain health and disease. We report that the core circadian clock protein BMAL1 regulates astrogliosis in a synergistic manner via a cell-autonomous mechanism and a lesser non-cell-autonomous signal from neurons. Astrocyte-specific Bmal1 deletion induces astrocyte activation and inflammatory gene expression in vitro and in vivo, mediated in part by suppression of glutathione-S-transferase signaling. Functionally, loss of Bmal1 in astrocytes promotes neuronal death in vitro. Our results demonstrate that the core clock protein BMAL1 regulates astrocyte activation and function in vivo, elucidating a mechanism by which the circadian clock could influence many aspects of brain function and neurological disease.

Keywords: Bmal1; astrocyte; astrogliosis; circadian; glutathione; neuroinflammation; rhythm.

Figure 1.. Light-Induced Circadian Disruption or Neuron-Specific Bmal1 Deletion Partially Recapitulates the Astrocyte Activation Phenotype Observed following Brain-Specific Bmal1 Deletion

(A) Cortical qPCR from 6-week-old WT mice housed in 12 hr:12 hr or 10 hr:10 hr light-dark cycles for 6 weeks, harvested every 6 hr from CT6 to CT24, reveals blunting of circadian gene oscillations in 10 hr:10 hr mice. (B) Total Gfap mRNA levels for mice from (A). **p < 0.01 by 2-tailed t test. (C) Representative images showing GFAP immunostaining of hippocampus (top) and cingulate cortex (bottom) of neuron-specific Bmal1 KO mice (Cam2a-iCre+;Bmal1^f/f) and brain-specific (neurons+astrocytes) Bmal1 KO mice (Nestin-Cre+;Bmal1^f/f) at 4 months. Scale bar, 100 μm. (D) Quantification of GFAP immunoreactivity in two brain regions of Cre−, Cam2a-iCre+;Bmal1^f/f, and Nestin-Cre+;Bmal1^f/f mice. n = 3–4 mice per genotype. Each data point represents 1 mouse. *p < 0.05 by 1-way ANOVA. (E) Quantification of astrocyte activation transcripts by qPCR in the cortex of Cre−;Bmal1^f/f, Cam2a-iCre+;Bmal1^f/f, and Nestin-Cre+;Bmal1^f/f mice. The clock-controlled gene Nr1d1 is shown to illustrate loss of BMAL1-mediated transcription. *p < 0.05 by 2-way ANOVA with Dunnett’s post-test for multiple comparisons. All data represent mean ± SEM.

Figure 2.. Bmal1 Deficiency Induces Cell-Autonomous Astrocyte Activation In Vitro and In Vivo

(A) qPCR of Gfap and Aqp4 mRNA in primary astrocyte-enriched cultures from WT or global Bmal1 KO mice. n = 8–12 samples from 3 independent experiments. (B) Western blot showing increased GFAP protein levels in cultured astrocyte cell lysates from Bmal1 KO mice. p42/44 ERK protein is shown as a loading control. (c) Western blot showing knockdown of BMAL1 and increased GFAP protein in WT astrocyte cultures 7 days after treatment with non-targeting siRNA (siSCR) or siRNA targeting Bmal1 (siBmal1). p42/44 ERK protein is shown as a loading control. (D) Densitometric quantification of blots in (C). n = 4 samples/group. (E) Representative trace depicting detrended circadian oscillations in luminescence seen in primary astrocyte cultures from Per2-Luc reporter mice treated with siRNA (siSCR, blue; or siBmal1, red). Similar results were obtained in 3 separate cultures. (F) Representative confocal images from cerebral cortex of Bmal1^f/f mice 5 months after intracerebroventricular (i.c.v.) injection at age P2 (postnatal day 2) with viral vectors inducing astrocyte-specific expression of EGFP (AAV8-GFAP-EGFP) or Cre (AAV8-GFAP-Cre^GFP). AAV8-GFAP-GFP-infected cells show whole-cell GFP expression and persistent colocalized nuclear BMAL1 staining, while AAV8-GFAP-Cre infected cells show nuclear GFP expression, increased GFAP expression, and loss of nuclear BMAL1. Scale bar, 30 μm. (G) Representative images (left) and quantification (right) illustrating increased percent area covered by GFAP+ astrocytes in cortex in micefrom (F) injected with AAV8-GFAP-Cre, as compared to AAV8-GFAP-GFP-injected controls. Note that AAV8-GFAP-Cre virus encodes a Cre^GFP fusion protein that is nuclear localized and more difficult to see (F). Scale bar, 100 μm. Each data point represents 1 mouse. (H) Representative images and quantification of percent area of GFAP staining from hippocampus of Aldh1l1-Cre^ERT2+;Bmal1^f/f and Aldh1 l1-Cre^ERT2+;Bmal1^wt/flox mice at 3 months(2 months after tamoxifen treatment), CAG-Cre^ERT2+;Bmal1^f/f mice at 4 months (2 months after tamoxifen treatment), and Bmal1^f/f controls. Scale bar, 100 μm. All data represent mean + SEM. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p<0.0001 by 2-tailed t tests(C) and (G) or 1-way ANOVA(H) with Holm-Sidak correction for multiple comparisons when applicable.

Figure 3.. BMAL1 Regulates Astrocyte Activation via a Glutathionylation-Dependent Mechanism

(A) Astrocyte-specific genes upregulated in Nestin-Cre⁺;Bmal1^f/f(NBmal1 KO) cortexand changes to the same genes in Per1/2^mut mice. Transcripts upregulated at least 2-fold in NBmal1 KO were compared to the top 200 astrocyte-specific genes (Zhang et al., 2014), and overlapping genes are displayed. Other notable genes implicated in astrocyte activation and function were upregulated in NBmal1 KO cortex (bottom). The clock-controlled genes Dbp and Nr1d1 are direct Bmal1 transcriptional targets. (B) Expression of neurotoxic, neurotrophic, and pan-reactive astrocyte activation genes (Liddelow et al., 2017) in 5-month-old global Bmal1 KO versus WT littermate hippocampus assayed by microfluidic qPCR. Each data point represents the average of 4 Bmal1 KO mice normalized to the average of 4 WT littermates. (C) Results from Ingenuity Pathway Analysis canonical pathway analysis of NBmal1 KO microarray data. Pathways related to glutathione homeostasis are colored blue. (D) Expression levels for downregulated glutathione-S-transferases in NBmal1 KO cortex versus Bmal1^f/fcontrols. Expression levels in Per1/2^mut cortex included for comparison (based on microarray data). (E) qPCR illustrating effect of glutathione manipulation on Gfap transcript levels following Bmal1 knockdown. WT primary astrocytes treated with control siRNA (siSCR, gray bars) or siBmal1 (blue bars), with or without N-acetyl-cysteine (NAC; striped bars) to increase glutathione levels, or buthionine sulfoxime (BSO; speckled bars) to deplete glutathione. n = 9 samples/condition from 3 separate experiments. (F) WT astrocytes transfected with siSCR, siBmal1, or siSCR +BSO (BSO) were treated with biotin-linked glutathione ethyl ester (bioGEE) to assess glutathionylation. Increased band intensity indicates decreased endogenous glutathionylation. Arrows denote altered bands. NT, not treated with bioGEE, showing nonspecific labeling. (G) qPCR depicting effects of NAC treatment on Gfap transcript levels in cortex of inducible Bmal1 KO mice (CAG-Cre^ERT2+;Bmal1^f/f). Inducible Bmal1 KO mice and Cre− control littermates were treated with NAC before, during, and after 5-day tamoxifen treatment to delete Bmal1. Mice were harvested 9 days after the start of tamoxifen. n = 6–10 mice per group. All data represent mean + SEM. *p < 0.05, ***p < 0.001 by t test with Holm-Sidak correction for multiple comparisons.

Figure 4.. Loss of Bmal1 Impairs Astrocytic Support of Neurons

(A) Diagram depicting co-culture experimental design. DIV, days in vitro; TAM, tamoxifen. (B) Representative images showing NeuN+ WT neuronal nuclei at DIV 1 and 7, grown on primary astrocytes from Cre− control mice, or inducible Bmal1 kO mice (iCAG-Cre+) treated with tamoxifen in vitro. Scale bar, 100 μm. (C) Quantification of NeuN+ nuclei from (B) normalized to NeuN counts from Cre− condition, n = 8 replicates from 2 independent experiments. (D) Representative images of MAP2+ WT neurons grown as in (B) at DIV 12. Lower panels show neurons from adjacent wells treated for 24 hr with hydrogen peroxide at DIV 11 (H₂O₂, 100 μM). Scale bar, 100 μm. (E) Quantification of MAP2 immunoreactivity (percent area) from (D). n = 9 replicates from 3 independent experiments. All data represent mean ± SEM. *p < 0.05, **p < 0.01 by 2-tailed t test.