Circadian clock protein BMAL1 broadly influences autophagy and endolysosomal function in astrocytes

Abstract

An emerging role for the circadian clock in autophagy and lysosome function has opened new avenues for exploration in the field of neurodegeneration. The daily rhythms of circadian clock proteins may coordinate gene expression programs involved not only in daily rhythms but in many cellular processes. In the brain, astrocytes are critical for sensing and responding to extracellular cues to support neurons. The core clock protein BMAL1 serves as the primary positive circadian transcriptional regulator and its depletion in astrocytes not only disrupts circadian function but also leads to a unique cell-autonomous activation phenotype. We report here that astrocyte-specific deletion of Bmal1 influences endolysosome function, autophagy, and protein degradation dynamics. In vitro, Bmal1-deficient astrocytes exhibit increased endocytosis, lysosome-dependent protein cleavage, and accumulation of LAMP1- and RAB7-positive organelles. In vivo, astrocyte-specific Bmal1 knockout (aKO) brains show accumulation of autophagosome-like structures within astrocytes by electron microscopy. Transcriptional analysis of isolated astrocytes from young and aged Bmal1 aKO mice indicates broad dysregulation of pathways involved in lysosome function which occur independently of TFEB activation. Since a clear link has been established between neurodegeneration and endolysosome dysfunction over the course of aging, this work implicates BMAL1 as a key regulator of these crucial astrocyte functions in health and disease.

Keywords: Bmal1; astrocyte; autophagy; circadian; lysosome.

Fig. 1.

Bmal1 deletion in astrocytes induces upregulation of endolysosomal pathways. RNAseq analysis comparing isolated astrocytes from control (Cre−) and Bmal1 aKO brains. Young mice (n = 3 control, n = 5 Bmal1 aKO) were harvested at 7 mo of age and aged mice (n = 4 per group) were harvested at 20 mo of age. (A) Volcano plots showing differential gene expression comparing young (Left) or aged (Right) astrocytes from control and Bmal1 aKO brains (Log2 fold change cutoff = 1.0, −Log10pvalue cutoff = 2.0, corresponds to P value of 0.01). (B) Top 20 upregulated GO cellular component pathways obtained from GAGE pathway analysis. Pathways involved in the endolysosome system are in bold.

Fig. 2.

Astrocytes lacking Bmal1 exhibit elevated levels of lysosome markers. (A) Lysotracker Green in Bmal1 knockdown and scramble siRNA-treated astrocytes. (Scale bars, 100 µm.) (B) Lysotracker %area above threshold per image, normalized to Hoechst cell count per well. N = 23 to 25 wells per condition, representative of three independent experiments. **** = P < 0.0001 by t test. (C) Flow cytometry of Lysotracker in astrocytes collected at listed time points after synchronization by media change. N = 3 to 4 pups over two independent experiments (some overlapping points not visible), MFI values normalized to each pup’s siScramble 24-h value. (D and E) Confocal imaging and quantification for LAMP1 and RAB7 in siBmal1 and siScramble-treated astrocytes. N = 10 wells per condition, representative of three independent experiments. (Scale bars, 50 µm.) * = P < 0.05, ** = P < 0.005, *** =P < 0.0005, **** =P < 0.0001 by t test. (F) Flow cytometry of Lysosensor Yellow/Blue DND-160 in siBmal1 and siScramble-treated astrocytes. N = 3 independent experiments, MFI values normalized per experiment to siScramble controls. *** =P < 0.0005 by t test. (G) Flow cytometry of Lysotracker in siScramble and siBmal1 astrocytes treated for 3 h with either serum-free medium, 20 µM chloroquine (CQ), or 50 nM Bafilomycin (BAF). N = 3 independent experiments, MFI values normalized per experiment to serum-free media-treated siScramble controls. * =P < 0.05, **** =P < 0.0001 by two-way ANOVA with Sidak’s multiple comparisons test.

Fig. 3.

Bmal1 deletion in astrocytes boosts extracellular protein uptake and degradation. (A) 10X epifluorescence live cell imaging of siScramble and siBmal1 astrocytes treated with 1 µg/mL BSA-647 and DQ-BSA for 3 h. (Scale bars, 100 µm.) (B) Example 40X confocal live cell imaging of siScramble and siBmal1 astrocytes treated with 1 µg/mL BSA-647 and DQ-BSA for 3 h. (Scale bars, 50 µm.) (C) Quantification from epifluorescence imaging of BSA-647 and DQ-BSA uptake in live siScramble and siBmal1 astrocytes, %area above threshold normalized to Hoechst cell count per field of view. N = 23 to 25 wells per condition, representative of two independent experiments. (D) Flow cytometry quantification of BSA-647 and DQ-BSA uptake in live siScramble and siBmal1 astrocytes treated with serum-free medium or 50nM Bafilomycin for 3 h. N = 3 independent experiments, MFI normalized per experiment to serum-free media-treated siScramble controls. (E) Flow cytometry quantification of Lysotracker, BSA-647, and DQ-BSA in siScramble and siBmal1 astrocytes treated with either serum-free medium, 100 µM Dynasore, or 200 µM Dynasore for 3 h during BSA incubation. N = 3 to 4 independent experiments, MFI normalized per experiment to serum-free media-treated siScramble controls. (D and E) * =P < 0.05, ** =P < 0.005, *** =P < 0.0005, **** =P < 0.0001 by two-way ANOVA with Sidak multiple comparisons test.

Fig. 4.

Bmal1 deletion in astrocytes induces autophagy without increasing degradation. (A and B) Western blot of siScramble and siBmal1 astrocytes treated with either vehicle (DMSO) or 50nM Bafilomycin in serum-free media for 6 h. (A) LC3 blot image and quantification. N = 3 to 5 independent experiments, density quantifications normalized per experiment to vehicle-treated siScramble controls. β-tubulin is shown as a loading control. ** = P < 0.005 compared to control by two-way ANOVA with Dunnett’s multiple comparison test. (B) p62 blot image and quantification. N = 3 independent experiments, density quantifications normalized per experiment to vehicle-treated siScramble controls. β-tubulin is shown as a loading control. * =P < 0.05 main effect of bafilomycin compared to untreated groups by two-way ANOVA. (C) Schematic of LC3 eGFP/RFP reporter signal in autophagosomes. Created with Biorender.com (D) Fixed cell confocal imaging of LC3 eGFP/RFP primary astrocytes transfected with siScramble or siBmal1 and treated with serum-free medium alone or with 50 nM Bafilomycin for 3 h. For each treatment, left images are 20× confocal images, scale bars, 100 µm and right images are insets, scale bars, 50 µm. (E) Quantification of confocal imaging from D, plotted as LC3 RFP or eGFP mean intensity per LC3 RFP thresholded ROI, or as the LC3 RFP intensity: eGFP intensity ratio per image. (F) LC3 RFP:eGFP ratio from flow cytometry experiments of primary astrocytes treated as in D and E. N = 3 independent experiments, MFI normalized per experiment to serum-free media-treated siScramble controls. (E and F) * =P < 0.05, ** =P < 0.005, *** =P < 0.0005, **** =P < 0.0001, b = bafilomycin main effect of P < 0.0001 by two-way ANOVA with Sidak multiple comparison test.

Fig. 5.

Bmal1 deletion leads to autophagosome accumulation in astrocytes in vivo. (A) Transmission electron microscopy images of astrocytes from Cre- control (Left) and Bmal1 aKO (Right) hippocampus (Scale bars, 1 µm). Example images of electrolucent (endosomes), electron-dense (lysosomes), and multilamellar (autophagosomes) structures (Scale bars, 0.5 µm). Plots show counts of each organelle per cell. N = 15 to 19 cells from three to four mice per group. ** =P < 0.005 by t test.