Cell-type specific circadian transcription factor BMAL1 roles in excitotoxic hippocampal lesions to enhance neurogenesis

Abstract

Circadian clocks, generating daily rhythms in biological processes, maintain homeostasis in physiology, so clock alterations are considered detrimental. Studies in brain pathology support this by reporting abnormal circadian phenotypes in patients, but restoring the abnormalities by light therapy shows no dramatic effects. Recent studies on glial clocks report the complex effects of altered clocks by showing their beneficial effects on brain repairs. However, how neuronal clocks respond to brain pathology is elusive. This study shows that neuronal BMAL1, a core of circadian clocks, reduces its expression levels in neurodegenerative excitotoxicity. In the dentate gyrus of excitotoxic hippocampal lesions, reduced BMAL1 in granule cells precedes apoptosis. This subsequently reduces BMAL1 levels in neighbor neural stem cells and progenitors in the subgranular zone, enhancing proliferation. This shows the various BMAL1 roles depending on cell types, and its alterations can benefit brain repair. Thus, cell-type-specific BMAL1 targeting is necessary to treat brain pathology.

Keywords: Cell biology; Neuroscience; Sensory neuroscience.

Graphical abstract

Figure 1

NMDA-induced excitotoxicity mimics neurodegenerative conditions in the adult hippocampus (A) Confocal images of Thy1-YFP transgenic mouse brains in control and excitotoxicity on day 1 (Excito-1D) and day 3 (Excito-3D). Tiled overview scans of the brains (Low mag.). PBS and NMDA were stereotaxically injected into either side of the hippocampus as control and excitotoxicity. White arrows indicate reduced YFP⁺ neurons in the CA1 and DG of excitotoxicity. DAPI (blue) was used as a nuclear counterstain. (B) Cropped and amplified images of (A) in high magnification (High mag.). (C) The quantification of CA1 areas in (B) (mean ± SD; a dot indicates one mouse brain; two-tailed t-test). Control (Ctrl) and excitotoxicity (Excito) on day 1 (1D) and day 3 (3D). (D) The quantification of DG areas in (B) (mean ± SD; a dot indicates one mouse brain; two-tailed t-test). (E) Confocal micrographs of control and excitotoxic hippocampal lesions, stained with TUNEL (red). DAPI (blue) was used as a nuclear counterstain. White arrows indicate amplified images of CA1 areas in the dashed rectangles. White dashed lines and red arrows indicate the DG. Images from maximum intensity projections. (F) The quantification of CA1 areas in (E) (mean ± SD; a dot indicates one mouse brain; two-tailed t-test). ∗p < 0.01; ∗∗p < 0.001; ∗∗∗p < 0.0001. See also Figures S1.

Figure 2

Excitotoxicity reduces BMAL1 levels in hippocampal neurons (A) Confocal images of control and excitotoxic hippocampal lesions, stained with antibodies against BMAL1 (green) and pCREB (red). DAPI (blue) was used as a nuclear counterstain. White and red arrowheads indicate BMAL1⁺/pCREB⁻ and BMAL1^-/pCREB⁺ cells, respectively. (B) The quantification of CA1 areas in (A) (mean ± SD; a dot indicates one mouse brain; two-tailed t-test). Control (Ctrl) and excitotoxicity (Excito) on day 1 (1D) and day 3 (3D). (C) The quantification of DG areas in (A) (mean ± SD; a dot indicates one mouse brain; two-tailed t-test). ∗p < 0.01; ∗∗p < 0.001; ∗∗∗p < 0.0001. See also Figures S2 and S3.

Figure 3

Reduced BMAL1 in excitotoxic DG lesions does not affect BMAL1 levels in the CA3 (A) Simplified schematic neuronal projections in the hippocampus. White arrow indicates PBS or NMDA injection site. (B) Confocal images of control (PBS injection) and excitotoxic hippocampal lesions on day 1 (Excito-1D) and day 3 (Excito-3D), stained with anti-BMAL1(green) antibodies. Tiled overview scans of the hippocampi. The dashed rectangles in the lower magnification images (Low mag.) indicate amplified areas in the high magnification images (High mag.). CA3 and DG areas are amplified in the lower images. DAPI (blue) was used as a nuclear counterstain. (C) The quantification of nuclear BMAL1 fluorescence intensity in CA3 and DG areas in (B) (mean ± SD; a dot indicates one mouse brain; two-tailed t-test). ∗p < 0.05.

Figure 4

Granule cells with reduced BMAL1 in excitotoxic DG lesions subsequently reduce SGZ BMAL1 to induce SGZ cell proliferation (A) Confocal micrographs of Nestin-EGFP transgenic mice in control and excitotoxicity on day 1 (Excito-1D) and day 3 (Excito-3D), stained with anti-BMAL1 (red) antibodies. Overview scans of the DG in low magnification (Low mag.) and cropped and amplified images in high magnification (High mag.). The white arrowhead in the control indicates BMAL1⁺/EGFP⁺ cells. White arrowheads in Excito-1D indicate BMAL1^-/EGFP⁺ cells. Red arrowheads in Excito-3D indicate BMAL1⁺/EGFP⁺ cells. DAPI (blue) was used as a nuclear counterstain. (B) The quantification of the SGZ in (A) (mean ± SD; a dot indicates one mouse brain; two-tailed t-test). Control (Ctrl) and excitotoxicity (Excito) on day 1 (1D) and day 3 (3D). (C) The quantification of the DG in (A) (mean ± SD; a dot indicates one mouse brain; two-tailed t-test). (D) The quantification of the DG in (A) (mean ± SD; a dot indicates one mouse brain; two-tailed t-test). ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.0001. See also Figures S4.

Figure 5

Reduced BMAL1 in SGZ cells induces cell proliferation and differentiation (A) Confocal micrographs of the DG of control (Ctrl) and Nestin-Cre:Bmal1^f/f (N-C:B^f/f) mice, stained with TUNEL (red). DAPI (blue) was used as a nuclear counterstain. Images from maximum intensity projections. (B) Confocal images of the DG of control and Nestin-Cre:Bmal1^f/f mice, stained with antibodies against NESTIN (red) and Ki67 (green). DAPI (blue) was used as a nuclear counterstain. Images from maximum intensity projections. (C) The quantification of the SGZ in (B) (mean ± SD; a dot indicates one mouse brain; two-tailed t test). (D) The quantification of the DG in (B) (mean ± SD; a dot indicates one hemisphere of the brain; two-tailed t test). ∗p < 0.005; ∗∗p < 0.0001. See also Figure S5.