Article Figures & Data
Figures
- Figure 1.
Redox responses in the LCns diverge in response to short-term and extended wakefulness. A, SOD2 mRNA copy numbers, normalized to 18S RNA copy number and to the Rest value. Shown are the mean ± SE values for Rest (light gray), 3 h short-term sleep loss (Sh Wake, black), and sleep loss extended to 8 h/d for 3 consecutive days (Ext Wake, dark gray; n = 6–10/group). Lines denote significant differences across groups by one-way ANOVA, Bonferroni corrected. **p < 0.01. B, Catalase mRNA copy numbers, normalized and analyzed similarly for the same groups (n = 6–10/group). **p < 0.01. C, Representative LC protein immunoblots for SOD2, catalase, and corresponding β-tubulin for Rest, Sh Wake, and Ext Wake conditions. D, E, SOD2 (D) and catalase (E) LC protein responses across Rest, Sh Wake, and Ext Wake (n = 8–10/group), normalized to β-tubulin and mean rested condition value, analyzed with one-way ANOVA, Bonferroni corrected. *p < 0.05; **p < 0.01. F, Mean ± SE integrated density of catalase immunofluorescent intensity within LCns across Rest, Sh Wake, and Ext Wake conditions (n = 5/group). *p < 0.01. G, Representative confocal images at mid-LC. TH (red) delineates LCns. Catalase (green) is present largely in nuclei of LCns, particularly in response to Sh Wake. Scale bar, 25 μm. H, Mid-LC nucleus confocal images (1 μm) of autofluorescence (excitation and emission, 488 and >590 nm) from oxidized DHE for detection of O2−· in representative LC sections from Rest, Sh Wake, and Ext Wake mice. Arrows highlight LCns with nuclear/nucleolar DHE-autofluorescent labeling. Scale bar, 25 μm. I, Group DHE-integrated density (mean ± SE) signal for LCns (n = 5/group), one-way ANOVA. **p < 0.01.
- Figure 2.
SirT3 responses in LC vary with sleep loss duration, while corticosterone is not affected. A, Plasma corticosterone levels at ZT11 in mice exposed to Rest, Sh Wake, and Ext Wake conditions (n = 8–14/group). No significant differences were observed with one-way ANOVA despite 94% statistical power to detect an increase of 100 ng/ml. B, Mean ± SE LC SirT3 mRNA copies, normalized to 18S RNA and mean Rest values for Rest, Sh Wake, and Ext Wake conditions (n = 8–10/group) were analyzed with one-way ANONA, Bonferroni corrected. Bars highlight group differences: *p < 0.05; **p < 0.01; ***p < 0.001. C, LC SirT3 protein for Rest, Sh Wake, and Ext Wake conditions (n = 8–10/group) normalized to β-tubulin and mean Rest values are presented as mean ± SE. Data were analyzed with one-way ANOVA: *p < 0.05; **p < 0.01. D, Representative Western blots for LC SirT3 across the three conditions and loading control. E, Representative LC images across wake conditions (Rest, Sh Wake, and Ext Wake) showing SirT3 (green) alone in the top and SirT3 (green) with tyrosine hydroxylase (red) delineating LCns in the bottom. Scale bar, 25 μm. F, Summary data, mean ± SE, for SirT3 confocal integrated densities in LCns (n = 5/group). **p < 0.01; ***p < 0.001.
- Figure 3.
Mitochondrial lysine acetylation varies with wake duration and SirT3. A, Representative immunoblots of LC lysine-acetylated proteins and loading controls (VDAC1 and tubulin) in mitochondrial (mito) and cytoplasmic (cyto) enriched samples from SirT3wt mice exposed to Rest (R), Sh Wake (Sh), and Ext Wake (Ext) conditions. B, Summary data (mean ± SE) integrated densities for LC mitochondrial isolate-acetylated protein normalized to VDAC1 for the three conditions in SirT3wt LC samples (n = 20/group). One-way ANOVA identified significant differences. **p < 0.01. C, Mean ± SE integrated densities for LC cytoplasmic isolate-acetylated protein normalized to β-tubulin for the same conditions (n = 20/group). D, Representative Western blots of SirT3wt LC mitochondrial isolate acetyl-lysine on immunoprecipitate of complex I proteins (left) and complex I protein loading controls used in normalization (right). E, Mean ± SE normalized integrated densities for acetyl (Ac)-lysine (Lys) on complex I immunoprecipitates from mitochondria across the three wake conditions (n = 10/group). **p < 0.01. F, Representative immunoblots of LC lysine-acetylated proteins and loading controls (VDAC1 and tubulin) in mitochondrial (mito) and cytoplasmic (cyto) enriched samples from SirT3−/− mice exposed to Rest and Ext Wake (Ext) conditions. G, Mean ± SE integrated densities for SirT3−/− LC mitochondrial isolate-acetylated protein normalized to VDAC1 for the two conditions (n = 20/group), with nonsignificant differences across wake conditions. H, Mean ± SE integrated densities for SirT3−/− LC cytoplasmic isolate-acetylated protein normalized to β-tubulin for the same conditions (n = 20/group). I, Immunoblot examples for Ctx lysine-acetylated proteins and loading control (VDAC1) in mito samples in SirT3wt (left) and SirT3−/− (right) mice exposed to Rest, Sh Wake (Sh), and Ext Wake (Ext) conditions. J, K, Summary (mean ± SE) normalized integrated densities for LC mitochondrial isolate-acetylated protein normalized to VDAC1 for SirT3wt (J) and SirT3−/− (K) mice, analyzed with one-way ANOVA. *p < 0.05.
- Figure 4.
NAMPT in LCn and cortical NAD+ vary with wake duration. A, Representative confocal images within the LC nucleus across wake conditions. Top shows NAMPT only (green), and bottom shows NAMPT (green) in tyrosine hydroxylase (red)-labeled LCns, where the signal is predominantly nuclear. Scale bar, 50 μm. B, Mean ± SE NAMPT-integrated densities within LCns normalized to background (n = 5/group), analyzed with one-way ANOVA. *p < 0.05. C, NAD+ levels in cortical tissue were measured across Rest and Ext Wake conditions using a modified cycling assay in both SirT3wt and SirT3−/− mice. Rest (light gray) and Ext Wake (dark gray) are presented as the mean ± SE for SirT3wt and SirT3−/− mice (n = 10/group). Data were analyzed with two-way ANOVA: **p < 0.01; ***p < 0.001; ****p < 0.0001.
- Figure 5.
SirT3 is essential for the Sh Wake antioxidant response and redox homeostasis in LCns. A, Mean ± SE LC SOD2 mRNA copies, normalized to 18S RNA for Rest (gray) and Sh Wake (black) conditions in SirT3wt and SirT3−/− mice (n = 8–10/group). Data were analyzed with two-way ANOVA, Bonferroni corrected: *p < 0.05; **p < 0.01. B, Mean ± SE LC catalase mRNA copies, normalized to 18S RNA for Rest (gray) and Sh Wake (black) conditions in SirT3wt and SirT3−/− mice (n = 8–10/group). **p < 0.01. C, LC SOD2 protein for Rest (gray) and Sh Wake (black) conditions in SirT3wt and SirT3−/− mice (n = 8–10/group). **p < 0.01. D, Typical LC nucleus immunoblots for SOD2 and loading control tubulin for SirT3wt and SirT3−/− mice across Rest and Sh Wake (Sh) conditions. E, Confocal images of DHE autofluorescence in SirT3wt and SirT3−/− mice across Rest and Sh Wake conditions. Scale bar, 25 μm. Arrows highlight nuclei/nucleolar ox-DHE labeling. F, Mean ± SE percentages of LCns with DHE labeling for SirT3wt and SirT3−/− mice across Rest and Sh Wake conditions (n = 10/group). Data were analyzed with two-way ANOVA. ****p < 0.0001. G, Scattergrams of individual data for confocal autofluorescence integrated density over LCns across the four groups (n = 10/group) with mean and 95% confidence interval bars. Data were analyzed with two-way ANOVA; bars delineate *p < 0.05.
- Figure 6.
SirT3 is essential for LCn nuclear FoxO3a and upregulation of PGC-1α in response to Sh Wake. A, Mid-LC confocal images of TH+ (red) LCns with FoxO3a (green) across three sleep conditions. FOX03a is evident in nuclei and somata of LCns. B, Mean ± SE percentage of TH+ LCns with nuclear FoxO3a. Data were analyzed by two-way ANOVA. ***p < 0.001. C, Mean ± SE integrated intensity of the nuclear FoxO3a signal in LCns (n = 5 mice/group), analyzed similarly. **p < 0.01, ****p < 0.0001. D, Mean ± SE normalized PGC-1α immunointensities for the groups (n = 6–9/group). *p < 0.05. E, Immunoblots of PGC-1α and loading control β-tubulin in LC micropunches from SirT3wt and SirT3−/− mice across the three sleep conditions.
- Figure 7.
Extended sleep loss results in LCn dendrite and somata loss, and upregulation of apoptosis in remaining LCns. A, Representative ventral lateral LCn images from 60 μm sections with TH (DAB, brown) and Giemsa (blue) labeling demonstrate the effects of sleep loss and SirT3 genotype on LCn dendrites and somata. SirT3−/− mice exposed to Ext Wake reveal prominent beading of dendrites with vacuolization. B, Mean ± SE group data (n = 5/group) for dendrite complexity in Rest and Ext Wake for SirT3wt and SirT3−/− mice analyzed by two-way ANOVA. ***p < 0.001; ****p < 0.0001. C, Stereological LCn estimates (bilateral) using optical fractionator for complete LC bilaterally. Data were analyzed with two-way ANOVA (n = 5/group). *p < 0.05; **p < 0.01. D, Representative confocal image (0.7 μm) to localize CC3 (green) within the nuclei of LCns (TH, red). Arrows delineate examples of CC3 labeling within nuclei. Scale bar, 50 μm. E, Mean ± SE group data (n = 5/group) for CC3 in Rest (gray) and Ext Wake (black) conditions for SirT3wt and −/− mice also analyzed with two-way ANOVA. **p < 0.01.
- Figure 8.
Mice lacking SirT3 show impairments in wakefulness and NREM sleep homeostasis. A, Group average sleep latencies (mean ± SE, n = 8/group) in a murine MSLT during Zeitgeber hours (relative to lights-on) ZT6–ZT8 and ZT12–ZT14 for SirT3wt (gray) and SirT3−/− (black) as analyzed with two-way ANOVA. *p < 0.05; **p < 0.01. B, Shown are mean ± SE relative theta power in slower and faster frequencies: 5–7 Hz (gray bars) and 7–10 Hz (black bars), respectively, in response to cage change for SirT3wt and SirT3−/− mice; two-way ANOVA. *p < 0.05; **p < 0.01. C, Total time per 24 h spent in the Wake, NREMS, and REMS conditions for SirT3wt (gray) and SirT3−/− (black). Two-way ANOVA, NS. D, Average time for each behavioral state bout (minutes) across 24 h for Wake, NREMS, and REMS conditions for SirT3wt (gray) and SirT3−/− (black), NS E, Group (mean±SE) relative NREMS delta power for each zeitgeber hour across the lights-on period normalized to the delta power of the last hour of the lights-on period for SirT3wt (blue closed circles) and SirT3−/− mice (red closed squares). The NREM sleep homeostatic responses to 6 h wake are shown for SirT3−/− mice (open red squares) and SirT3wt mice (open blue circles) for the first 6 h of rebound sleep: *p < 0.05, Bonferroni-corrected differences across baseline; °p < 0.05, for differences across recovery sleep. F, Summary data for the percentage of TH+ neurons in LC nucleus with nuclear c-fos labeling for conditions Rest (light gray) and Sh Wake (black) in both SirT3wt and SirT3−/− mice. Data were analyzed with two-way ANOVA. ***p < 0.001. G, Representative images of c-fos in the LC. Shown are confocal images 1 μm thick to delineate nuclear labeling of c-fos (red) within TH (green)-labeled LCns.
Tables
Primary antibodies Catalog number/company IHC dilution WB dilution WB band size (kDa) Acetyl-Lysine 9441S/Cell Signaling Technology N/A 1:500 N/A Catalase ab1877/Abcam 1:250 1:1500 65 c-fos Ab-5/EMD Millipore 1:2000 N/A N/A CC3 9664/Cell Signaling Technology 1:100 N/A N/A Complex I Ab09798/Abcam N/A 1:1000 4–98 FoxO3a 9467/Cell Signaling Technology 1:500 1:500 82:97 NAMPT Ab58640/Abcam 1:250 N/A N/A PGC-1α NBP 1 04676/Novus Biologicals N/A 1:500 91 Sirt3 S4072/Sigma-Aldrich 1:1000 N/A N/A Sirt3 5490S/Cell Signaling Technology N/A 1:1000 28 SOD2 NB 100–1992/NOVUS N/A 1:1000 25 β-Tubulin SC-8035/Santa Cruz Biotechnology N/A 1:1000 52 TH Ab23763/Abcam 1:2000 N/A N/A VDAC1 Ab14734/Abcam N/A 1:1000 39 N/A, Not applicable; WB, Western blot; IHC, immunohistochemistry.
