Sirtuin3 ensures the metabolic plasticity of neurotransmission during glucose deprivation

Abstract

Neurotransmission is an energetically expensive process that underlies cognition. During intense electrical activity or dietary restrictions, the glucose level in the brain plummets, forcing neurons to utilize alternative fuels. However, the molecular mechanisms of neuronal metabolic plasticity remain poorly understood. Here, we demonstrate that glucose-deprived neurons activate the CREB and PGC1α transcriptional program, which induces expression of the mitochondrial deacetylase Sirtuin 3 (Sirt3) both in vitro and in vivo. We show that Sirt3 localizes to axonal mitochondria and stimulates mitochondrial oxidative capacity in hippocampal nerve terminals. Sirt3 plays an essential role in sustaining synaptic transmission in the absence of glucose by providing metabolic support for the retrieval of synaptic vesicles after release. These results demonstrate that the transcriptional induction of Sirt3 facilitates the metabolic plasticity of synaptic transmission.

Figure S1.

(Related to Fig. 1 and Fig. 2). Neuronal viability is sustained during glucose deprivation and hippocampal Sirt3 expression is not altered by overnight fasting. (A) Fraction of dead cells following glucose deprivation (3 h) as determined by propidium iodide staining (PI+). Mean (% total) ± SEM: +glucose (3 h), 1.76 ± 0.30; −glucose (3 h), 3.76 ± 0.53. (B) Serum glucose levels in mice fed ad lib. or fasted overnight. Serum glucose levels ± SEM (mg/dl): ad lib., 71.65 ± 10.59; fasted overnight, 16.63 ± 3.91. n = 12 mice/condition. (C) Hippocampal tissues from mice fed ad lib. or fasted overnight were immunoblotted for Sirt3 and mitochondrial ATPase5β. (D) Sirt3 band intensity normalized to ATPase5β and expressed relative to ad lib samples. Average normalized Sirt3 band intensity ± SEM: ad lib., 1.00 ± 0.13; overnight fasted, 1.354 ± 0.22. n = 11 mice/condition. Mann–Whitney U test (A, B, and D). Source data are available for this figure: SourceData FS1.

Figure 1.

Transcriptional reprogramming of neuronal metabolism during glucose deprivation. (A) Schematic for glucose deprivation of rat cortical neurons. A subset of ∼250 genes significantly upregulated during glucose deprivation (P value <0.01) were selected for pathway analysis using annotated gene sets from the Molecular Signatures Database (MSigDB). n = 3 cortical samples from one rat litter. (B) Enrichment of genes in MSigDB Reactome pathways with adjusted P value <0.05. (C) Enrichment of the target genes of the CREB transcription factor in glucose-deprived neurons (adjusted P value <0.05). The target gene Pgc1α (PPARGC1A) is highlighted in red. (D) Immunostaining of cortical neuronal cultures (treated as in A) with an anti-phospho-CREB antibody and Hoechst nuclear stain. Arrowheads denote pCREB-positive nuclei. (E) Fraction of nuclei with positive p-CREB staining in fields of view (FOV), determined as described in Materials and methods. % total ± SEM: +glucose (3 h), 14.87 ± 2.29, −glucose (3 h), 27.41 ± 3.3. n = 17–22 FOVs. (F) Relative mRNA expression of Pgc1α in neuronal cultures treated as in A, with or without the AMPK inhibitor, dorsomorphin. Values are normalized to β-actin mRNA and expressed relative to the +glucose condition. Average normalized mRNA level ± SEM: −glucose (1 h), 2.83 ± 0.42; −glucose (3 h), 3.67 ± 0.46; −glucose + dorsomorphin (3 h), 1.32 ± 0.22. n = 3–10 cortical samples. Bar graphs are plotted as mean ± SEM. Mann–Whitney U test (E), one-way ANOVA (F). See Table 1.

Figure 2.

Glucose deprivation stimulates neuronal Sirt3 expression and deacetylation of mitochondrial proteins. (A) Relative Sirt3 mRNA expression in control and glucose-deprived neurons. Values are normalized to β-actin mRNA and expressed relative to control (+glucose). n = 3–10 cortical samples. Average normalized mRNA level ± SEM: −glucose (1 h), 2.58 ± 0.62; −glucose (3 h), 2.20 ± 0.27; −glucose + dorsomorphin (3 h), 1.12 ± 0.08. n = 3–11 cortical samples. (B) Relative Sirt3 mRNA expression in cultures transduced with adenoviral particles encoding GFP (control) and GFP-PGC1α, normalized to 18s rRNA and expressed relative to control. Average normalized mRNA level ± SEM: GFP-PGC1α, 1.93 ± 0.30. n = 16 cortical samples/condition. (C) Immunoblotting of Sirt3 protein expression in cortical neurons with antibodies against Sirt3 and the cytosolic and mitochondrial controls, β-actin, and ATPase5β, respectively. (D) Sirt3 band intensity normalized to the ATPase5β and expressed relative to control. Average normalized Sirt3 band intensity ± SEM: +glucose, 0.99 ± 0.05; −glucose, 1.74 ± 0.16. n = 7 cortical samples/condition. (E) A paradigm for the analysis of Sirt3 expression in hippocampi of mice fed ad libitum (ad lib) or alternate-day fasted for 6 mo (ADF). Schematic created with https://biorender.com. (F) Immunoblotting of Sirt3 protein in mouse hippocampal lysates. (G) Sirt3 band intensity normalized to the ATPase5β band and expressed relative to the ad lib mice. Average normalized Sirt3 band intensity ± SEM: ad lib, 1 ± 0.24; ADF (6 mo), 1.88 ± 0.19. n = 6 mice/condition. (H) Mitochondrial and cytosolic fractions isolated from cortical neuronal cultures maintained for 3 h with (+) or without (−) glucose. High and low exposures were 60 and 25 s, respectively. (I) Intensity of lysine acetylation bands normalized to α-tubulin or ATPase5β plotted relative to control. Average normalized Ac-K intensity: cytosolic fraction (−glucose), 1.21 ± 0.15; mitochondrial fraction (−glucose), 0.66 ± 0.08. n = 5 cortical samples. One-way ANOVA (A), two-tailed, unpaired t test (B), Mann–Whitney U test (D and G), one sample t test (I). See Table 1. Source data are available for this figure: SourceData F2.

Figure 3.

Sirt3 is expressed in neuronal mitochondria and is present in presynaptic terminals. (A) Control hippocampal neurons and neurons transduced with lentivirus expressing Sirt3 shRNA (Sirt3 KD) were immunostained with anti-Sirt3 antibody and the Hoechst nuclear stain. (B) Quantification of Sirt3 immunofluorescence in control and Sirt3 KD neurons. Normalized mean Sirt3 intensity per coverslip ± SEM: Control, 1.02 ± 0.19; Sirt3 KD, 0.34 ± 0.04. n = 4–5 coverslips. (C) Co-immunostaining of neurons with antibodies against Sirt3 and the mitochondrial marker TOMM20. (D) Quantification of Sirt3 colocalization with TOMM20 or Hoechst in neuronal cell bodies. Mean Pearson’s correlation coefficient (r) ± SEM: Sirt3/TOMM20, r = 0.55 ± 0.01, Sirt3/Hoechst: −0.09 ± 0.02. n = 78 cell bodies. (E–G) Coimmunostaining of neurons with antibodies against the neurite marker Tuj1 (E) or presynaptic marker vGLUT1 (F and G). (G) Magnification of the boxed area in F. Arrowheads indicate colocalization of Sirt3 with presynaptic terminals. (H) Quantification of Sirt3 colocalization with vGLUT1 or Hoechst across fields of view (FOVs). Mean Pearson’s correlation coefficient ± SEM: Sirt3/vGLUT1: r = 0.60 ± 0.02, Sirt3/Hoechst: −0.01 ± 0.02. n = 27 FOVs. Scale bars, 10 μm. Mann–Whitney U test (B, D, and H). See Table 1.

Figure S2.

(Related to Fig. 3). Sirt3 regulates mitochondrial morphology in hippocampal axons. (A) Relative expression of sirt3 mRNA in control and Sir3 KD cortical neurons. Values are normalized to β-actin mRNA and expressed relative to the control. Average normalized mRNA level ± SEM: Sirt3 KD, 0.19 ± 0.07. n = 4 cortical samples. (B) Hippocampal axons from control and Sirt3 KD neurons expressing the mitochondrial marker mito^4x-RFP. (C) Mitochondria length in control and Sirt3 KD axons. Mean length per neuronal axon (μm) ± SEM: control, 2.0 ± 0.03, Sirt3 KD, 1.6 ± 0.03. n = 31–42 neurons. Two-tailed, unpaired t test (A and C).

Figure 4.

Sirt3 stimulates oxidative ATP production and sustains synaptic transmission in hippocampal nerve terminals. (A) Luminescence and fluorescence (physin-mCherry) images of hippocampal nerve terminals expressing Syn-ATP. (B) Normalized and pH-corrected presynaptic ATP traces in control and Sirt3 KD neurons were supplied with lactate and pyruvate and stimulated with 600 AP at 10 Hz. (C) Normalized ATP level before and after electrical stimulation. Average ATP level: control (prestimulation), 1.00 ± 0.06, Sirt3 KD (prestimulation), 0.61 ± 0.19, control (poststimulation), 1.10 ± 0.35, Sirt3 KD (poststimulation), 0.65 ± 0.18. n = 10–32 neurons. (D) Schematic for determination of SV release probability (P_r) using vGLUT1-pH in neurons stimulated with 100 AP at 1 Hz, or 10 Hz. Representative images from successful and failed release events are shown in green and grey, respectively. (E) P_r in control and Sirt3 KD terminals at different stimulation frequencies. Average P_r ± SEM: control (1 Hz), 0.079 ± 0.005, Sirt3 KD (1 Hz), 0.081 ± 0.003, control (10 Hz), 0.112 ± 0.007, Sirt3 KD (10 Hz), 0.116 ± 0.011. n = 9 coverslips. (F) Sample vGLUT1-pH traces of control, Sirt3 KD, and neurons expressing shRNA-resistant Sirt3 (rescue) stimulated with 100 AP at 10 Hz in the presence of glucose or lactate/pyruvate. Inset: Semi-log plot of vGLUT1-pH traces following stimulation. (G) Fractional retrieval block of vGLUT1-pH was calculated as described in Materials and methods. n = 6–13 neurons. Average retrieval block ± SEM: control (lact+pyr), 0.14 ± 0.02, Sirt3 KD (lact+pyr), 0.40 ± 0.05, rescue (lact+pyr), 0.19 ± 0.01, Sirt3 KD (glucose), 0.18 ± 0.04. n = 6–13 neurons. (H) Sample vGLUT1-pH traces of control and Sirt3 KD neurons stimulated with 100 AP at 10 Hz in the presence of β-hydroxybutyrate (BHB). (I) Fractional retrieval block of vGLUT1-pH. n = 19 neurons. Average retrieval block ± SEM: control, 0.13 ± 0.03, Sirt3 KD, 0.36 ± 0.05. Gray bars and arrows denote electrical stimulation. The box-whisker plots denote median (line), 25th–75th percentile (box), and min-max (whiskers). Kruskal–Wallis test (C and G), two-sample t test (E), Mann–Whitney U test (I). See Table 1.

Figure S3.

(Related to Fig. 4). Quantification of presynaptic ATP, cytosolic pH, mitochondrial and cytosolic calcium dynamics in control and Sirt3 KD nerve terminals. (A) Average traces of raw luminescence/fluorescence values of control and Sirt3 KD hippocampal nerve terminals expressing Syn-ATP, electrically stimulated with 600 AP at 10 Hz. n = 10–32 neurons. (B) Average traces of cytosolic pH in control and Sirt3 KD terminals expressing cyto-pHluorin, electrically stimulated with 600 AP at 10 Hz. n = 7–8 neurons. (C) Representative vGLUT1-pH traces of a neuron supplied with 5 mM glucose, with or without incubation with 2 µM oligomycin. (D) Fractional retrieval block of vGLUT1-pH. Average retrieval block ± SEM: Glucose, 0.14 ± 0.02, Glucose + Oligomycin 0.40 ± 0.05. n = 8 neurons. (E) Average traces of mito^4xi-GCaMP6f showing mitochondrial Ca²⁺ uptake in control and Sirt3 KD axons stimulated with 20 AP at 20 Hz. (F) Peak responses of Mito^4xi - GCaMP6f (ΔF/F) following stimulation. Mean ΔF/F ± SEM: control, 0.34 ± 0.04, Sirt3 KD, 0.26 ± 0.02. n = 19–26 neurons. (G) Average traces of physin-GCaMP6f showing cytosolic Ca²⁺ uptake in control and Sirt3 KD axons stimulated with 1AP. (H) Peak responses of physin-GCaMP6f (ΔF/F) to stimulation. Mean ΔF/F ± SEM: control, 0.27 ± 0.06, Sirt3 KD, 0.21 ± 0.04. n = 13–20 neurons. (I) Average traces of physin-GCaMP6f showing cytosolic Ca²⁺ uptake in control and Sirt3 KD axons stimulated with 10 AP at 10 Hz. (J) Peak responses of physin-GCaMP6f (ΔF/F). Mean ΔF/F ± SEM: control, 2.5 ± 0.7, Sirt3 KD, 2.7 ± 0.4. n = 13–20 neurons. Grey bars or arrows denote electrical stimulation. Error bars are SEM. Wilcoxon test (D), Two-tailed, unpaired t test (F, H, and J).