Synaptic Activity Drives a Genomic Program That Promotes a Neuronal Warburg Effect

Abstract

Synaptic activity drives changes in gene expression to promote long lasting adaptations of neuronal structure and function. One example of such an adaptive response is the buildup of acquired neuroprotection, a synaptic activity- and gene transcription-mediated increase in the resistance of neurons against harmful conditions. A hallmark of acquired neuroprotection is the stabilization of mitochondrial structure and function. We therefore re-examined previously identified sets of synaptic activity-regulated genes to identify genes that are directly linked to mitochondrial function. In mouse and rat primary hippocampal cultures, synaptic activity caused an up-regulation of glycolytic genes and a concomitant down-regulation of genes required for oxidative phosphorylation, mitochondrial biogenesis, and maintenance. Changes in metabolic gene expression were induced by action potential bursting, but not by glutamate bath application activating extrasynaptic NMDA receptors. The specific and coordinate pattern of gene expression changes suggested that synaptic activity promotes a shift of neuronal energy metabolism from oxidative phosphorylation toward aerobic glycolysis, also known as the Warburg effect. The ability of neurons to up-regulate glycolysis has, however, been debated. We therefore used FACS sorting to show that, in mixed neuron glia co-cultures, activity-dependent regulation of metabolic gene expression occurred in neurons. Changes in gene expression were accompanied by changes in the phosphorylation-dependent regulation of the key metabolic enzyme, pyruvate dehydrogenase. Finally, increased synaptic activity caused an increase in the ratio of l-lactate production to oxygen consumption in primary hippocampal cultures. Based on these data we suggest the existence of a synaptic activity-mediated neuronal Warburg effect that may promote mitochondrial homeostasis and neuroprotection.

Keywords: calcium; energy metabolism; gene expression; neurobiology; synapse.

FIGURE 1.

Synaptic activity alters the expression levels of metabolism-related genes. A and B, qRT-PCR analysis of expression of indicated genes in primary mouse hippocampal cultures 4 h after induction of AP bursting with 50 μm bicuculline (Bic). The cells were infected with rAAVs that express the nuclear Ca²⁺/calmodulin inhibitor CaMBP4-mCherry or mCherry alone or were left uninfected. The p values were determined by repeated measures one-way ANOVA with Bonferroni's multiple comparison test (n = 3 independent experiments, df = 4) and are as follows: a, 0.0007; b, 0.26; c, 0.004; d, 0.014; e, 0.012; f, 0.021; g, 0.007; h, 0.004; i, 0.0007; j, <0.0001; k, 0.0005; l, 0.007; m, 0.02; n, 0.026; o, <0.0001; p, 0.91; q, 0.003; r, 0.09; s, 0.0006; t, 0.056; u, 0.012; v, 0.1; w, <0.0001; x, 0.009. C, qRT-PCR analysis of mitochondrial DNA levels at the indicated times after induction of AP bursting (two independent experiments). D, immunoblot analysis of TOM20 protein levels at the indicated times after induction of AP bursting. Repeated measures one-way ANOVA with a post test for linear trend revealed a significant reduction of TOM20 protein over time (slope = −0.03, p = 0.0054, n = 3 independent experiments). A representative immunoblot is shown in the inset. Positions of molecular mass markers (kDa) are indicated. E, qRT-PCR analysis of expression of indicated genes in primary mouse hippocampal cultures 4 h after bath application of 20 μm glutamate (summary of three independent experiments). F and G, qRT-PCR analysis of expression of indicated genes in adult mouse hippocampus 4 h after i.p. injection of PBS or KA. The p values are indicated above each pair of columns (*, p < 0.001) and were determined by two-tailed t test (one experiment; PBS, n = 5 animals; KA, n = 6 animals; df = 9). H, qRT-PCR analysis of expression of indicated genes in adult rat hippocampus 4 h after i.p. injection of PBS or KA. The p values are indicated above each pair of columns (*, p < 0.001) and were determined by two-tailed t test (one experiment, n = 8 animals per condition, df = 14). All graphs show mean, individual values, and 95% CI. Ctrl or ctrl, control.

FIGURE 2.

Synaptic activity does not cause an increase in HIF-1α protein. A, representative immunoblot that shows HIF-1α levels in primary mouse hippocampal cultures that were left untreated (ctrl), stimulated for 4 h with 50 μm bicuculline (AP bursting), or deprived of oxygen supply for 4 h (oxygen depr.). AP bursting-mediated gene expression was verified by detection of c-FOS protein. The positions of molecular mass markers (kDa) are indicated. B, quantification of HIF-1α levels in control and stimulated cultures. The HIF-1α signal of positive control samples was usually saturated at exposure times that were appropriate for quantification of control and stimulated samples. The positive control was therefore not quantified. The graph shows mean, individual values, and 95% CI (n = 3 independent experiments).

FIGURE 3.

Synaptic activity-mediated changes in the expression of metabolism-related genes can be detected in purified neurons. A, representative immunofluorescence staining of primary rat hippocampal culture on DIV12. EGFP expression is indicated in green. NeuN immunolabeling is indicated in magenta. Hoechst 33258-labeled nuclei are indicated in blue. Scale bar, 50 μm. Arrows point to EGFP- and NeuN-negative astrocytes. B, qRT-PCR analysis of the expression of astrocytic marker mRNAs Gfap and Aqp4 in primary rat hippocampal cultures versus neurons that were purified from these cultures by FACS sorting. C, qRT-PCR analysis of the expression of indicated genes 4 h after the induction of AP bursting. Gene expression changes (Bic/ctrl) were analyzed in mixed rat hippocampal cultures and purified neurons. D and E, qRT-PCR analysis of the expression of indicated genes in glia-free primary mouse cortical cultures 4 h after the induction of AP bursting. All graphs show mean, individual values, and 95% CI.

FIGURE 4.

Long lasting synaptic activity induces a biphasic change in the phosphorylation of PDH. A, representative immunoblot analysis of PDH phosphorylation at Ser-293 at the indicated times after the induction of AP bursting with 50 μm bicuculline (Bic). The positions of molecular mass markers (kDa) are indicated. B, quantification of PDH phosphorylation at the indicated times after the induction of AP bursting. The p values were determined by repeated measures one-way ANOVA with Bonferroni's multiple comparison test (n = 4 independent experiments, df = 9). The graph shows mean, individual values, and 95% CI.

FIGURE 5.

Short term and long term stimulation of neurons differently affects cellular energy metabolism. A, quantification of OCR in primary rat hippocampal cultures immediately after induction of AP bursting with 50 μm bicuculline (Bic) or after silencing of electrical activity with 1 μm TTX. The p values (compared with basal OCR) are indicated above the bar graphs and were determined by two-tailed paired t test (n = 5 independent experiments, df = 4). B, quantification of a Warburg Index determined by the ratio of LPR to OCR in primary rat hippocampal cultures 6 h after application of vehicle (control, Ctrl) or application of three brief pulses of 25 mm KCl to depolarize the cells and induce calcium-dependent gene transcription (see “Experimental Procedures” for details). The p value was determined by two-tailed paired t test (n = 4 independent experiments, df = 3). C, qRT-PCR analysis of the expression of activity-regulated genes in primary rat hippocampal cultures 3 h after application of three brief pulses of 25 mm KCl. As expected, fold changes after three brief KCl pulses are smaller than after 4 h of continuous AP bursting (cf. Fig. 1). All graphs show mean, individual values, and 95% CI.

FIGURE 6.

Synaptic maturation in primary hippocampal cultures is accompanied by a shift from oxidative phosphorylation to aerobic glycolysis. A, qRT-PCR analysis of the expression of indicated activity-regulated genes in primary rat hippocampal cultures on DIV5 and DIV12. The p values were determined by two-tailed paired t test (n = 4 independent experiments, df = 3). B, quantification of a Warburg Index determined by the ratio of LPR to OCR in primary rat hippocampal cultures on DIV5 and DIV12. The Warburg Index is independent of cell density and energy consumption rate and thus directly reflects the mode of energy metabolism, i.e. aerobic glycolysis versus oxidative phosphorylation. The p value was determined by two-tailed paired t test (n = 5 independent experiments, df = 4). C and D, quantification of GFAP immunofluorescence to determine the number of astrocytes (C, percentage of total cells) and the surface area covered by astrocytes (D, percentage of total area) in primary rat hippocampal cultures on DIV5 and DIV12. The p values were determined by two-tailed paired t test (n = 3 independent experiments, df = 2). E, representative immunofluorescence staining of primary rat hippocampal cultures on DIV5 and DIV12. GFAP labeling is indicated in grayscale, and Hoechst 33258-labeled nuclei are indicated in blue. Scale bar, 100 μm. F, quantification of PFKFB3 protein expression by immunoblot analysis in primary rat hippocampal cultures on DIV5 and DIV12. A representative immunoblot is shown in the inset. The p value was determined by two-tailed paired t test (n = 3 independent experiments, df = 2). All graphs show mean, individual values, and 95% CI.

FIGURE 7.

Schematic representation of the main findings of this study. A, schematic illustration of the major steps of glucose flux through glycolysis and the TCA cycle. Genes that were found in this study to be up-regulated by synaptic activity are indicated in bold blue. They are located at strategic positions within the metabolic network to control the mode of ATP generation. B, proposed model of a synaptic activity-mediated neuronal Warburg effect. Synaptic activity causes an influx of Ca²⁺ that leads to enhanced mitochondrial respiration via Ca²⁺-dependent activation of mitochondrial dehydrogenases. Excitation transcription coupling causes a delayed increase in expression of the Warburg gene program that results in enhanced aerobic glycolysis and reduced mitochondrial respiration. The neuronal Warburg effect thus keeps mitochondrial activity in a sustainable range and ensures sufficient spare respiratory capacity to allow neurons to quickly respond to further increases in energy demand. Both mechanisms are proposed to protect neurons from oxidative damage and metabolic failure and thus provide a neuroprotective effect.