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. 2023 May;165(4):521-535.
doi: 10.1111/jnc.15745. Epub 2023 Jan 9.

Mitochondrial sodium/calcium exchanger NCLX regulates glycolysis in astrocytes, impacting on cognitive performance

João Victor Cabral-Costa  1   2 , Carlos Vicente-Gutiérrez  2   3   4 , Jesús Agulla  2   4 , Rebeca Lapresa  2   4 , John W Elrod  5 , Ángeles Almeida  2   4 , Juan P Bolaños  2   3   4 , Alicia J Kowaltowski  1
Affiliations

Mitochondrial sodium/calcium exchanger NCLX regulates glycolysis in astrocytes, impacting on cognitive performance

João Victor Cabral-Costa et al. J Neurochem. 2023 May.

Abstract

Intracellular Ca2+ concentrations are strictly controlled by plasma membrane transporters, the endoplasmic reticulum, and mitochondria, in which Ca2+ uptake is mediated by the mitochondrial calcium uniporter complex (MCUc), while efflux occurs mainly through the mitochondrial Na+ /Ca2+ exchanger (NCLX). RNAseq database repository searches led us to identify the Nclx transcript as highly enriched in astrocytes when compared with neurons. To assess the role of NCLX in mouse primary culture astrocytes, we inhibited its function both pharmacologically or genetically. This resulted in re-shaping of cytosolic Ca2+ signaling and a metabolic shift that increased glycolytic flux and lactate secretion in a Ca2+ -dependent manner. Interestingly, in vivo genetic deletion of NCLX in hippocampal astrocytes improved cognitive performance in behavioral tasks, whereas hippocampal neuron-specific deletion of NCLX impaired cognitive performance. These results unveil a role for NCLX as a novel modulator of astrocytic glucose metabolism, impacting on cognition.

Keywords: NCLX; astrocyte; brain metabolism; calcium transport; energy metabolism; glycolysis; lactate; metabolic regulation; mitochondrial metabolism; sodium transport; sodium-calcium exchange.

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Conflict of interest statement

CONFLICT OF INTEREST

Dr. Juan P. Bolaños is a handling editor for the Journal of Neurochemistry. None of the other authors have any conflict of interest to declare.

Figures

FIGURE 1
FIGURE 1
Nclx (Slc8b1, Slc24a6) transcript is enriched in astrocytes. (a) Schematic illustration of NCLX exchanging extramitochondrial Na+ with matrix Ca2+, in a manner inhibited by its pharmacological modulator CGP-37157 (CGP). RNA-seq data extracted from Zhang et al. (2014) indicating (b) fragments per kilobase million (FPKM) values of Nclx transcript from astrocytes and neurons isolated from mouse cerebral cortex, and (c) FPKM value ratios between astrocytes and neurons from selected transcripts, average ± SD. RNA-seq data extracted from Chai et al. (2017) and Srinivasan et al. (2016) indicating FPKM values for the Nclx transcript from isolated astrocytes and respective hippocampal (d) or cortical (F) tissues, and ratio of FPKM values from selected transcripts between astrocytes and input tissue in the hippocampus (e) and (g) cortex. Bars indicate mean ± SEM.
FIGURE 2
FIGURE 2
NCLX inhibition changes intracellular Ca2+ homeostasis. Primary mouse astrocytes were incubated with the membrane-permeable cytosolic Ca2+ probe Fura-2-AM and imaged using a fluorescence microscope. (a) Representative trace from a Fura-2 imaging experiment (shadowed areas represent the confidence interval; continuous lines indicate mean value from 65 individual cells), indicating incubation with CGP-37157 (or DMSO as control) and ATP to induce a Ca2+ wave, and (b) an excerpt highlighting the slope after the peak (dashed lines). (c) Cytosolic [Ca2+] peak, (d) slope after reaching peak, and (e) area under the curve (AUC) of the ATP peak. *p < 0.05, paired Student’s t test, n = 4 independent cell culture preparations with 55–125 cells each. Paired values are connected by lines, with a bar indicating the mean.
FIGURE 3
FIGURE 3
NCLX inhibition increases glycolytic ATP production rates in primary mouse astrocytes. Primary mouse astrocytes incubated with the NCLX inhibitor CGP-37157 (CGP) or DMSO had their oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) monitored in a seahorse ATP production rate assay. Representative traces of (a) DMSO- and (b) CGP-treated astrocytes stimulated with ATP and followed by oligomycin (Oligo) and rotenone plus antimycin a (Rot/AA) inhibition, average ± SEM; basal and ATP-induced (c) total ATP production rate, and proportional (d) oxidative phosphorylation (OxPhos)- and (e) glycolytic-associated ATP production rate. Average values (±SEM) of the proportional difference between CGP- and DMSO-treated groups were calculated and presented in their respective conditions (c–e). *p < 0.05, **p < 0.01, paired two-way ANOVA followed by Holm–Šidak’s post hoc test, n = 4 independent cell culture preparations. Lines and error bars indicate mean and SD, respectively (a, b); paired values are connected by lines, with a bar indicating the mean (c–e).
FIGURE 4
FIGURE 4
NCLX inhibition increases astrocytic glycolytic flux in a Ca2+-dependent manner. Primary mouse astrocytes were co-incubated with the NCLX inhibitor CGP-37157 (CGP) and marked D-[3-3H]-glucose for 4 h. Derived tritiated water was measured to estimate glucose metabolism through glycolysis. Glucose consumption (b) and lactate secretion (c) were assessed in parallel experiments. (d) Primary astrocytes derived from NclxloxP/loxP mice were transduced with an adenoviral vector to express Cre-recombinase and achieve genetic deletion (NCLX KO); lactate secretion was measured for 1 h. Astrocytes were treated with the cytosolic Ca2+ chelator BAPTA-AM, followed by incubation with CGP-37157 or DMSO as a control, similar to Figure 3c. Lactate secretion (d) was then assessed. *p < 0.05, ***p < 0.001, paired (b–d) or ratio-paired (a) Student’s t test, or paired two-way ANOVA followed by Holm–Šidak’s post hoc test (e), n = 3–4 independent cell culture preparations. Paired values are connected by lines, with a bar indicating the mean.
FIGURE 5
FIGURE 5
In vivo cell-specific NCLX deletion in astrocytes or neurons has opposite behavioral effects. (a) Schematic depiction of the experimental design: NclxloxP/loxP mice were injected with cell-targeted vectors stereotaxically in the hippocampus to induce astrocytic or neuronal NCLX deletion, followed by behavioral assessment. (b) Illustration of the viral constructs used to induce astrocyte- (AAV/5) or neuron-specific (AAV/rh10) Cre recombinase expression. (c,d) open field spatiotemporal quantitative heatmaps showing average occupation of the arena area, and calculation of proportional time in the central area. Not significant, unpaired Student’s t test. (e,f) novel object recognition spatiotemporal quantitative heatmaps, showing average occupation in the arena during the recognition test, and the discrimination index calculated from entries in novel and familiar object areas. *p < 0.05, one sample Wilcoxon test with theoretical mean = 0.0. (g,h) Y-maze spatiotemporal quantitative heatmaps, showing average occupation of the arena, and calculation of the proportion of spontaneous alternations in respect to total entries. Not significant, unpaired Student’s t test, n = 6–13 mice. Boxes indicate upper and lower quartiles and the median (line), whiskers represent min and max values.
FIGURE 6
FIGURE 6
Schematic overview. In astrocytes, (1) inhibition/deletion of mitochondrial Na+/Ca2+ exchanger (NCLX) activity leads to (2) augmented cytosolic Ca2+ clearance. This results in (3) increased glycolytic flux; and (4) slightly decreased mitochondrial oxidative phosphorylation, leading to (5) increased lactate dehydrogenase (LDH)-mediated reduction of pyruvate to lactate. The resulting increased lactate in astrocytes (6) is secreted (7) and may contribute to enhanced behavioral performance in vivo. (ETC: Electron transport chain).
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