Mitochondrial pyruvate transport regulates presynaptic metabolism and neurotransmission

Abstract

Glucose has long been considered the primary fuel source for the brain. However, glucose levels fluctuate in the brain during sleep or circuit activity, posing major metabolic stress. Here, we demonstrate that the mammalian brain uses pyruvate as a fuel source, and pyruvate can support neuronal viability in the absence of glucose. Nerve terminals are sites of metabolic vulnerability, and we show that mitochondrial pyruvate uptake is a critical step in oxidative ATP production in hippocampal terminals. We find that the mitochondrial pyruvate carrier is post-translationally modified by lysine acetylation, which, in turn, modulates mitochondrial pyruvate uptake. Our data reveal that the mitochondrial pyruvate carrier regulates distinct steps in neurotransmission, namely, the spatiotemporal pattern of synaptic vesicle release and the efficiency of vesicle retrieval-functions that have profound implications for synaptic plasticity. In summary, we identify pyruvate as a potent neuronal fuel and mitochondrial pyruvate uptake as a critical node for the metabolic control of neurotransmission in hippocampal terminals.

Fig. 1.. Pyruvate is efficiently oxidized in intact brain and is a metabolic fuel for primary neuronal cultures.

(A) Schematic of ¹³C₃ pyruvate perfusion into mouse jugular vein and its metabolic tracing in the serum, brain, and liver using LC-MS. Created with Biorender.com. (B) Pathways of pyruvate metabolism including gluconeogenesis and oxidation via the TCA cycle. (C) Enrichment of ¹³C-labeled (m + 1 to m + 6) intermediates of gluconeogenesis and the TCA cycle in different tissues. ¹³C-labeled metabolite levels were normalized to the amount of circulating tracer. Serum glucose-6-phosphate (G6P), FBP, and succinate were excluded due to lack of reliable measurement. n = 4 mice. (D) Schematic for survival analysis and metabolic profiling of primary neurons at 8 DIV supplied with various fuel types, with glucose (5 mM), no glucose, or an equimolar mix of lactate and pyruvate (5 mM each) denoted as lac/pyr. (E) Survival rate of neurons relative to control (+glucose). n = 108 (wells), 3 (cultures). (F) Pyruvate consumption rate (μM/hour per μg of lysate) in neurons treated as in (E). n = 3 (wells). (G) Steady-state levels of TCA metabolites in neurons treated as in (D) and plotted as fold change relative to +glucose condition = 3 (wells). Unpaired t test [(F) and (G)] and one-way analysis of variance (ANOVA) [(C) and (E)]. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, *q < 0.01, **q < 0.001, and ***P < 0.0001. Error bars are SEM.

Fig. 2.. MPC is essential for pyruvate metabolism in nerve terminals.

(A and C) Co-immunostaining of hippocampal neurons with antibodies against MPC1 (A) or MPC2 (C) and the presynaptic protein vGLUT1. Scale bars, 10 μm. (B and D) Magnification of the boxed areas in (A) and (C). Colocalization denoted with arrowheads. Scale bars, 10 μm (B) and 5 μm (D). (E) Representative images of axonal mitochondria expressing a mitochondrial pyruvate sensor, CamKII-mtPyronicSF, showing an increase in fluorescence intensity with perfusion of 10 mM extracellular pyruvate compared to 0 mM. Scale bar, 10 μm. (F) Average traces of CamKII-mtPyronicSF (ΔF/F₀) showing inhibition of mitochondrial pyruvate uptake in neurons expressing shRNA against mpc1 (MPC1 KD). F: fluorescence intensity. n = 12 to 18 (neurons). (G) Maximal CamKII-mtPyronicSF fluorescence response in 10 mM pyruvate as determined from traces in (F). (H) Schematic of the pyruvate sensor CaMKII-mtPyronicSF and the ATP indicator Syn-ATP expressed in nerve terminals. (I) Presynaptic ATP traces in terminals supplied with lactate and pyruvate (lac/pyr) after incubation with the MPC inhibitor UK5099 (100 μM), normalized to preincubation levels. n = 9 (neurons). (J) Presynaptic ATP level after application of MPC inhibitor UK5099 normalized to ATP level before. The box-whisker plots denote median (line), 25th to 75th percentile (box), and minimum-maximum (whiskers). Man-Whitney U test (G) and one sample t test (J). Error bars are SEM.

Fig. 3.. Mitochondrial pyruvate uptake regulates the spatiotemporal properties of SV release and retrieval.

(A) Two examples of AZs with the localization of release events without (left) or with (right) UK5099. Representative images of individual events evoked at 1 Hz are shown for each AZ. (B) Average release Pr of events evoked by 1-Hz stimulation for 200 s in the two conditions. Sample number (cultures/coverslips/synapses): Control: 3/16/691, UK5099: 3/9/230. (C) Average number of clusters (release sites) in an individual AZ. Sample number: Control: 3/16/732, UK5099: 3/9/220. (D) Number of release events detected per release site in the two conditions (plotted as fraction of total events). The inset shows the cumulative distribution plot. Sample number (cultures/coverslips/release sites): Control: 3/16/7005, UK5099: 3/9/1705 for cultures/coverslips/synapses/release sites. (E) Distribution of distances from release events to the center of the AZ (plotted as fraction of total events). The inset shows the cumulative distribution plot. Sample number (cultures/coverslips/release sites): Control: 3/16/9097, UK5099: 3/9/2786 for cultures/dishes/events. (F) Distribution of distances between consecutive events over time (plotted as fraction of total events). The inset shows the cumulative distribution plot. Sample number (cultures/coverslips/events): Control: 3/16/9097, UK5099: 3/9/2786 for cultures/dishes/events. (G and H) Average (G), normalized (H), and raw [(H) inset] representative traces of vesicle release evoked by high-frequency stimulation (50 AP at 40 Hz) in the two conditions. a.u., arbitrary units. (I and J) Rates of vesicle exocytosis (I) and endocytosis (J) in the two conditions calculated by linear fitting of the rise and decay components of the train responses during high-frequency stimulation. Sample number (cultures/coverslips/synapses): Control: 3/16/691, UK5099: 3/9/230. All experiments were performed in the presence of lactate and pyruvate (no glucose). Kolmogorov-Smirnov test [(B) to (F), (I), and (J)]. Error bars are SEM. **P < 0.001.

Fig. 4.. Sirt3 modulates mitochondrial pyruvate uptake and MPC acetylation.

(A) Representative images of an HEK293 cell expressing mtPyronicSF in media containing 0 and 10 mM pyruvate. (B) Average traces of mtPyronicSF showing differential mitochondrial pyruvate accumulation in control HEK293 cells and cells expressing shRNA against sirt3 (Sirt3 KD) or mpc1 (MPC1 KD). (C) Peak values of mtPyronicSF (ΔF/F₀) in response to 10 mM pyruvate, as determined from traces in (B). n = 24 to 57 fields of view (FOV), five to six cells per FOV. (D) Immunoprecipitation (IP) of endogenous MPC1 from Sirt3^+/+and Sirt3^−/− mouse brain lysates with antibodies against MPC1 and rabbit immunoglobulin G (IgG) (control) followed by immunoblotted (IB) with antibodies against MPC1 (top) and Ac-K (bottom). Asterix denotes a nonspecific band in the IgG immunoprecipitate. (E) Intensity of Ac-K band normalized to MPC1 band from (D), expressed relative to Sirt3^+/+ control. n = 4 (IP blots). Raw intensities of Ac-K normalized to MPC1 in Sirt3^+/+and Sirt3^−/− brains were used for statistical comparison. (F) Alignment of rat (Rattus norvegicus, R. n.), mouse (Mus musculus, M. m.), and human (Homo sapiens, H. s.) MPC1 protein sequences showing conservation of an acetylation motif (boxed in blue). A nonacetylatable form was constructed by mutation of K45/46 to A45/A46. (G) Immunoprecipitation of wild-type (WT) MPC1 or acetyl mutant (MPC1-AA) from Sirt3-deficient (KD) HEK293 cells, immunoblotted for FLAG (top) and Ac-K (bottom). (H) Intensity of Ac-K band normalized to FLAG intensity from panel G, expressed relative to WT-MPC1. n = 6 (IP blots). Kruskal-Wallis test (C), paired t test (E), and one sample t test (H). Error bars are SEM. IB, immunoblot. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig. 5.. An acetyl mimetic MPC1 mutant impairs mitochondrial pyruvate uptake and SV retrieval.

(A) Schematic of a HEK293 cell expressing mtPyronicSF. (B) Average traces of mtPyronicSF expressed in HEK293 cells showing differential mitochondrial pyruvate accumulation in control, MPC1 KD, MPC1 KD + WT MPC1, or MPC1 KD + MPC1-QQ (acetyl mimetic mutant). Control and MPC1 KD traces are the same as in Fig. 4B. (C) Peak values of mtPyronicSF (ΔF/F₀) in response to perfusion of 10 mM pyruvate, as determined from traces in (B). n = 22 to 57 FOV, five to six cells per FOV. (D) Schematic of a hippocampal neuron-expressing vGLUT1-pH. (E) Sample normalized vGLUT1-pH traces in hippocampal terminals electrically stimulated with 100 AP at 10 Hz in control neurons, or neurons expressing MPC1 KD, MPC1 KD + WT MPC1, or MPC1 KD + MPC1-QQ (acetyl mimetic mutant). (F) SV retrieval quantified as fractional retrieval block calculated from traces in (E). n = 16 to 81 (neurons). Black bar denotes electrical stimulation. Kruskal-Wallis test [(C) and (F)]. Error bars are SEM. ***P < 0.001; ****P < 0.0001.