This is a tribute to Sebastián Cerdán, a brilliant and innovative NMR spectroscopist whose studies contributed greatly to the fundamental information to the understanding of brain metabolism, particularly in regard to multinuclear magnetic resonance spectroscopy (MRS) techniques. Sebastián Cerdán sadly passed away in May 2022. He was a wonderful mentor and colleague who will be greatly missed.

(A) Quantitative PCR analysis of RBP1 expression of the 49 tumors included in the study compared to controls comprising five tumors carrying mutation in Krebs cycle genes and five cases carrying mutations in RET or NF1. RU: relative units. (B) SDHB immunohistochemistry of tumor_4 compared to a positive control tumor carrying a non-SDH mutation. The scale bars represent 50μm. (C) Representation of DNA methylation (M_values) of the CpG island probes located within the SDHC locus of tumor_4 compared to a blood sample from the same patient and a control DNA. (D) Immunohistochemical staining of 5-hmC in IDH3B-mutated tumor. Nuclear 5-hmC was observed only in sustentacular and some stromal cells. The scale bar represents 50μm.

Legends of the Supplementary Figures

Calcium is a major regulator of cellular metabolism. Calcium controls mitochondrial respiration, and calcium signaling is used to meet cellular energetic demands through energy production in the organelle. Although it has been widely assumed that Ca2+-actions require its uptake by mitochondrial calcium uniporter (MCU), alternative pathways modulated by cytosolic Ca2+ have been recently proposed. Recent findings have indicated a role for cytosolic Ca2+ signals acting on mitochondrial NADH shuttles in the control of cellular metabolism in neurons using glucose as fuel. It has been demonstrated that AGC1/Aralar, the component of the malate/aspartate shuttle (MAS) regulated by cytosolic Ca2+, participates in the maintenance of basal respiration exerted through Ca2+-fluxes between ER and mitochondria, whereas mitochondrial Ca2+-uptake by MCU does not contribute. Aralar/MAS pathway, activated by small cytosolic Ca2+ signals, provides in fact substrates, redox equivalents and pyruvate, fueling respiration. Upon activation and increases in workload, neurons upregulate OxPhos, cytosolic pyruvate production and glycolysis, together with glucose uptake, in a Ca2+-dependent way, and part of this upregulation is via Ca2+ signaling. Both MCU and Aralar/MAS contribute to OxPhos upregulation, Aralar/MAS playing a major role, especially at small and submaximal workloads. Ca2+ activation of Aralar/MAS, by increasing cytosolic NAD+/NADH provides Ca2+-dependent increases in glycolysis and cytosolic pyruvate production priming respiration as a feed-forward mechanism in response to workload. Thus, except for glucose uptake, these processes are dependent on Aralar/MAS, whereas MCU is the relevant target for Ca2+ signaling when MAS is bypassed, by using pyruvate or β-hydroxybutyrate as substrates.

(A) GOT2 western blot of HeLa cells stably silenced for GOT2 expression by shRNA transfection compared to non-silenced scrambled (Scr) control cells. β-actin was used as a loading control. (B) Number of GOT2 KD HeLa cells after transfection with empty vector (EV), GOT2- WT cDNA, and GOT2- c.357A>T. Cells were seeded into 12-well plates and incubated for various times, as indicated. The counts are reported as means (n=3). A t-test was applied to test for differences. n.s.: not significant.

The deficiency of CITRIN, the liver mitochondrial aspartate–glutamate carrier (AGC), is the cause of four human clinical phenotypes, neonatal intrahepatic cholestasis caused by CITRIN deficiency (NICCD), silent period, failure to thrive and dyslipidemia caused by CITRIN deficiency (FTTDCD), and citrullinemia type II (CTLN2). Clinical symptoms can be traced back to disruption of the malate-aspartate shuttle due to the lack of citrin. A potential therapy for this condition is the expression of aralar, the AGC present in brain, to replace citrin. To explore this possibility we have first verified that the NADH/NAD+ ratio increases in hepatocytes from citrin(−/−) mice, and then found that exogenous aralar expression reversed the increase in NADH/NAD+ observed in these cells. Liver mitochondria from citrin (−/−) mice expressing liver specific transgenic aralar had a small (~ 4–6 nmoles x mg prot−1 x min−1) but consistent increase in malate aspartate shuttle (MAS) activity over that of citrin(−/−) mice. These results support the functional replacement between AGCs in the liver. To explore the significance of AGC replacement in human therapy we studied the relative levels of citrin and aralar in mouse and human liver through absolute quantification proteomics. We report that mouse liver has relatively high aralar levels (citrin/aralar molar ratio of 7.8), whereas human liver is virtually devoid of aralar (CITRIN/ARALAR ratio of 397). This large difference in endogenous aralar levels partly explains the high residual MAS activity in liver of citrin(−/−) mice and why they fail to recapitulate the human disease, but supports the benefit of increasing aralar expression to improve the redox balance capacity of human liver, as an effective therapy for CITRIN deficiency.

Genes included in the targeted next-generation sequencing panel

DNA methylation (M-values) of the CpG island probes located within six PCC/PGL susceptibility genes encoding Krebs cycle enzymes (SDHA, SDHAF2, SDHB, SDHD, FH, and MDH2) in the 11 analyzed tumors, compared to in vitro methylated DNA (IVD).

(A) Aspartate/glutamate ratios assessed by LC-MS in GOT2 KD Hela cells transfected with empty vector (EV), GOT2 wild-type (WT) cDNA, GOT2- c.223T>G cDNA, and GOT2- c.357A>T cDNA. The ratios were reported as means (n=3). Error bars represent standard deviations. A t-test was applied to test for differences between GOT WT and GOT2- c.223T>G and - c.357A>T transfected cells. n.s.: not significant. (B) Succinate/fumarate ratios assessed by LC-MS in OGDHL KD Hela cells transfected with EV, OGDHL WT cDNA, and OGDHL- c.750G>T cDNA. The ratios were reported as means (n=3). Error bars represent standard deviations. A t-test was applied to test for differences. n.s.: not significant

AGC1/Aralar/Slc25a12 is the mitochondrial carrier of aspartate-glutamate, the regulatory component of the NADH malate-aspartate shuttle (MAS) that transfers cytosolic redox power to neuronal mitochondria. The deficiency in AGC1/Aralar leads to the human rare disease named "early infantile epileptic encephalopathy 39" (EIEE 39, OMIM # 612949) characterized by epilepsy, hypotonia, arrested psychomotor neurodevelopment, hypo myelination and a drastic drop in brain aspartate (Asp) and N-acetylaspartate (NAA). Current evidence suggest that neurons are the main brain cell type expressing Aralar. However, paradoxically, glial functions such as myelin and Glutamine (Gln) synthesis are markedly impaired in AGC1 deficiency. Herein, we discuss the role of the AGC1/Aralar-MAS pathway in neuronal functions such as Asp and NAA synthesis, lactate use, respiration on glucose, glutamate (Glu) oxidation and other neurometabolic aspects. The possible mechanism triggering the pathophysiological findings in AGC1 deficiency, such as epilepsy and postnatal hypomyelination observed in humans and mice, are also included. Many of these mechanisms arise from findings in the aralar-KO mice model that extensively recapitulate the human disease including the astroglial failure to synthesize Gln and the dopamine (DA) mishandling in the nigrostriatal system. Epilepsy and DA mishandling are a direct consequence of the metabolic defect in neurons due to AGC1/Aralar deficiency. However, the deficits in myelin and Gln synthesis may be a consequence of neuronal affectation or a direct effect of AGC1/Aralar deficiency in glial cells. Further research is needed to clarify this question and delineate the transcellular metabolic fluxes that control brain functions. Finally, we discuss therapeutic approaches successfully used in AGC1-deficient patients and mice.

Aralar/AGC1/Slc25a12, the mitochondrial aspartate-glutamate carrier expressed in neurons, is the regulatory component of the NADH malate-aspartate shuttle. AGC1 deficiency is a neuropediatric rare disease (OMIM #612949, also named early infantile epileptic encephalopathy 39) characterized by hypomyelination, hypotonia, developmental arrest and epilepsy. In mice, the global aralar knockout shows hyperreactivity, growth retardation, motor discoordination, seizures and hypomyelination; thus, recapitulating the pathology of the human disease. Aralar brain expression is mainly restricted to neurons, while glial expression of Aralar has been demonstrated to be scarce. However, Aralar deficiency manifests alteration in fundamental glial functions such as glutamine and myelination synthesis by astrocytes and oligodendrocytes respectively. In order to dissect the role of neuronal Aralar in the disease mechanism of AGC1 deficiency, we have ablated the expression of Aralar in mature (from PND30 onward) excitatory cortical and hippocampal neurons using the CaMKIIα-driven Cre recombinase expression in Aralarlox/lox mice. Here, we present the characterization of the mature neuron-specific aralar knock out mice. We demonstrated that the excision of exon 3 aralar starts from PND30 and continues to occur during mouse adulthood. Consequently, Aralar protein levels gradually decay across the 2nd to 6th month up to a 70- 75% decrease in the cortex and a 50% decrease in the hippocampus. The drop in Aralar levels drive an alteration in cortical metabolites causing a 25% and 50% decrease in aspartate and serine levels, the metabolites most affected in the brain of the global aralar-KO mice. This partial decreases in ARALAR and metabolites caused by cortical CaMKIIα-driven aralar ablation are not accompanied by growth or behavioral alterations in a battery of motor, memory and cognitive tests. The results suggest that the absence of Aralar in CaMKIIα expressing neurons (cortical and hippocampal mature excitatory neurons), does not mimic the alterations caused by global deficiency of Aralar/AGC1. However, since the levels of cortical ARALAR in this model resemble those of healthy aralar+/- mice, further experiments are being carried out to explore the origin (or location) of the ARALAR protein remnants and the possible metabolic compensation by other neurons and/or cell types in these mice. Without ruling out either that the ARALAR-MAS pathway could play a more critical role during neurodevelopment than adulthood in brain physiology and metabolism.

Calcium is an important second messenger regulating a bioenergetic response to the workloads triggered by neuronal activation. In embryonic mouse cortical neurons using glucose as only fuel, activation by NMDA elicits a strong workload (ATP demand)-dependent on Na + and Ca 2+ entry, and stimulates glucose uptake, glycolysis, pyruvate and lactate production, and oxidative phosphorylation (OXPHOS) in a Ca 2+ -dependent way. We find that Ca 2+ upregulation of glycolysis, pyruvate levels, and respiration, but not glucose uptake, all depend on Aralar/AGC1/Slc25a12, the mitochondrial aspartate-glutamate carrier, component of the malate-aspartate shuttle (MAS). MAS activation increases glycolysis, pyruvate production, and respiration, a process inhibited in the presence of BAPTA-AM, suggesting that the Ca 2+ binding motifs in Aralar may be involved in the activation. Mitochondrial calcium uniporter (MCU) silencing had no effect, indicating that none of these processes required MCU-dependent mitochondrial Ca 2+ uptake. The neuronal respiratory response to carbachol was also dependent on Aralar, but not on MCU. We find that mouse cortical neurons are endowed with a constitutive ER-to-mitochondria Ca 2+ flow maintaining basal cell bioenergetics in which ryanodine receptors, RyR2, rather than InsP 3 R, are responsible for Ca 2+ release, and in which MCU does not participate. The results reveal that, in neurons using glucose, MCU does not participate in OXPHOS regulation under basal or stimulated conditions, while Aralar-MAS appears as the major Ca 2+ -dependent pathway tuning simultaneously glycolysis and OXPHOS to neuronal activation. SIGNIFICANCE STATEMENT Neuronal activation increases cell workload to restore ion gradients altered by activation. Ca 2+ is involved in matching increased workload with ATP production, but the mechanisms are still unknown. We find that glycolysis, pyruvate production, and neuronal respiration are stimulated on neuronal activation in a Ca 2+ -dependent way, independently of effects of Ca 2+ as workload inducer. Mitochondrial calcium uniporter (MCU) does not play a relevant role in Ca 2+ stimulated pyruvate production and oxygen consumption as both are unchanged in MCU silenced neurons. However, Ca 2+ stimulation is blunt in the absence of Aralar, a Ca 2+ -binding mitochondrial carrier component of Malate-Aspartate Shuttle (MAS). The results suggest that Ca 2+ -regulated Aralar-MAS activation upregulates glycolysis and pyruvate production, which fuels mitochondrial respiration, through regulation of cytosolic NAD + /NADH ratio.

Introduction: The regulation of adenine nucleotide levels in the mitochondria and the cytosol is essential for cell metabolism. To sustain mitochondrial respiration, ATP generated by ATP synthase must be exported to the cytosol, whereas mitochondria must be refilled with ADP and Pi. Adenine Nucleotide Translocases (ANTs), which catalyze the electrogenic ATP4-/ADP3- exchange, have been proposed to work together with ATP synthase and the mitochondrial phosphate carrier to support mitochondrial oxidative phosphorylation by forming the so-called ATP synthasome. Nevertheless, disruption of the main ANT isoform or pharmacological inhibition of ANTs in cancer cells does not decrease oxidative phosphorylation. Furthermore, the import of cytosolic ATP to the mitochondria when oxidative phosphorylation is inhibited (required, for example, for the maintenance of the ΔΨm) is neither mediated by ANTs in cancer cells. Here, we study the possibility that calcium-regulated mitochondrial ATP-Mg2+/Pi or ADP/Pi carriers, (also called SCaMCs), might participate in the mitochondrial transport of adenine nucleotides in cancer cells. Material and methods: To study the function of SCaMC-1, the main isoform in cancer cells, we have generated SCaMC-1 KO HeLa cells using CRISPR-Cas9 genome editing. These cells have been characterized in terms of OCR (with Seahorse XF24), mitochondrial membrane potential (with TMRM) and mitochondrial ATP levels (with mitoGoATeam genetic probe) in basal conditions and in the presence of ETC inhibitors. Results and conclusions: We show that SCaMC-1, the isoform that is abundant in tumor cells, mediates ATP import to the mitochondria after histamine stimulation of HeLa cells, probably to buffer calcium entry to the mitochondria. However, SCaMC-1 does not participate in the transport of cytosolic ATP for the maintenance of ΔΨm after OXPHOS inhibition, neither decreases mitochondrial respiration. Future studies based on the deletion of the other SCaMCs isoforms (SCaMC-2, SCaMC-3, SCaMC-3 like) together with simultaneous deletion of ANTs and SCaMCs will be required to shed light on the control of adenine nucleotides transport between the mitochondria and the cytosol in cancer cells.