Regulation of neuronal energy metabolism by calcium: Role of MCU and Aralar/malate-aspartate shuttle.

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 ß-hydroxybutyrate as substrates.

Exogenous aralar/slc25a12 can replace citrin/slc25a13 as malate aspartate shuttle component in liver.

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.

A Ca2+-Dependent Mechanism Boosting Glycolysis and OXPHOS by Activating Aralar-Malate-Aspartate Shuttle, upon Neuronal Stimulation.

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 Ca2+ entry, and stimulates glucose uptake, glycolysis, pyruvate and lactate production, and oxidative phosphorylation (OXPHOS) in a Ca2+-dependent way. We find that Ca2+ 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 Ca2+ 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 Ca2+ 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 Ca2+ flow maintaining basal cell bioenergetics in which ryanodine receptors, RyR2, rather than InsP3R, are responsible for Ca2+ 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 Ca2+-dependent pathway tuning simultaneously glycolysis and OXPHOS to neuronal activation.SIGNIFICANCE STATEMENT Neuronal activation increases cell workload to restore ion gradients altered by activation. Ca2+ 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 Ca2+-dependent way, independently of effects of Ca2+ as workload inducer. Mitochondrial calcium uniporter (MCU) does not play a relevant role in Ca2+ stimulated pyruvate production and oxygen consumption as both are unchanged in MCU silenced neurons. However, Ca2+ stimulation is blunt in the absence of Aralar, a Ca2+-binding mitochondrial carrier component of Malate-Aspartate Shuttle (MAS). The results suggest that Ca2+-regulated Aralar-MAS activation upregulates glycolysis and pyruvate production, which fuels mitochondrial respiration, through regulation of cytosolic NAD+/NADH ratio.

Ca2+-regulated mitochondrial carriers of ATP-Mg2+/Pi: Evolutionary insights in protozoans.

In addition to its uptake across the Ca2+ uniporter, intracellular calcium signals can stimulate mitochondrial metabolism activating metabolite exchangers of the inner mitochondrial membrane belonging to the mitochondrial carrier family (SLC25). One of these Ca2+-regulated mitochondrial carriers (CaMCs) are the reversible ATP-Mg2+/Pi transporters, or SCaMCs, required for maintaining optimal adenine nucleotide (AdN) levels in the mitochondrial matrix representing an alternative transporter to the ADP/ATP translocases (AAC). This CaMC has a distinctive Calmodulin-like (CaM-like) domain fused to the carrier domain that makes its transport activity strictly dependent on cytosolic Ca2+ signals. Here we investigate about its origin analysing its distribution and features in unicellular eukaryotes. Unexpectedly, we find two types of ATP-Mg2+/Pi carriers, the canonical ones and shortened variants lacking the CaM-like domain. Phylogenetic analysis shows that both SCaMC variants have a common origin, unrelated to AACs, suggesting in turn that recurrent losses of the regulatory module have occurred in the different phyla. They are excluding variants that show a more limited distribution and less conservation than AACs. Interestingly, these truncated variants of SCaMC are found almost exclusively in parasitic protists, such as apicomplexans, kinetoplastides or animal-patogenic oomycetes, and in green algae, suggesting that its lost could be related to certain life-styles. In addition, we find an intricate structural diversity in these variants that may be associated with their pathogenicity. The consequences on SCaMC functions of these new SCaMC-b variants are discussed.

The Response to Stimulation in Neurons and Astrocytes.

The brain uses mainly glucose as fuel with an index of glucose to oxygen utilization close to 6, the maximal index if all glucose was completely oxidized. However, this high oxidative index, contrasts with the metabolic traits of the major cell types in the brain studied in culture, neurons and astrocytes, including the selective use of the malate-aspartate shuttle (MAS) in neurons and the glycerol-phosphate shuttle in astrocytes. Metabolic interactions among these cell types may partly explain the high oxidative index of the brain. In vivo, neuronal activation results in a decrease in the oxygen glucose index, which has been attributed to a stimulation of glycolysis and lactate production in astrocytes in response to glutamate uptake (astrocyte-neuron lactate shuttle, ANLS). Recent findings indicate that this is accompanied with a stimulation of pyruvate formation and astrocyte respiration, indicating that lactate formation is not the only astrocytic response to neuronal activation. ANLS proposes that neurons utilize lactate produced by neighboring astrocytes. Indeed, neurons can use lactate to support an increase in respiration with different workloads, and this depends on the Ca2+ activation of MAS. However, whether this activation operates in the brain, particularly at high stimulation conditions, remains to be established.