bOHB protective pathways in Aralar-Ko neurons and brain: An alternative to ketogenic diet

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 characterized by hypomyelination, hypotonia, developmental arrest, and epilepsy. We have investigated whether b-hydroxybutyrate (bOHB), the main ketone body (KB) produced in ketogenic diet (KD), is neuroprotective in aralar-knock-out (KO) neurons and mice. We report that bOHB efficiently recovers aralar-KO neurons from deficits in basal-stimulated and glutamate-stimulated respiration, effects requiring bOHB entry into the neuron, and protects from glutamate excitotoxicity. Aralar-deficient mice were fed a KD to investigate its therapeutic potential early in development, but this approach was unfeasible. Therefore, aralar-KO pups were treated without distinction of gender with daily intraperitoneal injections of bOHB during 5d. This treatment resulted in a recovery of striatal markers of the dopaminergic system including dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC)/DA ratio, and vesicular monoamine transporter 2 (VMAT2) protein. Regarding postnatal myelination, myelin basic protein (MBP) and myelin-associated glycoprotein (MAG) myelin proteins were markedly increased in the cortices of bOHB-treated aralar-KO mice. Although brain Asp and NAA levels did not change by bOHB administration, a 4-d bOHB treatment to aralar-KO, but not to control, neurons led to a substantial increase in Asp (3-fold) and NAA (4-fold) levels. These results suggest that the lack of increase in brain Asp and NAA is possibly because of its active utilization by the aralar-KO brain and the likely involvement of neuronal NAA in postnatal myelination in these mice. The effectiveness of bOHB as a therapeutic treatment in AGC1 deficiency deserves further investigationThis work was supported by Ministerio de Economía Grants SAF2014-56929R (to J.S. and B.P.) and SAF2017-82560R (AEI/FEDER, UE; to B.P.); the Centro de Investigación Biomédica en Red de Enfermedades Raras, an initiative of the Instituto de Salud Carlos III (ISCIII); a grant from the Fundación Ramon Areces (J.S.); the Irycis Chromatographic Services and Nervous System Markers Unit, UCS (2018/0135; to M.J.C.); and an institutional grant from the Fundación Ramon Areces to the Centro de Biología Molecular Severo Ochoa. I.P.-L. is the recipient of Contrato Predoctoral de Formación de Personal Investigador (FPI MINECO). We thank Dr. Antonio S. Herranz for his inputs as an expert in amino acid analysis by HPLC-UV, Dr. Araceli del Arco for critical reading of the manuscript, and Isabel Manso and Barbara Sesé for technical support. All experiments were conducted in compliance with the ARRIVE guideline

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 β-hydroxybutyrate as substrate

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.This work was supported by Spanish Ministry of Science, Innovation and Universities SAF2014-56929R to J.S. and B.P.; SAF2017-82560-R to A.d.A. and B.P.; Fundación Ramón Areces to J.S.; and Fundación Ramón Areces institutional grant to Centro de Biología Molecular Severo Ochoa (CBMSO). I.P.-L. and L.G.-M. received predoctoral fellowships from MINECO. P.G.-S. received a postdoctoral research contract from Comunidad de Madri

Treatment with tocilizumab or corticosteroids for COVID-19 patients with hyperinflammatory state: a multicentre cohort study (SAM-COVID-19)

Objectives: The objective of this study was to estimate the association between tocilizumab or corticosteroids and the risk of intubation or death in patients with coronavirus disease 19 (COVID-19) with a hyperinflammatory state according to clinical and laboratory parameters. Methods: A cohort study was performed in 60 Spanish hospitals including 778 patients with COVID-19 and clinical and laboratory data indicative of a hyperinflammatory state. Treatment was mainly with tocilizumab, an intermediate-high dose of corticosteroids (IHDC), a pulse dose of corticosteroids (PDC), combination therapy, or no treatment. Primary outcome was intubation or death; follow-up was 21 days. Propensity score-adjusted estimations using Cox regression (logistic regression if needed) were calculated. Propensity scores were used as confounders, matching variables and for the inverse probability of treatment weights (IPTWs). Results: In all, 88, 117, 78 and 151 patients treated with tocilizumab, IHDC, PDC, and combination therapy, respectively, were compared with 344 untreated patients. The primary endpoint occurred in 10 (11.4%), 27 (23.1%), 12 (15.4%), 40 (25.6%) and 69 (21.1%), respectively. The IPTW-based hazard ratios (odds ratio for combination therapy) for the primary endpoint were 0.32 (95%CI 0.22-0.47; p < 0.001) for tocilizumab, 0.82 (0.71-1.30; p 0.82) for IHDC, 0.61 (0.43-0.86; p 0.006) for PDC, and 1.17 (0.86-1.58; p 0.30) for combination therapy. Other applications of the propensity score provided similar results, but were not significant for PDC. Tocilizumab was also associated with lower hazard of death alone in IPTW analysis (0.07; 0.02-0.17; p < 0.001). Conclusions: Tocilizumab might be useful in COVID-19 patients with a hyperinflammatory state and should be prioritized for randomized trials in this situatio

CARB-ES-19 Multicenter Study of Carbapenemase-Producing Klebsiella pneumoniae and Escherichia coli From All Spanish Provinces Reveals Interregional Spread of High-Risk Clones Such as ST307/OXA-48 and ST512/KPC-3

ObjectivesCARB-ES-19 is a comprehensive, multicenter, nationwide study integrating whole-genome sequencing (WGS) in the surveillance of carbapenemase-producing K. pneumoniae (CP-Kpn) and E. coli (CP-Eco) to determine their incidence, geographical distribution, phylogeny, and resistance mechanisms in Spain.MethodsIn total, 71 hospitals, representing all 50 Spanish provinces, collected the first 10 isolates per hospital (February to May 2019); CPE isolates were first identified according to EUCAST (meropenem MIC &gt; 0.12 mg/L with immunochromatography, colorimetric tests, carbapenem inactivation, or carbapenem hydrolysis with MALDI-TOF). Prevalence and incidence were calculated according to population denominators. Antibiotic susceptibility testing was performed using the microdilution method (EUCAST). All 403 isolates collected were sequenced for high-resolution single-nucleotide polymorphism (SNP) typing, core genome multilocus sequence typing (cgMLST), and resistome analysis.ResultsIn total, 377 (93.5%) CP-Kpn and 26 (6.5%) CP-Eco isolates were collected from 62 (87.3%) hospitals in 46 (92%) provinces. CP-Kpn was more prevalent in the blood (5.8%, 50/853) than in the urine (1.4%, 201/14,464). The cumulative incidence for both CP-Kpn and CP-Eco was 0.05 per 100 admitted patients. The main carbapenemase genes identified in CP-Kpn were blaOXA–48 (263/377), blaKPC–3 (62/377), blaVIM–1 (28/377), and blaNDM–1 (12/377). All isolates were susceptible to at least two antibiotics. Interregional dissemination of eight high-risk CP-Kpn clones was detected, mainly ST307/OXA-48 (16.4%), ST11/OXA-48 (16.4%), and ST512-ST258/KPC (13.8%). ST512/KPC and ST15/OXA-48 were the most frequent bacteremia-causative clones. The average number of acquired resistance genes was higher in CP-Kpn (7.9) than in CP-Eco (5.5).ConclusionThis study serves as a first step toward WGS integration in the surveillance of carbapenemase-producing Enterobacterales in Spain. We detected important epidemiological changes, including increased CP-Kpn and CP-Eco prevalence and incidence compared to previous studies, wide interregional dissemination, and increased dissemination of high-risk clones, such as ST307/OXA-48 and ST512/KPC-3

Adelante / Endavant

Séptimo desafío por la erradicación de la violencia contra las mujeres del Institut Universitari d’Estudis Feministes i de Gènere "Purificación Escribano" de la Universitat Jaume

The mitochondrial aspartate-glutamate carrier Aralar/AGC1 controls neuronal respiration and (beta)-Hydroxybutyrate rescues brain defects caused by AGC1 deficiency

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 30-06-2020Esta tesis tiene embargado el acceso al texto completo hasta el 30-12-2021La regulación de la respiración neuronal se ha atribuido tradicionalmente al transportador de Ca2+ mitocondrial (MCU), ya que el Ca2+ mitocondrial (Ca2+-mit) activa las deshidrogenasas del ciclo de Krebs. Sin embargo, los transportadores mitocondriales regulados por Ca2+ (CaMCs) tienen un papel muy relevante regulando la respiración en respuesta a estímulos que producen pequeñas señales de Ca2+ citosólicas. Aralar/AGC1/Slc25a12 es el transportador de aspartato-glutamato neuronal, y el punto de regulación de la lanzadera de NADH malato-aspartato (MAS). En este estudio se demuestra que la activación por Ca2+ de Aralar, y no MCU/Ca2+-mit, es esencial para la estimulación de la respiración en respuesta a la estimulación por NMDA. La activación por Ca2+ de Aralar-MAS en neuronas que usan glucosa es necesaria para estimular la síntesis de piruvato, activando su transporte a la mitocondria y la respiración y la glucolisis, algo que no ocurre en ausencia de Ca2+. Además, se demuestra que las neuronas aumentan la captura de glucosa de forma Ca2+-dependiente e independiente de Aralar tras la estimulación por NMDA. Sorprendentemente, el silenciamiento de Mcu no sólo no disminuye la respiración estimulada por NMDA, sino que tiene un efecto potenciador en parte atribuible a los efectos tóxicos del Ca2+ mitocondrial. La deficiencia en Aralar/AGC1 causa una enfermedad rara del neurodesarrollo caracterizada por retraso en el crecimiento, epilepsia e hipomielinización. Los ratones aralar-KO tienen una corta esperanza de vida y alteraciones motoras asociadas a una grave afección en el estriado. En humanos, la dieta cetogénica (KD) revierte la epilepsia y mejora parcialmente la hipomielinización. Este estudio revela que el principal cuerpo cetónico sintetizado tras el tratamiento con KD, β-Hidroxibutirato (βOHB) es beneficioso en la deficiencia en Aralar. βOHB recupera la respiración mitocondrial en glucosa en neuronas aralar-KO y las protege de la excitotoxicidad inducida por glutamato. In vivo, βOHB recupera parcialmente marcadores dopaminérgicos estriatales, como el contenido en dopamina, el transportador vesicular VMAT2 y el mediador postsináptico DARPP-32. βOHB también aumenta la síntesis de proteínas de mielina. Aunque no se recuperan los niveles de aspartato y NAA cerebrales, βOHB los aumenta en neuronas aralar-KO, sugiriendo que su uso está aumentado en el ratón, reforzando el papel de NAA como precursor en la síntesis de mielina en oligodendrocitos. A pesar de las limitaciones impuestas por el fenotipo tan severo de los ratones aralar-KO, este estudio demuestra que el βOHB es un sustrato alternativo efectivo e independiente de aralar que revierte las principales alteraciones de esta enfermedad. Un estudio más profundo de sus funciones neuroprotectoras, también a través de vías de señalización, sería muy relevante para identificar terapias sustitutivas de la dieta cetogénica en humanosRegulation of neuronal respiration has been traditionally attributed to mitochondrial Ca2+ uniporter (MCU), as it allows Ca2+ activation of matrix dehydrogenases and TCA cycle. However, Ca2+-regulated mitochondrial carriers (CaMCs) have revealed an important role in stimulating neuronal respiration in response to agents evoking of small cytosolic Ca2+ signals. Aralar/AGC1/Slc25a12 is the aspartate-glutamate carrier expressed in neurons, and the regulatory step in the malate-aspartate NADH shuttle (MAS). This study demonstrates that Ca2+ activation of Aralar, rather than MCU/mitochondrial Ca2+ (Ca2+- mit), controls the upregulation of respiration in response to NMDA in neurons using glucose. Ca2+ activation of Aralar-MAS is necessary to increase endogenous pyruvate synthesis, activating its transport into mitochondria and upregulating respiration and glycolysis, neither of which occurring in the absence of Ca2+. In addition, this study shows that following NMDA stimulation, neurons upregulate glucose uptake in a Ca2+-dependent and Aralar-independent way. Surprisingly, Mcu silencing not only fails to decrease NMDAstimulated respiration but in fact has a potentiator effect, probably due to suppression of the toxic effect of mitochondrial Ca2+. Aralar/AGC1 deficiency causes a neurodevelopmental rare disease characterized by growth retardation, epilepsy and secondary postnatal hypomyelination. Aralar-KO mice have reduced life expectancy and altered motor coordination with severe affectation of striatum. In human patients, ketogenic diet (KD) alleviates epilepsy and partially improves myelination. This study reveals that the main ketone body synthesized during treatment with KD, β-hydroxybutyrate (βOHB), reports beneficial effects in Aralar/AGC1 deficiency. βOHB rescues impaired mitochondrial respiration on glucose in aralar-KO primary cortical neurons, and also protects them from glutamate-induced excitotoxicity. In vivo, βOHB administration partially rescues striatal dopaminergic system in aralar-KO mice, increasing the levels of neuronal markers as dopamine, VMAT2 and DARPP-32. βOHB administration also increases the synthesis of myelin proteins, but not aspartate and NAA levels. However βOHB supply to aralar-KO neurons resulted in increased synthesis of aspartate and NAA, suggesting their increased use in the aralar-KO brain and reinforcing neuronal NAA as an essential precursor for myelin lipid synthesis in oligodendrocytes. In spite of the limitations imposed by the strong phenotype of aralar- KO mice, this study demonstrates that βOHB is an effective alternative substrate that bypasses Aralar rescuing the main hallmarks of Aralar/AGC1 deficiency. A deeper study of the neuroprotective roles of βOHB, also acting through signaling pathways, would be very interesting with the aim of finding an effective therapy that substitutes KD in human patient

L-lactate-mediated neuroprotection against glutamate- induced excitotoxicity requires ARALAR/AGC1

ARALAR/AGC1/Slc25a12, the aspartate-glutamate carrier from brain mitochondria, is the regulatory step in the malate-aspartate NADH shuttle, MAS. MAS is used to oxidize cytosolic NADH in mitochondria, a process required to maintain oxidative glucose utilization. The role of ARALAR was analyzed in two paradigms of glutamate-induced excitotoxicity in cortical neurons: glucose deprivation and acute glutamate stimulation. ARALAR deficiency did not aggravate glutamate-induced neuronal death in vitro, although glutamate-stimulated respiration was impaired. In contrast, the presence of L-lactate as an additional source protected against glutamate-induced neuronal death in control, but not ARALAR-deficient neurons. L-Lactate supplementation increased glutamate-stimulated respiration partially prevented the decrease in the cytosolic ATP/ADP ratio induced by glutamate and substantially diminished mitochondrial accumulation of 8-oxoguanosine, a marker of reactive oxygen species production, only in the presence, but not the absence, of ARALAR. In addition, L-lactate potentiated glutamate-induced increase in cytosolic Ca, in a way independent of the presence of ARALAR. Interestingly, in vivo, the loss of half-a-dose of ARALAR inaralar mice enhanced kainic acid-induced seizures and neuronal damage with respect to control animals, in a model of excitotoxicity in which increased L-lactate levels and L-lactate consumption have been previously proven. These results suggest that, in vivo, an inefficient operation of the shuttle in the aralar hemizygous mice prevents the protective role of L-lactate on glutamate excitotoxiciy and that the entry and oxidation of L-lactate through ARALAR-MAS pathway is required for its neuroprotective function. SIGNIFICANCE STATEMENT Lactate now stands as a metabolite necessary for multiple functions in the brain and is an alternative energy source during excitotoxic brain injury. Here we find that the absence of a functional malate-aspartate NADH shuttle caused by aralar/AGC1 disruption causes a block in lactate utilization by neurons, which prevents the protective role of lactate on excitotoxicity, but not glutamate excitotoxicity itself. Thus, failure to use lactate is detrimental and is possibly responsible for the exacerbated in vivo excitotoxicity in aralar mice.Peer Reviewe

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 Ca-actions require its uptake by mitochondrial calcium uniporter (MCU), alternative pathways modulated by cytosolic Ca have been recently proposed. Recent findings have indicated a role for cytosolic Ca 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 Ca, participates in the maintenance of basal respiration exerted through Ca-fluxes between ER and mitochondria, whereas mitochondrial Ca-uptake by MCU does not contribute. Aralar/MAS pathway, activated by small cytosolic Ca 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 Ca-dependent way, and part of this upregulation is via Ca signaling. Both MCU and Aralar/MAS contribute to OxPhos upregulation, Aralar/MAS playing a major role, especially at small and submaximal workloads. Ca activation of Aralar/MAS, by increasing cytosolic NAD/NADH provides Ca-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 Ca signaling when MAS is bypassed, by using pyruvate or β-hydroxybutyrate as substrates.This work has been funded by grants from the Spanish Ministry of Science, Innovation and Universities SAF2017-82560-R and PID2020-114499RB-I00 (to AdelA and BP), and by an institutional grant from the Fundación Ramón Areces to the Centro de Biología Molecular Severo Ochoa (CBMSO)