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Background: At present, the etiology and pathogenesis of Moyamoya disease (MMD) are not completely clear. Patients are usually diagnosed after cerebrovascular events. Therefore, it is of great clinical significance to explore the predictive factors of MMD. Objective: This study aimed to investigate the serum level of CoQ10B, the amount of endothelial progenitor cells (EPCs), and mitochondrial function of EPCs in MMD patients. Methods: Forty-one MMD patients and 20 healthy controls were recruited in this study. Patients with MMD were divided into two groups: Ischemic type (n=23) and hemorrhagic type (n=18). Blood samples were collected from the antecubital vein and analyzed by CoQ10B ELISA and flow cytometry. Measures of mitochondrial function of EPCs include oxygen consumption rate (OCR), mitochondrial membrane potential, Ca2+ concentration, adenosine triphosphatases activity and ROS level. Results: The serum CoQ10B level in MMD patients was significantly lower than that in healthy controls (p

La principal característica de los tumores malignos de crecimiento rápido es el metabolismo de tipo Warburg, que está directamente relacionado con una cantidad extremadamente alta de hexocinasa (HK) unida a los canales de aniones dependientes de voltaje (VDAC) en la membrana externa mitocondrial. Anteriormente, explicamos el metabolismo de tipo Warburg como resultado de la supresión eléctrica de las mitocondrias debido al cierre de VDAC libres que no están unidos a HK. Aquí, proponemos un posible nuevo mecanismo de actividad anticancerígena de vitamina C (ascorbato) en dosis altas estimado con un modelo computacional simplificado. De acuerdo con la hipótesis propuesta y el modelo, la oxidación del ascorbato en las mitocondrias conduce a la generación del potencial de membrana externa (PMO) negativo de signo opuesto al PMO positivo generado por los complejos VDAC-HK en las células cancerosas. la reapertura de los VDAC eléctricamente cerrados, reprogramando así el metabolismo energético de las células. Según la hipótesis, los mediadores redox, que aumentan la tasa de oxidación del ascorbato en las mitocondrias, deberían aumentar sinérgicamente los efectos anticancerígenos del ascorbato en dosis altas de acuerdo con los datos experimentales registrados en el literatura. El modelo muestra que incluso pequeños cambios en la sensibilidad al voltaje VDAC y/o la cantidad de complejos VDAC-HK, que se sabe que son causados ​​por varios factores fisiológicos, podrían influir fuertemente en el mecanismo mitocondrial propuesto de la actividad anticancerígena del ascorbato. The main feature of fast-growing malignant tumors is the Warburg-type metabolism, which is directly related to an extremely high quantity of hexokinase (HK) bound to the voltage-dependent anion channels (VDACs) in the mitochondrial outer membrane. Previously, we explained the Warburg type metabolism as a result of the electrical suppression of mitochondria due to the closure of free VDACs that are not bound to HK. Here, we propose a possible new mechanism of high-dose vitamin C (ascorbate) anticancer activity estimated with a simplified computational model. According to the proposed hypothesis and the model, ascorbate oxidation in mitochondria leads to the generation of the negative outer membrane potential (OMP) of opposite sign to the positive OMP generated by the VDAC-HK complexes in cancer cells. The model demonstrates that negative OMP generated by any mechanism, even of relatively low magnitudes, leads to the reopening of the electrically closed VDACs, thus reprogramming cell energy metabolism. According to the hypothesis, redox mediators, which increase the rate of ascorbate oxidation in mitochondria, should synergistically increase anticancer effects of high-dose ascorbate in accordance with experimental data recorded in the literature. The model shows that even small changes in the VDAC-voltage sensitivity and/or quantity of the VDAC-HK complexes, known to be caused by various physiological factors, might strongly influence the proposed mitochondrial mechanism of ascorbate anticancer activity

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de Lectura: 11-04-2022, Metabolic pathologies and their complications are one of the main causes of death in western high-income countries. Mitochondria are key controllers of metabolism and the skeletal muscle (Skm) structure. However, whether Skm mitochondrial dysfunction is a cause or consequence of metabolic disorders is still controversial. In order to unveil the role of mitochondria in the onset and progression of these pathologies, in this thesis we have developed and characterized the first mouse model of conditional and Skm-specific inhibition of the mitochondrial H+-ATP synthase by the overexpression of its constitutively active inhibitor, ATPIF1|H49K. 6-month-old mice presenting the inhibition of the mitochondrial oxidative phosphorylation (OXPHOS) show a higher body weight compared to control littermates, due to deep alterations in glucose, lipid and amino acid metabolism in both muscle and white adipose tissue. Upon mitochondrial inhibition, Skm increases lactate production, reduces fatty acid β-oxidation, and relies on branched-chain amino acids catabolism as major source of acetyl-CoA for the de novo lipid synthesis. Moreover, acetyl-CoA accumulates in muscle and feedbacks the mitochondrial dysfunction by increasing the production of reactive oxygen species. As a result, mice with restrained OXPHOS become more prone to develop insulin resistance when fed a high-fat diet. Furthermore, Skm mitochondrial dysfunction alters motor behaviour, an effect that increases with ageing. 18-month-old mice presenting a chronic inhibition of the H+-ATP synthase show deep alterations in Skm organization, developing a myopathy characterized by the presence of tubular aggregates (TA), honeycomb-like arrays of sarcoplasmic-reticulum (SR) tubules that disorganize the sarcomere structure. The long-term OXPHOS inhibition reprograms soleus oxidative fibers to glycolytic ones, and increases mitochondrial fission and mitophagy, presumably to recycle defective mitochondria. TA appeared as the result of SR tubulation aimed at increasing mitochondria/SR contact sites to buffer calcium overloads due to prolonged OXPHOS dysfunction. Hence, enhancing OXPHOS activity appears as a promising strategy for therapeutic intervention in metabolic and muscle disorders. With this aim, we screened 702 drugs and identified edaravone as a new mitochondrial antioxidant and enhancer. Remarkably, in vivo edaravone treatment restores a healthy phenotype both in 6-month and 18-month old mice. Altogether, these results suggest that Skm mitochondrial alterations contribute to the setting of metabolic and muscular disorders, proposing edaravone as a potential treatment for these pathologies, This work has been supported by a predoctoral fellowship from the Spanish Ministerio de Economía y Competitividad (BES-2017-079909). The project was funded by the Spanish Ministerio de Economía y Competitividad (Grants SAF2016-76028- R and PID2019- 104241RB-I00)

Fil: Balparda, Manuel. University of Bonn. Molecular Plant Physiology; Alemania Fil: Elsasse, Marlene. University of Bonn. Institute for Cellular and Molecular Botany. Molecular Evolution; Alemania Fil: Elsasse, Marlene. University of Munster. Institute of Plant Biology and Biotechnology. Plant Energy Biology; Alemania Fil: Badía, Mariana B. Heinrich Heine University. Institute of Developmental and Molecular Biology of Plants. Plant Molecular Physiology and Biotechnology. Cluster of Excellence on Plant Sciences; Alemania Fil: Badía, Mariana B. Pontificia Universidad Católica Argentina. Facultad de Química e Ingeniería del Rosario; Argentina Fil: Giese, Jonas. University of Munster. Institute of Plant Biology and Biotechnology. Plant Physiology; Alemania Fil: Bovdilova, Anastasiia. Heinrich Heine University. Institute of Developmental and Molecular Biology of Plants. Plant Molecular Physiology and Biotechnology. Cluster of Excellence on Plant Sciences; Alemania Fil: Hüdig, Meike. University of Bonn. Molecular Plant Physiology; Alemania Fil: Hüdig, Meike. Heinrich Heine University. Institute of Developmental and Molecular Biology of Plants. Plant Molecular Physiology and Biotechnology. Cluster of Excellence on Plant Sciences; Alemania Fil: Reinmuth, Lisa. University of Bonn. Institute for Cellular and Molecular Botany. Molecular Evolution; Alemania Fil: Eirich, Jürgen. University of Munster. Institute of Plant Biology and Biotechnology. Plant Physiology; Alemania Fil: Schwarzlander, Markus. University of Munster. Institute of Plant Biology and Biotechnology. Plant Energy Biology; Alemania Fil: Finkemeier, Iris. University of Munster. Institute of Plant Biology and Biotechnology. Plant Physiology; Alemania Fil: Schallenberg Rüdinger, Mareike. University of Bonn. Institute for Cellular and Molecular Botany. Molecular Evolution; Alemania Fil: Maurino, Verónica G. University of Bonn. Molecular Plant Physiology; Alemania Fil: Maurino, Verónica G. Heinrich Heine University. Institute of Developmental and Molecular Biology of Plants. Plant Molecular Physiology and Biotechnology. Cluster of Excellence on Plant Sciences; Alemania Summary: Plants need to rapidly and flexibly adjust their metabolism to changes of their immediate environment. Since this necessity results from the sessile lifestyle of land plants, key mechanisms for orchestrating central metabolic acclimation are likely to have evolved early. Here, we explore the role of lysine acetylation as a post-translational modification to directly modulate metabolic function. We generated a lysine acetylome of the moss Physcomitrium patens and identified 638 lysine acetylation sites, mostly found in mitochondrial and plastidial proteins. A comparison with available angiosperm data pinpointed lysine acetylation as a conserved regulatory strategy in land plants. Focusing on mitochondrial central metabolism, we functionally analyzed acetylation of mitochondrial malate dehydrogenase (mMDH), which acts as a hub of plant metabolic flexibility. In P. patens mMDH1, we detected a single acetylated lysine located next to one of the four acetylation sites detected in Arabidopsis thaliana mMDH1. We assessed the kinetic behavior of recombinant A. thaliana and P. patens mMDH1 with site-specifically incorporated acetyl-lysines. Acetylation of A. thaliana mMDH1 at K169, K170, and K334 decreases its oxaloacetate reduction activity, while acetylation of P. patens mMDH1 at K172 increases this activity. We found modulation of the malate oxidation activity only in A. thaliana mMDH1, where acetylation of K334 strongly activated it. Comparative homology modeling of MDH proteins revealed that evolutionarily conserved lysines serve as hotspots of acetylation. Our combined analyses indicate lysine acetylation as a common strategy to fine-tune the activity of central metabolic enzymes with likely impact on plant acclimation capacity.

[eng] SLIMP (Seryl-tRNA Synthetase-Like Insect Mitochondrial Protein) was identified during the generation of a mitochondrial human disease fly model. SLIMP was described as a previously uncharacterized paralog of the Drosophila mitochondrial seryl-tRNA synthetase (SerRS2), which became an essential protein, universally distributed in insects, echinoderms, and molluscs. Notably, SLIMP constitutes an aaRS-like protein that can bind to tRNAs, but it has lost the aminoacylation activity (Guitart, Bernardo, Sagalés, et al., 2010). Interestingly, we recently reported that SLIMP plays an essential role in the mitochondria by simultaneously regulating the mitochondrial protein synthesis and mtDNA copy number through interacting with SerRS2 and LON, respectively (Picchioni, Antolin-Fontes, et al., 2019). On the other hand, two reported studies pointed to SLIMP as an essential cell cycle regulator (Ambrus et al., 2009; Liang et al., 2014). Previous experiments in our group showed a G2 accumulation phenotype in SLIMP-KD cells, which can be rescued by overexpressing SLIMP without the mitochondria-targeting signal. Additionally, transcriptional upregulation of a core set of E2F1-target genes was detected upon SLIMP depletion. All these data point to a cell cycle-related role of SLIMP, potentially carried out from outside the organelle. Interestingly, here we first experimentally characterized the SLIMP MSP and proved that it is essential for driving the protein into the organelle. Moreover, we discovered an extra-mitochondrial population of SLIMP potentially shuttling between the cytosol and the nucleus, presumably in charge of the cell cycle role of SLIMP. Furthermore, our results suggest a role of SLIMP repressing the transition from the G1 to S phase during the cell cycle progression in an E2F1-independent pathway. Finally, interestingly, we demonstrate that the CDK1-P levels are increased upon SLIMP depletion, suggesting an activation of the G2/M checkpoint. In balance, the data presented in this thesis reinforces the idea of SLIMP playing a non-canonical function outside the mitochondria regulating the cell cycle progression near the R point. Thus, SLIMP would represent a single molecule at the crossroad between the mitochondrial homeostasis and cell cycle regulation., [cat] SLIMP (Seryl-tRNA Synthetase-Like Insect Mitochondrial Protein) es va identificar com un paraleg prèviament desconegut de la seril-tRNA sintetasa mitocondrial de Drosòfila (SerRS2), durant la generació d’un model de malaltia mitocondrial humana. Segurament SLIMP és fruit d’una duplicació gènica a la base dels animals i s’ha convertit en una proteïna essencial i universalment distribuïda en artròpodes, mol·luscs i equinoderms. A més, SLIMP ha perdut l’activitat aminoaciladora, tot i que encara conserva la capacitat d’unir-se a ARNs de transferència (Guitart, Bernardo, Sagalés, et al., 2010). El nostre laboratori ha demostrat recentment que SLIMP desenvolupa una funció clau a la mitocòndria, regulant simultàniament la traducció gènica a l’orgànul i el número de copies de ADN mitocondrial, a traves de la interacció amb SerRS2 i LON respectivament (Picchioni, Antolin-Fontes, et al., 2019). D’altre banda, dos estudis publicats en els últims anys vinculen SLIMP amb la regulació del cicle cel·lular (Ambrus et al., 2009; Liang et al., 2014). En aquest sentit, experiments prèviament realitzats al grup demostren que quan els nivells d’SLIMP baixen, les cèl·lules s’acumulen en la fase G2 del cicle. Curiosament, aquest fenotip es pot revertir sobre-expressant un forma truncada d’SLIIMP que no pot entrar a la mitocòndria. A més, en aquestes condicions, també s’observa una pujada a nivell transcripcional d’alguns gens regulats per E2F1. Conjuntament aquestes dades apunten a una possible funció d’SLIMP relacionada amb la regulació del cicle cel·lular potencialment portada a terme des de fora de la mitocòndria. En aquesta tesi, nosaltres, primer determinem la seqüència del pèptid senyal mitocondrial d’SLIMP experimentalment, i demostrem que es essencial i suficient per dirigir la proteïna cap a l’orgànul. A més, detectem una petita població d’SLIMP fora de la mitocòndria, present tant en el nucli com en el citoplasma. És important remarcar, que varis experiments presentats en aquesta tesi suggereixen que SLIMP estaria reprimint la progressió del cicle cel·lular a nivell de la transició G1-S, mitjançant un mecanisme alternatiu a E2F1. Finalment, demostrem que els nivells de CDK1-P son mes elevats en les cèl·lules que els hi manca SLIMP, cosa que indica una activació del punt de control de G2/M i que podria explicar la acumulació en G2 observada en aquestes cèl·lules. En conjunt les dades presentades en aquesta tesi, reforcen la idea que SLIMP desenvolupa una funció no canònica des de fora de la mitocòndria, reprimint l’entrada en la fase S del cicle cel·lular. Per tant, SLIMP representaria una molècula que vincula directament la homeòstasi mitocondrial i la regulació del cicle cel·lular.

Protein kinase Cε (PKCε) is highly expressed in nociceptor neurons and its activation has been reported as pro-nociceptive. Intriguingly, we previously demonstrated that activation of the mitochondrial PKCε substrate aldehyde dehydrogenase-2 (ALDH2) results in anti-nociceptive effects. ALDH2 is a major enzyme responsible for the clearance of 4-hydroxy-2-nonenal (4-HNE), an oxidative stress byproduct accumulated in inflammatory conditions and sufficient to induce pain hypersensitivity in rodents. Here we determined the contribution of the PKCε-ALDH2 axis during 4-HNE-induced mechanical hypersensitivity. Using knockout mice, we demonstrated that PKCε is essential for the nociception recovery during 4-HNE-induced hypersensitivity. We also found that ALDH2 deficient knockin mice display increased 4-HNE-induced nociceptive behavior. As proof of concept, the use of a selective peptide activator of PKCε (ΨεHSP90), which favors PKCε translocation to mitochondria and activation of PKCε-ALDH2 axis, was sufficient to block 4-HNE-induced hypersensitivity in WT, but not in ALDH2-deficient mice. Similarly, ΨεHSP90 administration prevented mechanical hypersensitivity induced by endogenous production of 4-HNE after carrageenan injection. These findings provide evidence that selective activation of mitochondrial PKCε-ALDH2 axis is important to mitigate aldehyde-mediated pain in rodents, suggesting that ΨεHSP90 and small molecules that mimic it may be a potential treatment for patients with pain.

[eng] With the most absolute purpose of understanding what the “simple” concept of “growth regulation” implies, the present thesis is a compilation of original works that aims to deepen in how the growth and metabolism of fish, and most concretely, its muscle, behaves in different metabolically challenging conditions. Hopefully, the knowledge generated in these works and integrated in this multi-perspective thesis, can contribute to somehow help to improve the Aquaculture sector in the Mediterranean Sea and abroad. This ambitious goal is structured in different objective: 1) To establish the regulation of the ghrelinergic system in bream through the fasting and feedback model: a) Characterization of the sequences of Preproghrelin, Ghsr1a and Ghsr1b in gilthead sea bream (Article 1). b) Characterizing Preproghrelin and its two receptors in acute and long-term feeding responses and its interaction with the Gh/Igf1 axis (Article 1). 2) To deepen in the beneficial effects of exercise on growth and its modulation through changes in the dietary protein/lipid ratio: a) Determining how this dietary modification alters different growth parameters, muscular proximal composition and reserves turnover (Article 3). b) Determining how the mitochondrial proteins in red and white muscle and in the liver respond to this dietary modification in order to infer changes in the nutritional efficiency and growth improvement (Article 3). 3) To make a comparative study about the response of the main musculoskeletal growth regulation systems under two different challenging experimental models, the fasting and refeeding and the sustained exercise under two different diets. a) Studying the gene expression of specific proteins of the ontogenetic and growth pathways (myogenesis and osteogenesis), as well as the degradation pathways (proteolysis and osteoclastogenesis) in white muscle and bone (Articles 2 and 4). b) Determining its effects on white muscle growth (myogenesis/proteolysis balance) and meat quality (Article 4). 4) To complete the characterization of the new Myomaker and Myomixer fusogens in rainbow trout and gilthead sea bream and its involvement in the myogenic process in vivo and in vitro and in the in vivo muscle regeneration. a) Characterization of the sequence of Myomixer in rainbow trout (Article 5) and Muomaker and Myomixer in gilthead sea bream (Article 6). b) To study the involvement of Myomaker (in gilthead sea bream) and Myomixer (in rainbow trout and gilthead sea bream) in the myogenic process in vivo and in vitro as well as during the muscle regeneration in vivo (Article 5 and 6).

Trypanosoma cruzi, the etiological agent of Chagas disease in humans, infects a wide variety of vertebrates. Trypomastigotes, the parasite infective forms, invade mammalian cells by a still poorly understood mechanism. Adhesion of tissue culture- derived trypomastigotes to the extracellular matrix (ECM) prior to cell invasion has been shown to be a relevant part of the process. Changes in phosphorylation, S-nitrosylation, and nitration levels of proteins, in the late phase of the interaction (2 h), leading to the reprogramming of both trypomastigotes metabolism and the DNA binding profile of modified histones, were described by our group. Here, the involvement of calcium signaling at a very early phase of parasite interaction with ECM is described. Increments in the intracellular calcium concentrations during trypomastigotes-ECM interaction depends on the Ca2+ uptake from the extracellular medium, since it is inhibited by EGTA or Nifedipine, an inhibitor of the L-type voltage gated Ca2+ channels and sphingosine-dependent plasma membrane Ca2+ channel, but not by Vanadate, an inhibitor of the plasma membrane Ca2+-ATPase. Furthermore, Nifedipine inhibits the invasion of host cells by tissue culture- derived trypomastigotes in a dose-dependent manner, reaching 95% inhibition at 100 µM Nifedipine. These data indicate the importance of both Ca2+ uptake from the medium and parasite-ECM interaction for host-cell invasion. Previous treatment of ECM with protease abolishes the Ca2+ uptake, further reinforcing the possibility that these events may be connected. The mitochondrion plays a relevant role in Ca2+ homeostasis in trypomastigotes during their interaction with ECM, as shown by the increment of the intracellular Ca2+ concentration in the presence of Antimycin A, in contrast to other calcium homeostasis disruptors, such as Cyclopiazonic acid for endoplasmic reticulum and Bafilomycin A for acidocalcisome. Total phosphatase activity in the parasite decreases in the presence of Nifedipine, EGTA, and Okadaic acid, implying a role of calcium in the phosphorylation level of proteins that are interacting with the ECM in tissue culture- derived trypomastigotes. In summary, we describe here the increment of Ca2+ at an early phase of the trypomastigotes interaction with ECM, implicating both nifedipine-sensitive Ca2+ channels in the influx of Ca2+ and the mitochondrion as the relevant organelle in Ca2+ homeostasis. The data unravel a complex sequence of events prior to host cell invasion itself.

Objective: We aimed to determine the effect of different low-temperature range interventions at different time-points in a rat model of pressure injury (PI) produced by Ischemia/Reperfusion (I/R) injury. Methods: Sprague-Dawley rats were randomly assigned to blank control, injury control, and temperature intervention groups. Rats in the injury control and temperature intervention groups (involving exposure to different temperature range at different time-points) were subjected to three cycles of I/R injury with 2-h ischemia and 0.5-h reperfusion to induce PI. Results: The muscle tissues exhibited degenerative changes after compression. Low temperature intervention of 16–18°C in the ischemia period resulted in the lowest degree of tissue damage and significantly decreased levels of Bcl-2-associated X protein (Bax), caspase-9, and caspase-3. Moreover, it resulted in the highest expression level of B-cell lymphoma 2 (Bcl-2) and lowest expression levels of Bax, caspase-9, and caspase-3 in muscle tissues among all intervention groups. Conclusion: Low-temperature intervention at 16–18°C during the ischemia period showed optimal effects on the expressions of apoptotic factors during the development of PI with I/R-induced tissue damage. RESUMO Objetivo: Nosso objetivo foi determinar o efeito de diferentes intervenções na faixa de baixa temperatura em diferentes pontos do tempo em um modelo de lesão por pressão (IP) de rato produzida por lesão de isquemia/reperfusão (I/R). Métodos: Ratos Sprague-Dawley foram aleatoriamente designados para grupos de controle em branco, controle de lesão e intervenção por temperatura. Ratos nos grupos de controle de lesão e intervenção de temperatura (envolvendo exposição a diferentes faixas de temperatura em diferentes momentos) foram submetidos a três ciclos de lesão de I/R com isquemia de 2 h e reperfusão de 0,5 h para induzir IP. Resultados: Os tecidos musculares exibiram alterações degenerativas após a compressão. A intervenção em baixa temperatura de 16–18°C no período de isquemia resultou no menor grau de dano ao tecido e diminuiu significativamente os níveis de proteína X associada a Bcl-2 (Bax), caspase-9 e caspase-3. Além disso, resultou no nível de expressão mais alto de linfoma de células B 2 (Bcl-2) e níveis de expressão mais baixos de Bax, caspase-9 e caspase-3 em tecidos musculares entre todos os grupos de intervenção. Conclusão: A intervenção em baixa temperatura de 16–18°C durante o período de isquemia mostrou efeitos ótimos nas expressões de fatores apoptóticos durante o desenvolvimento de IP com dano tecidual induzido por I/R. RESUMEN Objetivo: Nuestro objetivo fue determinar el efecto de diferentes intervenciones de rango de temperatura baja en diferentes puntos de tiempo en un modelo de rata de lesión por presión (IP) producida por lesión por isquemia/reperfusión (I/R). Métodos: Se asignaron aleatoriamente ratas Sprague-Dawley a grupos de control en blanco, control de lesiones e intervención de temperatura. Las ratas en los grupos de control de lesiones e intervención de temperatura (que implican exposición a diferentes rangos de temperatura en diferentes puntos de tiempo) se sometieron a tres ciclos de lesión I/R con isquemia de 2 h y reperfusión de 0,5 h para inducir IP. Resultados: Los tejidos musculares presentaron cambios degenerativos después de la compresión. La intervención a baja temperatura de 16–18°C en el período de isquemia resultó en el grado más bajo de daño tisular y niveles significativamente reducidos de proteína X asociada a Bcl-2 (Bax), caspasa-9 y caspasa-3. Además, dio como resultado el nivel de expresión más alto de linfoma de células B 2 (Bcl-2) y los niveles de expresión más bajos de Bax, caspasa-9 y caspasa-3 en los tejidos musculares entre todos los grupos de intervención. Conclusión: La intervención a baja temperatura a 16–18°C durante el período de isquemia mostró efectos óptimos sobre la expresión de factores apoptóticos durante el desarrollo de IP con daño tisular inducido por I/R.

The optimization of conditions to promote the stemness of pluripotent cells in vitro is instrumental for their use in advanced therapies. We show here that exposure of human iPSCs and human ESCs to low concentrations of the chemical NO donor DETA/NO leads to stabilization of hypoxia-inducible factors (HIF-1α and HIF-2α) under normoxia, with this effect being dependent on diminished Pro 402 hydroxylation and decreased degradation by the proteasome. Moreover, the master genes of pluripotency, NANOG and OCT-4, were upregulated. NO also induces a shift in the metabolic profile of PSCs, with an increased expression of hypoxia response genes in glycolysis. Furthermore, a reduction in the mitochondrial membrane potential with lower oxygen consumption and increased expression of mitochondrial fusion regulators, such as DRP1, was observed. The results reported here indicate that NO mimics hypoxia response in human PSCs and enhances their stemness properties when cultured under normoxic conditions.

Abnormal trinucleotide expansions cause rare disorders that compromise quality of life and, in some cases, lifespan. In particular, the expansions of the CGG-repeats stretch at the 5'-UTR of the Fragile X Mental Retardation 1 (FMR1) gene have pleiotropic effects that lead to a variety of Fragile X-associated syndromes: the neurodevelopmental Fragile X syndrome (FXS) in children, the late-onset neurodegenerative disorder Fragile X-associated tremor-ataxia syndrome (FXTAS) that mainly affects adult men, the Fragile X-associated primary ovarian insufficiency (FXPOI) in adult women, and a variety of psychiatric and affective disorders that are under the term of Fragile X-associated neuropsychiatric disorders (FXAND). In this review, we will describe the pathological mechanisms of the adult "gain-of-function" syndromes that are mainly caused by the toxic actions of CGG RNA and FMRpolyG peptide. There have been intensive attempts to identify reliable peripheral biomarkers to assess disease progression and onset of specific pathological traits. Mitochondrial dysfunction, altered miRNA expression, endocrine system failure, and impairment of the GABAergic transmission are some of the affectations that are susceptible to be tracked using peripheral blood for monitoring of the motor, cognitive, psychiatric and reproductive impairment of the CGG-expansion carriers. We provided some illustrative examples from our own cohort. Understanding the association between molecular pathogenesis and biomarkers dynamics will improve effective prognosis and clinical management of CGG-expansion carriers. Yes

This study was funded by grants from the Ministerio de Economia, Industria y Competitividad y por el Fondo de Desarrollo Regional FEDER, Spain nº SAF2013-49019, SAF2017-85903-P, and from the Consejería de Innovación, Ciencia y Empresa, Junta de Andalucía (P07- CTS- 03135, P10- CTS- 5784, and CTS- 101), Spain. J.F. and L.M. have FPU fellowships from the Ministerio de Educación Cultura y Deporte, Spain. C.R.S. was a schorlarship holder from the Plan Propio de Investigación of the University of Granada., We wish to thank Michael O’Shea for proofreading the paper., Metabolic reprogramming, which is characteristic of cancer cells that rapidly adapt to the hypoxic microenvironment and is crucial for tumor growth and metastasis, is recognized as one of the major mechanisms underlying therapeutic resistance. Mitochondria, which are directly involved in metabolic reprogramming, are used to design novel mitochondria-targeted anticancer agents. Despite being targeted by melatonin, the functional role of mitochondria in melatonin's oncostatic activity remains unclear. In this study, we aim to investigate the role of melatonin in mitochondrial metabolism and its functional consequences in head and neck cancer. We analyzed the effects of melatonin on head and neck squamous cell carcinoma (HNSCC) cell lines (Cal-27 and SCC-9), which were treated with 100, 500, and 1500 mu M of melatonin for 1, 3, and 5 days, and found a connection between a change of metabolism following melatonin treatment and its effects on mitochondria. Our results demonstrate that melatonin induces a shift to an aerobic mitochondrial metabolism that is associated with changes in mitochondrial morphology, function, fusion, and fission in HNSCC. We found that melatonin increases oxidative phosphorylation (OXPHOS) and inhibits glycolysis in HNSCC, resulting in increased ROS production, apoptosis, and mitophagy, and decreased cell proliferation. Our findings highlight new molecular pathways involved in melatonin's oncostatic activity, suggesting that it could act as an adjuvant agent in a potential therapy for cancer patients. We also found that high doses of melatonin, such as those used in this study for its cytotoxic impact on HNSCC cells, might lead to additional effects through melatonin receptors., Ministerio de Economia, Industria y Competitividad y por el Fondo de Desarrollo Regional FEDER, Spain SAF2013-49019 SAF2017-85903-P, Junta de Andalucia P07-CTS-03135 P10-CTS-5784 CTS-101, Ministerio de Educacion Cultura y Deporte, Spain, Plan Propio de Investigacion of the University of Granada

Currently, the drugs used for toxoplasmosis have severe toxicity to human cells, and the treatment still lacks effective and safer alternatives. The search for novel drug targets is timely., Toxoplasmosis, a protozoan infection caused by Toxoplasma gondii, is estimated to affect around 2.5 billion people worldwide. Nevertheless, the side effects of drugs combined with the long period of therapy usually result in discontinuation of the treatment. New therapies should be developed by exploring peculiarities of the parasite’s metabolic pathways, similarly to what has been well described in cancer cell metabolism. An example is the switch in the metabolism of cancer that blocks the conversion of pyruvate into acetyl coenzyme A in mitochondria. In this context, dichloroacetate (DCA) is an anticancer drug that reverts the tumor proliferation by inhibiting the enzymes responsible for this switch: the pyruvate dehydrogenase kinases (PDKs). DCA has also been used in the treatment of certain symptoms of malaria; however, there is no evidence of how this drug affects apicomplexan species. In this paper, we studied the metabolism of T. gondii and demonstrate that DCA also inhibits T. gondii’s in vitro infection with no toxic effects on host cells. DCA caused an increase in the activity of pyruvate dehydrogenase followed by an unbalanced mitochondrial activity. We also observed morphological alterations frequently in mitochondria and in a few apicoplasts, essential organelles for parasite survival. To date, the kinases that potentially regulate the activity of pyruvate metabolism in both organelles have never been described. Here, we confirmed the presence in the genome of two putative kinases (T. gondii PDK [TgPDK] and T. gondii branched-chain α-keto acid dehydrogenase kinase [TgBCKDK]), verified their cellular localization in the mitochondrion, and provided in silico data suggesting that they are potential targets of DCA. IMPORTANCE Currently, the drugs used for toxoplasmosis have severe toxicity to human cells, and the treatment still lacks effective and safer alternatives. The search for novel drug targets is timely. We report here that the treatment of T. gondii with an anticancer drug, dichloroacetate (DCA), was effective in decreasing in vitro infection without toxicity to human cells. It is known that PDK is the main target of DCA in mammals, and this inactivation increases the conversion of pyruvate into acetyl coenzyme A and reverts the proliferation of tumor cells. Moreover, we verified the mitochondrial localization of two kinases that possibly regulate the activity of pyruvate metabolism in T. gondii, which has never been studied. DCA increased pyruvate dehydrogenase (PDH) activity in T. gondii, followed by an unbalanced mitochondrial activity, in a manner similar to what was previously observed in cancer cells. Thus, we propose the conserved kinases as potential regulators of pyruvate metabolism and interesting targets for new therapies.

Recently, it was proposed that the thiophene ring is capable of promoting mitochondrial accumulation when linked to fluorescent markers. As a noncharged group, thiophene presents several advantages from a synthetic point of view, making it easier to incorporate such a side moiety into different molecules. Herein, we confirm the general applicability of the thiophene group as a mitochondrial carrier for drugs and fluorescent markers based on a new concept of nonprotonable, noncharged transporter. We implemented this concept in a medicinal chemistry application by developing an antitumor, metabolic chimeric drug based on the pyruvate dehydrogenase kinase (PDHK) inhibitor dichloroacetate (DCA). The promising features of the thiophene moiety as a noncharged carrier for targeting mitochondria may represent a starting point for the design of new metabolism-targeting drugs. ispartof: PHARMACEUTICS vol:13 issue:2 ispartof: location:Switzerland status: published

Oxidative distress and mitochondrial dysfunction, are key factors involved in the pathophysiology of Parkinson's disease (PD). The pleiotropic hormone insulin-like growth factor II (IGF-II) has shown neuroprotective and antioxidant effects in some neurodegenerative diseases. In this work, we demonstrate the protective effect of IGF-II against the damage induced by 1-methyl-4-phenylpyridinium (MPP+) in neuronal dopaminergic cell cultures and a mouse model of progressive PD. In the neuronal model, IGF-II counteracts the oxidative distress produced by MPP + protecting dopaminergic neurons. Improved mitochondrial function, increased nuclear factor (erythroid-derived 2)-like2 (NRF2) nuclear translocation along with NRF2-dependent upregulation of antioxidative enzymes, and modulation of mammalian target of rapamycin (mTOR) signalling pathway were identified as mechanisms leading to neuroprotection and the survival of dopaminergic cells. The neuroprotective effect of IGF-II against MPP + -neurotoxicity on dopaminergic neurons depends on the specific IGF-II receptor (IGF-IIr). In the mouse model, IGF-II prevents behavioural dysfunction and dopaminergic nigrostriatal pathway degeneration and mitigates neuroinflammation induced by MPP+. Our work demonstrates that hampering oxidative stress and normalising mitochondrial function through the interaction of IGF-II with its specific IGF-IIr are neuroprotective in both neuronal and mouse models. Thus, the modulation of the IGF-II/IGF-IIr signalling pathway may be a useful therapeutic approach for the prevention and treatment of PD., Graphical abstract Image 1, Highlights • IGF-II hampers oxidative damage and promotes survival in a cellular model of PD. • IGF-II avoids mitochondrial damage in dopaminergic cells in a model of PD. • IGF-II receptor mediates the neuroprotective effect of IGF-II in a cellular model of PD. • IGF-II prevents nigrostriatal degeneration and inflammation in a mice model of PD. • IGF-II prevents behavioural dysfunction in a mice model of PD.

Producción Científica, The FASTK family of proteins have been recently reported to play a key role in the post-transcriptional regulation of mitochondrial gene expression, including mRNA stability and translation. Accumulated studies have provided evidence that the expression of some FASTK genes is altered in certain types of cancer, in agreement with the central role of mitochondria in cancer development. Here, we obtained a pan-cancer overview of the genomic and transcriptomic alterations of FASTK genes. FASTK, FASTKD1, FASTKD3 and FASTKD5 showed the highest rates of genetic alterations. FASTK and FASTKD3 alterations consisted mainly of amplifications that were seen in more than 8% of ovarian and lung cancers, respectively. FASTKD1 and FASTKD5 were the most frequently mutated FASTK genes, and the mutations were identified in 5–7% of uterine cancers, as well as in 4% of melanomas. Our results also showed that the mRNA levels of all FASTK members were strongly upregulated in esophageal, stomach, liver and lung cancers. Finally, the protein-protein interaction network for FASTK proteins uncovers the interaction of FASTK, FASTKD2, FASTKD4 and FASTKD5 with cancer signaling pathways. These results serve as a starting point for future research into the potential of the FASTK family members as diagnostic and therapeutic targets for certain types of cancer., Junta de Castilla y León (grants GRS 1971/A/19 and GRS 2201/A/2020), Junta de Castilla y León (grant VA172P20), Ministerio de Ciencia e Innovación (Severo Ochoa SVP-2014–068895)

The Papillon&ndash, Lef&egrave, vre syndrome (PLS) is a rare autosomal recessive disorder caused by mutations in the Cathepsin C (CTSC) gene, characterized by periodontitis and palmoplantar hyperkeratosis. The main inflammatory deficiencies include oxidative stress and autophagic dysfunction. Mitochondria are the main source of reactive oxygen species, their impaired function is related to skin diseases and periodontitis. The mitochondrial function has been evaluated in PLS and mitochondria have been targeted as a possible treatment for PLS. We show for the first time an important mitochondrial dysfunction associated with increased oxidative damage of mtDNA, reduced CoQ10 and mitochondrial mass and aberrant morphologies of the mitochondria in PLS patients. Mitochondrial dysfunction, determined by oxygen consumption rate (OCR) in PLS fibroblasts, was treated with CoQ10 supplementation, which determined an improvement in OCR and a remission of skin damage in a patient receiving a topical administration of a cream enriched with CoQ10 0.1%. We provide the first evidence of the role of mitochondrial dysfunction and CoQ10 deficiency in the pathophysiology of PLS and a future therapeutic option for PLS.

Ca2+ signaling has been involved in controling critical cellular functions such as activation of proteases, cell death, and cell cycle control. The endoplasmatic reticulum plays a significant role in Ca2+ storage inside the cell, but mitochondria have long been recognized as a fundamental Ca2+ pool. Protozoan parasites such as Plasmodium falciparum, Toxoplasma gondii, and Trypanosoma cruzi display a Ca2+ signaling toolkit with similarities to higher eukaryotes, including the participation of mitochondria in Ca2+-dependent signaling events. This review summarizes the most recent knowledge in mitochondrial Ca2+ signaling in protozoan parasites, focusing on the mechanism involved in mitochondrial Ca2+ uptake by pathogenic protists.

This study was supported by the grant #3650 managed by Fundacion General EmpresaUniversidad de Granada and by the investigation group CTS-454 "Impacto fisiologico del estres oxidativo, deporte, actividad fisica y salud"., We tested whether physiological doses of hydroxytyrosol (HT) may alter the mRNA transcription of key metabolic genes in exercised skeletal muscle. Two groups of exercise-trained Wistar rats, HTlow and HTmid, were supplemented with 0.31 and 4.61 mg/kg/d of HT, respectively, for 10 weeks. Another two groups of rats were not supplemented with HT; one remained sedentary and the other one was exercised. After the experimental period, the soleus muscle was removed for qRT-PCR and western blot analysis. The consumption of 4.61 mg/kg/d of HT during exercise increased the mRNA expression of important metabolic proteins. Specifically, 4.61 mg/kg/d of HT may upregulate long-chain fatty acid oxidation, lactate, and glucose oxidation as well as mitochondrial Krebs cycle in trained skeletal muscle. However, a 4.61 mg/kg/d of HT may alter protein translation, as in spite of the increment showed by CD36 and GLUT4 at the mRNA level this was not translated to higher protein content., 3650 CTS-454

Purpose To evaluate the effects of treatment with Indigo Carmine (IC) on rat livers subjected to ischemia-reperfusion injury. Methods The animals were subdivided into 4 groups: 1.SHAM group(SH) - saline; 2.SHAM group with IC-2mg/Kg(SHIC); 3.IR group - rats submitted to ischemia and reperfusion with saline(IR); 4.IR group with IC-2mg/Kg(IRIC). The IR protocol consists of liver exposure and administration of drug or saline intravenously, followed by 60 minutes of ischemia and 15 of reperfusion. Liver samples were collected for biochemical analysis. Results State 3 of mitochondrial respiration showed a significant worsening of the IRIC group in relation to all others. State 4 showed a difference between IRIC and SHIC. The Respiratory Control Ratio showed statistical decrease in IR and IRIC versus Sham. The osmotic swelling showed significant difference between SHxIR; SHICxIRIC and SHxIRIC. There was a significant increase in ALT in the IRIC group in relation to all the others. Concerning the nitrate dosage, there was a decrease in the group treated with IC(IRxIRIC). There was no difference regarding the dosage of Malondialdehyde. Conclusion IC was not able to protect mitochondria from IR injury and proved to be a potentiating agent, acting in synergy with the IR injury promoting damage to the hepatocyte membranes.

Cancer stem cells (CSC) are associated with the mechanisms of chemoresistance to different cytotoxic drugs or radiotherapy, as well as with tumor relapse and a poor prognosis. Various studies have shown that mitochondria play a central role in these processes because of the ability of this organelle to modify cell metabolism, allowing survival and avoiding apoptosis clearance of cancer cells. Thus, the whole mitochondrial cycle, from its biogenesis to its death, either by mitophagy or by apoptosis, can be targeted by different drugs to reduce mitochondrial fitness, allowing for a restored or increased sensitivity to chemotherapeutic drugs. Once mitochondrial misbalance is induced by a specific drug in any of the processes of mitochondrial metabolism, two elements are commonly boosted: an increment in reactive nitrogen/oxygen species and, subsequently, activation of the intrinsic apoptotic pathway. Yes

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 30-06-2020, Esta tesis tiene embargado el acceso al texto completo hasta el 30-12-2021, La 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 humanos, Regulation 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 patients

Objective. Describe the influence of Fetal bovine serum (FBS) on the survival, growth and expression of cellular organelles in rat dental epithelial cells. Material and methods. Cell cultures of rat dental epithelial cells were carried out at 37°C in a humid atmosphere, in the absence and at a concentration of 10% FBS. Morphological evaluation was performed during the proliferation and confluence of cell in culture. Double immunofluorescence labels were made using anti-Actin, anti-TOMM20A, and anti-LAMP1 antibodies. Results. Circular or ovoid dental epithelial cells with bulky nuclei were evidenced during proliferation and confluences in a similar manner in culturing cells in the presence and absence of FBS. The lack of FBS negatively impacts the proliferation of epithelial cells. No alterations were observed in the localization of the anti-actin, anti-TOMM20 and anti-LAMP1 immunomarkers in both conditions of experimental cultures. Conclusions. FBS suppression in rat dental epithelial cells decreased survival, proliferation and suggests not having an impact on the organelles evaluated., Objetivo. Describir la influencia del Suero Fetal Bovino (SFB) en la supervivencia, crecimiento y expresión de organelas celulares en las células epiteliales dentales de rata. Materiales y métodos. Cultivos de células epiteliales dentales de rata fueron llevados a cabo a 37°C en una atmosfera húmeda, en ausencia y a una concentración de 10% de SFB. Una evaluación morfológica fue realizada durante la proliferación y confluencia de las células en cultivo. Dobles marcajes por inmunofluorencia fueron efectuados haciendo uso de anticuerpos anti-actina, anti-TOMM20 y anti-LAMP1. Resultados. Se evidenciaron células epiteliales dentales circulares u ovoides con núcleos voluminosos durante la proliferación y confluencias de manera similar en las células cultivas en presencia y ausencia de SFB. La carencia de SFB impactó negativamente la proliferación de las células epiteliales. No fueron observadas alteraciones en la localización de los inmunomarcajes anti-actina, anti-TOMM20 y anti-LAMP1 en las dos condiciones de cultivos experimentales. Conclusiones. La supresión del SFB en el cultivo de células epiteliales dentales de rata disminuyó la supervivencia, proliferación y sugiere no tener un impacto sobre las organelas evaluadas.

[eng] OPA1 is a dynamin-related protein that is responsible for the fusion of the internal mitochondrial membrane and essential to control the morphology of the mitochondrial cristae. Thus, it directly impacts the efficiency of OxPhos and the stability of mitochondrial DNA. In this study, we have explored the effects of hepatic deletion of OPA1 on mitochondrial function and metabolism. We have shown that the ablation of OPA1 in the liver produces a mitochondrial dysfunction characterized by alterations in mitochondrial cristae structure, concomitant with a reduced respiratory capacity and mtDNA copy number, and perturbed mitochondrial proteostasis. The mitochondrial dysfunction caused by the ablation of OPA1 in liver triggers the activation of a mitochondrial stress response, including mitochondrial unfolded stress response (UPRmt) that is probably mediated by the transcription factor ATF5. Interestingly, we have observed that OPA1 deficiency in the liver causes better glucose tolerance and protects against diet-induced obesity and insulin resistance concomitant to a high circulating levels of the metabolic modulator FGF21. Here we suggest that the systemic protective effects associated to OPA1 ablation are due to the action of FGF21, probably mediated by the mitochondrial stress response associated to OPA1 loss-of-function via ATF5 activation., [spa] OPA1 es una proteína relacionada con la dinamina que es responsable de la fusión de la membrana mitocondrial interna y esencial para controlar la morfología de las crestas mitocondriales, afectando directamente la eficiencia de OxPhos y la estabilidad del DNA mitocondrial. En este estudio, hemos explorado los efectos de la eliminación hepática de OPA1 sobre la función mitocondrial y el metabolismo. Hemos demostrado que la ablación de OPA1 en el hígado produce una disfunción mitocondrial caracterizada por alteraciones en la estructura de las crestas mitocondriales, concomitante con una capacidad respiratoria reducida y menor número de copias de DNA miocondrial, y perturbación en la proteostasis mitocondrial. La disfunción mitocondrial causada por la ablación de OPA1 en el hígado desencadena la activación de una respuesta al estrés mitocondrial, incluyendo la respuesta a las proteínas mal plegadas de la mitocondria, que probablemente es mediada por el factor de transcripción ATF5. Curiosamente, hemos observado que la deficiencia de OPA1 en el hígado causa una mejor tolerancia a la glucosa y protege contra la obesidad y resistencia a la insulina inducida por la dieta, en paralelo con un aumento de los niveles de FGF21 circulante, un factor involucrado en la modulación metabólica. Con este estudio proponemos que los efectos sistémicos protectores asociados a la ablación de OPA1 se deben a la acción de FGF21 que probablemente es mediada por la respuesta al estrés mitocondrial asociada a la pérdida de función de OPA1 a través de la activación de ATF5.

Glutaminase (GA) catalyzes the first step in mitochondrial glutaminolysis playing a key role in cancer metabolic reprogramming. Humans express two types of GA isoforms: GLS and GLS2. GLS isozymes have been consistently related to cell proliferation, but the role of GLS2 in cancer remains poorly understood. GLS2 is repressed in many tumor cells and a better understanding of its function in tumorigenesis may further the development of new therapeutic approaches. We analyzed GLS2 expression in HCC, GBM and neuroblastoma cells, as well as in monkey COS-7 cells. We studied GLS2 expression after induction of differentiation with phorbol ester (PMA) and transduction with the full-length cDNA of GLS2. In parallel, we investigated cell cycle progression and levels of p53, p21 and c-Myc proteins. Using the baculovirus system, human GLS2 protein was overexpressed, purified and analyzed for posttranslational modifications employing a proteomics LC-MS/MS platform. We have demonstrated a dual targeting of GLS2 in human cancer cells. Immunocytochemistry and subcellular fractionation gave consistent results demonstrating nuclear and mitochondrial locations, with the latter being predominant. Nuclear targeting was confirmed in cancer cells overexpressing c-Myc- and GFP-tagged GLS2 proteins. We assessed the subnuclear location finding a widespread distribution of GLS2 in the nucleoplasm without clear overlapping with specific nuclear substructures. GLS2 expression and nuclear accrual notably increased by treatment of SH-SY5Y cells with PMA and it correlated with cell cycle arrest at G2/M, upregulation of tumor suppressor p53 and p21 protein. A similar response was obtained by overexpression of GLS2 in T98G glioma cells, including downregulation of oncogene c-Myc. Furthermore, human GLS2 was identified as being hypusinated by MS analysis, a posttranslational modification which may be relevant for its nuclear targeting and/or function. Our studies provide evidence for a tumor suppressor role of GLS2 in certain types of cancer. The data imply that GLS2 can be regarded as a highly mobile and multilocalizing protein translocated to both mitochondria and nuclei. Upregulation of GLS2 in cancer cells induced an antiproliferative response with cell cycle arrest at the G2/M phase.

MitoBlue is a fluorescent bisamidine that can be used to easily monitor the changes in mitochondrial degradation processes in different cells and cellular conditions. MitoBlue staining pattern is exceptional among mitochondrial dyes and recombinant fluorescent probes, allowing the dynamic study of mitochondrial recycling in a variety of situations in living cells. MitoBlue is a unique tool for the study of these processes that will allow the detailed characterization of communication between mitochondria and lysosomes We are thankful for the support given by the Spanish grants BFU2013-43513-R, SAF2016-76689-R, CTQ2015-70698-R, RTI2018-099877-B-I00, CTQ2013-49317-EXP, CTQ2013-41339-P, CTQ2016-75870-P, CTQ2015-71896-REDT (Red de Fotoquímica Biológica) and Instituto de Salud Carlos III (ISCIII; RETIC ARADYALRD16/0006/0012), Junta de Andalucía (PI0250-2016 and UMA18-FEDERJA-007), Orfeo-cinqa network CTQ2016-81797-REDC; the Xunta de Galicia (2015-CP082, ED431C 2017/19, Centro Singular de investigación de Galicia accreditation 2019–2022, ED431G 2019/03), the European Union (European Regional Development Fund - ERDF), and the European Research Council (Advanced Grant No. 340055) are gratefully acknowledged. M.E.V. also acknowledges the support provided by the Fundación AECC (IDEAS197VAZQ grant) SI

[eng] G proteins transduce a myriad of signals from receptors at the plasma membrane. Recent reports point to a novel localization of G proteins at the mitochondria and other endomembranes where they regulate the physiology of these organelles. In particular, the Gαq subfamily is required to keep the proper balance between mitochondria fusion and fission acting at both outer and inner membrane, among other functions. In order to unveil the putative effectors of Gαq that mediate those effects at the mitochondria, our group has undertaken a mass-spectrometry analysis based on Gαq immunoprecipitates from cellular endomembranes. The “mito- interactome” study provided evidence of Gαq interaction with the armadillo domain-containing proteins Alex3 and Armc10. Subsequent immunoprecipitation and pull-down studies demonstrated a specific interaction of Gαq with the mitochondrial Rho GTPase 1 (Miro1) and both milton adaptor proteins TRAK1 and 2, that couple mitochondria to kinesin and dynein motor proteins and constitute the main regulators of mitochondrial transport in neurons. To analyze the physiological role of those interactions, we have performed tracking analysis of mitochondria along the axons of hippocampal neurons overexpressing Gαq or its constitutive-active mutant, GαqR183C, as well as activating a Gαq-specific GPCR (DREADD) with its specific agonist. The results of these studies reveal a significant increase in anterograde movement upon Gαq expression, whereas Gαq activation by either expressing the active-mutant or activating the Gαq-specific GPCR induces mitochondrial arrest. In contrast, depletion of Gαq using short-hairpin RNAs increases the number of motile mitochondria and their speed and promotes retrograde transport. Both activation of Gαq or its depletion alter mitochondrial dynamics including fusion/fission events, whereas expression of active-Gαq also alters neuronal physiology by reducing their complexity and dendritic branching. In summary, our group postulates a new non-canonical mitochondrial function of Gαq acting as a molecular switch through its association with Alex3, Miro1 and TRAK2. Gαq would associate to Alex3 and Miro1 to allow mitochondrial movement, whereas its GTP-bound conformation would associate to TRAK2 to halt motility. This process would be regulated by Alex3, which could play crucial roles as an adaptor for the protein complex and Gαq transactivation.

This article was submitted to Cellular Endocrinology, a section of the journal Frontiers in Endocrinology Volumen 11 Atículo 319. Secretion is an energy consuming process that plays a relevant role in cell communication and adaptation to the environment. Among others, endocrine cells producing hormones, immune cells producing cytokines or antibodies, neurons releasing neurotransmitters at synapsis, and more recently acknowledged, senescent cells synthesizing and secreting multiple cytokines, growth factors and proteases, require energy to successfully accomplish the different stages of the secretion process. Calcium ions (Ca2+) act as second messengers regulating secretion in many of these cases. In this setting, mitochondria appear as key players providing ATP by oxidative phosphorylation, buffering Ca2+ concentrations and acting as structural platforms. These tasks also require the concerted actions of the mitochondrial dynamics machinery. These proteins mediate mitochondrial fusion and fission, and are also required for transport and tethering of mitochondria to cellular organelles where the different steps of the secretion process take place. Herein we present a brief overview of mitochondrial energy metabolism, mitochondrial dynamics, and the different steps of the secretion processes, along with evidence of the interaction between these pathways. We also analyze the role of mitochondria in secretion by different cell types in physiological and pathological settings. Agencia Nacional de Investigación e Innovación FCE_1_2017_1_136021