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Fil: Pandi Perumal, Seithikurippu R. Somnogen Canada Inc; Canadá Fil: Cardinali, Daniel Pedro. Pontificia Universidad Católica Argentina. Facultad de Ciencias Médicas; Argentina Fil: Reiter, R. UT Health San Antonio. Department of Cell Systems and Anatomy; Estados Unidos Fil: Brown, Gregory M. Univeridad de Toronto. Centro para la Adicción y la Salud Mental. Ciencias Moleculares del Cerebro; Canadá Abstract: That the pineal gland is a source of melatonin is widely known; however, by comparison, few know of the much larger pool of extrapineal melatonin. That pool is widely distributed in all animals, including those that do not have a pineal gland, e.g., insects. Extrapineal melatonin is not released into the blood but is used locally to function as an antioxidant, anti-inflammatory agent, etc. A major site of action of peripherally-produced melatonin is the mitochondria where it neutralizes reactive oxygen species (ROS) that are generated during oxidative phosphorylation. Its role also includes major actions as an immune modulator reducing overreactions to foreign agents while simultaneously boosting immune processes. During a pandemic such as coronavirus disease 2019 (COVID-19), caused by the virus SARS-CoV-2, melatonin is capable of suppressing the damage inflicted by the cytokine storm. The implications of melatonin in susceptibility and treatment of COVID-19 disease are discussed.

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.

Blastocladiella emersonii is an early diverging fungus of the phylum Blastocladiomycota. During the life cycle of the fungus, mitochondrial morphology changes significantly, from a fragmented form in sessile vegetative cells to a fused network in motile zoospores. In this study, we visualize these morphological changes using a mitochondrial fluorescent probe and show that the respiratory capacity in zoospores is much higher than in vegetative cells, suggesting that mitochondrial morphology could be related to the differences in oxygen consumption. While studying the respiratory chain of the fungus, we observed an antimycin A and cyanide-insensitive, salicylhydroxamic (SHAM)-sensitive respiratory activity, indicative of a mitochondrial alternative oxidase (AOX) activity. The presence of AOX was confirmed by the finding of a B. emersonii cDNA encoding a putative AOX, and by detection of AOX protein in immunoblots. Inhibition of AOX activity by SHAM was found to significantly alter the capacity of the fungus to grow and sporulate, indicating that AOX participates in life cycle control in B. emersonii.

Cells respond to stress by activating a variety of defense signaling pathways, including cell survival and cell death pathways. Although cell survival signaling helps the cell to recover from acute insults, cell death or senescence pathways induced by chronic insults can lead to unresolved pathologies. Arterial hypertension results from chronic physiological maladaptation against various stressors represented by abnormal circulating or local neurohormonal factors, mechanical stress, intracellular accumulation of toxic molecules, and dysfunctional organelles. Hypertension and aging share common mechanisms that mediate or prolong chronic cell stress, such as endoplasmic reticulum stress and accumulation of protein aggregates, oxidative stress, metabolic mitochondrial stress, DNA damage, stress-induced senescence, and proinflammatory processes. This review discusses common adaptive signaling mechanisms against these stresses including unfolded protein responses, antioxidant response element signaling, autophagy, mitophagy, and mitochondrial fission/fusion, STING (signaling effector stimulator of interferon genes)-mediated responses, and activation of pattern recognition receptors. The main molecular mechanisms by which the vasculature copes with hypertensive and aging stressors are presented and recent advancements in stress-adaptive signaling mechanisms as well as potential therapeutic targets are discussed.

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.

The COVID-19 pandemic brought attention to studies about viral infections and their impact on the cell machinery. SARS-CoV-2, for example, invades the host cells by ACE2 interaction and possibly hijacks the mitochondria. To better understand the disease and to propose novel treatments, crucial aspects of SARS-CoV-2 enrolment with host mitochondria must be studied. The replicative process of the virus leads to consequences in mitochondrial function, and cell metabolism. The hijacking of mitochondria, on the other hand, can drive the extrusion of mitochondrial DNA (mtDNA) to the cytosol. Extracellular mtDNA evoke robust proinflammatory responses once detected, that may act in different pathways, eliciting important immune responses. However, few receptors are validated and are able to detect and respond to mtDNA. In this review, we propose that the mtDNA and its detection might be important in the immune process generated by SARS-CoV-2 and that this mechanism might be important in the lung pathogenesis seen in clinical symptoms. Therefore, investigating the mtDNA receptors and their signaling pathways might provide important clues for therapeutic interventions.

Lymphoma is the most common type of blood cancer today and, as its name suggests, it begins in the lymphatic system. The origin of this disease is related to mitochondrial defects, generated by mutations in the PNPase enzyme or polynucleotide phosphorylase, whose main functions are import and degradation mitochondrial RNA. For this reason, the main objective of the present work was to carry out a bibliographic review of scientific publications that described the important role of this enzyme in relation to mitochondria as the cause of lymphatic cancer. The methodology used consisted of a bibliometric analysis based on the use of different databases, in which search equations formed from keywords were introduced. Then, the selection of articles related to the study topic and published in the last 20 years was carried out. Subsequently, the journals were analyzed, based on the h-index, in order to determine which supported the hypothesis of the role of PNPase in lymphoma and which stated the opposite. The results showed that a total of 441,288 scientific publications were obtained, of which 133 were selected to carry out this work. As for the journals, those with the highest h-index were Nature and Cell . It can be concluded that PNPase plays a very important role in the transport of mitochondrial RNA and that the nuclear transcription factor (NF-Y) is involved in the control of cell growth, therefore, both have a crucial role in the development of this disease. Therefore, research on both PNPase and NF-Y is essential to establish the specific genetic characteristics that define the early lesions of lymphatic cancer and the consequent determination of their treatment. El linfoma es el tipo de cáncer de sangre más común en la actualidad y, como su nombre indica, comienza en el sistema linfático. El origen de esta enfermedad está relacionado con defectos mitocondriales, generados por mutaciones en la enzima PNPasa o polinucleótido fosforilasa, cuyas principales funciones son las de importación y degradación del ARN mitocondrial. Por este motivo, el objetivo principal del presente trabajo fue la realización de una revisión bibliográfica acerca de publicaciones científicas, que hicieran relevancia al papel de esta enzima, en relación con la mitocondria como causante del cáncer linfático. La metodología empleada consistió en un análisis bibliométrico basado en la utilización de diferentes bases de datos, en las que se introdujeron ecuaciones de búsqueda formadas a partir de palabras clave. Después, se llevó a cabo la selección de los artículos relacionados con el tema de estudio y publicados en los últimos 20 años. Posteriormente, se analizaron las revistas, en función del índice H, para poder observar cuáles apoyaban la hipótesis del papel de PNPasa en linfoma y cuáles decían lo contrario. Los resultados mostraron que se obtuvo un total de 441.288 publicaciones científicas, de las que fueron seleccionadas 147 para poder llevar a cabo este trabajo. En cuanto a las revistas, las que presentaron un índice H mayor fueron Natura y Cell. Se puede concluir que la PNPasa desempeña un papel muy importante en el transporte del ARN mitocondrial, y que el factor NF-Y está involucrado en el control del crecimiento de las células, por lo que, ambos presentan una función crucial en el desarrollo de esta enfermedad. Por ello, la investigación, tanto de PNPasa como de NF- Y, es fundamental para establecer las características genéticas concretas, que definan las lesiones tempranas del cáncer linfático y la consiguiente determinación de su tratamiento. Ciencias Experimentales

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)

Made available in DSpace on 2022-04-29T08:30:43Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-01-01 The mitogenome of the South American parthenogenetic lizard Loxopholis percarinatum Müller, 1923 (Squamata: Gymnophthalmidae), a uni-bisexual species complex, was recovered for three individuals from Rio Negro region, Amazonas, Brazil. The content and order of genes are typical for vertebrate mitochondrial genomes, and we recovered 13 protein-coding genes, 22 tRNA, and two rRNA (12S and 16S), in addition to partial fragments of the Control Region. A maximum likelihood phylogenetic analysis with mitogenomes of selected lizard families recovered L. percarinatum with Iphisa elegans Gray, 1851, the only other Gymnophthalmidae species available in GenBank. Departamento de Zoologia Instituto de Biociências Universidade de São Paulo Departamento de Biodiversidade e Centro de Aquicultura Instituto de Biociências Universidade Estadual Paulista (UNESP) Amphibian and Reptile Diversity Research Center and Department of Biology University of Texas at Arlington Departamento de Ecologia e Biologia Evolutiva Universidade Federal de São Paulo Departamento de Biodiversidade e Centro de Aquicultura Instituto de Biociências Universidade Estadual Paulista (UNESP)

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.

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.

Mitochondria contribute to shape intraneuronal Ca2+ signals. Excessive Ca2+ taken up by mitochondria could lead to cell death. Amyloid beta (A beta) causes cytosolic Ca2+ overload, but the effects of A beta on mitochondrial Ca2+ levels in Alzheimer's disease (AD) remain unclear. Using a ratiometric Ca2+ indicator targeted to neuronal mitochondria and intravital multiphoton microscopy, we find increased mitochondrial Ca2+ levels associated with plaque deposition and neuronal death in a transgenic mouse model of cerebral beta -amyloidosis. Naturally secreted soluble A beta applied onto the healthy brain increases Ca2+ concentration in mitochondria, which is prevented by blockage of the mitochondrial calcium uniporter. RNA-sequencing from post-mortem AD human brains shows downregulation in the expression of mitochondrial influx Ca2+ transporter genes, but upregulation in the genes related to mitochondrial Ca2+ efflux pathways, suggesting a counteracting effect to avoid Ca2+ overload. We propose lowering neuronal mitochondrial Ca2+ by inhibiting the mitochondrial Ca2+ uniporter as a novel potential therapeutic target against AD. Calvo-Rodriguez et al. show elevated calcium levels in neuronal mitochondria in a mouse model of cerebral beta -amyloidosis after plaque deposition, which precede rare neuron death events in this model. The mechanism involves toxic extracellular A beta oligomers and the mitochondrial calcium uniporter.

We developed a highly sensitive oxygen consumption scanning microscopy system using platinized platinum disc microelectrodes. The system is capable of reliably detecting single-cell respiration, responding to classical regulators of mitochondrial oxygen consumption activity as expected. Comparisons with commercial multi-cell oxygen detection systems show that the system has comparable errors (if not smaller), with the advantage of being able to monitor inter and intra-cell heterogeneity in oxygen consumption characteristics. Our results uncover heterogeneous oxygen consumption characteristics between cells and within the same cell´s microenvironments. Single Cell Oxygen Mapping (SCOM) is thus capable of reliably studying mitochondrial oxygen consumption characteristics and heterogeneity at a single-cell level.

Mitochondrial oxidative stress accumulates with aging and age-related diseases and induces alterations in mitochondrial DNA (mtDNA) content. Since mtDNA qualitative alterations are also associated with aging, repair of mtDNA damage is of great importance. The most relevant form of DNA repair in this context is base excision repair (BER), which removes oxidized bases such as 8-oxoguanine (8-oxoG) and thymine glycol through the action of the mitochondrial isoform of the specific 8-oxoG DNA glycosylase/apurinic or apyrimidinic (AP) lyase (OGG1) or the endonuclease III homolog (NTH1). Mouse strains lacking OGG1 (OGG1−/−) or NTH1 (NTH1−/−) were analyzed for mtDNA alterations. Interestingly, both knockout strains presented a significant increase in mtDNA content, suggestive of a compensatory mtDNA replication. The mtDNA “common deletion” was not detected in either knockout mouse strain, likely because of the young age of the mice. Formamidopyrimidine DNA glycosylase (Fpg)-sensitive sites accumulated in mtDNA from OGG1−/− but not from NTH1−/− mice. Interestingly, the D-loop region was most severely affected by the absence of OGG1, suggesting that this region may be a hotspot for oxidative damage. Thus, we speculate that mtDNA alterations may send a stress message to evoke cell changes through a retrograde mitochondrial−nucleus communication.

Fil: Godoy, Juan A. Pontificia Universidad Católica de Chile. Facultad de Ciencias Biológicas. Departamento de Biología Celular y Molecular. Centro de Envejecimiento y Regeneración; Cihle Fil: Godoy, Juan A. Universidad de Magallanes. Centro de Excelencia en Biomedicina de Magallanes; Chile Fil: Valdivieso, Ángel Gabriel. Pontificia Universidad Católica Argentina. Facultad de Ciencias Médicas. Instituto de Biología Celular y Molecular; Argentina Investigaciones Biomédicas. Laboratorio de Fil: Valdivieso, Ángel Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Inestrosa, Nibaldo C. Pontificia Universidad Católica de Chile. Facultad de Ciencias Biológicas. Departamento de Biología Celular y Molecular. Centro de Envejecimiento y Regeneración; Cihle Fil: Inestrosa, Nibaldo C. Universidad de Magallanes. Centro de Excelencia en Biomedicina de Magallanes; Chile Fil: Inestrosa, Nibaldo C. University of New South Wales. Faculty of Medicine. School of Psychiatry. Centre for Healthy Brain Ageing; Australia Fil: Inestrosa, Nibaldo C. Pontificia Universidad Católica de Chile. Biomedical Center; Chile Abstract: Mitochondria are widely recognized as fundamental organelles for cellular physiology and constitute the main energy source for different cellular processes. The location, morphology, and interactions of mitochondria with other organelles, such as the endoplasmic reticulum (ER), have emerged as critical events capable of determining cellular fate. Mitochondria-related functions have proven particularly relevant in neurons; mitochondria are necessary for proper neuronal morphogenesis and the highly energy-demanding synaptic transmission process. Mitochondrial health depends on balanced fusion-fission events, termed mitochondrial dynamics, to repair damaged organelles and/or improve the quality of mitochondrial function, ATP production, calcium homeostasis, and apoptosis, which represent some mitochondrial functions closely related to mitochondrial dynamics. Several neurodegenerative disorders, such as Alzheimer's, Parkinson's, and Huntington's diseases, have been correlated with severe mitochondrial dysfunction. In this regard, nicotine, which has been associated with relevant neuroprotective effects mainly through activation of the nicotinic acetylcholine receptor (nAChR), exerts its effects at least in part by acting directly on mitochondrial physiology and morphology. Additionally, a recent description of mitochondrial nAChR localization suggests a nicotine-dependent mitochondrial function. In the present work, we evaluated in cultured hipocampal neurons the effects of nicotine on mitochondrial dynamics by assessing mitochondrial morphology, membrane potential, as well as interactions between mitochondria, cytoskeleton and IP3R, levels of the cofactor PGC-1α, and fission-fusion-related proteins. Our results suggest that nicotine modulates mitochondrial dynamics and influences mitochondrial association from microtubules, increasing IP3 receptor clustering showing modulation between mitochondria-ER communications, together with the increase of mitochondrial biogenesis.

INTRODUCTION: Several mitochondrial DNA mutations have been reported to be associated with nonsyndromic hearing loss in several families. However, little is known about the prevalence of these mutations in sporadic patients with nonsyndromic sensorineural hearing loss. OBJECTIVE: The purpose of our study was to investigate the incidence of these mitochondrial DNA mutations in such population. METHODS: A total of 178 sporadic patients with nonsyndromic sensorineural hearing loss were enrolled in this study. Genomic DNA was extracted from the peripheral blood sample. We employed the SNaPshot(r) sequencing method to detect five mitochondrial DNA mutations, including A1555G and A827G in 12S rRNA gene and A7445G, 7472insC, and T7511C in tRNASerUCN gene. Meanwhile, we used polymerase chain reaction and sequenced the products to screen GJB2 gene mutations in patients carrying mitochondrial DNA mutations. RESULTS: We failed to detect the presence of A1555G mutation in 12S rRNA gene, and of A7445G, 7472insC, T7511C mutations in tRNASerUCN gene in our population. However, we found that 6 patients (3.37%) were carriers of a homozygous A827G mutation and one of them also carried homozygous GJB2 235delC mutation. CONCLUSION: Our findings in the present study indicate that even in sporadic patients with nonsyndromic sensorineural hearing loss, mitochondrial DNA mutations might also contribute to the clinical phenotype. Resumo Introdução: Diversas mutações do DNA mitocondrial tem sido descritas, em diferentes famílias, associadas à deficiência auditiva não sindrômica. No entanto, pouco se sabe sobrea prevalência dessas mutações em pacientes esporádicos com deficiência auditiva sensorioneural não sindrômica. Objetivo: A finalidade do nosso estudo foi investigar a incidência dessas mutações no DNA mitocondrial nessa população. Método: No total, 178 pacientes esporádicos com deficiência auditiva sensorioneural não sindrômica foram recrutados para participação no estudo. O DNA genômico foi extraído de amostra, de sangue periférico. Utilizamos o método de sequenciamento SNaPshot(r) para detecção de cinco mutações do DNA mitocondrial: A1555G e A827G no gene 12S rRNA e A7445G, 7472insCe T7511C no gene tRNASerUCN. Paralelamente, utilizamos a reação de polimerase em cadeia e sequenciamos os produtos para triagem das mutações no gene GJB2 nos pacientes portadores de mutações no DNA mitocondrial. Resultados: Em nossa população, não conseguimos detectar a presença da mutação A1555G no gene 12S rRNA e nem as mutações A7445G, 7472insC e T7511C no gene tRNASerUCN. Entretanto, constatamos que seis pacientes (3,37%) eram portadores da mutação homozigota A827G; e um deles também portava a mutação homozigota GJB2 235delC. Conclusão: Nossos achados no presente estudo indicam que, mesmo em pacientes esporádicos com deficiência auditiva sensorioneural não sindrômica, as mutações do DNA mitocondrial também podem contribuir para o fenótipo clínico.

Clinical conditions associated with obesity can be improved by daily intake of conjugated linoleic acid (CLA) or extra virgin olive oil (EVOO). Here we investigated whether dietary supplementation with CLA and EVOO, either alone or in combination, changes body metabolism associated with mitochondrial energetics. Male C57Bl/6 mice were divided into one of four groups: CLA (1:1 cis-9, trans-11:trans-10, cis-12; 18:2 isomers), EVOO, CLA plus EVOO or control (linoleic acid). Each mouse received 3 g/kg body weight of the stated oil by gavage on alternating days for 60 days. Dietary supplementation with CLA, alone or in combination with EVOO: (a) reduced the white adipose tissue gain; (b) increased body VO2 consumption, VCO2 production and energy expenditure; (c) elevated uncoupling protein (UCP)-2 expression and UCP activity in isolated liver mitochondria. This organelle, when energized with NAD(+)-linked substrates, produced high amounts of H2O2 without inducing oxidative damage. Dietary supplementation with EVOO alone did not change any metabolic parameter, but supplementation with CLA itself promoted insulin resistance and elevated weight, lipid content and acetyl-CoA carboxylase-1 expression in liver. Interestingly, the in vivo antioxidant therapy with N-acetylcysteine abolished the CLA-induced rise of body metabolism and liver UCP expression and activity, while the in vitro antioxidant treatment with catalase mitigated the CLA-dependent UCP-2 expression in hepatocytes; these findings suggest the participation of an oxidative-dependent pathway. Therefore, this study clarifies the mechanisms by which CLA induces liver UCP expression and activity, and demonstrates for the first time the beneficial effects of combined CLA and EVOO supplementation.

Mouse skin mesenchymal stem cells (msMSCs) are dermis CD105+CD90+CD73+CD29+CD34− mesodermal precursors which, after in vitro induction, undergo chondro, adipo, and osteogenesis. Extensive metabolic reconfiguration has been found to occur during differentiation, and the bioenergetic status of a cell is known to be dependent on the quality and abundance of the mitochondrial population, which may be regulated by fusion and fission. However, little is known regarding the impact of mitochondrial dynamics on the differentiation process. We addressed this knowledge gap by isolating MSCs from Swiss female mice, inducing these cells to differentiate into osteo, chondro, and adipocytes and measuring changes in mass, morphology, dynamics, and bioenergetics. Mitochondrial biogenesis was increased in adipogenesis, as evaluated through confocal microscopy, citrate synthase activity, and mtDNA content. The early steps of adipo and osteogenesis involved mitochondrial elongation, as well as increased expression of mitochondrial fusion proteins Mfn1 and 2. Chondrogenesis involved a fragmented mitochondrial phenotype, increased expression of fission proteins Drp1, Fis1, and 2, and enhanced mitophagy. These events were accompanied by profound bioenergetic alterations during the commitment period. Moreover, knockdown of Mfn2 in adipo and osteogenesis and the overexpression of a dominant negative form of Drp1 during chondrogenesis resulted in a loss of differentiation ability. Overall, we find that mitochondrial morphology and its regulating processes of fission/fusion are modulated early on during commitment, leading to alterations in the bioenergetic profile that are important for differentiation. We thus propose a central role for mitochondrial dynamics in the maintenance/commitment of mesenchymal stem cells.

Beef from Bos taurus indicus is associated with toughness compared to Bos taurus taurus, suggesting there is antagonism between adaptability to heat and beef quality. Resistance to cellular stress in muscle may be protective postmortem, thereby delaying its conversion to meat. Therefore, our objective was to determine pH decline, calpain-1 and caspase 3 activation, and proteolysis in different biological cattle types. Angus, Brangus, and Brahman steers (n = 18) were harvested, and Longissimus lumborum were assessed postmortem for pH decline, ATP content, protease activation, and calpastatin content; and myofibrillar protein degradation was evaluated in beef aged to 14d. Brahman Longissimus lumborum exhibited resistance to pH decline, greater ATP content at 1 h, and delayed calpain-1 autolysis. Although content of caspase-3 zymogen was lower in Brahman, there was no evidence of caspase-3 mediated proteolysis. Greater resistance to energetic and pH changes early postmortem in Brahman Longissimus lumborum are associated with calpain-1 autolysis but not mitochondria mediated apoptosis.

Oxidative stress has been implicated in the pathogenesis of cardiac hypertrophy and associated heart failure. Cardiac tissue grows in response to pressure or volume overload, leading to wall thickening or chamber enlargement. If sustained, this condition will lead to a dysfunctional cardiac tissue and oxidative stress. Calorie restriction (CR) is a powerful intervention to improve health and delay aging. Here, we investigated whether calorie restriction in mice prevented isoproterenol-induced cardiac hypertrophy in vivo by avoiding reactive oxygen species (ROS) production and maintaining antioxidant enzymatic activity. Additionally, we investigated the involvement of mitochondrial ATP-sensitive K+ channels (mitoKATP) in cardiac hypertrophy. CR was induced by 40% reduction in daily calorie ingestion. After 3 weeks on CR or ad libitum (Control) feeding, Swiss mice were treated intraperitoneally with isoproterenol (30 mg/kg per day) for 8 days to induce hypertrophy. Isoproterenol-treated mice had elevated heart weight/tibia length ratios and cardiac protein levels. These gross hypertrophic markers were significantly reduced in CR mice. Cardiac tissue from isoproterenol-treated CR mice also produced less H2O2 and had lower protein sulfydryl oxidation. Additionally, calorie restriction blocked hypertrophic-induced antioxidant enzyme (catalase, superoxide dismutase and glutathione peroxidase) activity repression during cardiac hypertrophy. MitoKATP opening was repressed in isolated mitochondria from hypertrophic hearts, in a manner sensitive to calorie restriction. Finally, mitoKATP inhibition significantly blocked the protective effects of calorie restriction. Altogether, our results suggest that CR improves intracellular redox balance during cardiac hypertrophy and prevents this process in a mechanism involving mitoKATP activation.

FAPESP Background: We have recently demonstrated that palmitoleic acid (16:1n7) increases lipolysis, glucose uptake and glucose utilization for energy production in white adipose cells. In the present study, we tested the hypothesis that palmitoleic acid modulates bioenergetic activity in white adipocytes. Methods: For this, 3 T3-L1 pre-adipocytes were differentiated into mature adipocytes in the presence (or absence) of palmitic (16:0) or palmitoleic (16:1n7) acid at 100 or 200 mu M. The following parameters were evaluated:lipolysis, lipogenesis, fatty acid (FA) oxidation, ATP content, oxygen consumption, mitochondrial mass, citrate synthase activity and protein content of mitochondrial oxidative phosphorylation (OXPHOS) complexes. Results: Treatment with 16:1n7 during 9 days raised basal and isoproterenol-stimulated lipolysis, FA incorporation into triacylglycerol (TAG), FA oxidation, oxygen consumption, protein expression of subunits representing OXPHOS complex II, III, and V and intracellular ATP content. These effects were not observed in adipocytes treated with 16:0. Conclusions: Palmitoleic acid, by concerted action on lipolysis, Gamma A esterification, mitochondrial Gamma A oxidation, oxygen consumption and ATP content, does enhance white adipocyte energy expenditure and may act as local hormone. Univ Fed Sao Paulo, Inst Environm Sci Chem & Pharmaceut, Dept Biol Sci, 210 Sao Nicolau St, BR-09913030 Diadema, Brazil Univ Sao Paulo, Inst Biomed Sci, Dept Physiol & Biophys, Sao Paulo, Brazil Cruzeiro do Sul Univ, Inst Phys Act Sci & Sports, Interdisciplinary Postgrad Program Hlth Sci, Sao Paulo, Brazil Univ Fed Espirito Santo, Hlth Sci Ctr, Dept Nursing, Vitoria, Spain Univ Fed Sao Paulo, Inst Environm Sci Chem & Pharmaceut, Dept Biol Sci, 210 Sao Nicolau St, BR-09913030 Diadema, Brazil FAPESP: 2011/51627-8 FAPESP: 2011/51701-3 Web of Science

A criopreservação de sêmen de Curimba (Prochilodus lineatus) é de importância ecológica e comercial. O objetivo deste estudo foi avaliar o efeito de diferentes concentrações (2, 5, 8 e 11%) de dimetilsulfóxido (DMSO) diluído em Betsville Thawing Solution (BTS) sobre a qualidade do sêmen de Curimba pós-descongelamento. Foram analisadas a taxa e período de motilidade, viabilidade espermática, integridade da membrana plasmática e do DNA, funcionalidade mitocondrial, e taxa de fertilização e eclosão. A membrana plasmática e a integridade do DNA a uma concentração de DMSO de 11% obtiveram melhores resultados que na concentração de 5% (p .

The processes that control the number and shape of the mitochondria (mitochondrial dynamics) and the removal of damaged mitochondria (mitophagy) have been the subject of intense research. Recent work indicates that these processes may contribute to the pathology associated with cardiac diseases. This review describes some of the key proteins that regulate these processes and their potential as therapeutic targets for cardiac diseases.

Mitochondria are central key players in cell metabolism, and mitochondrial DNA (mtDNA) instability has been linked to metabolic changes that contribute to tumorigenesis and to increased expression of pro-tumorigenic genes. Here, we use melanoma cell lines and metastatic melanoma tumors to evaluate the effect of mtDNA alterations and the expression of the mtDNA packaging factor, TFAM, on energetic metabolism and pro-tumorigenic nuclear gene expression changes. We report a positive correlation between mtDNA copy number, glucose consumption, and ATP production in melanoma cell lines. Gene expression analysis reveals a down-regulation of glycolytic enzymes in cell lines and an up-regulation of amino acid metabolism enzymes in melanoma tumors, suggesting that TFAM may shift melanoma fuel utilization from glycolysis towards amino acid metabolism, especially glutamine. Indeed, proliferation assays reveal that TFAM-down melanoma cell lines display a growth arrest in glutamine-free media, emphasizing that these cells rely more on glutamine metabolism than glycolysis. Finally, our data indicate that TFAM correlates to VEGF expression and may contribute to tumorigenesis by triggering a more invasive gene expression signature. Our findings contribute to the understanding of how TFAM affects melanoma cell metabolism, and they provide new insight into the mechanisms by which TFAM and mtDNA copy number influence melanoma tumorigenesis.

Studies have reported the importance of mitochondria in sperm functionality. However, for some species, the glycolytic pathway appears to be as important as oxidative phosphorylation in ATP synthesis and sperm kinetics. These mechanisms have not been fully elucidated for bovine spermatozoa. Therefore, the aim of this study was to evaluate the role of mitochondria and the glycolytic pathway in ATP synthesis, sperm movement patterns, and oxidative homeostasis of epididymal spermatozoa in bovine specimens. We observed that mitochondrial uncoupling with protonophores significantly reduced ATP levels. However, these levels were reestablished after stimulation of the glycolytic pathway. We verified the same pattern of results for sperm kinetic variables and the production of reactive oxygen species (ROS). Thus, we suggest that, after its appropriate stimulation, the glycolytic pathway is capable of maintaining ATP levels, sperm kinetic patterns, and oxidative balance of bovine epididymal spermatozoa submitted to mitochondrial uncoupling.

Cystic Fibrosis (CF) is a disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Previously, we found several genes showing a differential expression in CFDE cells (epithelial cells derived from a CF patient). One corresponded to c-Src; its expression and activity was found increased in CFDE cells, acting as a signaling molecule between the CFTR activity and MUC1 overexpression. Here we report that bronchial IB3-1 cells (CF cells) also showed increased c-Src activity compared to ‘CFTR-corrected’ S9 cells. In addition, three different Caco-2 cell lines, each stably transfected with a different CFTR-specific shRNAs, displayed increased c-Src activity. The IL-1β receptor antagonist IL1RN reduced the c-Src activity of Caco-2/pRS26 cells (expressing a CFTR-specific shRNA). In addition, increased mitochondrial and cellular ROS levels were detected in Caco-2/pRS26 cells. ROS levels were partially reduced by incubation with PP2 (c-Src inhibitor) or IL1RN, and further reduced by using the NOX1/4 inhibitor GKT137831. Thus, IL-1β→c-Src and IL-1β→NOX signaling pathways appear to be responsible for the production of cellular and mitochondrial ROS in CFTR-KD cells. In conclusion, IL-1β constitutes a new step in the CFTR signaling pathway, located upstream of c-Src, which is stimulated in cells with impaired CFTR activity. Fil: Massip Copiz, María Macarena. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; Argentina Fil: Clauzure, Mariangeles. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; Argentina Fil: Valdivieso, Ángel Gabriel. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; Argentina Fil: Santa Coloma, Tomás Antonio. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; Argentina

Many physiological changes occur in response to endurance exercise in order to adapt to the increasing energy needs, mitochondria biogenesis, increased reactive oxygen species (ROS) production and acute inflammatory responses. Mitochondria are organelles within each cell that are crucial for ATP production and are also a major producer of ROS and reactive nitrogen species during intense exercise. Recent evidence shows there is a bidirectional interaction between mitochondria and microbiota. The gut microbiota have been shown to regulate key transcriptional co-activators, transcription factors and enzymes involved in mitochondrial biogenesis such as PGC-1a, SIRT1, and AMPK genes. Furthermore, the gut microbiota and its metabolites, such as short chain fatty acids and secondary bile acids, also contribute to host energy production, ROS modulation and inflammation in the gut by attenuating TNFa- mediated immune responses and inflammasomes such as NLRP3. On the other hand, mitochondria, particularly mitochondrial ROS production, have a crucial role in regulating the gut microbiota via modulating intestinal barrier function and mucosal immune responses. Recently, it has also been shown that genetic variants within the mitochondrial genome, could affect mitochondrial function and therefore the intestinal microbiota composition and activity. Diet is also known to dramatically modulate the composition of the gut microbiota. Therefore, studies targeting the gut microbiota can be useful for managing mitochondrial related ROS production, pro-inflammatory signals and metabolic limits in endurance athletes. Muchos cambios fisiológicos ocurren en respuesta al ejercicio de resistencia para adaptarse a las crecientes necesidades energéticas, la biogénesis de las mitocondrias, el aumento de la producción de especies reactivas de oxígeno (ROS) y las respuestas inflamatorias agudas. Las mitocondrias son orgánulos dentro de cada célula que son cruciales para la producción de ATP y también son un importante productor de ROS y especies reactivas de nitrógeno durante el ejercicio intenso. La evidencia reciente muestra que hay una interacción bidireccional entre las mitocondrias y la microbiota. Se ha demostrado que la microbiota intestinal regula los coactivadores transcripcionales clave, los factores de transcripción y las enzimas implicadas en la biogénesis mitocondrial, como los genes PGC-1a, SIRT1 y AMPK. Además, la microbiota intestinal y sus metabolitos, como los ácidos grasos de cadena corta y los ácidos biliares secundarios, también contribuyen a la producción de energía del huésped, la modulación de ROS y la inflamación en el intestino al atenuar las respuestas inmunes mediadas por TNFa e inflamasomas como NLRP3. Por otro lado, las mitocondrias, particularmente la producción de ROS mitocondrial, tienen un papel crucial en la regulación de la microbiota intestinal mediante la modulación de la función de barrera intestinal y la respuesta inmune de la mucosa. Recientemente, también se ha demostrado que las variantes genéticas dentro del genoma mitocondrial podrían afectar la función mitocondrial y, por lo tanto, la composición y actividad de la microbiota intestinal. También se sabe que la dieta modula drásticamente la composición de la microbiota intestinal. Por lo tanto, los estudios dirigidos a la microbiota intestinal pueden ser útiles para controlar la producción de ROS relacionada con la mitocondria, las señales proinflamatorias y los límites metabólicos en los atletas de resistencia. Molts canvis fisiològics tenen lloc en resposta a l'exercici de resistència per adaptar-se a les creixents necessitats energètiques, la biogènesi de les mitocòndries, l'augment de la producció d'espècies reactives d'oxigen (ROS) i les respostes inflamatòries agudes. Els mitocondris són orgànuls dins de cada cèl·lula que són crucials per a la producció d'ATP i també són un important productor de ROS i espècies reactives de nitrogen durant l'exercici intens. L'evidència recent mostra que hi ha una interacció bidireccional entre les mitocòndries i la microbiota. S'ha demostrat que la microbiota intestinal regula els coactivadors transcripcionals clau, els factors de transcripció i els enzims implicats en la biogènesi mitocondrial, com els gens PGC-1a, SIRT1 i AMPK. A més, la microbiota intestinal i els seus metabòlits, com els àcids grassos de cadena curta i els àcids biliars secundaris, també contribueixen a la producció d'energia de l'hoste, la modulació de ROS i la inflamació a l'intestí a atenuar les respostes immunes intervingudes per TNFa i inflamasomas com NLRP3. D'altra banda, les mitocòndries, particularment la producció de ROS mitocondrial, tenen un paper crucial en la regulació de la microbiota intestinal mitjançant la modulació de la funció de barrera intestinal i la resposta immune de la mucosa. Recentment, també s'ha demostrat que les variants genètiques dins el genoma mitocondrial podrien afectar la funció mitocondrial i, per tant, la composició i activitat de la microbiota intestinal. També se sap que la dieta modula dràsticament la composició de la microbiota intestinal. Per tant, els estudis dirigits a la microbiota intestinal poden ser útils per controlar la producció de ROS relacionada amb la mitocòndria, els senyals proinflamatòries i els límits metabòlics en els atletes de resistència.

[Background]:Leishmaniasis is the world's second deadliest parasitic disease after malaria, and current treatment of the different forms of this disease is far from satisfactory. Alkylphospholipid analogs (APLs) are a family of anticancer drugs that show antileishmanial activity, including the first oral drug (miltefosine) for leishmaniasis and drugs in preclinical/clinical oncology trials, but their precise mechanism of action remains to be elucidated., [Methodology/Principal Findings]:Here we show that the tumor cell apoptosis-inducer edelfosine was the most effective APL, as compared to miltefosine, perifosine and erucylphosphocholine, in killing Leishmania spp. promastigotes and amastigotes as well as tumor cells, as assessed by DNA breakdown determined by flow cytometry. In studies using animal models, we found that orally-administered edelfosine showed a potent in vivo antileishmanial activity and diminished macrophage pro-inflammatory responses. Edelfosine was also able to kill Leishmania axenic amastigotes. Edelfosine was taken up by host macrophages and killed intracellular Leishmania amastigotes in infected macrophages. Edelfosine accumulated in tumor cell mitochondria and Leishmania kinetoplast-mitochondrion, and led to mitochondrial transmembrane potential disruption, and to the successive breakdown of parasite mitochondrial and nuclear DNA. Ectopic expression of Bcl-XL inhibited edelfosine-induced cell death in both Leishmania parasites and tumor cells. We found that the cytotoxic activity of edelfosine against Leishmania parasites and tumor cells was associated with a dramatic recruitment of FOF1-ATP synthase into lipid rafts following edelfosine treatment in both parasites and cancer cells. Raft disruption and specific FOF1-ATP synthase inhibition hindered edelfosine-induced cell death in both Leishmania parasites and tumor cells. Genetic deletion of FOF1-ATP synthase led to edelfosine drug resistance in Saccharomyces cerevisiae yeast., [Conclusions/Significance]:The present study shows that the antileishmanial and anticancer actions of edelfosine share some common signaling processes, with mitochondria and raft-located FOF1-ATP synthase being critical in the killing process, thus identifying novel druggable targets for the treatment of leishmaniasis., This work was supported by grants from the Spanish Ministerio de Economia y Competitividad (SAF2014-59716-R and BIO2014-56930-P), Instituto de Salud Carlos III (RD12/0036/0065 from Red TemaÂtica de Investigación Cooperativa en Cáncer, cofunded by the EU's European Regional Development Fund ± FEDER),European Community's Seventh Framework Programme FP7-2007-2013 (grant HEALTH-F2-2011-256986, PANACREAS), and Spain-UK International Joint Project grant from The Royal Society-CSIC (2004GB0032). CG was supported by the RamoÂn y Cajal Program from the Spanish Ministerio de Ciencia e Innovación.AÂCM was recipient of Formación de Profesorado Universitario predoctoral fellowship from the Spanish Ministerio de Ciencia e Innovación.

Mitochondria supply cellular energy and also perform a role in the adaptation to metabolic stress. In mammals, the ataxia-telangiectasia mutated (ATM) kinase acts as a redox sensor controlling mitochondrial function. Subsequently, transcriptomic and genetic studies were utilized to elucidate the role played by a fungal ATM homolog during carbon starvation. In Aspergillus nidulans, AtmA was shown to control mitochondrial function and glucose uptake. Carbon starvation responses that are regulated by target of rapamycin (TOR) were shown to be AtmA-dependent, including autophagy and hydrolytic enzyme secretion. AtmA also regulated a p53-like transcription factor, XprG, inhibiting starvation-induced XprG-dependent protease secretion and cell death. Thus, AtmA possibly represents a direct or indirect link between mitochondrial stress, metabolism, and growth through the influence of TOR and XprG function. The coordination of cell growth and division with nutrient availability is crucial for all microorganisms to successfully proliferate in a heterogeneous environment. Mitochondria supply cellular energy but also perform a role in the adaptation to metabolic stress and the cross-talk between prosurvival and prodeath pathways. The present study of Aspergillus nidulans demonstrated that AtmA also controlled mitochondrial mass, function, and oxidative phosphorylation, which directly or indirectly influenced glucose uptake. Carbon starvation responses, including autophagy, shifting metabolism to the glyoxylate cycle, and the secretion of carbon scavenging enzymes were AtmA-dependent. Transcriptomic profiling of the carbon starvation response demonstrated how TOR signaling and the retrograde response, which signals mitochondrial dysfunction, were directly or indirectly influenced by AtmA. The AtmA kinase was also shown to influence a p53-like transcription factor, inhibiting starvation-induced XprG-dependent protease secretion and cell death. Therefore, in response to metabolic stress, AtmA appears to perform a role in the regulation of TOR signaling, involving the retrograde and SnfA pathways. Thus, AtmA may represent a link between mitochondrial function and cell cycle or growth, possibly through the influence of the TOR and XprG function.

Cardiolipin, the main anionic phospholipid in the inner mitochondrial membrane, provides shape, charge and osmotic support to this membrane due to its biophysical properties. In addition, it helps form respiratory supercomplexes and provides functionality to mitochondrial proteins. Defects in the biosynthesis or remodeling of cardiolipin have been related to severe diseases, such as Barth syndrome. Opi1p, a transcriptional repressor for most enzymes in phospholipid biosynthesis found in Saccharomyces cerevisiae, has been demonstrated not to affect the biosynthesis of this mitochondrial phospholipid. However, we found that opi1 deletion compromises mitochondrial metabolism producing severe respiratory defects. The mechanism producing this phenotype was explored and found to be a mitochondrial cardiolipin depletion of almost 50%, resulting in low cytochrome content and high mitochondrial DNA instability. The origin of this low cardiolipin content strongly correlated with the overproduction of inositol, an intrinsic phenotype of this mutation. Overall, our results show that adequate regulation of phospholipid synthesis is essential for the maintenance of mitochondrial function.

Mitochondrial cholesterol has been reported to be increased under specific pathological conditions associated with enhanced oxidative stress parameters. In this scenario, cholesterol oxidation would be increased, leading to the production of reactive aldehydes, including cholesterol carboxyaldehyde (ChAld). By using SDS micelles as a mitochondrial mimetic model, we have demonstrated that ChAld covalently modifies cytochrome c (cytc), a protein known to participate in electron transport and apoptosis signaling. This mimetic model induces changes in cytc structure in the same way as mitochondrial membranes do. Tryptic digestion of the cytc-ChAld adduct followed by MALDI-TOF/TOF analyses revealed that modifications occur at Lys residues (K22) localized at cytc site L, a site involved in protein-protein and protein-membrane interactions. Interestingly, ChAld ligation prevented cytc detachment from liposomes even under high ionic strength conditions. Overall, it can be concluded that ChAld ligation to Lys residues at site L creates a hydrophobic tail at cytc, which promotes cytc anchoring to the membrane. Although not investigated in detail in this study, cytc adduction to cholesterol derived aldehydes could have implications in cytc release from mitochondria under apoptotic stimuli.

Fil: Cardinali, Daniel P. Pontificia Universidad Católica Argentina. Facultad de Ciencias Médicas; Argentina Fil: Pagano, Eleonora S. Pontificia Universidad Católica Argentina. Facultad de Ciencias Médicas; Argentina Fil: Scacchi Bernasconi, Pablo A. Pontificia Universidad Católica Argentina. Facultad de Ciencias Médicas; Argentina Fil: Reynoso, Roxana. Pontificia Universidad Católica Argentina. Facultad de Ciencias Médicas; Argentina Fil: Scacchi, Pablo. Pontificia Universidad Católica Argentina. Facultad de Ciencias Médicas; Argentina Abstract: Cell death and survival are critical events for neurodegeneration, mitochondria being increasingly seen as important determinants of both. Mitochondrial dysfunction is considered a major causative factor in Alzheimer’s disease (AD), Parkinson’s disease (PD) and Huntington’s disease (HD). Increased free radical generation, enhanced mitochondrial inducible nitric oxide (NO) synthase activity and NO production, and disrupted electron transport system and mitochondrial permeability transition, have all been involved in impaired mitochondrial function. Melatonin, the major secretory product of the pineal gland, is an antioxidant and an effective protector of mitochondrial bioenergetic function. Both in vitro and in vivo, melatonin was effective to prevent oxidative stress/nitrosative stress-induced mitochondrial dysfunction seen in experimental models of AD, PD and HD. These effects are seen at doses 2-3 orders of magnitude higher than those required to affect sleep and circadian rhythms, both conspicuous targets of melatonin action. Melatonin is selectively taken up by mitochondria, a function not shared by other antioxidants. A limited number of clinical studies indicate that melatonin can improve sleep and circadian rhythm disruption in PD and AD patients. More recently, attention has been focused on the development of potent melatonin analogs with prolonged effects which were employed in clinical trials in sleep-disturbed or depressed patients in doses considerably higher than those employed for melatonin. In view that the relative potencies of the analogs are higher than that of the natural compound, clinical trials employing melatonin in the range of 50-100 mg/day are needed to assess its therapeutic validity in neurodegenerative disorders.

Background Cell senescence is a process of central importance to the understanding of aging as well as to the development of new drugs. It is related with genomic instability, which has been shown to occur in the presence of autophagy deficiency. Yet, the mechanism that triggers genomic instability and senescence from a condition of autophagy deficiency remains unknown. By analyzing the consequences of treating human keratinocytes (HaCaT) with the pentacyclic triterpenoid Betulinic Acid (BA) we were able to propose that cell senescence can develop as a response to parallel damage in the membranes of mitochondria and lysosome. Methods We performed biochemical, immunocytochemical and cytometric assays after challenging HaCaT cells with BA. We also evaluated membrane leakage induced by BA in liposomes and giant unilamellar vesicles. Results By destabilizing lipid bilayers of mitochondria and lysosomes, BA triggers the misbalance in the mitochondrial-lysosomal axis leading to perceived autophagy impairment, lipofuscinogenesis, genomic instability and cell senescence. The progressive accumulation of mitochondria and lipofuscin, which comes from imperfect mitophagy triggered by BA, provides a continuous source of reactive species further damaging lysosomes and leading to cell aging. Conclusions This work reveals that the initial trigger of cell senescence can be the physical damage in the membranes of lysosomes and mitochondria. General significance This concept will help in the search of new drugs that act as senescence-inductors. BA is under evaluation as chemotherapeutic agent against several types of tumors and induction of cell senescence should be considered as one of its main mechanisms of action.

Genetic mitochondrial disorders are responsible for the most common inborn errors of metabolism, caused by mutations in either nuclear genes or in mitochondrial DNA. This article presents the prokaryotic origin of the organelle and the relation between nuclear and mitochondrial genomes, as well as current evolutionary models for such mechanisms. It also addresses the structure of mitochondrial genes, their expression pattern, clinical features of gene defects, risk of transmission and current techniques to avoid these events in assisted human reproduction. Finally, it discusses the ethical implications of these possibilities.RESUMOAs doenças genéticas mitocondriais são responsáveis pelos erros inatos do metabolismo mais comuns, causados por mutações tanto em genes nucleares como no DNA mitocondrial. Este artigo apresenta a origem procariótica dessa organela, e a relação entre os genomas nuclear e mitocondrial, bem como modelos evolutivos correntes para esses mecanismos. Também trata da estrutura dos genes mitocondriais, seu padrão de expressão, características clínicas de defeitos genéticos, riscos de transmissão e técnicas atualmente utilizadas para evitar esses eventos em reprodução humana assistida. Finalmente, discute as implicações éticas dessas possibilidades.

Studies have demonstrated the importance of mitochondria to sperm functionality, as the main source of ATP for cellular homoeostasis and motility. However, the role of mitochondria on sperm metabolism is still controversial. Studies indicate that, for some species, glycolysis may be the main mechanism for sperm energy production. For ram sperm, such pathway is not clear. Thus, we evaluated ram sperm in response to mitochondrial uncoupling and glycolysis inhibition aiming to assess the importance of each pathway for sperm functionality. Statistical analysis was performed by the SAS System for Windows, using the General Linear Model Procedure. Data were tested for residue normality and variance homogeneity. A p

Human metabolic diseases can be mimicked in rodents by using dietary interventions such as high fat diets (HFD). Nonalcoholic fatty liver disease (NAFLD) develops as a result of HFD and the disease may progress in a manner involving increased production of oxidants. The main intracellular source of these oxidants are mitochondria, which are also responsible for lipid metabolism and thus widely recognized as important players in the pathology and progression of steatosis. Here, we review publications that study redox and bioenergetic effects of HFD in the liver. We find that dietary composition and protocol implementations vary widely, as do the results of these dietary interventions. Overall, all HFD promote steatosis, changes in β-oxidation, generation and consequences of oxidants, while effects on body weight, insulin signaling and other bioenergetic parameters are more variable with the experimental models adopted. Our review provides a broad analysis of the bioenergetic and redox changes promoted by HFD as well as suggestions for changes and specifications in methodologies that may help explain apparent disparities in the current literature., Graphical abstract fx1, Highlights • High fat diets (HFDs) induce steatosis, even with no weight changes . • HFDs activate β-oxidation. • HFDs promote oxidative imbalance.

Mutations in the POLG gene, encoding the mitochondrial DNA (mtDNA) polymerase subunit gamma-1, have been identified in severe mtDNA depletion syndromes and mtDNA deletion disorders which include ataxia neuropathy spectrum disorders and AR and AD forms of progressive external ophthalmoplegia (PEO) and PEO-plus disorders. We report on a family with exercise intolerance. The proband was a 50-year-old man with severe muscle pain and premature fatigue after exercise of mild to moderate intensity. Serum CK ranged from 400 to 4500 U/L. Sin financiación 2.969 JCR (2016) Q2, 72/194 Clinical Neurology, 124/259 Neurosciences 1.421 SJR (2016) Q1, 69/380 Neurology (clinical), 32/313 Pediatrics, Perinatology and Child Health; Q2, 39/103 Genetics (clinical), 44/166 Neurology No data IDR 2016 UEM

Producción Científica, The Fas-activated serine/threonine kinase (FASTK) family of proteins has recently emerged as a central regulator of mitochondrial gene expression through the function of an unusual RNA-binding domain named RAP (for RNA-binding domain abundant in Apicomplexans), shared by all six members of the family. Here we describe the role of one of the less characterized members, FASTKD3, in mitochondrial RNA metabolism. First, we show that, in contrast to FASTK, FASTKD2, and FASTKD5, FASTKD3 does not localize in mitochondrial RNA granules, which are sites of processing and maturation of mtRNAs and ribosome biogenesis. Second, we generated FASTKD3 homozygous knock-out cell lines by homologous recombination and observed that the absence of FASTKD3 resulted in increased steady-state levels and half-lives of a subset of mature mitochondrial mRNAs: ND2, ND3, CYTB, COX2, and ATP8/6. No aberrant processing of RNA precursors was observed. Rescue experiments demonstrated that RAP domain is required for FASTKD3 function in mRNA stability. Besides, we describe that FASTKD3 is required for efficient COX1 mRNA translation without altering mRNA levels, which results in a decrease in the steady-state levels of COX1 protein. This finding is associated with reduced mitochondrial complex IV assembly and activity. Our observations suggest that the function of this family of proteins goes beyond RNA processing and ribosome assembly and includes RNA stability and translation regulation within mitochondria., Junta de Castilla y León (grants BIO/ VA20/15 and BIO/VA21/15), Swiss National Science Foundation (grant 310030B_160257/1)

The research leading to these results was funded by the Spanish Ministry of Health (FIS-PI11/00610, FIS-PI041436, FIS-PI081151, FIS-PI042018, and FIS-PI09/02311), the European Union (EU) (FP7-ENV-2011 cod 282957 and HEALTH.2010.2.4.5-1), the Instituto de Salud Carlos III (Red INMA G03/176, CB06/02/0041, FIS-FEDER 03/1615, 04/1509, 04/1112, 04/1931, 05/1079, 05/1052, 06/1213, 07/0314, 09/02647, 11/01007, 11/02591, CP11/00178, FIS-PI06/0867, and FIS-PS09/00090), the Conselleria de Sanitat Generalitat Valenciana, the Generalitat de Catalunya-CIRIT (1999SGR 00241), the Obre Social Cajastur, the Universidad de Oviedo, the Department of Health of the Basque Government (2005111093 and 2009111069), and the Provincial Government of Gipuzkoa (DFG06/004 and DFG08/001). The ENVIRONAGE cohort is supported by the EU Program “Ideas” (ERC-2012-StG 310898) and by the Flemish Fund for Scientific Research (FWO 1516112N and G073315N)., Clemente, D.B.P., Casas, M., Vilahur, N., Begiristain, H., Bustamante, M., Carsin, A., Fernández, M.F., Fierens, F., Gyselaers, W., Iñiguez, C., Janssen, B.G., Lefebvre, W., Llop, S., Olea, N., Pedersen, M., Pieters, N., Marina, L.S., Souto, A., Tardón, A., Vanpoucke, C., Vrijheid, M., Sunyer, J., Nawrot, T.S.

O cobre (Cu) é um metal essencial à vida, porém é toxico quando em altas concentrações. Seus efeitos incluem redução de crescimento e alteração no metabolismo energético. Portanto, o objetivo do presente estudo foi avaliar os efeitos da exposição crônica ao Cu sobre o metabolismo mitocondrial e a regulação do eixo somatotrópico em diferentes tecidos do teleósteo Poecilia vivipara através da avaliação da expressão dos genes que codificam o GH, GHR1, GHR2, IGF1, IGF2, COX I, COXII, COXIII e ATP5A1, bem como através da análise da concentração de GH e GHR. Peixes recém-nascidos (

Producción Científica, Aging is associated to cognitive decline and susceptibility to neuron death, two processes related recently to subcellular Ca2+ homeostasis. Memory storage relies on mushroom spines stability that depends on store-operated Ca2 + entry (SOCE). In addition, Ca2+ transfer from endoplasmic reticulum (ER) to mitochondria sustains energy production but mitochondrial Ca2+ overload promotes apoptosis. We have addressed whether SOCE and ER-mitochondria Ca2+ transfer are influenced by culture time in long-term cultures of rat hippocampal neurons, a model of neuronal aging. We found that short-term cultured neurons show large SOCE, low Ca2+ store content and no functional coupling between ER and mitochondria. In contrast, in long-term cultures reflecting aging neurons, SOCE is essentially lost, Stim1 and Orai1 are downregulated, Ca2+ stores become overloaded, Ca2+ release is enhanced, expression of the mitochondrial Ca2+ uniporter (MCU) increases and most Ca2 + released from the ER is transferred to mitochondria. These results suggest that neuronal aging is associated to increased ER-mitochondrial cross talking and loss of SOCE. This subcellular Ca2+ remodeling might contribute to cognitive decline and susceptibility to neuron cell death in the elderly., Ministerio de Economía, Industria y Competitividad (grants BFU2012-37146 and BFU2015-70131R), Junta de Castilla y León (grants VA145U13 and BIO/VA33/13)

Capybara is the largest rodent in the world and displays a seasonally dependent herbivore feeding behavior. Here, we present an anatomical contribution for understand this fact, by light, scanning, and transmission electron microscopy methodologies for tongue tissue analysis. The histological preparations revealed filiform, fungiform, vallate, and foliate papillae on the dorsal mucosa of the capybara tongue. The epithelial layer exhibited a lining of keratinized stratified squamous epithelial cells. The lamina propria was characterized by a dense connective tissue composed of the primary and secondary papillar projections. We also revealed the original aspects of the connective papillae. The shapes of the papillae varied by region of the tongue, and filiform, fungiform, vallate, and foliate papillae and subjacent layers of muscular fibers were observed. Pyriform taste buds occupying the epithelial layer of fungiform, vallate and foliate papillae were identified and the intracellular components of the taste buds and the intracorpuscular amyelinated nerve fibers were observed. The taste buds were characterized by the distribution of granular endoplasmic reticulum throughout the perinuclear area, the Golgi apparatus, and mitochondrial assemblies of various distinct diameters. Mitochondrial accumulation was also observed in the collagen bundle-surrounded amyelinated nerve fibers beside the basal cells. Therefore, these peculiar anatomical descriptions may contribute to understanding the adaptation of the feeding behavior of capybaras in a seasonally changing environment.

CHCHD10-related diseases include mitochondrial DNA instability disorder, frontotemporal dementia-amyotrophic lateral sclerosis (FTD-ALS) clinical spectrum, late-onset spinal motor neuropathy (SMAJ), and Charcot-Marie-Tooth disease type 2 (CMT2). Here, we show that CHCHD10 resides with mitofilin, CHCHD3 and CHCHD6 within the mitochondrial contact site and cristae organizing system (MICOS) complex. CHCHD10 mutations lead to MICOS complex disassembly and loss of mitochondrial cristae with a decrease in nucleoid number and nucleoid disorganization. Repair of the mitochondrial genome after oxidative stress is impaired in CHCHD10 mutant fibroblasts and this likely explains the accumulation of deleted mtDNA molecules in patient muscle. CHCHD10 mutant fibroblasts are not defective in the delivery of mitochondria to lysosomes suggesting that impaired mitophagy does not contribute to mtDNA instability. Interestingly, the expression of CHCHD10 mutant alleles inhibits apoptosis by preventing cytochrome c release., We acknowledge Pasteur-IRCAN Cellular and Molecular Imaging platform (PICMI). This work was made possible by grants to VP-F from the Fondation pour la Recherche Medicale (FRM) and the Association Francaise contre les Myopathies (AFM), to HS from National Institutes of Health (GM089853) and to J-ER from la Fondation ARC pour la Recherche sur le Cancer. PYWM is supported by a Clinician Scientist Fellowship Award (G1002570) from the Medical Research Council (UK) and also receives funding from Fight for Sight (UK) and the UK National Institute of Health Research (NIHR) as part of the Rare Diseases Translational Research Collaboration. CV-B is supported by the Spanish Instituto de Salud Carlos III (CP11/00046). MP is granted by an FRM fellowship (DEA20130727390).

7,8-Dihydro-8-oxoguanine DNA glycosylase (OGG1) is a major DNA glycosylase involved in base-excision repair (BER) of oxidative DNA damage to nuclear and mitochondrial DNA (mtDNA). We used OGG1-deficient (OGG1−/–) mice to examine the possible roles of OGG1 in the vulnerability of neurons to ischemic and oxidative stress. After exposure of cultured neurons to oxidative and metabolic stress levels of OGG1 in the nucleus were elevated and mitochondria exhibited fragmentation and increased levels of the mitochondrial fission protein dynamin-related protein 1 (Drp1) and reduced membrane potential. Cortical neurons isolated from OGG1−/– mice were more vulnerable to oxidative insults than were OGG1+/+ neurons, and OGG1−/– mice developed larger cortical infarcts and behavioral deficits after permanent middle cerebral artery occlusion compared with OGG1+/+ mice. Accumulations of oxidative DNA base lesions (8-oxoG, FapyAde, and FapyGua) were elevated in response to ischemia in both the ipsilateral and contralateral hemispheres, and to a greater extent in the contralateral cortex of OGG1−/– mice compared with OGG1+/+ mice. Ischemia-induced elevation of 8-oxoG incision activity involved increased levels of a nuclear isoform OGG1, suggesting an adaptive response to oxidative nuclear DNA damage. Thus, OGG1 has a pivotal role in repairing oxidative damage to nuclear DNA under ischemic conditions, thereby reducing brain damage and improving functional outcome.

Aims In the present work we investigated the in vitro effect of cis -4-decenoic acid, the pathognomonic metabolite of medium-chain acyl-CoA dehydrogenase deficiency, on various parameters of bioenergetic homeostasis in rat brain mitochondria. Main methods Respiratory parameters determined by oxygen consumption were evaluated, as well as membrane potential, NAD(P)H content, swelling and cytochrome c release in mitochondrial preparations from rat brain, using glutamate plus malate or succinate as substrates. The activities of citric acid cycle enzymes were also assessed. Key findings cis -4-decenoic acid markedly increased state 4 respiration, whereas state 3 respiration and the respiratory control ratio were decreased. The ADP/O ratio, the mitochondrial membrane potential, the matrix NAD(P)H levels and aconitase activity were also diminished by cis -4-decenoic acid. These data indicate that this fatty acid acts as an uncoupler of oxidative phosphorylation and as a metabolic inhibitor. cis -4-decenoic acid also provoked a marked mitochondrial swelling when either KCl or sucrose was used in the incubation medium and also induced cytochrome c release from mitochondria, suggesting a non-selective permeabilization of the inner mitochondrial membrane. Significance It is therefore presumed that impairment of mitochondrial homeostasis provoked by cis -4-decenoic acid may be involved in the brain dysfunction observed in medium-chain acyl-CoA dehydrogenase deficient patients.

To evaluate the cytotoxicity of PDT (photodynamic therapy) with Photogem associated to blue LED (light-emitting diode) on L929 and MDPC-23 cell cultures, 30000 cells/cm2 were seeded in 24-well plates for 48 h, incubated with Photogem (10, 25 or 50 mg/l) and irradiated with an LED source (460+/-3 nm; 22 mW/cm2) at two energy densities (25.5 or 37.5 J/cm2). Cell metabolism was evaluated by the MTT (methyltetrazolium) assay (Dunnet's post hoc tests) and cell morphology by SEM (scanning electron microscopy). Flow cytometry analysed the type of PDT-induced cell death as well and estimated intracellular production of ROS (reactive oxygen species). There was a statistically significant decrease of mitochondrial activity (90% to 97%) for all Photogem concentrations associated to blue LED, regardless of irradiation time. It was also demonstrated that the mitochondrial activity was not recovered after 12 or 24 h, characterizing irreversible cell damage. PDT-treated cells presented an altered morphology with ill-defined limits. In both cell lines, there was a predominance of necrotic cell death and the presence of Photogem or irradiation increased the intracellular levels of ROS. PDT caused severe toxic effects in normal cell culture, characterized by the reduction of the mitochondrial activity, morphological alterations and induction of necrotic cell death.

Fe2+/ascorbate, hydrogen peroxide (H2O2), and hypoxanthine/xanthine oxidase (XOD) are commonly used for inducing oxidative stress on spermatozoa. A comparative study of these agents was carried out on thawed spermatozoa from red deer. First, we tested a high, medium, and low concentration of each agent: 100, 10, and 1 μM Fe2+ (hydroxyl radical generator); 1 mM, 100, and 10 μM H2O2; and 100, 10, and 1 mU/ml XOD (superoxide and H2O2 generator), incubated at 37 °C for 180 min. Intracellular reactive oxygen species (ROS; H2DCFDA) increased with dose and time similarly for the three systems at each concentration level. Motility and mitochondrial membrane potential (Δψm) were considerably decreased by H2O2 (1 mM and 100 μM) and XOD (100 and 10 mU/ml). Only 1 mM H2O2 reduced viability. The antioxidant Trolox (10 μM) reduced intracellular ROS, but could not prevent the H2O2 or XOD effects. In a second experiment, YO-PRO-1 and M540 were used as apoptotic and membrane stability markers respectively. Only H2O2 increased the proportion of apoptotic and membrane-destabilized spermatozoa. Catalase added to XOD prevented Δψm loss, confirming that H2O2 was the causative agent, not superoxide. In a third experiment, caspase activation was tested using the (FAM-VAD-FMK) probe. Viable spermatozoa with activated caspases could be detected in untreated samples, and only H2O2 increased their proportion after 60 min. There were important differences between ROS generators, H2O2 being the most cytotoxic. Although H2O2 and XOD caused Δψm dissipation, this was not reflected in increasing apoptotic markers.

β-cells quickly adjust insulin secretion to oscillations in nutrients carried by the blood, acting as fuel sensors. However, most studies of β-cell responses to nutrients do not discriminate between fuel levels and signaling components present in the circulation. Here we studied the effect of serum from calorie-restricted rats versus serum from rats fed ad libitum, diluted tenfold in the medium, which did not contribute significantly to the pool of nutrients, on β-cell mitochondrial function and dynamics under regular and high-nutrient culture conditions. Insulin secreting beta-cell derived line (INS1) cells incubated with serum from calorie-restricted rats (CR serum) showed higher levels of peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α) and active nitric oxide synthase. The expression of mitofusin-2 (Mfn-2) and optic atrophy 1 (OPA-1), proteins involved in mitochondrial fusion, was increased, while the levels of the mitochondrial fission mediator dynamin related protein 1 (DRP-1) were reduced. Consistent with changes in mitochondrial dynamics protein levels, CR serum treatment increased mitochondrial fusion rates, as well as their length and connectivity. These changes in mitochondrial morphology were associated with prolonged glucose-stimulated insulin secretion and mitochondrial respiration. When combining CR serum and high levels of glucose and palmitate (20 and 0.4 mm, respectively), an in vitro model of type II diabetes, we observed that signaling promoted by CR serum was enough to overcome glucolipotoxicity, as indicated by CR-mediated prevention of mitochondrial fusion arrest and reduced respiratory function in INS1 cells under glucolipotoxicity. Overall, our results provide evidence that non-nutrient factors in serum have a major impact on β-cell mitochondrial adaptations to changes in metabolism.

Intermittent fasting (IF) is a dietary intervention often used as an alternative to caloric restriction (CR) and characterized by 24 hour cycles alternating ad libitum feeding and fasting. Although the consequences of CR are well studied, the effects of IF on redox status are not. Here, we address the effects of IF on redox state markers in different tissues in order to uncover how changes in feeding frequency alter redox balance in rats. IF rats displayed lower body mass due to decreased energy conversion efficiency. Livers in IF rats presented increased mitochondrial respiratory capacity and enhanced levels of protein carbonyls. Surprisingly, IF animals also presented an increase in oxidative damage in the brain that was not related to changes in mitochondrial bioenergetics. Conversely, IF promoted a substantial protection against oxidative damage in the heart. No difference in mitochondrial bioenergetics or redox homeostasis was observed in skeletal muscles of IF animals. Overall, IF affects redox balance in a tissue-specific manner, leading to redox imbalance in the liver and brain and protection against oxidative damage in the heart.

The role of autophagy in cell death is still controversial and a lot of debate has concerned the transition from its pro-survival to its pro-death roles. The similar structure of the triterpenoids Betulinic (BA) and Oleanolic (OA) acids allowed us to prove that this transition involves parallel damage in mitochondria and lysosome. After treating immortalized human skin keratinocytes (HaCaT) with either BA or OA, we evaluated cell viability, proliferation and mechanism of cell death, function and morphology of mitochondria and lysosomes and the status of the autophagy flux. We also quantified the interactions of BA and OA with membrane mimics, both in-vitro and in-silico. Essentially, OA caused mitochondrial damage that relied on autophagy to rescue cellular homeostasis, which failed upon lysosomal inhibition by Chloroquine or Bafilomycin-A1. BA caused parallel damage on mitochondria and lysosome, turning autophagy into a destructive process. The higher cytotoxicity of BA correlated with its stronger efficiency in damaging membrane mimics. Based on these findings, we underlined the concept that autophagy will turn into a destructive outcome when there is parallel damage in mitochondrial and lysosomal membranes. We trust that this concept will help the development of new drugs against aggressive cancers.