Mitochondrial protective effects caused by the administration of mefenamic acid in sepsis.

The pathophysiology of sepsis may involve the activation of the NOD-type receptor containing the pyrin-3 domain (NLPR-3), mitochondrial and oxidative damages. One of the primary essential oxidation products is 8-oxoguanine (8-oxoG), and its accumulation in mitochondrial DNA (mtDNA) induces cell dysfunction and death, leading to the hypothesis that mtDNA integrity is crucial for maintaining neuronal function during sepsis. In sepsis, the modulation of NLRP-3 activation is critical, and mefenamic acid (MFA) is a potent drug that can reduce inflammasome activity, attenuating the acute cerebral inflammatory process. Thus, this study aimed to evaluate the administration of MFA and its implications for the reduction of inflammatory parameters and mitochondrial damage in animals submitted to polymicrobial sepsis. To test our hypothesis, adult male Wistar rats were submitted to the cecal ligation and perforation (CLP) model for sepsis induction and after receiving an injection of MFA (doses of 10, 30, and 50 mg/kg) or sterile saline (1 mL/kg). At 24 h after sepsis induction, the frontal cortex and hippocampus were dissected to analyze the levels of TNF-α, IL-1ß, and IL-18; oxidative damage (thiobarbituric acid reactive substances (TBARS), carbonyl, and DCF-DA (oxidative parameters); protein expression (mitochondrial transcription factor A (TFAM), NLRP-3, 8-oxoG; Bax, Bcl-2 and (ionized calcium-binding adaptor molecule 1 (IBA-1)); and the activity of mitochondrial respiratory chain complexes. It was observed that the septic group in both structures studied showed an increase in proinflammatory cytokines mediated by increased activity in NLRP-3, with more significant oxidative damage and higher production of reactive oxygen species (ROS) by mitochondria. Damage to mtDNA it was also observed with an increase in 8-oxoG levels and lower levels of TFAM and NGF-1. In addition, this group had an increase in pro-apoptotic proteins and IBA-1 positive cells. However, MFA at doses of 30 and 50 mg/kg decreased inflammasome activity, reduced levels of cytokines and oxidative damage, increased bioenergetic efficacy and reduced production of ROS and 8-oxoG, and increased levels of TFAM, NGF-1, Bcl-2, reducing microglial activation. As a result, it is suggested that MFA induces protection in the central nervous system early after the onset of sepsis.

Load-matched acute and chronic exercise induce changes in mitochondrial biogenesis and metabolic markers.

We investigated the effects of acute and chronic exercise, prescribed in different intensity zones, but with total load-matched on mitochondrial markers (cytochrome C oxidase subunit IV (COX-IV), mitochondrial transcription factor A (Tfam), and citrate synthase (CS) activity in skeletal muscles, heart, and liver), glycogen stores, aerobic capacity, and anaerobic index in swimming rats. For this, 2 experimental designs were performed (acute and chronic efforts). Load-matched exercises were prescribed below, above, and on the anaerobic threshold (AnT), determined by the lactate minimum test. In chronic programs, 2 training prescription strategies were assessed (monotonous and linear periodized model). Results show changes in glycogen stores but no modification in the COX-IV and Tfam contents after acute exercises. In the chronic protocols, COX-IV and Tfam proteins and CS adaptations were intensity- and tissue-dependent. Monotonous training promoted better adaptations than the periodized model. Training at 80% of the AnT improved both performance variables, emphasizing the anaerobic index, concomitant to CS and COX-IV improvement (soleus muscle). The aerobic capacity and CS activity (gastrocnemius) were increased after 120% AnT training. In conclusion, acute exercise protocol did not promote responses in mitochondrial target proteins. An intensity and tissue dependence were reported in the chronic protocols, highlighting training at 80 and 120% of the AnT. Novelty: Load-matched acute exercise did not enhance COX-IV and Tfam contents in skeletal muscles, heart, and liver. In chronic exercise, COX-IV, Tfam, and CS activity adaptations were intensity- and tissue-dependent. Monotonous training was more efficient than the periodized linear model in adaptations of target proteins and enzymatic activity.

NEK5 interacts with LonP1 and its kinase activity is essential for the regulation of mitochondrial functions and mtDNA maintenance.

Little is known about Nima-related kinase (NEKs), a widely conserved family of kinases that have key roles in cell-cycle progression. Nevertheless, it is now clear that multiple NEK family members act in networks, not only to regulate specific events of mitosis, but also to regulate metabolic events independently of the cell cycle. NEK5 was shown to act in centrosome disjunction, caspase-3 regulation, myogenesis, and mitochondrial respiration. Here, we demonstrate that NEK5 interacts with LonP1, an AAA+ mitochondrial protease implicated in protein quality control and mtDNA remodeling, within the mitochondria and it might be involved in the LonP1-TFAM signaling module. Moreover, we demonstrate that NEK5 kinase activity is required for maintaining mitochondrial mass and functionality and mtDNA integrity after oxidative damage. Taken together, these results show a new role of NEK5 in the regulation of mitochondrial homeostasis and mtDNA maintenance, possibly due to its interaction with key mitochondrial proteins, such as LonP1.

HEK293T Cells with TFAM Disruption by CRISPR-Cas9 as a Model for Mitochondrial Regulation.

The mitochondrial transcription factor A (TFAM) is considered a key factor in mitochondrial DNA (mtDNA) copy number. Given that the regulation of active copies of mtDNA is still not fully understood, we investigated the effects of CRISPR-Cas9 gene editing of TFAM in human embryonic kidney (HEK) 293T cells on mtDNA copy number. The aim of this study was to generate a new in vitro model by CRISPR-Cas9 system by editing the TFAM locus in HEK293T cells. Among the resulting single-cell clones, seven had high mutation rates (67-96%) and showed a decrease in mtDNA copy number compared to control. Cell staining with Mitotracker Red showed a reduction in fluorescence in the edited cells compared to the non-edited cells. Our findings suggest that the mtDNA copy number is directly related to TFAM control and its disruption results in interference with mitochondrial stability and maintenance.

Mitochondria Transcription Factor A: A Putative Target for the Effect of Melatonin on U87MG Malignant Glioma Cell Line.

The disruption of mitochondrial activity has been associated with cancer development because it contributes to regulating apoptosis and is the main source of reactive oxygen species (ROS) production. Mitochondrial transcription factor A (TFAM) is a protein that maintains mitochondrial DNA (mtDNA) integrity, and alterations in its expression are associated with mitochondrial damage and cancer development. In addition, studies have shown that mitochondria are a known target of melatonin, the pineal gland hormone that plays an important anti-tumorigenic role. Thus, we hypothesized that melatonin decreases the expression of TFAM (RNA and protein) in the human glioblastoma cell line U87MG, which disrupts mtDNA expression and results in cell death due to increased ROS production and mitochondrial damage. Our results confirm the hypothesis, and also show that melatonin reduced the expression of other mitochondrial transcription factors mRNA (TFB1M and TFB2M) and interfered with mtDNA transcription. Moreover, melatonin delayed cell cycle progression and potentiated the reduction of cell survival due to treatment with the chemotherapeutic agent temozolomide. In conclusion, elucidating the effect of melatonin on TFAM expression should help to understand the signaling pathways involved in glioblastoma progression, and melatonin could be potentially applied in the treatment of this type of brain tumor.

Association of a TFAM haplotype with aggressive prostate cancer in overweight or obese Mexican Mestizo men.

BACKGROUND: Mitochondrial dysfunction has been associated with the development of cancer and obesity, being prostate cancer more aggressive in obese men. It has been suggested that the mitochondrial transcription factor A (TFAM) plays a central role in these events. OBJECTIVE: The aim of this study was to analyze the possible association of 3 TFAM polymorphisms, as well as their haplotypes, with the development of aggressive prostate cancer in overweight or obese Mexican Mestizo men. SUBJECTS AND METHODS: A total of 257 unrelated men with histologically confirmed prostate cancer, of Mexican Mestizo ethnic origin, were included. Body mass index was determined and the degree of prostate cancer aggressiveness was demarcated by the D'Amico classification. DNA was obtained from blood leukocytes. The rs1937, rs1049432, and rs11006132, as well as their haplotypes, were studied by real-time polymerase chain reaction allelic discrimination. Deviations from Hardy-Weinberg equilibrium were tested. Pairwise linkage disequilibrium between single nucleotide polymorphisms was calculated; haplotype analysis was performed. RESULTS: A higher risk (D'Amico classification) was documented in 56 patients (21.8%). We did not find a significant association among those polymorphisms analyzed; however, one haplotype was significantly associated with cancer aggressiveness. CONCLUSIONS: To our knowledge, this constitutes the first study regarding the relationship of 3 TFAM polymorphisms, as well as their haplotypes, and the aggressiveness of prostate cancer in overweight or obese men; the most frequent haplotype was associated with cancer aggressiveness.

Brain Region Specificity of Mitochondrial Biogenesis and Bioenergetics Response to Nrf2 Knockdown: A Comparison Among Hippocampus, Prefrontal Cortex and Amygdala of Male Rat Brain

ABSTRACT Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) has been identified as the well-known coordinator of intracellular antioxidant defense system. Herein, we aimed to evaluate the effects of Nrf2 silencing on mitochondrial biogenesis markers peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), nuclear respiratory factor-1(NRF-1), mitochondrial transcription factor A (TFAM) and cytochrome c as well activities of two enzymes citrate synthase (CS) and malate dehydrogenase (MDH) in three brain regions hippocampus, amygdala, and prefrontal cortex of male Wistar rats. Small interfering RNA (siRNA) targeting Nrf2 was injected in dorsal third ventricle. Next, western blot analysis and biochemical assays were used to evaluation of protein level of mitochondrial biogenesis factors and CS and MDH enzymes activity, respectively. Based on findings, whilst Nrf2-silencing led to notably reduction in protein level of mitochondrial biogenesis upstream PGC-1α in three brain regions compared to the control rats, the level of NRF-1, TFAM and cytochrome c remained unchanged. Furthermore, although Nrf2 silencing increased CS activity, activity of MDH significantly decreased in hippocampus and prefrontal cortex areas. Interestingly, CS and MDH activities in amygdala did not change after Nrf2 knockdown. In conclusion, the present findings highlighted complexity of interaction of Nrf2 and mitochondrial functions in a brain region-specific manner. However, by outlining the exact interaction between Nrf2 and mitochondria, it would be possible to find a new therapeutic strategies for neurological disorders related to oxidative stress.

Alterações metabólicas e o papel da mitocôndria no processo de tumorigênese de astrocitomas humanos / Metabolic alterations and the role of mitochondria in tumorigenic process of human astrocytomas

As mitocôndrias desempenham um papel fundamental na sobrevivência e morte celular. Alterações do DNA mitocondrial (DNAmt) - como, por exemplo, amplificação, mutação homoplásmica, deleção e depleção, bem como suas implicações clínico-patológicas, tem sido analisadas em inúmeras neoplasias humanas. No intuito de se pesquisar alterações mitocondriais associadas à tumorigênese, o presente trabalho teve como objetivos analisar a expressão de genes implicados no metabolismo energético e envolvidos na replicação e transcrição mitocondriais, quantificar o número de organelas mitocondriais e de cópias de DNAmt e analisar a expressão dos genes em astrocitomas de diferentes graus de malignidade (23 OMS grau I, 26 grau II, 18 grau III e 84 grau IV ou GBM) em relação ao tecido cerebral não tumoral (22 amostras). As expressões relativas dos genes selecionados, bem como as quantificações relativa e absoluta do DNA mitocondrial, foram realizadas por PCR em tempo real. O aumento de expressão relativa de genes-chave da via glicolítica, alterações nos níveis de expressão dos genes do ciclo dos ácidos tricarboxílicos e hipoexpressão de genes da fosforilação oxidativa detectados corroboraram o efeito Warburg. Foi demonstrado que a redução do número de cópias do DNAmt está associada com o grau de malignidade dos astrocitomas difusamente infiltrativos, sendo GBM o mais depletado e independente do número de organelas. As médias observadas para tecido não tumoral, astrocitoma grau I, grau II, grau III e GBM foram, respectivamente, 1,28, 0,26, 0,45, 0,42 e 0,17. Níveis aumentados de expressão relativa dos genes dos fatores de transcrição mitocondriais A (TFAM), B1 (TFB1M), B2 (TFB2M) e da subunidade catalítica da polimerase mitocondrial (POLG) foram detectados em todos os graus de astrocitomas, exceto TFB2M em astrocitoma grau II. Embora exista forte correlação entre os fatores de transcrição mitocondriais, somente os níveis de expressão de POLG se correlacionaram inversamente...

Mitochondria has a key role in cell survival and death. Mitochondrial DNA (mtDNA) alterations, for example, amplification, homoplasmic mutation, deletion and depletion, and their clinical and pathological implications have been analyzed in human malignancies. In order to search for mitochondrial alterations associated to tumorigenesis, this study aimed to analyze the expression levels of genes involved in energetic metabolism, and in mitochondrial replication and transcription, to quantify the number of mitochondrial organelle and mtDNA copy number in astrocytomas of different grades of malignancy (23 WHO grade I, 26 grade II, 18 grade III and 84 grade IV or GBM) related to non-neoplastic brain tissue (22 samples). The relative expression level of the selected genes as well as the relative and absolute quantification of mtDNA were performed by real-time PCR. Relative expression increase of glycolytic pathway key genes, change of citric acid cycle genes and hipoexpression of oxidative phosphorylation genes were detected, and confirmed the presence of Warburg effect. The reduced mtDNA copy number was associated to the grade of malignancy of diffusely infiltrating astrocytoma, being GBM the most depleted, and not related to parallel decrease in the number of organelle. The mean mtDNA copy number for non neoplastic tissue, astrocytoma grade I, grade II, grade III and GBM were respectively 1.28, 0.26, 0.45, 0.42 and 0.17. The increased relative gene expression of mitochondrial transcription factor A (TFAM), B1 (TFB1M), B2 (TFB2M) and the catalytic subunit of mitochondrial polymerase (POLG) were observed in all grades of astrocytoma, except TFB2M in grade II astrocytoma. Although a strong correlation was observed among the mitochondrial transcription factors, only the expression level of POLG correlated inversely to the mtDNA copy number. The overexpression of TFAM was associated with long-term survival in the GBM patients and interpreted as compensatory...