Changes In Mitochondrial Mass Research Articles

Application of Flow Cytometric Analysis for Measuring Multiple Mitochondrial Parameters in 3D Brain Organoids.

Mitochondrial dysfunction is a common primary or secondary contributor to many types of neurodegeneration, and changes in mitochondrial mass, mitochondrial respiratory chain (MRC) complexes, and mitochondrial DNA (mtDNA) copy number often feature in these processes. Human brain organoids derived from human induced pluripotent stem cells (iPSCs) recapitulate the brain's three-dimensional (3D) cytoarchitectural arrangement and offer the possibility to study disease mechanisms and screen new therapeutics in a complex human system. Here, we report a unique flow cytometry-based approach to measure multiple mitochondrial parameters in iPSC-derived cortical organoids. This report details a protocol for generating cortical brain organoids from iPSCs, single-cell dissociation of generated organoids, fixation, staining, and subsequent flow cytometric analysis to assess multiple mitochondrial parameters. Double staining with antibodies against the MRC complex subunit NADH: Ubiquinone Oxidoreductase Subunit B10 (NDUFB10) or mitochondrial transcription factor A (TFAM) together with voltage-dependent anion-selective channel 1 (VDAC 1) permits assessment of the amount of these proteins per mitochondrion. Since the quantity of TFAM corresponds to the amount of mtDNA, it provides an indirect estimation of the number of mtDNA copies per mitochondrial content. This entire procedure can be completed within a span of 2-3 h. Crucially, it allows for the concurrent quantification of multiple mitochondrial parameters, including both total and specific levels relative to the mitochondrial mass.

The αMSH-Dependent PI3K Pathway Supports Energy Metabolism, via Glucose Uptake, in Melanoma Cells.

Stimulation of melanocytes and murine melanoma cells with αMSH plus the PI3K inhibitor LY294002 resulted in ROS increase, oxidative DNA damage, and pigment retention. We performed cellular and molecular biology assays (Western blot, FACS, immunofluorescence analysis, scratch assay) on murine and human melanoma cells. Treatment with αMSH plus LY294002 altered cortical actin architecture. Given that cytoskeleton integrity requires energy, we next evaluated ATP levels and we observed a drop in ATP after exposure to αMSH plus LY294002. To evaluate if the αMSH-activated PI3K pathway could modulate energy metabolism, we focused on glucose uptake by analyzing the expression of the Glut-1 glucose translocator. Compared with cells treated with αMSH alone, those exposed to combined treatment showed a reduction of Glut-1 on the plasma membrane. This metabolic alteration was associated with changes in mitochondrial mass. A significant decrease of the cell migratory potential was also observed. We demonstrated that the αMSH-dependent PI3K pathway acts as a regulator of energy metabolism via glucose uptake, influencing the actin cytoskeleton, which is involved in melanosome release and cell motility. Hence, these results could constitute the basis for innovative therapeutical strategies.

Exposure of combination of environmental pollutant, lead (Pb) and β-amyloid peptides causes mitochondrial dysfunction and oxidative stress in human neuronal cells.

Exposure to the environmental pollutant lead (Pb) has been linked to Alzheimer's disease (AD), in which mitochondrial dysfunction is a pathological consequence of neuronal degeneration. The toxicity of Pb in combination with β-amyloid peptides (1-40) and (25-35) causes selective death in neuronal cells. However, the precise mechanism through which Pb induces Alzheimer's disease, particularly mitochondrial damage, is unknown. Changes in mitochondrial mass, membrane potential, mitochondrial complex activities, mitochondrial DNA and oxidative stress were examined in neuronal cells of human origin exposed to Pb and β-amyloid peptides (1-40) and (25-35) individually and in different combinations. The results showed depolarization of mitochondrial membrane potential, decrease in mitochondrial mass, ATP levels and mtDNA copy number in Pb and β-amyloid peptides (1-40) and (25-35) exposed cells. Also, significant reductions in the expression of mitochondrial electron transport chain (ETC) complex proteins (ATP5A, COXIV, UQCRC2, SDHB, NDUFS3), as well as down regulation of ETC complex gene expressions such as COXIV, ATP5F1 and NDUFS3 and antioxidant gene expressions like MnSOD and Gpx4 were observed in exposed cells. Furthermore, Pb and β-amyloid peptides exposure resulted in elevated mitochondrial malondialdehyde levels and a decrease in mitochondrial GSH levels. Our findings suggest that Pb toxicity could be one of the causative factors for the mitochondrial dysfunction and oxidative stress in Alzheimer's disease progression.

Deletion of AMPK Reduces Oxidative Metabolism in Gvhd-Causing Human T Cells

BACKGROUND Allogeneic hematopoietic stem cell transplantation (AlloHSCT) is a curative therapy for many hematological disorders including primary immunodeficiencies and relapsed/refractory leukemia. However, the therapeutic application of AlloHSCT is limited by acute graft-versus-host-disease (GVHD), where donor T cells recognize and destroy recipient tissues in the skin, liver, and gastrointestinal tract. GVHD occurs following 30-50% of allogeneic transplants and is a leading cause of non-relapse morbidity and mortality. Recent efforts to treat GVHD have improved but attempts to prevent GVHD remain an important area of ongoing investigation. Following transplantation, alloreactive T cells exhibit heightened energy requirements that present a promising area for therapeutic intervention. Previously, we have shown that murine T cells upregulate activity of the cellular energy sensor AMPK-activated protein kinase (AMPK) during GVHD initiation and furthermore, that transfer of AMPK-deficient T cells increased recipient survival while preserving leukemia clearance and immune reconstitution. Whether these findings translate to human cells is unknown. Here, we report successful targeting of AMPK in primary human T cells and demonstrate clear similarities in phenotype between murine and human T cells lacking AMPK. METHODS To delete AMPK, primary human T cells were isolated from healthy donors and electroporated with Cas 9 ribonuclear protein complexes targeting the AMPKa1 locus. Deletion of AMPK was assessed via DNA sequencing (TIDE analysis) and phosphorylation of downstream target Unc-51-like kinase (ULK1) by immunoblot. Oxidative metabolism and spare respiratory capacity (SRC) were determined via extracellular flux analysis (Agilent Seahorse). Mitochondrial transmembrane potential (TMRM) and cell phenotype (mitochondrial density, apoptosis) were evaluated by flow cytometry after cells stimulation. Intracellular cytokine expression was assessed following 72 hours in a mixed lymphocyte reaction (MLR), followed by re-stimulation with PMA/Ionomycin in the presence of Brefeldin A. AMPK-deficient murine T cells from AMPKa1/a2 dKO C57BL/6J mice were transplanted into allogeneic B6D2F1 recipients for 7 days for comparative analysis. RESULTS CRISPR treatment of primary human T cells efficiently deleted AMPK as measured by both DNA sequencing (92.3% insertion/deletion frequency by TIDE analysis) and immunoblot analysis of downstream ULK1 phosphorylation. When re-stimulated in physiological glucose (5.5 mM), AMPK-deficient human T cells significantly decreased mitochondrial respiration (Fig. 1A) as measured by both maximal oxygen consumption (174.6 ± 27.73 vs 277.3 ± 22.88, p<0.0001) and spare respiratory capacity (SRC, 126.9 ± 22.78 vs 207.1 ± 19.68, p<0.0001). This result was strikingly similar to the metabolism of post-transplant murine T cells lacking AMPK, which exhibited a proportional decrease in SRC (-6.499 ± 5.156 vs 29.11 ± 7.064, p<0.0001) and maximal oxygen consumption (46.82 ± 10.11 vs 80.01 ± 6.946, p<0.0001). AMPK-deficient human T cells also experienced decreased cellular viability (43.4% vs 51.8%) as well as limited mitochondrial membrane potential (Fig. 1B); however, no differences in mitochondrial density were observed. Upon co-culture with allogeneic antigen presenting cells, AMPK-deficient human CD4+ T cells produced lower levels of the pro-inflammatory cytokines TNFa (97.7 MFI vs 209 MFI) and IL-2 ( 391 vs 454 MFI). DISCUSSION Here, we demonstrate a method to effectively delete AMPK in human T cells, a modification which decreases oxidative metabolism identical to what is observed in AMPK-deficient murine T cells. Specifically, human T cells lacking AMPK decreased their maximal mitochondrial respiration and spare respiratory capacity, a result we attribute to decreased mitochondrial transmembrane potential without changes in mitochondrial mass. Together with decreased pro-inflammatory cytokine production, we predict these changes will limit the viability and function of alloreactive T cells in vivo. As such, future studies will assess the disease potential of AMPK-deficient human T cells in a modified model of xenogeneic GVHD. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Regulation of Adipocyte Metabolic Homeostasis by SR‐BI and PCPE2

Obesity continues to be an epidemic in the United States, placing individuals at a higher risk of developing other comorbidities such as type 2 diabetes and cardiovascular disease. Adipocytes accumulate excess triglycerides and undergo hypertrophic expansion as a protective mechanism to avoid lipotoxicity. Adipocytes are also highly dynamic cells that perform many key metabolic processes including lipid and glucose metabolism. Recent collaborative studies demonstrated that SR‐BI function may require the extracellular matrix protein procollagen C‐endopeptidase enhancer 2 (PCPE2) to regulate cholesterol transport in hepatocytes and adipocytes. However, the roles of SR‐BI and PCPE2 in other adipocyte metabolic processes and whether they maintain their cooperative partnership for these functions remains unknown. This knowledge gap prompted our novel hypothesis that PCPE2 facilitates SR‐BI’s ability to regulate metabolic processes in adipocytes. To test our hypothesis, we have optimized an adipocyte model system where mesenchymal stem cells isolated from the outer ears of wild‐type (WT), SR‐BI knockout (SR‐BI‐/‐), and PCPE2‐/‐ mice are differentiated into adipocyte‐like cells. We validated our adipocyte model system by demonstrating increased Oil‐Red‐O staining, mRNA expression of adipogenesis markers, and adiponectin secretion over a 9‐day post‐differentiation period for all three genotypes. This model system was then used to test the roles of SR‐BI and PCPE2 on various metabolic functions, including glucose, fatty acid, and mitochondrial metabolism. Preliminary data suggest that loss of SR‐BI and PCPE2 impaired glucose uptake, despite observing elevated levels of GLUT4 at the cell surface. PCPE2‐/‐ adipocytes also showed increased response to insulin through AKT signaling, which may be responsible for the increased expression of GLUT4 at the cell surface. However, we observed that loss of SR‐BI or PCPE2 had no effect on transport of palmitic acid in adipocytes, despite there being elevated expression of fatty acid transporters (FABP4, CD36). Adipocytes lacking SR‐BI or PCPE2 showed an inverse response to insulin compared to WT adipocytes when measuring phosphorylation status of proteins responsible for mediating fatty acid synthesis (AMPK, ACC). These data suggest that although SR‐BI and PCPE2 do not appear to mediate uptake of palmitic acid, they may have altered lipid metabolism. Lastly, adipocytes lacking SR‐BI or PCPE2 exhibit changes in mitochondrial mass and morphology, but their role in maintaining healthy mitochondria remains inconclusive. Altogether, these data suggest that SR‐BI and PCPE2 appear to have independent roles in various steps of adipocyte metabolism, but their required partnership remains unclear.

Characterizing Extracellular Vesicles and Particles Derived from Skeletal Muscle Myoblasts and Myotubes and the Effect of Acute Contractile Activity.

Extracellular vesicles (EVs), released from all cells, are essential to cellular communication and contain biomolecular cargo that can affect recipient cell function. Studies on the effects of contractile activity (exercise) on EVs usually rely on plasma/serum-based assessments, which contain EVs from many different cells. To specifically characterize skeletal muscle–derived vesicles and the effect of acute contractile activity, we used an in vitro model where C2C12 mouse myoblasts were differentiated to form myotubes. EVs were isolated from conditioned media from muscle cells at pre-differentiation (myoblasts) and post-differentiation (myotubes) and also from acutely stimulated myotubes (1 h @ 14 V, C-Pace EM, IonOptix, Westwood, MA, USA) using total exosome isolation reagent (TEI, ThermoFisher (Waltham, MA, USA), referred to as extracellular particles [EPs]) and differential ultracentrifugation (dUC; EVs). Myotube-EPs (~98 nm) were 41% smaller than myoblast-EPs (~167 nm, p < 0.001, n = 8–10). Two-way ANOVA showed a significant main effect for the size distribution of myotube vs. myoblast-EPs (p < 0.01, n = 10–13). In comparison, myoblast-EPs displayed a bimodal size distribution profile with peaks at <200 nm and 400–600, whereas myotube-Eps were largely 50–300 nm in size. Total protein yield from myotube-EPs was nearly 15-fold higher than from the myoblast-EPs, (p < 0.001 n = 6–9). Similar biophysical characteristics were observed when EVs were isolated using dUC: myotube-EVs (~195 nm) remained 41% smaller in average size than myoblast-EVs (~330 nm, p = 0.07, n = 4–6) and had comparable size distribution profiles to EPs isolated via TEI. Myotube-EVs also had 4.7-fold higher protein yield vs. myoblast EVs (p < 0.05, n = 4–6). Myotube-EPs exhibited significantly decreased expression of exosomal marker proteins TSG101, CD63, ALIX and CD81 compared with myoblast-EPs (p < 0.05, n = 7–12). Conversely, microvesicle marker ARF6 and lipoprotein marker APO-A1 were only found in the myotube-EPs (p < 0.05, n = 4–12). There was no effect of acute stimulation on myotube-EP biophysical characteristics (n = 7) or on the expression of TSG101, ARF6 or CD81 (n = 5–6). Myoblasts treated with control or acute stimulation–derived EPs (13 µg/well) for 48 h and 72 h showed no changes in mitochondrial mass (MitoTracker Red, ThermoFisher, Waltham, MA, USA), cell viability or cell count (n = 3–4). Myoblasts treated with EP-depleted media (72 h) exhibited ~90% lower cell counts (p < 0.01, n = 3). Our data show that EVs differed in size, distribution, protein yield and expression of subtype markers pre vs. post skeletal muscle–differentiation into myotubes. There was no effect of acute stimulation on biophysical profile or protein markers in EPs. Acute stimulation–derived EPs did not alter mitochondrial mass or cell count/viability. Further investigation into the effects of chronic contractile activity on the biophysical characteristics and cargo of skeletal muscle–specific EVs are warranted.

Reprogramming of mitochondria by proteolysis

Mitochondria are central metabolic hubs whose structure and function dynamically adapt to changing metabolic demands and environmental challenges. Metabolic reprogramming of mitochondria occurs during development, cell differentiation, in ageing and disease and is coupled to changes in mitochondrial mass and shape. Similarly, stress conditions and altered metabolic cues induce mitochondrial adaptations, altering proteostasis and lipid homeostasis of mitochondria. ATP-dependent AAA proteases residing in the inner membrane of mitochondria are emerging as central regulators of these adaptive responses.

Simulated Galactic Cosmic Rays Modify Mitochondrial Metabolism in Osteoclasts, Increase Osteoclastogenesis and Cause Trabecular Bone Loss in Mice.

Space is a high-stress environment. One major risk factor for the astronauts when they leave the Earth’s magnetic field is exposure to ionizing radiation from galactic cosmic rays (GCR). Several adverse changes occur in mammalian anatomy and physiology in space, including bone loss. In this study, we assessed the effects of simplified GCR exposure on skeletal health in vivo. Three months following exposure to 0.5 Gy total body simulated GCR, blood, bone marrow and tissue were collected from 9 months old male mice. The key findings from our cell and tissue analysis are (1) GCR induced femoral trabecular bone loss in adult mice but had no effect on spinal trabecular bone. (2) GCR increased circulating osteoclast differentiation markers and osteoclast formation but did not alter new bone formation or osteoblast differentiation. (3) Steady-state levels of mitochondrial reactive oxygen species, mitochondrial and non-mitochondrial respiration were increased without any changes in mitochondrial mass in pre-osteoclasts after GCR exposure. (4) Alterations in substrate utilization following GCR exposure in pre-osteoclasts suggested a metabolic rewiring of mitochondria. Taken together, targeting radiation-mediated mitochondrial metabolic reprogramming of osteoclasts could be speculated as a viable therapeutic strategy for space travel induced bone loss.

Improvement in mitochondrial oxidative phosphorylation of cardiomyocytes derived from human-induced pluripotent stem cells using micropatterned anisotropic substrates

Abstract Background/Introduction Alterations in cellular bioenergetic events owing to metabolic dysfunctions are well demonstrated in heart failure (HF). Cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CMs) have the potential to model metabolic alterations as observed in the human failing hearts, which however requires precise arrangements of the respiratory chain complexes. Purpose Develop a method to improve the hiPSC-CMs architecture by using a micropatterned substrate (repeated lines of 30 μm-width separated with a cell repellant) that promotes elongation and anisotropy. Methods Cardiomyocyte differentiation was performed using a chemically defined method and 35 days later the generated hiPSC-CMs were cultured on unpatterned vs. micropatterned substrates for 7 additional days. Energy metabolic profiles were then measured using the Seahorse analyzer and mitochondrial components were analyzed by fluorescence microscopy and western blot. Results Cultures on micropatterned anisotropic substrates resulted in the generation of elongated cell morphology, rod-shaped hiPSC-CMs with improved sarcomere organization scores, and more aligned Z-lines. We first found a specific increase in oxidation phosphorylation (OXPHOS) activity in micropatterned CMs with a significant increase in the oxygen consumption rate (OCR) linked to basal respiration (58.4±24.0 vs 40.6±21.0 pmol/min, p&amp;lt;0.01), ATP production (49.6±19.1 vs 33.2±18.8 pmol/min, p&amp;lt;0.001) and maximal respiration (239.1±119.0 vs 123.0±68.5 pmol/min, p&amp;lt;0.001). Micropatterned CMs showed a better electron transport chain activity that explains the significantly decreased glycolytic function observed by Seahorse (17.2±9.9 vs 23.9±7.2 mpH/min, p&amp;lt;0.001). Micropatterned CMs displayed a more organized mitochondrial network, without evident change in mitochondrial mass. Transcriptomic profiling of micropatterned vs. unpatterned CMs revealed no significant differences. We next evaluated the proteomic profiling and found no significant change in the micropatterned CMs OXPHOS proteins expression. Conclusion(s) We developed a novel method for modeling metabolic activity in hiPSC-CMs based on micropatterned cultures. Our data show that the linear architectural pattern drives the formation and efficiency of OXPHOS function in cardiomyocytes with a mitochondrial structural reorganization. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Agence Nationale de la Recherche

Spatiotemporal AMPK\u03b12 deletion in mice induces cardiac dysfunction, fibrosis and cardiolipin remodeling associated with mitochondrial dysfunction in males only

BackgroundThe AMP-activated protein kinase (AMPK) is a major regulator of cellular energetics which plays key role in acute metabolic response and in long-term adaptation to stress. Recent works have also suggested non-metabolic effects.MethodsTo decipher AMPK roles in the heart, we generated a cardio-specific inducible model of gene deletion of the main cardiac catalytic subunit of AMPK (Ampkα2) in mice. This allowed us to avoid the eventual impact of AMPK-KO in peripheral organs.ResultsCardio-specific Ampkα2 deficiency led to a progressive left ventricular systolic dysfunction and the development of cardiac fibrosis in males. We observed a reduction in complex I-driven respiration without change in mitochondrial mass or in vitro complex I activity, associated with a rearrangement of the cardiolipins and reduced integration of complex I into the electron transport chain supercomplexes. Strikingly, none of these defects were present in females. Interestingly, suppression of estradiol signaling by ovariectomy partially mimicked the male sensitivity to AMPK loss, notably the cardiac fibrosis and the rearrangement of cardiolipins, but not the cardiac function that remained protected.ConclusionOur results confirm the close link between AMPK and cardiac mitochondrial function, but also highlight links with cardiac fibrosis. Importantly, we show that AMPK is differently involved in these processes in males and females, which may have clinical implications for the use of AMPK activators in the treatment of heart failure.

A 1-Month Ketogenic Diet Increased Mitochondrial Mass in Red Gastrocnemius Muscle, but Not in the Brain or Liver of Middle-Aged Mice.

Alterations in markers of mitochondrial content with ketogenic diets (KD) have been reported in tissues of rodents, but morphological quantification of mitochondrial mass using transmission electron microscopy (TEM), the gold standard for mitochondrial quantification, is needed to further validate these findings and look at specific regions of interest within a tissue. In this study, red gastrocnemius muscle, the prefrontal cortex, the hippocampus, and the liver left lobe were used to investigate the impact of a 1-month KD on mitochondrial content in healthy middle-aged mice. The results showed that in red gastrocnemius muscle, the fractional area of both subsarcolemmal (SSM) and intermyofibrillar (IMM) mitochondria was increased, and this was driven by an increase in the number of mitochondria. Mitochondrial fractional area or number was not altered in the liver, prefrontal cortex, or hippocampus following 1 month of a KD. These results demonstrate tissue-specific changes in mitochondrial mass with a short-term KD and highlight the need to study different muscle groups or tissue regions with TEM to thoroughly determine the effects of a KD on mitochondrial mass.

Improvement in mitochondrial oxidative phosphorylation of cardiomyocytes derived from human-induced pluripotent stem cells using micropatterned anisotropic substrates

Cardiomyocytes derived from human-induced pluripotent stem cells (iPS-CMs) have the potential to model metabolic alterations observed in the human failing hearts, this however requires precise arrangements of the respiratory chain complexes. Improve iPS-CMs architecture by using a micropatterned anisotropic substrate (repeated lines of 30 μm-width separated with a cell repellant) Cardiomyocyte differentiation was performed using a chemically defined method. Thirty-five days later the generated iPS-CMs were cultured on unpatterned vs. micropatterned substrates for 7 days. Energy metabolic profiles were measured using the Seahorse analyzer and mitochondrial components analyzed by fluorescence microscopy and western blot. Micropatterned culture resulted in the generation of elongated, rod-shape iPS-CMs with improved sarcomere organization score and more aligned Z-lines. We found a specific increase in oxidation phosphorylation (OXPHOS) activity in micropatterned CMs with significant increase in the oxygen consumption rate linked to basal respiration (58.4 ± 24.0 vs. 40.6 ± 21.0 pmol/min, P < 0.01), ATP production (49.6 ± 19.1 vs. 33.2 ± 18.8 pmol/min, P < 0.001) and maximal respiration (239.1 ± 119.0 vs. 123.0 ± 68.5 pmol/min, P < 0.001). Micropatterned CMs displayed a more organized mitochondrial network, without evident change in mitochondrial mass. A transcriptomic profiling of micropatterned vs. unpatterned CMs revealed no significant differences. We next evaluated the proteomic profiling and found an upregulation of OXPHOS proteins in the micropatterned CMs with specific increase in complexes I though IV that explain the enhanced mitochondrial respiration and increased membrane potential observed by microscopy. We developed a novel method for modeling the metabolic activity in iPS-CMs based on micropatterned culture. Our data show that the linear architectural pattern drives the formation and efficiency of OXPHOS function in cardiomyocytes.

A ketogenic diet impacts markers of mitochondrial mass in a tissue specific manner in aged mice.

Declines in mitochondrial mass are thought to be a hallmark of mammalian aging, and a ketogenic diet (KD) may prevent the age-related decreases in mitochondrial content. The objective of this study was to investigate the impact of a KD on markers of mitochondrial mass. Mice were fed an isocaloric control diet (CD) or KD from 12 months of age. Tissues were collected after 1 month and 14 months of intervention, and a panel of commonly used markers of mitochondrial mass (mitochondrial enzyme activities and levels, mitochondrial to nuclear DNA ratio, and cardiolipin content) were measured. Our results showed that a KD stimulated activities of marker mitochondrial enzymes including citrate synthase, Complex I, and Complex IV in hindlimb muscle in aged mice. KD also increased the activity of citrate synthase and prevented an age-related decrease in Complex IV activity in aged brain. No other markers were increased in these tissues. Furthermore, the impacts of a KD on liver and kidney were mixed with no pattern indicative of a change in mitochondrial mass. In conclusion, results of the present study suggest that a KD induces tissue-specific changes in mitochondrial enzyme activities, or structure, rather than global changes in mitochondrial mass across tissues.

The program of renal fibrogenesis is controlled by microRNAs regulating oxidative metabolism

Excessive accumulation of extracellular matrix (ECM) is the hallmark of fibrotic diseases. In the kidney, it is the final common pathway of prevalent diseases, leading to chronic renal failure. While cytokines such as TGF-β play a fundamental role in myofibroblast transformation, recent work has shown that mitochondrial dysfunction and defective fatty acid oxidation (FAO), which compromise the main source of energy for renal tubular epithelial cells, have been proposed to be fundamental contributors to the development and progression of kidney fibrosis. MicroRNAs (miRNAs), which regulate gene expression post-transcriptionally, have been reported to control renal fibrogenesis. To identify miRNAs involved in the metabolic derangement of renal fibrosis, we performed a miRNA array screen in the mouse model of unilateral ureteral obstruction (UUO). MiR-150-5p and miR-495-3p were selected for their link to human pathology, their role in mitochondrial metabolism and their targeting of the fatty acid shuttling enzyme CPT1A. We found a 2- and 4-fold upregulation of miR-150-5p and miR-495-5p, respectively, in both the UUO and the folic acid induced nephropathy (FAN) models, while TGF-β1 upregulated their expressions in the human renal tubular epithelial cell line HKC-8. These miRNAs synergized with TGF-β regarding its pro-fibrotic effect by enhancing the fibrosis-associated markers Acta2, Col1α1 and Fn1. Bioenergetics studies showed a reduction of FAO-associated oxygen consumption rate (OCR) in HKC-8 cells in the presence of both miRNAs. Consistently, expression levels of their mitochondrial-related target genes CPT1A, PGC1α and the mitochondrial transcription factor A (TFAM), were reduced by half in renal epithelial cells exposed to these miRNAs. By contrast, we did not detect changes in mitochondrial mass and transmembrane potential (ΔѰm) or mitochondrial superoxide radical anion production. Our data support that miR-150 and miR-495 may contribute to renal fibrogenesis by aggravating the metabolic failure critically involved in tubular epithelial cells, ultimately leading to fibrosis.

Photon versus proton neurotoxicity: Impact on mitochondrial function and 8-OHdG base-excision repair mechanism in human astrocytes

This study assesses and compares the neurotoxic effects of proton and photon radiation on mitochondrial function and DNA repair capabilities of human astrocytes. Human astrocytes received either proton (0.5 Gy and 3 Gy), photon (0.5 Gy and 3 Gy), or sham-radiation treatment. The mRNA expression level of the DNA repair protein OGG1 was determined via RT-qPCR. The levels of 8-OHdG in the cell media were measured via ELISA. Real-time kinetic analysis of extracellular oxygen consumption rates was performed to assess mitochondrial function. Radiation-induced changes in mitochondrial mass and oxidative activity were assessed using fluorescent imaging with MitoTracker™ Green FM and MitoTracker™ Orange CM-H2TMRos dyes respectively. PCR was used to quantify the alteration in the mitochondrial DNA content, measured as the mitochondrial to nuclear DNA ratio. A significant increase in mitochondrial mass and levels of reactive oxygen species was observed after radiation treatment. Additionally, real-time PCR analysis indicated a significant depletion of mitochondrial DNA content in the irradiated cells when compared to the control. This was accompanied by a decreased gene expression of the DNA base-excision repair protein OGG1 and reduced clearance of 8-OHdG adducts from the genome. Photon radiation treatment was associated with a more detrimental cellular impact when compared to the same dose of proton radiation. These results are indicative of a radiation-induced dose-dependent decrease in mitochondrial function, an increase in senescence and astrogliosis, and impairment of the DNA repair capabilities in healthy glial cells. Photon irradiation was associated with a more significant disruption in mitochondrial function and base-excision repair mechanisms in vitro in comparison to proton treatment.

Adipocyte hypertrophy parallels alterations of mitochondrial status in a cell model for adipose tissue dysfunction in obesity

AimsAdipocyte hypertrophy is the main cause of obesity. A deeper understanding of the molecular mechanisms regulating adipocyte dysfunction may help to plan strategies to treat/prevent obesity and its metabolic complications. Here, we investigated in vitro the molecular alterations associated with early adipocyte hypertrophy, focusing on mitochondrial dysfunction. Main methodsAs model of adipocyte hypertrophy, we employed 3T3-L1 preadipocytes firstly differentiated into mature adipocytes, then cultured with long-chain fatty acids. As a function of differentiation and hypertrophy, we assessed triglyceride content, lipid droplet size, radical homeostasis by spectrophotometry and microscopy, as well as the expression of PPARγ, adiponectin and metallothioneins. Mitochondrial status was investigated by electron microscopy, oxygraph 2 k (O2K) high-resolution respirometry, fluorimetry and western blot. Key findingsCompared to mature adipocytes, hypertrophic adipocytes showed increased triglyceride accumulation and lipid peroxidation, larger or unique lipid droplet, up-regulated expression of PPARγ, adiponectin and metallothioneins. At mitochondrial level, early-hypertrophic adipocytes exhibited: (i) impaired mitochondrial oxygen consumption with parallel reduction in the mitochondrial complexes; (ii) no changes in citrate synthase and HSP60 expression, and in the inner mitochondrial membrane polarization; (iii) no stimulation of mitochondrial fatty acid oxidation. Our findings indicate that the content, integrity, and catabolic activity of mitochondria were rather unchanged in early hypertrophic adipocytes, while oxygen consumption and oxidant production were altered. SignificanceIn the model of early adipocyte hypertrophy exacerbated oxidative stress and impaired mitochondrial respiration were observed, likely depending on reduction in the mitochondrial complexes, without changes in mitochondrial mass and integrity.

Mitochondrial Potentiation Ameliorates Age-Related Heterogeneity in Hematopoietic Stem Cell Function.

Aging is associated with reduced fitness and increased myeloid bias of the hematopoietic stem cell (HSC) compartment, causing increased risk of immune compromise, anemia, and malignancy. We show that mitochondrial membrane potential (MMP) can be used to prospectively isolate chronologically old HSCs with transcriptional features and functional attributes characteristic of young HSCs, including a high rate of transcription and balanced lineage-affiliated programs. Strikingly, MMP is a stronger determinant of the quantitative and qualitative transcriptional state of HSCs than chronological age, and transcriptional consequences of manipulation of MMP in HSCs within their native niche suggest a causal relationship. Accordingly, we show that pharmacological enhancement of MMP in old HSCs invivo increases engraftment potential upon transplantation and reverses myeloid-biased peripheral blood output at steady state. Our results demonstrate that MMP is a source of heterogeneity in old HSCs, and its pharmacological manipulation can alter transcriptional programs with beneficial consequences for function.

Effect of antioxidants on the H2O2-induced premature senescence of human fibroblasts

The study was aimed at evaluation of the role of secondary oxidative stress in the stress-induced premature senescence (SIPS) of human fibroblasts induced by H2O2. Two fibroblast lines were used: lung MRC-5 and ear H8F2p25LM fibroblasts. The lines differed considerably in sensitivity to H2O2 (IC50 of 528 and 33.5 μM, respectively). The cells were exposed to H2O2 concentrations corresponding to IC50 and after 24 h supplemented with a range of antioxidants. Most of antioxidants studied slightly augmented the survival of fibroblasts at single concentrations or in a narrow concentration range, but the results were not consistent among the cell lines. Chosen antioxidants (4-amino-TEMPO, curcumin, caffeic acid and p-coumaric acid) did not restore the level of glutathione decreased by H2O2. Hydrogen peroxide treatment did not induce secondary production of H2O2 and even decreased it, decreased mitochondrial potential in both cell lines and induced changes in the mitochondrial mass inconsistent between the lines. Antioxidant protected mitochondrial potential only in H8F2p25LM cells, but attenuated changes in mitochondrial mass. These results speak against the intermediacy of secondary oxidative stress in the SIPS induced by H2O2 and suggest that the small protective action of antioxidants is due to their effects on mitochondria.

Using flow cytometry for mitochondrial assays

The understanding of how different cell types adapt their metabolism in the face of challenges has been attracting the attention of researchers for many years. Recently, immunologists also started to focus on how the metabolism of immune cells can impact the way that immunity drives its responses. The presence of a pathogen or damage in a tissue changes severely the way that the immune cells need to respond. When activated, immune cells usually shift their metabolism from a high energy demanding status using mitochondria respiration to a glycolytic based rapid ATP production. The diminished amount of respiration leads to changes in the mitochondrial membrane potential and, consequently, generation of reactive oxygen species. Here, we show how flow cytometry can be used to track changes in mitochondrial mass, membrane potential and superoxide (ROS) production in live immune cells.● This protocol suggests a quick way of evaluating mitochondrial fitness using flow cytometry. We propose using the probes MitoTraker Green and MitoTracker Red/ MitoSOX at the same time. This way, it is possible to evaluate different parameters of mitochondrial biology in living cells.● Flow cytometry is a highly used tool by immunologists. With the advances of studies focusing on the metabolism of immune cells, a simplified application of flow cytometry for mitochondrial studies and screenings is a helpful clarifying method for immunology.

Reductions in skeletal muscle mitochondrial mass are not restored following exercise training in patients with chronic kidney disease.

Patients with chronic kidney disease (CKD) exhibit reduced exercise capacity, poor physical function and symptoms of fatigue. The mechanisms that contribute to this are not clearly defined but may involve reductions in mitochondrial function, mass and biogenesis. Here we report on the effect of non-dialysis dependent CKD (NDD-CKD) on mitochondrial mass and basal expression of transcription factors involved in mitochondrial biogenesis compared to a healthy control cohort (HC). In addition, we sought to investigate the effect of a 12-week exercise-training programme on these aspects of mitochondrial dysfunction in a NDD-CKD cohort.For the comparison between NDD-CKD and HC populations, skeletal muscle biopsies were collected from the vastus lateralis (VL) of n=16 non-dialysis dependent CKD patient's stage 3b-5 (NDD-CKD) and n=16 healthy controls matched for age, gender and physical activity (HC). To investigate the effect of exercise training, VL biopsies were collected from n=17 NDD-CKD patients before and after a 12-week exercise intervention that was comprised of aerobic exercise (AE) or a combination of aerobic exercise and resistance training (CE). Mitochondrial mass was analysed by citrate synthase activity and mitochondrial protein content by Porin expression, whilst the expression of transcription factors involved in mitochondrial biogenesis were quantified by real-time qPCR. NDD-CKD patients exhibited a significant reduction in mitochondrial mass when compared to HC, coupled to a reduction in PGC-1α, NRF-1, Nrf2, TFam, mfn2 and SOD1/2 gene expression. 12-weeks of exercise training resulted in a significant increase in PGC-1α expression in both groups, with no further changes seen across indicators of mitochondrial biogenesis. No significant changes in mitochondrial mass were observed in response to either exercise programme. NDD-CKD patients exhibit reduced skeletal muscle mitochondrial mass and gene expression of transcription factors involved in mitochondrial biogenesis compared to HC. These reductions were not restored following 12-weeks of exercise training implying exercise resistance in this cohort. The reasons for this lack of improvement are currently unknown and require further investigation, as reversing the dysregulation of these processes in NDD-CKD may provide a therapeutic opportunity to improve muscle fatigue and dysfunction in this population.