Bioenergetic Alterations of Metabolic Redox Coenzymes as NADH,FAD and FMN by Means of Fluorescence Lifetime Imaging Techniques

Metabolic FLIM (fluorescence lifetime imaging) is used to image bioenergetic status in cells and tissue. Whereas an attribution of the fluorescence lifetime of coenzymes as an indicator for cell metabolism is mainly accepted, it is debated whether this is valid for the redox state of cells. In this regard, an innovative algorithm using the lifetime characteristics of nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD) to calculate the fluorescence lifetime induced redox ratio (FLIRR) has been reported so far. We extended the FLIRR approach and present new results, which includes FLIM data of the various enzymes, such as NAD(P)H, FAD, as well as flavin mononucleotide (FMN). Our algorithm uses a two-exponential fitting procedure for the NAD(P)H autofluorescence and a three-exponential fit of the flavin signal. By extending the FLIRR approach, we introduced FLIRR1 as protein-bound NAD(P)H related to protein-bound FAD, FLIRR2 as protein-bound NAD(P)H related to free (unbound) FAD and FLIRR3 as protein-bound NAD(P)H related to protein-bound FMN. We compared the significance of extended FLIRR to the metabolic index, defined as the ratio of protein-bound NAD(P)H to free NAD(P)H. The statistically significant difference for tumor and normal cells was found to be highest for FLIRR1.     

Plasma Gelsolin Reinforces the Diagnostic Value of FGF-21 and GDF-15 for Mitochondrial Disorders

Mitochondrial disorders (MD) comprise a group of heterogeneous clinical disorders for which non-invasive diagnosis remains a challenge. Two protein biomarkers have so far emerged for MD detection, FGF-21 and GDF-15, but the identification of additional biomarkers capable of improving their diagnostic accuracy is highly relevant. Previous studies identified Gelsolin as a regulator of cell survival adaptations triggered by mitochondrial defects. Gelsolin presents a circulating plasma isoform (pGSN), whose altered levels could be a hallmark of mitochondrial dysfunction. Therefore, we investigated the diagnostic performance of pGSN for MD relative to FGF-21 and GDF-15. Using ELISA assays, we quantified plasma levels of pGSN, FGF-21, and GDF-15 in three age- and gender-matched adult cohorts: 60 genetically diagnosed MD patients, 56 healthy donors, and 41 patients with unrelated neuromuscular pathologies (non-MD). Clinical variables and biomarkers’ plasma levels were compared between groups. Discrimination ability was calculated using the area under the ROC curve (AUC). Optimal cut-offs and the following diagnostic parameters were determined: sensitivity, specificity, positive and negative predictive values, positive and negative likelihood ratios, and efficiency. Comprehensive statistical analyses revealed significant discrimination ability for the three biomarkers to classify between MD and healthy individuals, with the best diagnostic performance for the GDF-15/pGSN combination. pGSN and GDF-15 preferentially discriminated between MD and non-MD patients under 50 years, whereas FGF-21 best classified older subjects. Conclusion: pGSN improves the diagnosis accuracy for MD provided by FGF-21 and GDF-15.     

Mitochondrial response of the lesser grain borer Rhyzopertha dominica (F.) to modified atmospheres

Rhyzopertha dominica is an insect pest that feeds on a wide variety of grains and stored cereals, producing economical losses. Bioenergetics studies on insects are necessary to eventually plan strategies to reduce pest infestation on stored grains with methods other than insecticides. Modified atmosphere treatments, where insects are exposed to either low O₂ or high CO₂ concentrations, or both, can be used as alternatives. In this research, R. dominica was subjected to a modified atmosphere environment (MA) consisting of 5% O₂ and 10% CO₂ for 24 h and was compared with normoxia insects (21% O₂ and 0.02% CO₂). Mitochondria from the whole insect were isolated and their protein content was analyzed by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) using OFFGEL Fractionator equipment and 12% polyacrylamide gels electrophoresis (SDS-PAGE). Lactate concentration, cytochrome c oxidase (COX) and ATPase activities were measured. Lactate concentration was 29% higher in R. dominica exposed to MA versus normoxia. 2-D PAGE and LC MS/MS revealed the presence of a heat shock protein (hsp60), an actin fragment, and ATP synthase α and β catalytic subunits. Activity levels of COX and ATPase were reduced by 58 and 59%, respectively, in MA versus normoxia. Therefore, R. dominica may have shifted into a hypo-metabolic state because oxygen intake was insufficient to maintain enzymatic activity. This scenario would ultimately result in a decrease in substrate concentration (oxygen) and a reduction in electron transport chain complex activity. Thus, eventually, this knowledge can be use to develop novel environmentally friendly alternatives to eradicate or reduce pests in stored grains.     

Mitochondrial Caseinolytic Protease P: A Possible Novel Prognostic Marker and Therapeutic Target in Cancer

Caseinolytic protease P (ClpP) is a mitochondrial serine protease. In mammalian cells, the heterodimerization of ClpP and its AAA+ ClpX chaperone results in a complex called ClpXP, which has a relevant role in protein homeostasis and in maintaining mitochondrial functionality through the degradation of mitochondrial misfolded or damaged proteins. Recent studies demonstrate that ClpP is upregulated in primary and metastatic human tumors, supports tumor cell proliferation, and its overexpression desensitizes cells to cisplatin. Interestingly, small modulators of ClpP activity, both activators and inhibitors, are able to impair oxidative phosphorylation in cancer cells and to induce apoptosis. This review provides an overview of the role of ClpP in regulating mitochondrial functionality, in supporting tumor cell proliferation and cisplatin resistance; finally, we discuss whether this protease could represent a new prognostic marker and therapeutic target for the treatment of cancer.     

Suppression of PGC-1α Drives Metabolic Dysfunction in TGFβ2-Induced EMT of Retinal Pigment Epithelial Cells

PGC-1α, a key orchestrator of mitochondrial metabolism, plays a crucial role in governing the energetically demanding needs of retinal pigment epithelial cells (RPE). We previously showed that silencing PGC-1α induced RPE to undergo an epithelial-mesenchymal-transition (EMT). Here, we show that induction of EMT in RPE using transforming growth factor-beta 2 (TGFβ2) suppressed PGC-1α expression. Correspondingly, TGFβ2 induced defects in mitochondrial network integrity with increased sphericity and fragmentation. TGFβ2 reduced expression of genes regulating mitochondrial dynamics, reduced citrate synthase activity and intracellular ATP content. High-resolution respirometry showed that TGFβ2 reduced mitochondrial OXPHOS levels consistent with reduced expression of NDUFB5. The reduced mitochondrial respiration was associated with a compensatory increase in glycolytic reserve, glucose uptake and gene expression of glycolytic enzymes (PFKFB3, PKM2, LDHA). Treatment with ZLN005, a selective small molecule activator of PGC-1α, blocked TGFβ2-induced upregulation of mesenchymal genes (αSMA, Snai1, CTGF, COL1A1) and TGFβ2-induced migration using the scratch wound assay. Our data show that EMT is accompanied by mitochondrial dysfunction and a metabolic shift towards reduced OXPHOS and increased glycolysis that may be driven by PGC-1α suppression. ZLN005 effectively blocks EMT in RPE and thus serves as a novel therapeutic avenue for treatment of subretinal fibrosis.     

Water-Soluble Gold(III)–Metformin Complex Alters Mitochondrial Bioenergetics in Breast Cancer Cells

Chemical control of mitochondrial dynamics and bioenergetics can unravel fundamental biological mechanisms and therapeutics for several diseases including, diabetes and cancer. We synthesized stable, water-soluble gold(III) complexes (Auraformin) supported by biguanide metformin or phenylmetformin for efficacious inhibition of mitochondrial respiration. The new compounds were characterized following the reaction of [C N]-cyclometalated gold(III) compounds with respective biguanides. Auraformin is solution stable in a physiologically relevant environment. We show that auraformin decreases mitochondrial respiration efficiently in comparison to the clinically used metformin by 100-fold. The compound displays significant mitochondrial uptake and induces antiproliferative activity in the micromolar range. Our results shed light on the development of new scaffolds as improved inhibitors of mitochondrial respiration.     

Mitochondrial Transfer in Cancer: A Comprehensive Review

Depending on their tissue of origin, genetic and epigenetic marks and microenvironmental influences, cancer cells cover a broad range of metabolic activities that fluctuate over time and space. At the core of most metabolic pathways, mitochondria are essential organelles that participate in energy and biomass production, act as metabolic sensors, control cancer cell death, and initiate signaling pathways related to cancer cell migration, invasion, metastasis and resistance to treatments. While some mitochondrial modifications provide aggressive advantages to cancer cells, others are detrimental. This comprehensive review summarizes the current knowledge about mitochondrial transfers that can occur between cancer and nonmalignant cells. Among different mechanisms comprising gap junctions and cell-cell fusion, tunneling nanotubes are increasingly recognized as a main intercellular platform for unidirectional and bidirectional mitochondrial exchanges. Understanding their structure and functionality is an important task expected to generate new anticancer approaches aimed at interfering with gains of functions (e.g., cancer cell proliferation, migration, invasion, metastasis and chemoresistance) or damaged mitochondria elimination associated with mitochondrial transfer.     

Metabolic Factors Affecting Tumor Immunogenicity: What Is Happening at the Cellular Level?

Immunotherapy has changed the treatment paradigm in multiple solid and hematologic malignancies. However, response remains limited in a significant number of cases, with tumors developing innate or acquired resistance to checkpoint inhibition. Certain “hot” or “immune-sensitive” tumors become “cold” or “immune-resistant”, with resultant tumor growth and disease progression. Multiple factors are at play both at the cellular and host levels. The tumor microenvironment (TME) contributes the most to immune-resistance, with nutrient deficiency, hypoxia, acidity and different secreted inflammatory markers, all contributing to modulation of immune-metabolism and reprogramming of immune cells towards pro- or anti-inflammatory phenotypes. Both the tumor and surrounding immune cells require high amounts of glucose, amino acids and fatty acids to fulfill their energy demands. Thus, both compete over one pool of nutrients that falls short on needs, obliging cells to resort to alternative adaptive metabolic mechanisms that take part in shaping their inflammatory phenotypes. Aerobic or anaerobic glycolysis, oxidative phosphorylation, tryptophan catabolism, glutaminolysis, fatty acid synthesis or fatty acid oxidation, etc. are all mechanisms that contribute to immune modulation. Different pathways are triggered leading to genetic and epigenetic modulation with consequent reprogramming of immune cells such as T-cells (effector, memory or regulatory), tumor-associated macrophages (TAMs) (M1 or M2), natural killers (NK) cells (active or senescent), and dendritic cells (DC) (effector or tolerogenic), etc. Even host factors such as inflammatory conditions, obesity, caloric deficit, gender, infections, microbiota and smoking status, may be as well contributory to immune modulation, anti-tumor immunity and response to immune checkpoint inhibition. Given the complex and delicate metabolic networks within the tumor microenvironment controlling immune response, targeting key metabolic modulators may represent a valid therapeutic option to be combined with checkpoint inhibitors in an attempt to regain immune function.