Research focused on succinate dehydrogenase (SDH) and its substrate, succinate, culminated in the 1950s accompanying the rapid development of research dedicated to bioenergetics and intermediary metabolism. This allowed researchers to uncover the implication of SDH in both the mitochondrial respiratory chain and the Krebs cycle. Nowadays, this theme is experiencing a real revival following the discovery of the role of SDH and succinate in a subset of tumors and cancers in humans. The aim of this review is to enlighten the many questions yet unanswered, ranging from fundamental to clinically oriented aspects, up to the danger of the current use of SDH as a target for a subclass of pesticides.

Background: In the last ten years, deficiencies in tricarboxylic acid cycle (TCAC) enzymes have been shown to cause a wide spectrum of human diseases, including malignancies and neurological and cardiac diseases. A prerequisite to the identification of disease-causing TCAC enzyme deficiencies is the availability of effective enzyme assays. Results: We developed three assays that measure the full set of TCAC enzymes. One assay relies on the sequential addition of reagents to measure succinyl-CoA ligase activity, followed by succinate dehydrogenase, fumarase and, finally, malate dehydrogenase. Another assay measures the activity of α-ketoglutarate dehydrogenase followed by aconitase and isocitrate dehydrogenase. The remaining assay measures citrate synthase activity using a standard procedure. We used these assays successfully on extracts of small numbers of human cells displaying various severe or partial TCAC deficiencies and on frozen heart homogenates from heterozygous mice harboring an SDHB gene deletion. Conclusion: This set of assays is rapid and simple to use and can immediately detect even partial defects, as the activity of each enzyme can be readily compared with one or more other activities measured in the same sample.

Mitochondria are the source of internal heat which influences all cellular processes. Hence, monitoring mitochondrial temperature provides a unique insight into cell physiology. Using a thermosensitive fluorescent probe MitoThermo Yellow (MTY), we have shown recently that mitochondria within human cells are maintained at close to 50 °C when active, increasing their temperature locally by about 10 °C. Initially reported in the HEK293 cell line, we confirmed this finding in the HeLa cell line. Delving deeper, using MTY and MTX (MitoThermo X), a modified version of MTY, we unraveled some caveats related to the nature of these charged fluorophores. While enabling the assessment of mitochondrial temperature in HEK and HeLa cell lines, the reactivity of MTY to membrane potential variations in human primary skin fibroblasts precluded local temperature monitoring in these cells. Chemical modification of MTY into MTX did not result in a temperature probe unresponsive to membrane potential variations that could be universally used in any cell type to determine mitochondrial temperature. Thus, the cell-type dependence of MTY in measuring mitochondrial temperature, which is likely due to the variable binding of this dye to specific internal mitochondrial components, should imply cautiousness while using these nanothermometers for mitochondrial temperature analysis.

In the last ten years, the use of fluorescent probes developed to measure oxygen has resulted in several marketed devices, some unreasonably expensive and with little flexibility. We have explored the use of the effective, versatile, and inexpensive Redflash technology to determine oxygen uptake by a number of different biological samples using various layouts. This technology relies on the use of an optic fiber equipped at its tip with a membrane coated with a fluorescent dye (www.pyro-science.com). This oxygen-sensitive dye uses red light excitation and lifetime detection in the near infrared. So far, the use of this technology has mostly been used to determine oxygen concentration in open spaces for environmental studies, especially in aquatic media. The oxygen uptake determined by the device can be easily assessed in small volumes of respiration medium and combined with the measurement of additional parameters, such as lactate excretion by intact cells or the membrane potential of purified mitochondria. We conclude that the performance of by this technology should make it a first choice in the context of both fundamental studies and investigations for respiratory chain deficiencies in human samples.

Research focused on succinate dehydrogenase (SDH) and its substrate, succinate, culminated in the 50’s accompanying the rapid development of research dedicated to bioenergetics and intermediary metabolism. This allowed to uncover the implication of the SDH in both the mitochondrial respiratory chain and the Krebs cycle. Nowadays this theme is experiencing a real revival following the discovery of the role of SDH and succinate in a subset of tumors and cancers in human. The aim of this review is to enlighten the many questions yet unanswered, ranging from fundamental to clinically oriented aspects, up to the danger of the current use of SDH as a target for a sub class of pesticides.

As to better understand and characterize the dramatic differences in antioxidant response of human cells harboring mutations in the frataxin gene responsible for Friedreich's ataxia (FRDA), we studied primary cultures of skin fibroblasts derived from five FRDA patients with different major frataxin gene expansion sizes. Since oxidative stress has been previously established to play a critical role in FRDA, among the many enzymes that may modulate oxidative stress sensitivity, we selected some that have previously been shown to be critical in oxidative stress. However, we were unable to identify a consistent index to rank individual cultures based on these measures. We therefore focused our study on FRDA fibroblast cell death and its modulation by different antioxidants under culture conditions that exacerbate oxidative stress sensitivity. Under conditions that require cells to rely on mitochondrial activity, we observed significant but variable effect on FRDA cell proliferation. These conditions were then used to test the efficacy to prevent or slow down cell death, of a panel of antioxidant molecules targeting different steps of the pro-oxidant cascade previously documented in FRDA, namely uridine, pyruvate, and pioglitazone. We observed a surprising variability in the response of patient fibroblasts to antioxidant molecules even using similar culture conditions. We conclude that the individual-specific response already discernible at the cellular level may well play an important role in the frequent difficulties encountered in reaching definitive conclusions when testing the ability of antioxidants to counteract the consequences of frataxin depletion, a conclusion that may apply to clinical trials as well.