Increased replicative longevity in Saccharomyces cerevisiae because of calorie restriction has been linked to enhanced mitochondrial respiratory activity. Here we have further investigated how mitochondrial respiration affects yeast life span. We found that calorie restriction by growth in low glucose increased respiration but decreased mitochondrial reactive oxygen species production relative to oxygen consumption. Calorie restriction also enhanced chronological life span. The beneficial effects of calorie restriction on mitochondrial respiration, reactive oxygen species release, and replicative and chronological life span could be mimicked by uncoupling agents such as dinitrophenol. Conversely, chronological life span decreased in cells treated with antimycin (which strongly increases mitochondrial reactive oxygen species generation) or in yeast mutants null for mitochondrial superoxide dismutase (which removes superoxide radicals) and for RTG2 (which participates in retrograde feedback signaling between mitochondria and the nucleus). These results suggest that yeast aging is linked to changes in mitochondrial metabolism and oxidative stress and that mild mitochondrial uncoupling can increase both chronological and replicative life span.

Mitochondria are essential organelles for eukaryotic homeostasis. Although these organelles possess their own DNA, the vast majority (>99%) of mitochondrial proteins are encoded in the nucleus. This situation makes systems that allow the communication between mitochondria and the nucleus a requirement not only to coordinate mitochondrial protein synthesis during biogenesis but also to communicate eventual mitochondrial malfunctions, triggering compensatory responses in the nucleus. Mitochondria-to-nucleus retrograde signaling has been described in various organisms, albeit with differences in effector pathways, molecules, and outcomes, as discussed in this review.

Changes in mitochondrial size and shape have been implicated in several physiologic processes, but their role in mitochondrial Ca ²⁺ uptake regulation and overall cellular Ca ²⁺ homeostasis is largely unknown. Here we show that modulating mitochondrial dynamics toward increased fusion through expression of a dominant negative (DN) form of the fission protein [dynamin‐related protein 1 (DRP1)] markedly increased both mitochondrial Ca ²⁺ retention capacity and Ca ²⁺ uptake rates in permeabilized C2C12 cells. Similar results were seen using the pharmacological fusion‐promoting M1 molecule. Conversely, promoting a fission phenotype through the knockdown of the fusion protein mitofusin (MFN)‐2 strongly reduced the mitochondrial Ca ²⁺ uptake speed and capacity in these cells. These changes were not dependent on modifications in mitochondrial calcium uniporter expression, inner membrane potentials, or the mitochondrial permeability transition. Implications of mitochondrial morphology modulation on cellular calcium homeostasis were measured in intact cells; mitochondrial fission promoted lower basal cellular calcium levels and lower endoplasmic reticulum (ER) calcium stores, as indicated by depletion with thapsigargin. Indeed, mitochondrial fission was associated with ER stress. Additionally, the calcium‐replenishing process of store‐operated calcium entry was impaired in MFN2 knockdown cells, whereas DRP1‐DN‐promoted fusion resulted in faster cytosolic Ca ²⁺ increase rates. Overall, our results show a novel role for mitochondrial morphology in the regulation of mitochondrial Ca ²⁺ uptake, which impacts cellular Ca ²⁺ homeostasis.—Kowaltowski, A. J., Menezes‐Filho, S. L., Assali, E. A., Gonçalves, I. G., Cabral‐Costa, J. V., Abreu, P., Miller, N., Nolasco, P., Laurindo, F. R. M., Bruni‐Cardoso, A., Shirihai, O. Mitochondrial morphology regulates organellar Ca ²⁺ uptake and changes cellular Ca ²⁺ homeostasis. FASEB J. 33, 13176–13188 (2019). www.fasebj.org

Replicative life span in Saccharomyces cerevisiae is increased by glucose (Glc) limitation [calorie restriction (CR)] and by augmented NAD+. Increased survival promoted by CR was attributed previously to the NAD+‐dependent histone deacetylase activity of sirtuin family protein Sir2p but not to changes in redox state. Here we show that strains defective in NAD+ synthesis and salvage pathways ( pnc1 Δ, npt1 Δ, and bna6 Δ) exhibit decreased oxygen consumption and increased mitochondrial H ₂ O ₂ release, reversed over time by CR. These null mutant strains also present decreased chronological longevity in a manner rescued by CR. Furthermore, we observed that changes in mitochondrial H ₂ O ₂ release alter cellular redox state, as attested by measurements of total, oxidized, and reduced glutathione. Surprisingly, our results indicate that matrix‐soluble dihydrolipoyl‐dehydrogenases are an important source of CR‐preventable mitochondrial reactive oxygen species (ROS). Indeed, deletion of the LPD1 gene prevented oxidative stress in npt1 Δ and bna6 Δ mutants. Furthermore, pyruvate and α‐ketoglutarate, substrates for dihydrolipoyl dehydrogenasecontaining enzymes, promoted pronounced reactive oxygen release in permeabilized wild‐type mitochondria. Altogether, these results substantiate the concept that mitochondrial ROS can be limited by caloric restriction and play an important role in S. cerevisiae senescence. Furthermore, these findings uncover dihydrolipoyl dehydrogenase as an important and novel source of ROS leading to life span limitation. Tahara, E. B., Barros, M. H., Oliveira, G. A., Netto, L. E. S., Kowaltowski, A. J. Dihydrolipoyl dehydrogenase as a source of reactive oxygen species inhibited by caloric restriction and involved in Saccharomyces cerevisiae aging. FASEB J. 21, 274–283 (2007)