Mechanisms Underlying Regulation of Yeast Longevity by Genetic and Pharmacological Interventions that Alter Mitochondrial Membrane Lipidome and Remodel Mitochondrial Respiratory Supercomplexes

Mechanisms Underlying Regulation of Yeast Longevity by Genetic and Pharmacological Interventions that Alter Mitochondrial Membrane Lipidome and Remodel Mitochondrial Respiratory Supercomplexes Olivia Roseline Koupaki, M.Sc. Recent studies in our laboratory demonstrated that in chronologically aging yeast grown under CR conditions LCA, a natural anti-aging compound, alters the age-related dynamics of changes in mitochondrial abundance and morphology, respiration, membrane potential, and ROS production. Cardiolipin (CL), a dimeric glycerophospholipid that is synthesized and almost exclusively localized in the inner mitochondrial membrane, has been shown to modulate mitochondria-governed processes whose dysfunction underlies aging and age-related pathologies. Phosphatidylethanolamine (PE) is another glycerophospholipid that is almost exclusively synthesized in the inner mitochondrial membrane, from which it is then distributed to various other cellular membranes. Hence, it is likely that the synthesis and stability of CL and, perhaps, PE in the inner mitochondrial membrane are important targets of longevity-extending and health-improving interventions. Because of the plausible importance of mitochondrially synthesized CL and PE in the longevity-extending effect of LCA, other graduate students in our laboratory elucidated how mutations eliminating nucleus-encoded mitochondrial proteins involved in the synthesis of CL and PE within the inner mitochondrial membrane influence the lifespan-extending efficacy of LCA in chronologically aging yeast grown under CR conditions. The results of this genetic analysis suggested that the synthesis of both these membrane lipids in mitochondria plays an essential role in the ability of LCA to extend longevity of yeast placed on a CR diet. All these findings prompted us to elucidate how LCA influences the composition of mitochondrial membrane lipids in chronologically aging yeast grown under CR conditions. To attain this objective, in experiments described in my thesis we used mass spectrometry (MS)-based lipidomics to elucidate the effect of LCA on the repertoire and quantities of membrane lipids in mitochondria that were purified from wild-type (WT) strain and from various long- and short-lived mutant strains impaired in different aspects of CL and PE metabolism. By correlating the effects of LCA on the age-related dynamics of changes in the composition and quantities of membrane lipids in mitochondria of these strains to the effects of this anti-aging compound on their chronological lifespan, we concluded that under CR conditions LCA extends yeast longevity by remodeling the composition of mitochondrial membrane lipids and thereby modulating longevity-defining processes confined to and governed by mitochondria. Specifically, findings described in my thesis strongly suggest that LCA extends longevity of WT yeast by (1) elevating the level of phosphatidylserine (PS; a precursor for the synthesis of PE in mitochondria) in the mitochondrial membrane, thereby enhancing its positive effect on longevity-defining processes in this membrane; (2) reducing the level of PE in the mitochondrial membrane, thereby weakening its negative effect on longevity-defining processes in this membrane; and (3) proportionally decreasing the levels of PE and CL in the mitochondrial membrane, thereby increasing PS/CL and PS/PE ratios but maintaining PE/CL ratio of mitochondrial membrane lipids and causing some longevity-extending changes in this membrane. It is important to emphasize that these LCA-induced alterations in mitochondrial membrane lipids can satisfactorily explain the observed implications of LCA treatment on mitochondrial structure and function, including (1) the ability of LCA to cause dramatic changes in the length and curvature of the inner mitochondrial membrane; and (2) the ability of LCA to activate protein machines involved in mitochondrial respiration, the maintenance of mitochondrial membrane potential, ROS production in mitochondria and mitochondrial fusion. Based on our recent findings and data of other researchers working in the field of mitochondrial biology, we hypothesized that, by altering the level of CL and other glycerophospholipids within the mitochondrial membrane, LCA could modulate the stoichiometry, composition and/or functional state of respiratory supercomplexes (respirasomes) in the inner mitochondrial membrane. To test the validity of this hypothesis, in studies described in my thesis we used a multistep method for recovery of intact respiratory complexes and supercomplexes from purified yeast mitochondria, their subsequent first-dimension electrophoretic separation using so-called blue-native gel electrophoresis (BN-PAGE), their resolution into individual protein components with the help of denaturing Tricine-SDS-PAGE, and the mass spectrometry (MS)-based identification of each of these individual protein components. Findings described in my thesis validate our hypothesis. Specifically, these findings revealed several ways of rearranging respiratory supercomplexes in the inner mitochondrial membrane of cells exhibiting altered mitochondrial membrane lipidome in response to LCA treatment or genetic manipulations impairing the synthesis of CL and other glycerophospholipids within the inner membrane of mitochondria. First, by altering the level of CL and other glycerophospholipids synthesized and residing in the inner mitochondrial membrane, LCA modulates the abundance of several major respiratory supercomplexes (respirasomes) in this membrane. Second, LCA- and genetic manipulations-driven changes in the inner mitochondrial membrane lipidome cause a recruitment of a number of new mitochondrial protein components, not previously known for being permanently associated with the ETC, into the remodeled respirasomes. Importantly, many of the proteins newly recruited into the remodeled respirasomes are known for their essential roles in mitochondria-confined processes that define longevity

Chemical genetic screen identifies lithocholic acid as an anti-aging compound that extends yeast chronological life span in a TOR-independent manner, by modulating housekeeping longevity assurance processes

In chronologically aging yeast, longevity can be extended by administering a caloric restriction (CR) diet or some small molecules. These life-extending interventions target the adaptable target of rapamycin (TOR) and cAMP/protein kinase A (cAMP/PKA) signaling pathways that are under the stringent control of calorie availability. We designed a chemical genetic screen for small molecules that increase the chronological life span of yeast under CR by targeting lipid metabolism and modulating housekeeping longevity pathways that regulate longevity irrespective of the number of available calories. Our screen identifies lithocholic acid (LCA) as one of such molecules. We reveal two mechanisms underlying the life-extending effect of LCA in chronologically aging yeast. One mechanism operates in a calorie availability-independent fashion and involves the LCA-governed modulation of housekeeping longevity assurance pathways that do not overlap with the adaptable TOR and cAMP/PKA pathways. The other mechanism extends yeast longevity under non-CR conditions and consists in LCA-driven unmasking of the previously unknown anti-aging potential of PKA. We provide evidence that LCA modulates housekeeping longevity assurance pathways by suppressing lipid-induced necrosis, attenuating mitochondrial fragmentation, altering oxidation-reduction processes in mitochondria, enhancing resistance to oxidative and thermal stresses, suppressing mitochondria-controlled apoptosis, and enhancing stability of nuclear and mitochondrial DNA

Mitochondrial membrane lipidome defines yeast longevity

Our studies revealed that lithocholic acid (LCA), a bile acid, is a potent anti‐aging natural compound that in yeast cultured under longevity‐extending caloric restriction (CR) conditions acts in synergy with CR to enable a significant further increase in chronological lifespan. Here, we investigate a mechanism underlying this robust longevity‐extending effect of LCA under CR. We found that exogenously added LCA enters yeast cells, is sorted to mitochondria, resides mainly in the inner mitochondrial membrane, and also associates with the outer mitochondrial membrane. LCA elicits an age‐related remodeling of glycerophospholipid synthesis and movement within both mitochondrial membranes, thereby causing substantial changes in mitochondrial membrane lipidome and triggering major changes in mitochondrial size, number and morphology. In synergy, these changes in the membrane lipidome and morphology of mitochondria alter the age‐related chronology of mitochondrial respiration, membrane potential, ATP synthesis and reactive oxygen species homeostasis. The LCA‐driven alterations in the age‐related dynamics of these vital mitochondrial processes extend yeast longevity. In sum, our findings suggest a mechanism underlying the ability of LCA to delay chronological aging in yeast by accumulating in both mitochondrial membranes and altering their glycerophospholipid compositions. We concluded that mitochondrial membrane lipidome plays an essential role in defining yeast longevity

Chemical genetic screen identifies lithocholic acid as an anti-aging compound that extends yeast chronological life span in a TOR-independent manner, by modulating housekeeping longevity assurance processes

In chronologically aging yeast, longevity can be extended by administering a caloric restriction (CR) diet or some small molecules. These life-extending interventions target the adaptable target of rapamycin (TOR) and cAMP/protein kinase A (cAMP/PKA) signaling pathways that are under the stringent control of calorie availability. We designed a chemical genetic screen for small molecules that increase the chronological life span of yeast under CR by targeting lipid metabolism and modulating housekeeping longevity pathways that regulate longevity irrespective of the number of available calories. Our screen identifies lithocholic acid (LCA) as one of such molecules. We reveal two mechanisms underlying the life-extending effect of LCA in chronologically aging yeast. One mechanism operates in a calorie availability-independent fashion and involves the LCA-governed modulation of housekeeping longevity assurance pathways that do not overlap with the adaptable TOR and cAMP/PKA pathways. The other mechanism extends yeast longevity under non-CR conditions and consists in LCA-driven unmasking of the previously unknown anti-aging potential of PKA. We provide evidence that LCA modulates housekeeping longevity assurance pathways by suppressing lipid-induced necrosis, attenuating mitochondrial fragmentation, altering oxidation-reduction processes in mitochondria, enhancing resistance to oxidative and thermal stresses, suppressing mitochondria-controlled apoptosis, and enhancing stability of nuclear and mitochondrial DNA

Interspecies Chemical Signals Released into the Environment May Create Xenohormetic, Hormetic and Cytostatic Selective Forces that Drive the Ecosystemic Evolution of Longevity Regulation Mechanisms

Various organisms (i.e., bacteria, fungi, plants and animals) within an ecosystem can synthesize and release into the environment certain longevity-extending small molecules. Here we hypothesize that these interspecies chemical signals can create xenohormetic, hormetic and cytostatic selective forces driving the ecosystemic evolution of longevity regulation mechanisms. In our hypothesis, following their release into the environment by one species of the organisms composing an ecosystem, such small molecules can activate anti-aging processes and/or inhibit pro-aging processes in other species within the ecosystem. The organisms that possess the most effective (as compared to their counterparts of the same species) mechanisms for sensing the chemical signals produced and released by other species and for responding to such signals by undergoing certain hormetic and/or cytostatic life-extending changes to their metabolism and physiology are expected to live longer then their counterparts within the ecosystem. Thus, the ability of a species of the organisms composing an ecosystem to undergo life-extending metabolic or physiological changes in response to hormetic or cytostatic chemical compounds released to the ecosystem by other species: 1) increases its chances of survival; 2) creates selective forces aimed at maintaining such ability; and 3) enables the evolution of longevity regulation mechanisms