Final Report Summary - SIRTUINS (Phenogenomics of sirtuin corepressor family)
The sirtuins, a small and evolutionary conserved family of proteins, are mainly known to catalyze deacetylation reactions that are tightly linked to cellular energy (NAD+) levels. The main goal of our work is to establish the function of this protein family in metabolic homeostasis by using a multi-pronged approach that involves a combination of molecular, cellular and in vivo studies. We made significant advances in delineating the activity of the individual sirtuins proteins using both pharmacological and genetic tools. In that context, we have generated genetically engineered mouse models (GEMMs) for all the sirtuins, except for SIRT4, which failed to transmit through the germline. We have furthermore imported a genetic reference population (GRP) consisting of 50 of the BXD mouse strains. Several of these SIRT deficient GEMMs and the BXD GRP are being used in various aspects of our research, which is summarized below.
Our work identified a novel link between two pathways that signal energy stress, i.e. AMP activated kinase (AMPK) and SIRT1. AMPK activation results in the modulation of the levels of NAD+, which critically determine SIRT1 activity. We furthermore showed that intact AMPK activity is required to induce SIRT1 action upon fasting, exercise and resveratrol administration. The effects of resveratrol on SIRT1 activity is therefore a downstream consequence of the increase in NAD+ promoted by AMPK and not the consequence of a direct activation of SIRT1, long time a central dogma in the field. An important implication of these results is that increases in NAD+ levels should translate in SIRT1 activation and boost oxidative metabolism. To test this, we analyzed PARP-1 KO mice, in which the absence of PARP-1, a major NAD+-consuming enzyme, increases NAD+ levels. The metabolic phenotype of these mice resembled that of SIRT1 stimulation and was characterized by a marked increase in mitochondrial oxidative metabolism. We have now extended our data on the importance of NAD+ for SIRT1 activation and have shown that boosting cellular NAD+ content, by using pharmacological PARP inhibitors or by supplementing the diet with NAD+ precursors has a similar beneficial metabolic effect. Furthermore, we speculate that the stimulation of oxidative metabolism, could contribute to the anti-tumoral effects of PARP inhiitors as they curb aerobic glycolysis, the hallmark of many cancers (aka the Warburg effect). In another line of research on SIRT1, we demonstrated that SIRT1 gene expression is induced by the activation of the cAMP-Response Element Binding protein (CREB) upon low nutrient availability, whereas in the absence of energetic stress, the Carbohydrate-Response Element Binding Protein (ChREBP) represses SIRT1 expression. The fact that SIRT1 expression is also tightly controlled at the transcriptional level by nutrient availability, hence highlights another layer of complexity in the regulation of SIRT1 activity. Finally, we also validated that SIRT1 is implicated in the regulation of energy metabolism and insulin sensitivity in humans.
As to the other sirtuins, we established that SIRT2 controls an essential polarity pathway in the spinal cord during myelin assembly. Furthermore, we have shown that despite a remarkable hyperacetylation of mitochondrial proteins, SIRT3-/- mice show no overt metabolic abnormalities. Together with the group of H. Lin at Cornell University, we identified protein lysine succinylation as a totally novel posttranslational modification that can be reversed by SIRT5 in vivo. Consistent with the importance of SIRT5 in desuccinylation, SIRT5-/- mice show increases in the level of succinylation on carbamoyl phosphate synthase 1, despite that these mice are again phenotypically normal. We are currently analyzing germline and tissue-specific SIRT6-/- and SIRT7-/- mice. To facilitate aging studies, we also identified a reliable set of in vivo biomarkers of aging, with decreased long chain acylcarnitines and amino acids together with increased free fatty acids being predictive of aging.
We used the mouse BXD genetic reference population (GRP), a highly diverse panel of ~150 recombinant inbred (RI) lines, descending from crosses between a C57BL/6J mother and a DBA/2J father, to study the link between sirtuins, mitochondrial function; and aging. Using the BXDs, we identified mitochondrial ribosomal proteins (Mrps5 and other Mrp family members) as integrators of mitochondrial protein synthesis and modulators of metabolism and lifespan. Reduced MRP expression and/or mitochondrial translation, which selectively decreases the 13 electron transport chain (ETC) proteins encoded by mtDNA, activates the mitochondrial protein quality control network, resulting in a robust mitochondrial unfolded protein response (UPRmt). Specific antibiotics, such as doxycycline, can pharmacologically inhibit mitochondrial translation and also induce UPRmt and longevity. Interestingly, exposing worms and cells to the well-known longevity compounds rapamycin and resveratrol, which are mitochondrial biogenesis inducers and, differently from mrps 5 and ETC downregulation, increase mitochondrial respiration, also induced UPRmt. Moreover, NAD+ boosters, such as NR and PARP inhibitors, which stimulate mitochondrial biogenesis via the SIRT1 pathway, also induced a mitonuclear protein imbalance resulting in UPRmt in mammalian cells and in C. elegans. In line with the beneficial and protective effects of UPRmt, the stimulation of the SIRT1 pathway also increased worm lifespan. This suggests that UPRmt, triggered by different mitochondrial stresses, i.e. by increasing protein folding workload during biogenesis or by mito nuclear proteostatic imbalance, could be the underlying mechanism leading to increased longevity. These data also underscore how studies in the BXDs can be used to efficiently nominate candidate genes and molecular/genetic networks that govern mitochondrial protein synthesis, UPRmt, health, and lifespan.
Our work identified a novel link between two pathways that signal energy stress, i.e. AMP activated kinase (AMPK) and SIRT1. AMPK activation results in the modulation of the levels of NAD+, which critically determine SIRT1 activity. We furthermore showed that intact AMPK activity is required to induce SIRT1 action upon fasting, exercise and resveratrol administration. The effects of resveratrol on SIRT1 activity is therefore a downstream consequence of the increase in NAD+ promoted by AMPK and not the consequence of a direct activation of SIRT1, long time a central dogma in the field. An important implication of these results is that increases in NAD+ levels should translate in SIRT1 activation and boost oxidative metabolism. To test this, we analyzed PARP-1 KO mice, in which the absence of PARP-1, a major NAD+-consuming enzyme, increases NAD+ levels. The metabolic phenotype of these mice resembled that of SIRT1 stimulation and was characterized by a marked increase in mitochondrial oxidative metabolism. We have now extended our data on the importance of NAD+ for SIRT1 activation and have shown that boosting cellular NAD+ content, by using pharmacological PARP inhibitors or by supplementing the diet with NAD+ precursors has a similar beneficial metabolic effect. Furthermore, we speculate that the stimulation of oxidative metabolism, could contribute to the anti-tumoral effects of PARP inhiitors as they curb aerobic glycolysis, the hallmark of many cancers (aka the Warburg effect). In another line of research on SIRT1, we demonstrated that SIRT1 gene expression is induced by the activation of the cAMP-Response Element Binding protein (CREB) upon low nutrient availability, whereas in the absence of energetic stress, the Carbohydrate-Response Element Binding Protein (ChREBP) represses SIRT1 expression. The fact that SIRT1 expression is also tightly controlled at the transcriptional level by nutrient availability, hence highlights another layer of complexity in the regulation of SIRT1 activity. Finally, we also validated that SIRT1 is implicated in the regulation of energy metabolism and insulin sensitivity in humans.
As to the other sirtuins, we established that SIRT2 controls an essential polarity pathway in the spinal cord during myelin assembly. Furthermore, we have shown that despite a remarkable hyperacetylation of mitochondrial proteins, SIRT3-/- mice show no overt metabolic abnormalities. Together with the group of H. Lin at Cornell University, we identified protein lysine succinylation as a totally novel posttranslational modification that can be reversed by SIRT5 in vivo. Consistent with the importance of SIRT5 in desuccinylation, SIRT5-/- mice show increases in the level of succinylation on carbamoyl phosphate synthase 1, despite that these mice are again phenotypically normal. We are currently analyzing germline and tissue-specific SIRT6-/- and SIRT7-/- mice. To facilitate aging studies, we also identified a reliable set of in vivo biomarkers of aging, with decreased long chain acylcarnitines and amino acids together with increased free fatty acids being predictive of aging.
We used the mouse BXD genetic reference population (GRP), a highly diverse panel of ~150 recombinant inbred (RI) lines, descending from crosses between a C57BL/6J mother and a DBA/2J father, to study the link between sirtuins, mitochondrial function; and aging. Using the BXDs, we identified mitochondrial ribosomal proteins (Mrps5 and other Mrp family members) as integrators of mitochondrial protein synthesis and modulators of metabolism and lifespan. Reduced MRP expression and/or mitochondrial translation, which selectively decreases the 13 electron transport chain (ETC) proteins encoded by mtDNA, activates the mitochondrial protein quality control network, resulting in a robust mitochondrial unfolded protein response (UPRmt). Specific antibiotics, such as doxycycline, can pharmacologically inhibit mitochondrial translation and also induce UPRmt and longevity. Interestingly, exposing worms and cells to the well-known longevity compounds rapamycin and resveratrol, which are mitochondrial biogenesis inducers and, differently from mrps 5 and ETC downregulation, increase mitochondrial respiration, also induced UPRmt. Moreover, NAD+ boosters, such as NR and PARP inhibitors, which stimulate mitochondrial biogenesis via the SIRT1 pathway, also induced a mitonuclear protein imbalance resulting in UPRmt in mammalian cells and in C. elegans. In line with the beneficial and protective effects of UPRmt, the stimulation of the SIRT1 pathway also increased worm lifespan. This suggests that UPRmt, triggered by different mitochondrial stresses, i.e. by increasing protein folding workload during biogenesis or by mito nuclear proteostatic imbalance, could be the underlying mechanism leading to increased longevity. These data also underscore how studies in the BXDs can be used to efficiently nominate candidate genes and molecular/genetic networks that govern mitochondrial protein synthesis, UPRmt, health, and lifespan.