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The Leiser lab takes a multi-organism and multi-system approach to studying aging
The Leiser lab takes a multi-organism and multi-system approach to studying aging
By studying the roundworm Caenorhabditis elegans, we are able to use large-scale screens and genetic engineering to manipulate animals and understand genes and processes that are important for promoting health and longevity. We then study conserved genes of interest in tissue culture models, looking for conserved function of the genes in cellular processes. We finally use studies in mice to validate the questions pertaining to the importance of genes, drugs, and/or biological processes that affect aging.
By studying the roundworm Caenorhabditis elegans, we are able to use large-scale screens and genetic engineering to manipulate animals and understand genes and processes that are important for promoting health and longevity. We then study conserved genes of interest in tissue culture models, looking for conserved function of the genes in cellular processes. We finally use studies in mice to validate the questions pertaining to the importance of genes, drugs, and/or biological processes that affect aging.
Our research, as shown in the triangle on the right, is not unidirectional, because we can take our findings in mice and extrapolate them to culture and to worms as well to better understand findings of interest in the mice. Most importantly, because the evolutionary distance between worms and mice is substantially greater than that of mice and humans, our resulting data describing conserved genes are very likely to also be conserved in people.
Our research, as shown in the triangle on the right, is not unidirectional, because we can take our findings in mice and extrapolate them to culture and to worms as well to better understand findings of interest in the mice. Most importantly, because the evolutionary distance between worms and mice is substantially greater than that of mice and humans, our resulting data describing conserved genes are very likely to also be conserved in people.
1. Understand the mechanisms of serotonin-based signaling in food perception and DR-mediated longevity
1. Understand the mechanisms of serotonin-based signaling in food perception and DR-mediated longevity
Recent studies in C. elegans identify multiple signaling pathways that improve longevity through cell non-autonomous mechanisms, including the heat shock response and insulin-like signaling. These pathways utilize sensory cells, frequently neurons, to signal to peripheral tissues and promote survival during the presence of external stress. Importantly, this neuronal activation of stress response pathways, either through genetic modification or through exposure to environmental stress is often sufficient to improve health and longevity. Our data show that the most well-studied longevity intervention, dietary restriction (DR), acts in part through a cell non-autonomous signaling pathway that is inhibited by the smell of food in C. elegans. We further find that DR leads to induction of an intestinal gene, flavin-containing monooxygenase-2 (fmo-2), that is both necessary and sufficient to improve healthspan, stress resistance, and longevity. We also observe that induction of fmo-2 and extension of lifespan both depend on the serotonergic signaling and can be recapitulated by the serotonin antagonist drug, mianserin. Interestingly, serotonin is also implicated in other cell non-autonomous signaling pathways, suggesting this conserved and important signaling molecule may play a critical role in regulating aging. However, the spatial and temporal response of serotonergic signaling during stressful stimuli, like DR, is not well-defined. With mounting evidence pointing to the nervous system’s role in regulating organismal health and longevity, and previous studies lacking detailed reconstruction of signaling pathways, this project will address this vital gap in our knowledge by identifying the critical serotonergic neuron(s), receiving cells and signaling components downstream of food perception.
Recent studies in C. elegans identify multiple signaling pathways that improve longevity through cell non-autonomous mechanisms, including the heat shock response and insulin-like signaling. These pathways utilize sensory cells, frequently neurons, to signal to peripheral tissues and promote survival during the presence of external stress. Importantly, this neuronal activation of stress response pathways, either through genetic modification or through exposure to environmental stress is often sufficient to improve health and longevity. Our data show that the most well-studied longevity intervention, dietary restriction (DR), acts in part through a cell non-autonomous signaling pathway that is inhibited by the smell of food in C. elegans. We further find that DR leads to induction of an intestinal gene, flavin-containing monooxygenase-2 (fmo-2), that is both necessary and sufficient to improve healthspan, stress resistance, and longevity. We also observe that induction of fmo-2 and extension of lifespan both depend on the serotonergic signaling and can be recapitulated by the serotonin antagonist drug, mianserin. Interestingly, serotonin is also implicated in other cell non-autonomous signaling pathways, suggesting this conserved and important signaling molecule may play a critical role in regulating aging. However, the spatial and temporal response of serotonergic signaling during stressful stimuli, like DR, is not well-defined. With mounting evidence pointing to the nervous system’s role in regulating organismal health and longevity, and previous studies lacking detailed reconstruction of signaling pathways, this project will address this vital gap in our knowledge by identifying the critical serotonergic neuron(s), receiving cells and signaling components downstream of food perception.
2. Cell non-autonomous control of aging by the hypoxic response
2. Cell non-autonomous control of aging by the hypoxic response
Hypoxia is a condition of low oxygen, which induces the hypoxia-inducible transcription factor (HIF) and extends lifespan in worms. Stabilization of HIF-1 in specific neurons is sufficient to extend lifespan of worms. HIF-1 in neurons cell non-autonomously induces a downstream target Flavin-containing monooxygenase-2 (FMO-2) in the intestine, which is sufficient to extend lifespan in worms. We are interested in mapping the neuronal circuit of the original neurons, the interneurons and the integrating neurons which pass the signaling by 5-HT release, neurotransmitters, or small peptides to reach the intestinal receptor and induce the transcription of stress effectors in the intestine to catalyze substrates for healthy aging and longevity. This neuronal circuit is not restricted to hypoxia, but could be shared by other interventions for longevity, which provides a potential method to promote whole organism longevity by targeting neuronal aging sensors.
Hypoxia is a condition of low oxygen, which induces the hypoxia-inducible transcription factor (HIF) and extends lifespan in worms. Stabilization of HIF-1 in specific neurons is sufficient to extend lifespan of worms. HIF-1 in neurons cell non-autonomously induces a downstream target Flavin-containing monooxygenase-2 (FMO-2) in the intestine, which is sufficient to extend lifespan in worms. We are interested in mapping the neuronal circuit of the original neurons, the interneurons and the integrating neurons which pass the signaling by 5-HT release, neurotransmitters, or small peptides to reach the intestinal receptor and induce the transcription of stress effectors in the intestine to catalyze substrates for healthy aging and longevity. This neuronal circuit is not restricted to hypoxia, but could be shared by other interventions for longevity, which provides a potential method to promote whole organism longevity by targeting neuronal aging sensors.
Knocking down tph-1, the rate-limiting enzyme in serotonin production, inhibits fmo-2 induction (*p<.05 compared to vector DR).
"Our lab uses the ProOx 110 oxygen controller and C-Chamber subchamber from BioSpherix to carry out in vivo studies in a nematode model system in order to understand the role that O2 levels play in regulating longevity. Learn more - https://biospherix.com/animal-products/"
"Our lab uses the ProOx 110 oxygen controller and C-Chamber subchamber from BioSpherix to carry out in vivo studies in a nematode model system in order to understand the role that O2 levels play in regulating longevity. Learn more - https://biospherix.com/animal-products/"
3. Small molecule screening for health and longevity in worms
3. Small molecule screening for health and longevity in worms
Flavin-containing monooxygenase-2 (FMO-2) induction by hypoxia-inducible transcription factor-1 (HIF-1) is sufficient to promote stress resistance and longevity without the detrimental effect of HIF-1 stabilization. We utilize a single copy fmo2p::mcherry transcriptional reporter to screen small molecules for FMO-2 transcription induction. Similar to the lifespan extension mechanism of FMO-2 induction by hypoxia, these target small molecules are potential replacement for hypoxia or genetically stabilization of HIF-1 to extend lifespan in worms or in humans in the future.
Flavin-containing monooxygenase-2 (FMO-2) induction by hypoxia-inducible transcription factor-1 (HIF-1) is sufficient to promote stress resistance and longevity without the detrimental effect of HIF-1 stabilization. We utilize a single copy fmo2p::mcherry transcriptional reporter to screen small molecules for FMO-2 transcription induction. Similar to the lifespan extension mechanism of FMO-2 induction by hypoxia, these target small molecules are potential replacement for hypoxia or genetically stabilization of HIF-1 to extend lifespan in worms or in humans in the future.
4. Establish how temperature, health and stress response intersect in C. elegans
4. Establish how temperature, health and stress response intersect in C. elegans
While there are numerous advantageous reasons to use nematodes, one critical reason is their ability to adapt and grow at a wide range of temperatures, including “room temperature” or 20° C. Out of convenience most survival assays are performed at 20° C. It’s well established that organisms that don’t regulate their own temperature, like nematodes, live longer as their ambient temperature decreases. Dr. Leiser previously found that lifespans of mutants in the hypoxic response pathway, when compared to control animals, are remarkably dependent on temperature. To follow-up these findings, lifespans of known longevity interventions were performed at 15°, 20°, and 25° C which captured a picture of the multiplex interaction between temperature and genetics. Collectively, these studies reveal that choosing which temperature survival assays are performed at may affect the outcome to a greater extent than was previously realized.
While there are numerous advantageous reasons to use nematodes, one critical reason is their ability to adapt and grow at a wide range of temperatures, including “room temperature” or 20° C. Out of convenience most survival assays are performed at 20° C. It’s well established that organisms that don’t regulate their own temperature, like nematodes, live longer as their ambient temperature decreases. Dr. Leiser previously found that lifespans of mutants in the hypoxic response pathway, when compared to control animals, are remarkably dependent on temperature. To follow-up these findings, lifespans of known longevity interventions were performed at 15°, 20°, and 25° C which captured a picture of the multiplex interaction between temperature and genetics. Collectively, these studies reveal that choosing which temperature survival assays are performed at may affect the outcome to a greater extent than was previously realized.
Data from Miller et al. 2017 Aging Cell.
5. Reduced insulin signaling extends longevity across taxa
5. Reduced insulin signaling extends longevity across taxa
In C. elegans, reduction of function mutations in the insulin/insulin-like-growth-factor receptor daf-2 increases longevity through a mechanism which requires increased activity of the FOXO transcription factor DAF-16. To identify the downstream mechanism through which DAF-16 activity promotes longevity, we used transcriptome profiling and genetic filtering to identify a short list of daf-16 targets associated with longevity or dal genes, then tested loss of function mutations in candidate dal genes for suppression of longevity in daf-2/IGFr mutants. We identified a conserved carboxylesterase family member, CEST-1, which is required for full lifespan extension in multiple contexts of reduced insulin signaling, and used CRISPR based mutation of a predicted catalytic residue to confirm that CEST-1 catalytic activity is required for lifespan extension in daf-2 mutants. Expression of GFP tagged CEST-1 under it’s endogenous promoter is induced by reduced insulin signaling, and localized to the luminal membrane of the intestine suggesting that CEST-1 may function in digestion or immunity. Overexpression of CEST-1 is sufficient to extend lifespan in the context of reduced insulin signaling, suggesting that CEST-1 activity may produce metabolites or signaling molecules that extend lifespan through a downstream pathway. Future work will focus on elucidating CEST-1 substrates using unbiased metabolomics.
In C. elegans, reduction of function mutations in the insulin/insulin-like-growth-factor receptor daf-2 increases longevity through a mechanism which requires increased activity of the FOXO transcription factor DAF-16. To identify the downstream mechanism through which DAF-16 activity promotes longevity, we used transcriptome profiling and genetic filtering to identify a short list of daf-16 targets associated with longevity or dal genes, then tested loss of function mutations in candidate dal genes for suppression of longevity in daf-2/IGFr mutants. We identified a conserved carboxylesterase family member, CEST-1, which is required for full lifespan extension in multiple contexts of reduced insulin signaling, and used CRISPR based mutation of a predicted catalytic residue to confirm that CEST-1 catalytic activity is required for lifespan extension in daf-2 mutants. Expression of GFP tagged CEST-1 under it’s endogenous promoter is induced by reduced insulin signaling, and localized to the luminal membrane of the intestine suggesting that CEST-1 may function in digestion or immunity. Overexpression of CEST-1 is sufficient to extend lifespan in the context of reduced insulin signaling, suggesting that CEST-1 activity may produce metabolites or signaling molecules that extend lifespan through a downstream pathway. Future work will focus on elucidating CEST-1 substrates using unbiased metabolomics.
6. Understand the effect of FMO perturbations on healthspan and lifespan in mice
6. Understand the effect of FMO perturbations on healthspan and lifespan in mice
Utilizing a translational approach to study aging, our lab focuses on the most intriguing results from C. elegans and tissue culture models to ask fundamental questions about the role of FMOs in mice. Preliminary results indicate that FMO proteins are implicated in altering the healthspan and lifespan of the primary C. elegans model, and play a conserved role in regulating stress resistance in both C. elegans and mammalian cells. To better understand this protein family in a mammalian system, we utilized Cas9-mediated (CRISPR) deletions to create two transgenic mouse strains, a knockout of FMO5 and a knockout of FMO1-4. This project will focus on the impact of altered FMO expression along with diet modification (high fat diet, calorie restriction) on metabolism, gut microbiome, stress resistance, cognitive function and ultimately aging in mice.
Utilizing a translational approach to study aging, our lab focuses on the most intriguing results from C. elegans and tissue culture models to ask fundamental questions about the role of FMOs in mice. Preliminary results indicate that FMO proteins are implicated in altering the healthspan and lifespan of the primary C. elegans model, and play a conserved role in regulating stress resistance in both C. elegans and mammalian cells. To better understand this protein family in a mammalian system, we utilized Cas9-mediated (CRISPR) deletions to create two transgenic mouse strains, a knockout of FMO5 and a knockout of FMO1-4. This project will focus on the impact of altered FMO expression along with diet modification (high fat diet, calorie restriction) on metabolism, gut microbiome, stress resistance, cognitive function and ultimately aging in mice.
7. The role of fmo-4 in longevity and stress response
7. The role of fmo-4 in longevity and stress response
The Leiser lab has previously shown that flavin-containing monooxygenase (fmo)-2 in C. elegans is required downstream of the hypoxic response and dietary restriction (DR) to extend lifespan and improve healthspan. Moreover, fmo-2 induction is sufficient to confer these benefits. As there are five C. elegans FMOs, we were curious to know if any of the other FMOs are also regulators of aging. This project focuses on C. elegans FMO-4 in the context of longevity and stress resistance. FMO-4 is structurally similar to FMO-2, with 88% conservation in catalytic residues, and we have found that it is also required for health benefits in multiple longevity pathways, including DR. Interestingly, fmo-4 is required for fmo-2-mediated longevity and is both required and sufficient to improve stress resistance downstream of fmo-2. However, while fmo-2 is required for the hypoxic response and DR-mediated longevity, fmo-4 is only required for some forms of DR-mediated longevity. Our most recent data show that fmo-4 is required for and downstream of atf-6-mediated longevity, which has implications in stress response and calcium signaling. Taken together, these data lead us to hypothesize that fmo-4 has both overlapping and distinct functions from fmo-2 and predict that fmo-4 promotes healthy aging by regulating calcium signaling and stress response. Going forward, this project will focus on fmo-4 and its interactions with fmo-2 as well as other longevity pathways, the distinct functions of fmo-4, and the downstream metabolic processes being altered by fmo-4 to regulate longevity.
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