Mair, William Worm Breeder's Gazette "A simple yet effective method to manipulate C. elegans in liquid"

Mair, William

[Worm Breeder's Gazette]

Mair, William et al. (2011) International Worm Meeting "Defining longevity-specific targets of AMPK."

Dillin, Andrew, Shaw, Reuben, Montminy, Marc, Manning, Gerard, Mair, William, Rodrigues, Ana, Morantte, Ianessa

[International Worm Meeting, 2011]

Dietary restriction increases lifespan in organisms from yeast to primates and reduces severity of a range of age-related pathologies. Targeting the molecular framework underpinning this phenomenon may therefore provide novel therapeutics that confer resistance to multiple diseases via a single intervention. AMP-activated protein kinase is a pro-longevity kinase activated when energy is low, which lies upstream of a variety of nutrient sensing pathways known to mediate organismal lifespan. We have shown that a critical longevity target of AMPK in C. elegans is the CREB-regulated transcriptional coactivator CRTC-1. CRTCs are a family of cofactors involved in diverse physiological processes including energy homeostasis, cancer and endoplasmic reticulum stress. CRTC-1 is a direct AMPK target, and interacts with the CREB homologue-1 (CRH-1) transcription factor in vivo. The pro-longevity effects of activating AMPK decrease CRTC-1 and CRH-1 activity and induce transcriptional responses similar to those of crh-1 null worms. Downregulation of crtc-1 increases lifespan in a crh-1-dependent manner and directly reducing crh-1 expression increases longevity, substantiating a role for CRTCs and CREB in aging. This finding allows us to uncouple causal longevity-specific processes regulated by AMPK from casually associated downstream targets. We demonstrate the specific role of CRTC-1 in the longevity function of AMPK, separable from the effect of AMPK on growth and reproduction. Further, we show that AMPK activates a family of genes involved in the unfolded protein response (UPRER) and that these targets modulate the effect of AMPK on lifespan. Together these data illustrate a molecular pathway activated under dietary restriction to induce transcriptional changes that up-regulate protein homeostasis and longevity.

William Mair et al. (2009) International Worm Meeting "The role of CRTC in worm longevity."

Andrew Dillin, Reuben Shaw, Ianessa Morantte, William Mair, Katherine Butler

[International Worm Meeting, 2009]

Mammalian CRTC2 is a coactivator of the transcription factor cAMP Responsive Binding Protein (CREB), which is involved in diverse biological processes from memory to gluconeogenesis and fat storage. CRTC2 is a target of the nutrient responsive AMP-activated protein kinase (AMPK), which phosphorylates CRTC2, resulting in its cytoplasmic translocation and inactivation. Conversely, dephosphorylation by the phosphatase Calcineurin leads to nuclear localization of CRTC2. In C. elegans, gain of function of AMPK (aak-2), and loss of function of Calcineurin (tax-6), have been shown to extend lifespan. Here we show that the sole worm CRTC orthologue is regulated via a mechanism similar to that seen in mammals that depends upon AMPK family kinases and tax-6. Furthermore, adult-onset reduction of both CREB and CRTC in worms extends lifespan, suggesting that perturbations to AMPK and Calcineurin may prolong life in part through down-regulation of CRTC/CREB target genes.

Escoubas, Caroline et al. (2015) International Worm Meeting "The role of CRTC-1 in Abeta induced transcriptional dysregulation."

Mair, William, Escoubas, Caroline

[International Worm Meeting, 2015]

The major risk factor for the development of Alzheimer's disease (AD) is age, and the biological hallmarks of AD are the accumulation of extracellular deposits of amyloid beta (Abeta) plaques as well as intracellular neurofibrillary tangles of phosphorylated tau. However, the role of Abeta in the development of the disease remains unclear, and efficacy of clinical trials targeting Abeta plaques has been low, leaving only symptomatic treatments available to patients. Dysregulation of calcium homeostasis due to enhanced intracellular calcium liberation from the endoplasmic reticulum has been suggested to link Abeta load to AD pathogenesis. This loss of calcium homeostasis dysregulates the function of calcium dependent proteins, such as the phosphatase calcineurin. We have shown previously in C. elegans that a key target of calcineurin and its effects on aging is the transcriptional regulator CREB regulated transcriptional coactivator (CRTC)-1. In mouse CRTC1 function is dysregulated in the brain in AD models and its overexpression in the hippocampus can reverse Abeta induced spatial learning and memory deficits. However, the specific genes targeted by CRTC1 to promote this effect have not been described. We aim to study the downstream transcriptional changes mediated by CRTC-1, before neuronal loss, during Abeta induced neurodegeneration, using a worm model of AD, which expresses Abeta 1-42 specifically in glutamatergic neurons, a neuronal subtype particularly vulnerable in AD. We have found that the knock out of tax-6/calcineurin impairs neuronal survival in the Abeta expression strain. This result is opposite to the effect of calcineurin on aging, as inhibition of tax-6 promotes longevity. We therefore hypothesize that the calcineurin/CRTC-1 pathway has opposing roles in aging and neurotoxicity. Here we will discuss our work aimed toward defining the transcriptional network downstream of calcineurin and CRTC-1 responsible for their effects on the pathogenicity of Abeta, with the goal of elucidating novel targets to treat AD. .

Jackson, Noel et al. (2021) International Worm Meeting "Defining a functional role for splicing factors in modulating longevity in C. elegans"

Heintz, Caroline, Howell, Porsha, Dutta, Sneha, Perez Matos, Maria, Jackson, Noel, Mair, William

[International Worm Meeting, 2021]

Aging is the decline in proliferative processes and organismal metabolism that promotes deterioration of cellular and physiological function and eventually leads to mortality. Biological aging can be uncoupled from chronological age and slowed by restricting food intake without malnutrition through dietary restriction (DR) and altering nutrient-sensing pathways, such as the mechanistic target of rapamycin (mTORC) pathway. The Mair lab has identified a conserved splicing factor, SFA-1/SF1, that modulates the longevity phenotype usually attained through dietary restriction (DR) and suppressed-mTORC1 activity in C. elegans. However, the mechanism that links DR and mTORC1 longevity to the regulation of SFA-1/SF1 is unknown. SFA-1 is the C. elegans homolog of mammalian splicing factor SF1, which coordinates with other splicing factors to initiate splicing of pre-mRNA transcripts. SF1 splicing efficiency is altered by phosphorylation events at conserved residues, and we have found that altering SFA-1 by inhibiting phosphorylation events in two distinct residues abolishes the suppressed-mTORC1 longevity phenotype in C. elegans. In future studies, I will characterize the functional role of SFA-1 and its downstream targets and interaction partners to identify their contribution to aging. Uncovering biological processes that are regulated by SFA-1 and drive lifespan extension could define novel RNA homeostatic modulators of longevity that may contribute to age-related diseases.

Zhang, Yue et al. (2013) International Worm Meeting "Delineating AMPK and TOR longevity."

Mair, William, Morantte, Ianessa, Zhang, Yue

[International Worm Meeting, 2013]

AMP-activated protein kinase (AMPK) and target of rapamycin (TOR) are two critical regulators of cellular nutrient-sensing pathways that mediate the aging process. Although mammalian AMPK and TOR are known to be linked mechanistically, whether this is also true in C. elegans - the model of choice for the bulk of the aging research - and whether AMPK and TOR function together or separately to increase longevity remains unclear. In mammals, AMPK and TOR negatively regulate each other: AMPK acts upstream of TOR, inhibiting TOR complex 1 (TORC1) indirectly via the TSC complex and directly by phosphorylation of the TORC1 component Raptor. However, recently TORC1 was also shown to act upstream of AMPK: TOR suppresses AMPK via the TORC1 target S6 Kinase, which phosphorylates AMPK alpha and inhibits its subsequent activation. AMPK and TORC1 also have antagonistic effects on lifespan: genetic or pharmacological activation of AMPK promotes C. elegans longevity, while inhibition of TOR extends lifespan. Here, we will examine whether AMPK and TOR modulate lifespan via shared or separable mechanisms, and whether lifespan extension via one, requires altered activity of the other. Suggesting that AMPK acts downstream of TORC1 to modulate aging, we show that lifespan extension via TOR/let-363 and S6 Kinase/rsks-1 inhibition requires the AMPK catalytic subunit AAK-2. Furthermore, we show that phosphorylation of the downstream longevity target of AMPK, CREB-regulated transcriptional coactivator-1 (CRTC-1), is required for lifespan extension by loss of S6K. Our results therefore suggest that the AMPK-CRTC-1 axis is a longevity-specific regulator downstream of the TOR pathway.

Sharma, Arpit et al. (2019) International Worm Meeting "Dissecting spatial and temporal requirement of TORC1 pathway components for longevity."

Sharma, Arpit, Zhang, Yue, Lanjuin, Anne, Mair, William

[International Worm Meeting, 2019]

AMPK and TORC1 show opposing responses to cellular energy and have antagonistic effects on several shared downstream processes, including longevity. While constitutive AMPK activation extends lifespan, TORC1 activation reduces lifespan. However, it is still unknown whether they coordinate to determine longevity or act via separate mechanisms. We found that neuronal AMPK activity is required for pro-longevity effects of global TORC1 inhibition via RAGA-1/RAGA RNAi. Furthermore, we found that lifespan extension by null mutations in genes encoding raga-1/RagA or rsks-1/S6K is reversed by the neuronal-specific rescue of these TORC1 components. Mechanistically, lifespan extension by the loss of RAGA-1 is mediated via maintenance of fused mitochondrial networks. Our results demonstrate that neuronal TORC1 activity is required for cell nonautonomous modulation of mitochondrial networks in the intestine to promote lifespan extension. These findings suggest that TORC1 activity in specific tissues, such as the neurons, can be targeted to promote longevity. We have now utilized an auxin-induced degradation (AID) system for inducible degradation of various TORC1 components, LET363/TOR, DAF-15/Raptor, RAGA-1, and RSKS-1, in neurons, intestine, germline, and muscle. We will present our ongoing investigation on the requirement of different TORC1 pathway proteins in various tissues for global lifespan extension. Furthermore, using the inducible deletion of TORC1 components during development, adulthood, midlife, and old-age, we plan to examine whether there is a temporal requirement for TORC1 inhibition for lifespan extension. Overall, we aim to dissect the spatial and temporal requirement of TORC1 components for longevity for prospective therapies.

Leslie, Jessica et al. (2021) International Worm Meeting "A fluorescent toolkit for live analysis of mitochondrial genome maintenance in C. elegans"

Vidyasagar, Nitin, Mair, William, Lewis, Samantha, Lanjuin, Anne, Leslie, Jessica, Yao, Pallas

[International Worm Meeting, 2021]

Mitochondria are energy-producing organelles that are essential for eukaryotic cell function. Central to their role is mitochondrial DNA (mtDNA), a small, circular genome present in multiple copies per cell that encodes proteins and RNAs necessary for oxidative phosphorylation. In multicellular organisms, mtDNA is continuously synthesized in both proliferating and post-mitotic cells and varies in copy number between tissue types. MtDNA distribution and synthesis are linked to mitochondrial fission and fusion dynamics in budding yeast and in cancer cell models, however, whether these processes are linked in post-mitotic somatic cells remains unclear. Here, we present molecular imaging tools for the study of mtDNA maintenance within live Caenorhabditis elegans. We have coupled Mitotracker and SYBR Gold stains with Airyscan superresolution microscopy to establish "ground truth" tissue-specific patterns of mtDNA distribution. Then, using transgenic strains in which the mtDNA binding protein hmg-5/TFAM has been fluorescently tagged with wrmScarlet at the endogenous locus we identify tissue-specific regimes of transcription factor distribution within mitochondria and relate them to fusion-fission dynamics. We find that some transgenic strategies, such as the overexpression of mtDNA binding factors via extrachromosomal array, perturb normal mitochondrial DNA levels; we present a set of best practices in strain construction. These validated imaging tools lay the foundation to test whether differences in mitochondrial structure between tissues are predictive of mtDNA abundance and/or distribution. This work has the potential to inform tissue-specific pathologies of mtDNA disease in humans.

Hwang, Ara B. et al. (2013) International Worm Meeting "Mitochondrial ROS promote longevity and innate immunity via a feedback loop involving HIF-1 and AMPK."

Lee, Seung-Jae, Hwang, Ara B., Artan, Murat, Ryu, Eun-A, Mair, William

[International Worm Meeting, 2013]

Mild inhibition of mitochondrial respiration promotes longevity across phyla. Recently we reported that reduced respiration lengthens lifespan by increasing reactive oxygen species (ROS) that activate the hypoxia-inducible factor 1 (HIF-1) in C. elegans. Here we elucidated a feedback-regulatory mechanism and functional significance of the ROS-induced longevity. We initially found that mutations in aak-2 (catalytic a subunit of AMP-activated protein kinase [AMPK]) suppressed the longevity induced by an ROS-generating chemical, paraquat (0.25 mM). In addition, we showed that mitochondrial ROS activated AMPK, and that paraquat did not further increase the longevity of gain-of-function AAK-2 transgenic animals. These data suggest that AMPK mediates the longevity induced by mitochondrial ROS. We then investigated the mechanisms by which AMPK contributes to the ROS-induced longevity. We demonstrated that AMPK acts as a negative feedback regulator of internal ROS levels, because aak-2 mutants were hyper-sensitive to ROS and contained increased ROS levels upon paraquat treatment. In contrast, HIF-1 was required for increasing internal ROS levels, suggesting that HIF-1 acts as a positive regulator of ROS. These data imply that feedback regulation by AMPK and HIF-1 ensures balance between the benefit and toxicity of ROS. Next, we determined the relationship between the ROS-induced longevity and innate immunity, because infection by E. coli is the major cause of death of aged C. elegans in laboratory. We found that feeding non-pathogenic bacteria increased the lifespan of wild type but not that of the long-lived mitochondrial isp-1 mutants, which display high levels of ROS. Moreover, isp-1 mutants were resistant to several pathogens, and both aak-2 and hif-1 were required for the enhanced pathogen resistance. Taken together, we propose that the feedback circuit involving AMPK and HIF-1 regulates ROS levels to maintain an optimal immunity and longevity.

Gheorghe, Ioana et al. (2021) International Worm Meeting "A genome-wide RNAi screen for factors of tissue growth coordination"

Smith, Hannah J., Lanjuin, Anne, Towbin, Benjamin D., Mair, William B., Stojanovski, Klement, Gheorghe, Ioana

[International Worm Meeting, 2021]

Reaching appropriate organ size proportions is crucial for organismal function. However, size proportions are intrinsically challenged by stochastic fluctuations in the growth rate of organs. Although, in principle, small heterogeneities in the growth rate can amplify to large differences in their size during development, organ size proportions are remarkably uniform among isogenic individuals. We therefore hypothesised that organs coordinate their growth and size with each other. To test this hypothesis and understand the mechanism involved, we used the AID/TIR1 to systematically inhibit mTOR signalling in individual tissues. The mTOR pathway is a conserved and central regulator of cellular growth that is thought to act cell autonomously. However, we find that tissue-specific inhibition of mTOR also has large systemic effects on other tissues. For example, pharynx-specific depletion of mTOR is associated with a global reduction of body growth. Depletion of mTOR in the pharynx led to a 2-fold reduction in the adult body size, but did not cause a deviation from the appropriate pharynx-to-body size proportions. To unravel the inter-cellular signalling network that coordinates organ growth, we are currently conducting a genome-wide high content RNAi screen for supressors and enhancers of this phenotype.