Carmona, Juan J. et al. (2009) International Worm Meeting "The C. elegans PNC-1 nicotinamidase increases stress resistance and mediates caloric restriction-induced longevity."

Hanna-Rose, Wendy, Vrablik, Tracy, Sleigh, James N., Carmona, Juan J., Hart, Anne C., Sinclair, David A., Michan, Shaday

[International Worm Meeting, 2009]

A common and powerful non-genetic method used to extend an organism''s lifespan is caloric restriction, yet the underlying molecular mechanisms of this increased longevity remain unclear. In yeast, Pnc1 (nicotinamidase), an enzyme in the NAD+ salvage pathway that converts nicotinamide to nicotinic acid to assist in regenerating NAD+, can mediate lifespan extension under caloric restriction and mild stresses. We have previously shown that Pnc1 is nuclear, cytoplasmic, and peroxisomal in yeast and that caloric restriction and stress, like salt, causes a significant up-regulation in expression (Anderson, 2003). To understand the impact of this nicotinamidase on longevity and stress resistance in multi-cellular organisms, we are using C. elegans as a model. We show that the worm ortholog PNC-1 also has robust nicotinamidase activity in vitro and that increased gene dosage in C. elegans protects against salt stress and extends lifespan. Interestingly, this longevity effect is sirtuin dependent. In addition, lifespan extension mediated by PNC-1 over-expression is not additive with caloric restriction-induced longevity, suggesting that PNC-1 is an important component of this pathway. Accordingly, inactivation of the pnc-1 gene leads to a defect in caloric restriction-induced longevity in C. elegans. Using a GFP reporter, we found that pnc-1 is expressed in the pharynx and nervous system, including the chemosensory ASK neurons. We are currently using cell-specific promoters to dissect the biological relevance of these sites of expression. Interestingly, invertebrate PNC-1 and the mammalian PBEF/Visfatin catalyze analogous biochemical steps; we are expressing the human PBEF/Visfatin in pnc-1 loss-of-function animals to test the functional conservation of these enzymes across species. Collectively, this study elucidates a functional role for PNC-1 in metazoans and expands our understanding of conserved pathways involved in longevity and stress resistance across organisms.

Juan Carmona et al. (2008) C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting "Functional characterization of PNC-1 in aging and stress"

Shaday Michan, Anne Hart, David Sinclair, Juan Carmona

[C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting, 2008]

In yeast cells, Pnc1 (nicotinamidase) is an evolutionarily conserved enzyme in the NAD+ salvage pathway and a master regulator of aging, as (1) expression is induced by environmental stresses and (2) is necessary and sufficient for lifespan extension by caloric restriction and stress. Yeast Pnc1 is thought to control the sirtuin family of NAD+-dependent deacetylases, enzymes previously implicated in aging and stress. Given this work, we hypothesized that PNC-1 in C. elegans may play a role in longevity and stress protection, and that specific tissues may be important for these biological effects. To study the enzymatic activity of PNC-1 in vitro, recombinant protein was expressed in bacterial cells, purified, assayed, and shown biochemically to have robust nicotinamidase activity. Moreover, site-directed mutagenesis of a conserved residue in the active site of PNC-1, as predicted by yeast Pnc1 crystallographic data, abolishes nicotinamidase enzymatic function. PNC-1 over-expressing transgenic animals were generated, employing a ubiquitous driver, to study the biological effects of over-expression in vivo. Using previously described dietary assays, PNC-1 over-expressing animals were tested for longevity and found to outlive controls. To assess a conserved role in stress responses, PNC-1 over-expressing animals were subjected to an established salt stress paradigm, to which they were remarkably resistant, suggesting that PNC-1 may play an important role in integrating nutritional signals and environmental conditions to determine lifespan. Lastly, to pinpoint sites of endogenous expression, a pnc-1 promoter fragment was cloned into a green fluorescent protein reporter system; green expression was observed in the pharynx and specific neurons. Also, epistasis studies are in progress to address how pnc-1 acts in relation to known regulators of aging. Collectively, all of these approaches will better define conserved pro-survival pathways across different organisms, which will contribute significantly to our basic understanding of the biology of aging.

Juan J. Carmona et al. (2007) International Worm Meeting "Functional Characterization of pnc-1 in C. elegans."

Juan J. Carmona, David A. Sinclair, Anne C. Hart

[International Worm Meeting, 2007]

In yeast, Pnc1 (nicotinamidase), an enzyme in the NAD+ salvage pathway that converts nicotinamide to nicotinic acid to assist in regenerating NAD+, has been shown to be necessary and sufficient for lifespan extension under caloric restriction and mild stresses. Furthermore, a Pnc1-GFP fusion reporter has shown that Pnc1 is nuclear, cytoplasmic, and peroxisomal in yeast and that caloric restriction and mild stress, like heat, causes a significant increase in fluorescence intensity. Given these data, therefore, attention has now turned to Pnc1 homologues in multi-cellular organisms, such as PNC-1 in C. elegans, specifically with the desire to study functional conservation of this enzyme with respect to longevity and stress pathways. Recently, it has been reported that RNAi-mediated knockdown of the C. elegans homologue pnc-1 significantly decreases adult lifespan and that increased gene dosage enhances survival during oxidative stress treatment. However, the tissue-specific expression pattern of PNC-1 remains unknown and a protective role for PNC-1 during heat shock, a common stress paradigm in C. elegans, has not been considered. To this end, we have generated GFP fusion constructs to pinpoint PNC-1 expression. Upon first examination, GFP is expressed in pharyngeal muscle and ASK neurons, with the latter playing a role in insulin signaling and longevity. Furthermore, we have generated polyclonal antibodies against PNC-1 and have determined by Western blot that stresses, in particular heat, increase PNC-1 protein levels. Finally, we have produced PNC-1 over-expressing lines which we are actively characterizing for a longevity phenotype. Our preliminary data suggest that these over-expressing lines are markedly thermotolerant; other forms of stress are under analysis. Collectively, it is anticipated that all of these studies will help to elucidate a functional role for pnc-1 in animals, to further expand our understanding of conserved longevity and stress pathways across organisms.

Forbes, Emily et al. (2009) International Worm Meeting "The Histone 3 Lysine 9 and 36 Tri-demethylase JMJD-2 Impacts C. elegans Longevity Through Genomic Integrity."

Carmona, Juan, Whetstine, Johnathan, Sleigh, James, Forbes, Emily, Hart, Anne

[International Worm Meeting, 2009]

Genomic stability is a major factor that influences aging and aging-related diseases. The integrity of the genome is influenced by histones and other chromosomal proteins that package and stabilize the genome as well as regulate gene expression. The posttranslational modifications of histones, especially lysine methylation, play an important role in these processes. The site and degree of methylation have specific affects on nuclear processes. For example, histone lysine 9 tri-methylation (H3K9me3) is a hallmark of chromosomal structures such as heterochromatin and telomeres and has distinct roles in gene repression. Consistent with H3K9me3 function in chromosomal stability, alterations in H3K9me3 levels have been shown to result in aging-related diseases (i.e., progeria and cancer). We have uncovered a new class of enzymes that are responsible for demethylating H3K9me3. The JMJD2 protein family is conserved from worm to human and plays an important role in stabilizing the genome. Using C. elegans, we have established an important role for this protein in maintaining the genomic stability of the germline and in lifespan. Our initial data suggest that DNA damage response within the germline plays a fundamental role in the reduced lifespan of the jmjd-2 loss of function allele. In addition, we have observed an increase in generational lifespan that may be associated with structural changes within the chromatin and/or changes in DNA damage response. Our data with the histone demethylase begins addressing the fundamental relationship between chromatin change, tissue homeostasis and lifespan.

Moroz, Natalie et al. (2015) International Worm Meeting "Dietary restriction involves NAD+-dependent mechanisms and a shift towards oxidative metabolism."

Hart, Anne, Sinclair, David, Blackwell, Keith, Anderson, Edward, Carmona, Juan, Moroz, Natalie

[International Worm Meeting, 2015]

Interventions that slow aging and prevent chronic disease may come from an understanding of how dietary restriction (DR) increases lifespan. Mechanisms proposed to mediate DR longevity include reduced mTOR signaling, activation of the NAD+-dependent deacylases known as sirtuins, and increases in NAD+ that derive from higher levels of respiration. Here we explored these hypotheses in Caenorhabditis elegans using a new liquid feeding protocol. DR lifespan extension depended upon a group of regulators that are involved in stress responses and mTOR signaling, and have been implicated in DR by some other regimens (DAF-16 (FOXO), SKN-1 (Nrf1/2/3), PHA-4 (FOXA), AAK-2 (AMPK)). Complete DR lifespan extension required the sirtuin SIR-2.1 (SIRT1), the involvement of which in DR has been debated. The nicotinamidase PNC-1, a key NAD+ salvage pathway component, was largely required for DR to increase lifespan but not two healthspan indicators: movement and stress resistance. Independently of pnc-1, DR increased the proportion of respiration that is coupled to ATP production but, surprisingly, reduced overall oxygen consumption. We conclude that stress response and NAD+-dependent mechanisms are each critical for DR lifespan extension, although some healthspan benefits do not require NAD+ salvage. Under DR conditions, NAD+-dependent processes may be supported by a DR-induced shift towards oxidative metabolism rather than an increase in total respiration. JC and EA contributed equally. AH, DS, and KB are co-corresponding authors.

Moroz, Natalie et al. (2011) International Worm Meeting "Exploring mechanisms of dietary restriction in C. elegans."

Anderson, Edward, Sinclair, David A., Carmona, Juan J., Moroz, Natalie, Hart, Anne, Blackwell, T. Keith

[International Worm Meeting, 2011]

Genetic variability and environmental conditions influence health and lifespan. A key component of our environment, our food intake, can dramatically affect longevity. The reduction of food consumption without malnutrition, called dietary restriction (DR), increases lifespan in essentially all eukaryotes, including yeast, Drosophila, C. elegans and mice. In C. elegans there are several methods of performing DR, including: diluting bacteria, using bacteria that are rich in different nutrients, using a chemically defined medium, or using a genetic mutant that has reduced pharyngeal pumping. Surprisingly, many of the current DR methods work independently of major longevity pathways. To further address how DR affects lifespan we developed a liquid DR protocol in C. elegans that 1) establishes bacteriostatic conditions, allowing bacteria to be kept at set concentrations for extended periods of time, and 2) is easy to maintain, in comparison to other methods that require daily maintenance and perturbation of the worms environment. We will discuss our findings regarding the role of key longevity factors in our protocol, including: all four homologs of the yeast NAD -dependent histone deacetylase SIR2, the nicotinamidase PNC-1, the low energy sensing kinase AMPK/AAK-2, the FoxO/DAF-16 transcription factor, and the Nrf/SKN-1 transcription factor. While some factors were found to be required, others play a partial role. Our results show that some key nutrient sensing pathways that are important for longevity do play a role in DR.

Moroz, Natalie et al. (2013) International Worm Meeting "Importance of Growth, Stress Defense, and NAD+-related Pathways for Dietary Restriction Longevity."

Moroz, Natalie, Anderson, Edward, Hart, Anne, Sinclair, David A., Carmona, Juan J., Blackwell, T. Keith

[International Worm Meeting, 2013]

The reduction of food consumption without malnutrition, called dietary restriction (DR), increases lifespan in essentially all eukaryotes. In C. elegans genetic requirements for lifespan extension vary among DR protocols, suggesting that C. elegans will be valuable for elucidating and analyzing processes involved in DR. Using a simple liquid DR protocol that minimizes maintenance and produces robust lifespan extension, we identified several mechanisms involved in DR. Some of these have been implicated by other DR protocols: the transcription factors Nrf/SKN-1, FoxA/PHA-4, and FoxO/DAF-16, and the low-energy sensing kinase AMPK/AAK-2. These factors have been linked to nutrient availability or growth, and implicated in stress defense. The importance of FoxO/DAF-16 is of particular interest because its role in DR has been controversial, and because this suggests a possible link between DR and insulin/IGF-1-like signaling. We also observed a partial but significant involvement of the conserved Sirtuin and NAD-dependent deacetylase SIR-2.1, for which a role in DR and aging has also been a subject of debate. The nicotinamidase NAMPT/PNC-1, which is required for nicotinamide metabolism and NAD+ synthesis through the salvage pathway, was largely required for DR to benefit lifespan but not two healthspan indicators: stress resistance and movement. Our findings support the view that growth- or nutrient-regulated defense mechanisms are of central importance in DR lifespan extension, that NAD+-dependent pathways also play an important role, and that some benefits of DR can be uncoupled from lifespan.

Juan Cabello et al. (2006) European Worm Meeting "The Wnt Pathway Controls Engulfment Of Apoptotic Corpses, Spindle Orientation And Migration Through CED-10/Rac"

Juan Cabello, Ralf Schnabel, Ufuk Gunesdogan

[European Worm Meeting, 2006]

Juan Cabello, Ufuk Gunesdogan and Ralf Schnabel. The recognition and engulfment of apoptotic cell corpses is achieved by at least two parallel pathways in C. elegans. These are defined by the proteins CED-1/SREC, CED-6/GULP, CED-7/ABCA1 and CED-2/CrkII, CED-5/Dock180, CED-12/ELMO. The pathways merge at CED-10, which codes for a Rac small GTPase. CED-1 is a scavenger receptor for the recognition of cell corpses in the first pathway. However, the receptor which regulates the second pathway has long remained elusive. Here we show that the receptor MOM-5, a Frizzled homolog of the Wnt pathway in C. elegans, functions upstream of the second pathway not only in engulfment but also in spindle orientation and gonadal migration. Therefore, in different developmental processes, CED-10/Rac links polar signals mediated by the Wnt pathway to the modulation of the cytoskeleton.

Juan Carlos Fierro Gonzalez et al. (2006) European Worm Meeting "C. elegans Thioredoxins Implicated in Nervous System Function and Aging"

Peter Swoboda, Antonio Miranda-Vizuete, Juan Carlos Fierro Gonzalez

[European Worm Meeting, 2006]

Juan Carlos Fierro Gonzlez1, Peter Swoboda1, Antonio Miranda-Vizuete2. Thioredoxins constitute a large family of proteins present in all organisms that catalyze thiol-disulfide redox reactions. They are involved in various cellular processes, such as DNA synthesis and repair, antioxidant defense, regulation of transcription factor DNA binding activity or longevity. Moreover, thioredoxins are also implicated in many important pathologies such as cancer, cardiovascular diseases, diabetes or Alzheimers and Parkinsons diseases.. The C. elegans genome encodes for two thioredoxin reductases and at least eight thioredoxins, many of which have clear orthologs in mammals. We report here the characterization of the first member of the thioredoxin family in metazoans that is mainly associated with neurons. The C. elegans gene B0228.5 encodes a thioredoxin (TRX-1) that is expressed in ASJ sensory neurons and, to some extent, also in the posterior-most intestinal cells. ASJ neurons regulate longevity and also the entry into and exit from the dauer stage, an endurance larval form triggered by adverse environmental conditions or by hormonal cues. A mutant worm strain carrying a null allele of the gene trx-1 displays a reproducible decrease in mean lifespan as compared to wild type, which implicates trx-1 in ASJ-dependent mechanisms that regulate longevity. On the other hand, the trx-1 null mutant is neither dauer formation defective (Daf-d) nor dauer formation constitutive (Daf-c), suggesting either that TRX-1 is only involved in ASJ functions that specifically influence longevity, or that potential TRX-1-dependent dauer phenotypes might only be revealed under sensitized (e.g., double mutant) backgrounds.. In this context, we are currently combining the trx-1 mutation with others in genes known to regulate longevity and/or dauer formation, such as daf-2, daf-16, daf-11 and daf-21. Obtaining double mutants of trx-1 with the last two genes is especially interesting with regard to searching for potentially synthetic phenotypes, as these genes have been reported to function in ASJ neurons. In parallel, by using GFP reporter fusion constructs, we are studying the expression patterns of all the thioredoxin and thioredoxin reductase genes in C. elegans, to identify genes associated with the nervous system and possibly interacting with trx-1. The outcome of these studies will help us clarify the implications of thioredoxins in C. elegans nervous system function and aging.