McReynolds, Melanie R. et al. (2013) International Worm Meeting "Exploring the Flexibility of NAD+ Biosynthesis in C. elegans."

Hanna-Rose, Wendy, McReynolds, Melanie R.

[International Worm Meeting, 2013]

NAD+ is an essential co-enzyme. It is necessary for electron transport in many metabolic reactions and it functions as a substrate for several enzymes known as NAD+ consumers. NAD+ biosynthetic pathways are well elucidated; however, the developmental and physiological roles of these pathways are not known. We focus on understanding the developmental and physiological role of NAD+ biosynthetic pathways as well as the questions: How is NAD+ metabolism controlled?, How do changes in NAD+ metabolism influence physiology? and How can NAD+ metabolism be manipulated for therapeutic benefit? Our previous studies have shown that mutations in the (nicotinamide) NAM to NAD+ salvage pathway enzyme, PNC-1, caused developmental and functional defects in the reproductive system of C. elegans. The developmental defects are more severe under nutritionally stressed conditions where animals are fed dead E. coli. We measured metabolite levels in wild-type and mutant animals and found that while NAM levels increased significantly and NA levels decreased significantly as expected, there was little change in NAD+ levels between pnc-1 mutants and wild-type. Therefore, we hypothesize that there are mechanisms compensating for the production and/or consumption of NAD+ in pnc-1 mutants. To address this hypothesis and to more fully investigate the relative contributions of all NAD+ biosynthetic pathways in development, we are studying the nicotinamide/nicotinic acid riboside (NR/NaR) pathway for the production of NAD+. We measured the transcription level of nrk-1 from N2 or pnc-1 worms grown on live and dead OP50. Our (q)RT-PCR analysis shows that while nrk-1 mRNA levels do not change in pnc-1 mutants when growing on live OP50, there is an up-regulation of nrk-1 transcription in the presence of nutritional stress. This data suggests that the NR/NaR pathway has a significant role in the compensation of NAD+ under stressed conditions. We are currently in the process of examining expression of nrk-1 other stress conditions. We are also creating nrk-1 transcriptional and translational GFP reporters, in order to visualize its gene expression and protein localization.

McReynolds, Melanie et al. (2017) International Worm Meeting "Eukaryotic de novo NAD+ biosynthesis from tryptophan in the absence of a QPRTase homolog."

Wang, Wenqing, Holleran, Lauren, Hanna-Rose, Wendy, McReynolds, Melanie

[International Worm Meeting, 2017]

NAD+ is found in all living cells, and is an essential coenzyme, which impacts the entire metabolome. Hence, NAD+ biosynthesis has proven to be an attractive and promising therapeutic target for influencing health-span and obesity-related phenotypes as well as tumor growth. A wide range of animals and yeast synthesize NAD+ via de novo synthesis from the degradation of tryptophan (Trp), via the kyneurine pathway. The C. elegans' genome encodes all the enzymes involved in the kynurenine pathway. However, the genome lacks the critical quinolinic acid phosphoribosyltransferase (QPRTase) that converts QA into NaMN for biosynthesis of NAD+. Because C. elegans lack an apparent QPRTase homolog, it's been assumed that this species lack active NAD+ de novo synthesis. We've uncovered evidence that suggests NAD+ de novo synthesis is active and contributing to NAD+ biosynthetic capacity in C. elegans. We previously found that phenotypes that are dependent on NAD+ levels could be reversed upon supplementation with QA and other kynurenine metabolites, suggesting that the kyneurine pathway may contribute to NAD+ biosynthesis. We have directly demonstrated that isotopic label from Trp is incorporated into NAD+. Furthermore, we show that loss of kynurenine pathway activity decreases global NAD+ levels and prevents incorporation of isotopic label supplied from Trp into NAD+, supporting the presence of de novo synthesis in C. elegans. Using genetics and isotope tracing experiments, we determined that the enzyme UMPS-1, (uridine monophosphate synthetase), is required for NAD+ biosynthesis from Trp in this organism. Finally, we connected the kyneurine pathway to normal fecundity. This evidence demonstrates that NAD+ de novo synthesis is active and contributes to NAD+ biosynthetic capacity and homeostasis in C. elegans. Intriguingly, a conserved enzyme is substituting for the missing QPRTase, raising questions about the relevance of similar underground metabolic activity in higher organisms.

McReynolds, Melanie et al. (2017) International Worm Meeting "Metabolic carbon tracing reveals that disrupted glycolysis arises from a loss of C. elegans salvage NAD+ synthesis."

Hanna-Rose, Wendy, McReynolds, Melanie

[International Worm Meeting, 2017]

As a key link connecting regulatory and bioenergetics process, NAD+ has emerged as a central metabolic co-factor playing a critical role in regulating cellular metabolism and energy homeostasis. Successfully recycling NAM, released from NAD+-consuming reactions, back to NAD+ is a major challenge for maintaining NAD+ metabolism and homeostasis. The first step in C. elegans' salvage NAD+ synthesis involves the nicotinamidase, pnc-1 (T L Vrablik, Huang, Lange, & Hanna-Rose, 2009). Via global metabolic profiling we previously linked loss of salvage NAD+ synthesis, via loss of pnc-1 activity, to disruptions in glycolysis and a subsequent reproductive developmental delay (Wang et al., 2015). However, the citric acid cycle was not perturbed and functions of the mitochondria were intact in pnc-1 mutants (Wang et al., 2015). This observation suggested that loss of salvage NAD+ biosynthesis affects the cytoplasm specifically. To investigate this model, we decided to directly test the effects of loss of pnc-1 on glycolytic and citric acid cycle flux via application of metabolic isotopic carbon tracing tools. After a short four-hour exposure with universally labeled glucose (13C6-Glucose), we were able to detect an isotopically labeled glucose pool in both wild-type animals and pnc-1 mutants. Metabolic carbon tracing confirmed that glycolysis is impeded at the NAD+-dependent step in pnc-1 mutants. Although glycolysis is compromised, we observed no change in the isotopic flow of label from pyruvate to lactate in pnc-1 mutants. Additionally, we observed an increase of isotopic label from pyruvate entering the TCA cycle in these mutants. Although trehalose steady state levels are increased in pnc-1 mutants, we did not observe increased shunting of glucose towards trehalose. This data bolsters the model that loss of NAD+ salvage biosynthesis preferentially affects cytoplasmic functions. Moreover, our work supports stable isotopic labeling as a robust technique to track the passage of metabolites through metabolic pathways in C. elegans.

McReynolds, Melanie et al. (2015) International Worm Meeting "Maintaining Global NAD+ Homeostasis Reveals Separable Functional and Compensatory Roles for NAD+ Biosynthetic Pathways in C. elegans."

Wang, Wenqing, Hanna-Rose, Wendy, McReynolds, Melanie

[International Worm Meeting, 2015]

NAD+ biosynthesis has proven to be a useful therapeutic target for influencing health-span and obseity-related phenotypes as well as tumor growth. The goal of our research is to understand how NAD+ homeostasis is maintained to support its core metabolic roles and its signaling and regulatory roles involving NAD+ consumers. NAD+ is synthesized via distinct routes including de novo synthesis from tryptophan, salvage synthesis from nicotinamide, which feeds into the Preiss-Handler pathway from nicotinic acid in C. elegans, and via the phosphorylation of nicotinamide or nicotinic acid ribosides using nicotinamide riboside kinase (NRK). We hypothesize that each pathway will have tissue-specific functions independent of the others and that they will also have compensatory roles in contributing to global NAD+ homeostasis when another pathway is compromised, creating a homeostatic network. We have previously shown, using pnc-1 nicotinamidase mutants, that salvage NAD+ biosynthesis is required for the appropriate temporal progression of reproductive development and for optimum muscle function. Here we examine functions of the NRK and the de novo pathways using genetics. We find that both pathways function to maintain muscle activity, but neither pathway has an independent role in gonad development. Using LC-MS we show that NAD+ is decreased when either pathway is blocked, as previously shown for PNC-1. This data supports the hypothesis that all pathways are required for maintenance of NAD+ levels and for muscle function but only the salvage pathway normally plays a role in gonad development. To examine homeostatic mechanisms, we investigated the function of the NRK and de novo pathways in a pnc-1 mutant background. Mutations in either pathway further exacerbate pnc-1-associated muscle defects, but not the reproductive development defect. We revealed using qRT-PCR that in the absence of salvage synthesis, key enzymes in de novo and NRK-mediated synthesis are up-regulated. We also observed reciprocal trends when the de novo or NRK pathways were blocked. Consistent with these results, supplementation with either nicotinamide riboside or de novo pathway metabolites rescues the gonad delay in pnc-1, indicating that boosting other pathways can restore gonad development when salvage synthesis is blocked, even though these pathways play no role in gonad development normally. We interpret these data as evidence that homeostatic mechanisms are indeed occurring between NAD+ biosynthetic pathways. Our work has implications for efforts to therapeutically target individual NAD+ biosynthetic pathways. .

McReynolds, Melanie et al. (2015) International Worm Meeting "Cytoplasmic-Specific NAD+ Deficiency Disrupts Glycolysis and Activates Amino Acid Catabolism Affecting Reproductive Development in C. elegans."

Hanna-Rose, Wendy, McReynolds, Melanie

[International Worm Meeting, 2015]

NAD+ is a key metabolite that impacts the entire metabolome because of its role in energy transfer in key metabolic pathways. We previously discovered that NAD+ salvage synthesis through the nicotinamidase PNC-1 is required for normal progression of gonad development in C. elegans. Global metabolic profiling suggested that glycolysis was perturbed in our pnc-1 mutants, which have lower global levels of NAD. We used stable isotope/mass spectrometric flux analysis in wild type and pnc-1 mutants to confirm that glycolysis is compromised when NAD+ salvage synthesis is blocked. In pnc-1 mutants, the turnover rate of universally isotope labeled glucose to phosphoenolpyruvate (PEP) is lower than in wild type, whereas, there was no change in the incorporation of isotope label in dihydroxyacetone (DHAP) between WT and pnc-1 mutants. Although glycolysis is impaired, we had evidence that mitochondrial functions were not changed in pnc-1. Metabolomics analysis showed that unlike other glycolytic intermediates pyruvate levels are not reduced in pnc-1 mutants. Therefore, we hypothesized that excessive use of amino acids as an energy source compensates for insufficient glycolytic flux in pnc-1 mutants. Using a targeted metabolomics approach, we revealed that both alanine and cysteine show a 2-fold increase in our pnc-1 mutants. alpha-ketoglutarate and glutamate levels are also significantly higher in pnc-1 mutants compared to WT. This data suggests that a steady reservoir of amino acids is in place for pyruvate production through protein degradation. We also observed an up-regulation of mRNA expression of important aminotransferase and other enzymes involved in amino acid catabolism in our pnc-1 mutants; supporting the notion that amino acid catabolism is indeed occurring. Our results suggest that compromised glycolysis activates amino acid catabolism to maintain functions of the mitochondria. However, this metabolic shift is not compatible with normal progression of reproductive development in C. elegans. To further investigate this hypothesis, we are extending our stable isotope/mass spectrometric flux experiments to the measurement of amino acid catabolism and are investigating the functional role of amino acid catabolic enzymes using RNAi. .

Wang, Wenqing et al. (2015) International Worm Meeting "Metabolic profiling and metabolic flux analyses in NAD+ salvage biosynthesis mutants."

Hanna-Rose, Wendy, Pacella, Marisa, McReynolds, Melanie, Braeckman, Bart, Detwiler, Ariana, Wang, Wenqing, Dhondt, Ineke, Lange, Stephanie, Goncalves, Jimmy, Shu, Muya, Kho, Kelvin

[International Worm Meeting, 2015]

Temporal developmental progression is highly coordinated in C. elegans. However, loss of nicotinamidase PNC-1 activity slows reproductive development, uncoupling it from its typical temporal progression relative to the soma. Nicotinamidases mediate salvage NAD+ synthesis. There has been significant recent interest in boosting NAD+ biosynthesis for therapeutic benefit in aging-related diseases and inhibiting biosynthesis for therapeutic benefit in cancer. However, we haven't fully elucidated how perturbing the availability of NAD+ impacts whole organism physiology. Using LC-MS we show that pnc-1 mutants do not salvage nicotinamide released by NAD+ consumers to resynthesize NAD+, and as a result NAD+ availability is reduced. By manipulating NAD+ levels using genetics, we show that a reduction in NAD+ bioavailability is incompatible with a normal pace of gonad development. We use the pnc-1 mutant phenotype to explore physiological requirements for reproductive development. First we demonstrate that the deficit in NAD+ production compromises NAD+ consumer activity as would be expected. Even though effects on NAD+ consumer activity have been the primary focus for probing the functional consequences of manipulation of NAD+ bioavailability, we revealed no functional link between loss of NAD+ consumer activity and reproductive development in genetic experiments. We used metabolomics profiling to seek explanations for the observed phenotype. The deficit in NAD+ availability from lack of salvage biosynthesis has wide-spread metabolic consequences, including perturbations in glycolysis. Using flux analysis, we demonstrate that glycolysis is blocked at the NAD+-dependent step, and we functionally link the block to the reproductive phenotype using genetics and pharmacological approaches. Interestingly, mitochondria are protected from the deficiency in NAD+ biosynthesis and the effects of reduced glycolytic output, also revealing compartment specific regulation of NAD+ biosynthesis. We suggest that compensatory metabolic processes that maintain mitochondrial activity in the absence of efficient glycolysis are incompatible with the cell division requirements for reproductive development. Our work has implications for understanding mechanisms that mediate therapies that target NAD+ salvage synthesis for the purposes of inhibiting tumor growth.

JA Waddle et al. (1999) International C. elegans Meeting "Non-destructive, 3-D imaging of GFP-tagged proteins throughout C. elegans embryogenesis"

JA Waddle, RH Waterston

[International C. elegans Meeting, 1999]

A powerful feature of C. elegans research is the ability to determine mutant phenotypes at the resolution of single cells. The advent of green fluorescent protein (GFP) provides means to carry the single cell analysis one step further, that is, to characterize critical events that take place during a short interval within a cell cycle. Improvements in computers and imaging devices motivated us to develop a 3-dimensional image acquisition system capable of merging traditional embryonic cell lineage analysis with the latest GFP technology. To gauge the utility of our microscope setup, we imaged a transgenic line carrying a fusion between GFP and an isoform of histone H1 (kindly provided by Melanie Dunn and Geraldine Seydoux). The most important outcome of our efforts is the ability to non-destructively (e.g the embryos hatch and are fertile) collect a 3-dimensional time lapse fluorescence image sequence starting at first cleavage and spanning more than 8 hours of development. The histone H1::GFP images greatly facilitate the tracking of nuclear movements and cell divisions in the bottom half of later stage embryos. The application of computational methods, to reduce the effects of out of focus light, greatly improves image detail and the signal-to-noise ratio. Current efforts are focused on custom software capable of tracking the 3-dimensional distribution and behavior of the histone H1::GFP labeled nuclei, that is, automated embryonic cell lineage analysis. In summary, several technologies have coalesced to provide non-damaging means to track the 3-dimensional distribution of important molecules during embryogenesis. Such technology will complement existing methods for phenotypic analysis in an era when both the embryonic cell lineage and the genome descriptions are complete.

Waddle JA et al. (1998) East Coast Worm Meeting "USING GREEN CHROMOSOMES TO AUTOMATE EMBRYONIC CELL LINEAGE ANALYSIS"

Waterston RH, Waddle JA

[East Coast Worm Meeting, 1998]

While early embryonic cell lineage analysis is routine (1,2,3), tracking small nuclei in the bottom half of >100 cell embryos is not robust and requires considerable time and expertise (4). We hope to exploit green fluorescent protein technology (5) to develop methods for more robust, faster and highly automated lineage analysis. One potential method relies on two key components. First, Melanie Dunn and Geraldine Seydoux provided a histone H1::GFP transgene which permits observation of chromosomes in living embryos. Second, we developed a microscope image acquisition system capable of taking 64 fluorescent optical sections, 10 times per cell cycle, throughout embryogenesis, at light levels commensurate with normal development. Navigation of simultaneously collected histone::GFP fluorescence and Nomarksi DIC 4D data sets reveals that fluorescence lineage analysis is much easier than scoring divisions by Nomarksi optics. The discrimination of individual nuclei, especially in the bottom half of late embryos, is greatly improved by image processing to reduce the effects of out of focus light. We took advantage of the improved image quality to write a program that will identify the number and 3-dimensional position of each nucleus at a given time point. We are now developing software that compares the position of nuclei at subsequent time points in order to detect cell deaths, divisions and migrations. The output from the tracking program will be in two forms: a traditional lineage diagram showing the time and relative orientation of each embryonic cell division or death (until movement at 400 min.); and a series of 3-dimensional coordinates over time that can be used to show a color coded animation of the recording from any perspective. Finally, an additional technical barrier to automated cell lineage analysis is to identify a set of histone H1 genes that, in composite, labels chromosomes in every embryonic cell. The existing histone H1::GFP fusion (his-24 gene) is not highly expressed until the 28-cell stage, and is not expressed in a small subset of cells, including the germline precursors. We plan to fuse GFP to the remaining histone H1 genes to determine which are expressed in the HIS-24::GFP negative cells. The automated lineage analysis system should greatly facilitate phenotypic analysis of cell fate mutants and the subsequent dissemination of such information in a viewable, database format. 1. Hird and White, JCB 121:1343(1994). 2. Fire, CABIOS 10:443(1994). 3. Thomas et al., Science 273:603(1996). 4. Schnabel et al., Dev. Biol. 184:234(1997). 5. Chalfie et al., Science 263:802(1994).

Waddle JA et al. (1998) Midwest Worm Meeting "USING GREEN CHROMOSOMES TO AUTOMATE EMBRYONIC CELL LINEAGE ANALYSIS"

Waterston RH, Waddle JA

[Midwest Worm Meeting, 1998]

While early embryonic cell lineage analysis is routine (1,2,3), tracking small nuclei in the bottom half of >100 cell embryos is not robust and requires considerable time and expertise (4). We hope to exploit green fluorescent protein technology (5) to develop methods for more robust, faster and highly automated lineage analysis. One potential method relies on two key components. First, Melanie Dunn and Geraldine Seydoux provided a histone H1::GFP transgene which permits observation of chromosomes in living embryos. Second, we developed a microscope image acquisition system capable of taking 64 fluorescent optical sections, 10 times per cell cycle, throughout embryogenesis, at light levels commensurate with normal development. Navigation of simultaneously collected histone::GFP fluorescence and Nomarksi DIC 4D data sets reveals that fluorescence lineage analysis is much easier than scoring divisions by Nomarksi optics. The discrimination of individual nuclei, especially in the bottom half of late embryos, is greatly improved by image processing to reduce the effects of out of focus light. We took advantage of the improved image quality to write a program that identifies the number and 3-dimensional position of each nucleus at a given time point. We are now developing software that compares the position of nuclei at subsequent time points in order to detect cell deaths, divisions and migrations. The output from the tracking program will be in two forms: a traditional lineage diagram showing the time and orientation of each embryonic cell division (until movement at 400 min.); and a series of 3-dimensional coordinates over time. The nuclear coordinates can be used to show a color coded animation of embryonic development from any perspective. Finally, an additional technical barrier to automated cell lineage analysis is to identify a set of histone H1 genes that, in composite, labels chromosomes in every embryonic cell. The existing histone H1::GFP fusion (his-24 gene) is not highly expressed until the 28-cell stage, and is not expressed in a small subset of cells, including the germline precursors. We plan to fuse GFP to the remaining histone H1 genes to determine which are expressed in the HIS-24::GFP negative cells. The automated lineage analysis system should greatly facilitate phenotypic analysis of cell fate mutants and the subsequent dissemination of such information in a viewable, database format. 1. Hird and White, JCB 121:1343(1994). 2. Fire, CABIOS 10:443(1994). 3. Thomas et al., Science 273:603(1996). 4. Schnabel et al., Dev. Biol. 184:234(1997). 5. Chalfie et al., Science 263:802(1994).