Characterization of the Novel Dietary Restriction Mimetic NPP1

1. Characterization of the Novel Dietary Restriction Mimetic NPP1 Theo Garrett, Mark Lucanic, Gordon Lithgow The Buck Institute for Research on Aging. Novato, CA Determination of Effective Dose of NPP1 Introduction Identification of NPP1 Affected Neuron The nematode C. elegansis an important model for aging research, due to its relative simplicity and short lifespan. Many of the pathways that determine C. elegans lifespan are also important for human aging and age-related disease. The Lithgow Lab has utilized chemical screens in C. elegans, in part as technique to identify novel molecular pathways that determine the lifespan of this model organism. Empirically we have found that the characterization of the biological activity of these new chemicals has allowed us to identify new mechanisms at play in lifespan determination. Here we describe our new work in characterizing the mechanism of a novel chemical, NPP1 which we identified in a screen for chemicals that extended the lifespan of C. elegans. The novel chemical NPP1 was found to extend lifespan through a dietary restriction type mechanism. We have additionally determined that the chemical decreases the pharyngeal pumping rate. Pharyngeal pumping is a feeding behavior, through which the animals take up food from their environment. We have found that both the lifespan extension response to the chemical and the pumping rate decrease response to the chemical require glutamatergic signaling. Glutamate is a major neurotransmitter that contributes to pharyngeal pumping. Our goal here was to determine which pharyngeal neurons are required for response to the chemical and also to determine the peak dose of the chemical for eliciting the lifespan and pumping effects. Lifespan Vs. NPP1 Dosage Lifespan of NSM Rescued eat-4 Mutant Figure 3. Glutamatergic Signaling is only Required in the NSM for the Response to NPP1. (A) eat-4 mutants that lack glutamatergic signals do not respond to NPP1 with lifespan extension. (B) eat-4; rfEx strains that express eat-4 only in the NSM neurons (glutamate signaling rescued) respond to NPP1 with lifespan extension. This data demonstrates that glutamate signaling must be active in the NSM for the lifespan extension induced by NPP1. Figure 1: Lifespan Dose Response to NPP1. Survivorship curves are shown for treatment with given concentrations of NPP1. A concentration of 50 micro-molar was found to induce the greatest extension in lifespan. Percent Decrease in Pumping Rate Hypothesis Model of Mechanism There will be a dose of NPP1 at which a maximum response will be elicited. NPP1 effect derives from altering the glutamatergic signaling from a single neuron (NSM) Methods Figure 4. A Model for the Mechanism of NPP1 Action. The NSM is associated with feeding. However its exact role remains unexplained. Our results indicate that the NSM signals positively (with glutamatergic neurotransmission) the absence of food. This causes a dietary restriction response, stress resistance and longevity. NPP1 triggers this response in the NSM, which simulates the absence of food, thereby acting like a true dietary restriction mimetic. Figure 2. Pumping Rate Dose Response to NPP1. After treating first day adults with NPP1 for 24 hours, animals were transferred to non-treated plates and the pumping rate was counted. NPP1 treated animals showed a significant decrease in pumping rate compared to the animals treated with the vehicle control and the response peaks around 100 micro-molar. Conclusion We have worked to further characterize the mechanism of the novel dietary restriction mimetic NPP1. In a dose response assay, we found that the dose which provoked the greatest lifespan extension was found to be 50 micro-molar. While higher doses decrease pumping rate, they do not provide a further extension in lifespan. Finally we find that the response to NPP1 is dependent on glutamatergic signaling from the NSM specifically. Our characterization of this chemicals effect has led us to identify a novel nutrient sensing pathway. Our model describes a previously unknown function for the NSM in nutrient detection as well as describing the novel biologically active chemical NPP1. Acknowledgements A thank you should be made to all of the fine researchers, administrators and support staff at The Buck Institute. Many thanks are also due to the donors that made the REU program possible. I would also like to give special thanks to members of the Lithgow Lab, who were all more than willing to help a confused undergrad whenever the opportunity arose. Last but by no means least, I extend many thanks to my mentor Mark Lucanic. I believe a good mentor must be able to tolerate many stupid questions, amateur mistakes, and moments of complete confusion, while accepting that the already slow and arduous research process will be further slowed by the presence of an intern. I put Mark to the test on all of these points. He passed.