Jennika Krisa

1. Genetic Mechanisms Responsible for Dietary Restriction-Dependent Lifespan Extension in Drosophila Melanogaster: A Role for Muscle Tissue? Jennika Krisa 1 The Buck Institute For Research on Aging, 8001 Redwood Blvd  Novato, CA 94945 Introduction Results A B Dietary Restriction (DR) is a robust intervention that is known to extend lifespan and increase spontaneous activity in multiple species. Whether activity increase plays a causal role in mediating the health protective benefits of DR remains unknown. The changes in physiology that occur in response to DR are partially mediated by the nutrient sensing TOR pathway and downstream signaling components, including eukaryotic translation initiation factor 4E-binding protein (4E-BP). To investigate this relationship, nutritional manipulations and laboratory selection for lifespan were simultaneously applied in the model organism, D. melanogaster. Laboratory selection was carried out using muscle-specific RNAi of genes which are known to be both responsive to Dietary Restriction and translationally dependent on 4E-BP. Three assays were used for screening and characterization of genes that may mediate the effects of DR in the muscle tissue: starvation resistance, spontaneous activity, and lifespan. Three downstream targets of 4E-BP were identified from screening: Nemo, Fumble, and Nedd2-like Caspase. A B D C C E • Figure 4. Inhibition of Two Candidate Genes Extends Lifespan upon DR in D. melanogaster. Median life span was calculated from Kaplan-Meier survival analysis of female flies upon RNAi of candidate genes in muscle tissue under DR (light red) and AL (dark red) conditions. Controls used were w1118; Mhc-Gal4/+ (blue, dashed lines) (A-C) and w1118; CG7892/+ (A), w1118; CG5725/+ (B), w1118; CG8091/+ (C) (green, dashed lines).( A) Nemo(CG7892-RNAi/Mhc-Gal4) extended lifespan in both the AL and DR conditions (B)Fumble (CG5725-RNAi/Mhc-Gal4) extended lifespan in both the AL and DR conditions when compared to one control(C)Nedd2-like Caspase(CG8091-RNAi/Mhc-Gal4) had no effect on lifespan Figure 1. Muscle-Specific Inhibition of Candidate Genes Influences Starvation Resistance in a Nutrient-Dependent Manner in D. melanogasterKaplan Meir survival analysis for starvation resistance in RNAi female flies (solid line) and control flies (dashed line). Control used was W1118 Mhc-Gal4/+ (A-E)(A) Nemo (CG7892-RNAi/Mhc-Gal4) decreased starvation resistance in both AL and DR conditions (B) Nemo with out-crossed driver (CG7892-RNAi/Mhc-Gal4)had no effect on starvation resistance (C) Fumble (CG5725-RNAi/Mhc-Gal4) decreased starvation resistance in both AL and DR conditions (D)Fumble with out-crossed driver (CG5725-RNAi/Mhc-Gal4)increased starvation resistance in the AL condition(E) Nedd2-like Caspase with out-crossed driver (CG8091-RNAi/Mhc-Gal4)increased starvation resistance in both AL and DR conditions Conclusion • Two of the three candidate genes, Fumble and Nemo, show significant lifespan extension when inhibited in the muscle tissue. • Fumble is involved in triglyceride homeostasis and may exert it’s effect on the three biological outputs that were measured through enhancing synthesis or breakdown of fat for energy utilization. • Nemo is involved in locomotor function and may exert it’s effect on the three biological outputs that were measured by enhancing neuromuscular dynamics. • Nedd2-like Caspasedid not extend lifespan, though it did impact starvation resistance and spontaneous activity • The two assays used for screening proved useful for finding genes which extend lifespan • While each of these genes holds promise for future studies in healthy aging, sources of variation in results must be controlled Neuromuscular Dynamics A Enhanced Activity Lifespan Extension B A model showing the proposed mechanisms by which muscle tissue mediates the lifespan extension effects of Dietary Restriction. DR Methods C MUSCLE Triglyceride Homeostasis Fumble Nedd2-like Caspase Nemo TOR For all experiments, Mhc-Gal4 females were mated with male transgenic and syngenic control flies, and the resulting female offspring analyzed in parallel by comparing transgene expressing flies with matched controls flies having the same genetic background. For longevity measurement, female flies were collected within 24 hours from eclosion and reared at standard density (25 flies per vial) on cornmeal/sucrose/yeast fly food at 25 degrees Celsius. Dead flies were counted every other day and food changed. For starvation treatments, flies were kept in normal vials with 1% agar as a water source for the period of time indicated. Spontaneous Activity was measured using the Drosophila Activity Monitor (Tri kinetics Inc.) Movement of flies is measured in the vertical direction and a three equidistant points over the length of the vial (approximately 2 cm, 5 cm, and 8 cm about food surface). The flies were transferred to fresh food in the morning by 12:00 pm and then moved to the monitors. Measurements for a 24 hour period began at 4:00 pm. Time points are plotted as Total Fly Activity/Day. Statistical analysis was performed with Excel (Microsoft) and p values were calculated with Student’s t tests . Autophagy 4E-BP Acknowledgements Figure 2.Muscle-specificInhibition of Candidate Genes Influences Spontaneous Activity in a Nutrient-Dependent Manner in D. melanogaster. Controls used were w1118; Mhc-Gal4/+ (A-C) and w1118; CG7892/+ (A), w1118; CG5725/+ (B), w1118; CG8091/+ (C). (A)Effect of Nmo (CG7892-RNAi/Mhc-Gal4) on spontaneous activity (B)Effect of Fbl (CG5725-RNAi/Mhc-Gal4) on spontaneous activity(C) Effect of Nc (CG8091-RNAi/Mhc-Gal4) on spontaneous activity I would like to thank Matthew Laye, PhD and Guiping Du, PhD for their technical assistance and PankajKapahi, PhD.forhis mentorship.

2. DR Muscle TOR 4E-BP Target Genes StarvationResistance Spontaneous Activity Lifespan Extension

4. Dietary Restriction TOR Fat Metabolism Fumble NC Nemo Autophagy 4E-BP Neuromuscular Dynamics Lifespan Extension Spontaneous Activity Starvation Resistance Muscle

5. A B D C

6. B A D C E

7. A B C

9. B A C

10. A B D C

12. Dietary Restriction Muscle Tissue Increased Activity Lifespan Extension ??? 4E-BP, targets of 4E-BP, other genes?