1. Inactive and couch potato mutants of Drosophila: Locomotor activity vs metabolism Inactive and couch potato mutants of Drosophila: Locomotor activity vs metabolism Sherry Lee Dr. Jadwiga Giebultowicz Dr. Natraj Krishnan HHMI Summer Program

2. Circadian Rhythm  Endogenous  Period (per) and Timeless (tim) genes  Entrained by exogenous cues – light  Drosophila – crepuscular Days Light jneurosci.org/content/vol26/issue9/images/large/zns0080614060002.jpeg Dark

3. Feedback Loop From Dr. Jadwiga Giebultowicz

4. Hypoactive Mutants -Inactive (iav): 70 and 78 -Couch potato (cpo): E023 flybase.bio.indiana.edu

5. Metabolism  Intracellular – mitochondria  Cellular respiration converts glucose to ATP C 6 H 12 O 6 (aq) + 6O 2 (g) → 6CO 2 (g) + 6H 2 O (l)  Waste products

6. www.sapphirebioscience.com Activity Links to ROS Production ↑ activity = ↑ respiration = ↑ reactive oxygen species (ROS)

7. Rhythmic ROS Production – wild-type Krishnan N., et al. 2008. Biochemical and Biophysical Research Communications END

8. Objectives  Rhythmic activity compared to wild-type controls (CS)  Rhythmic ROS and ATP productions compared to wild-type controls (CS)

9. Hypothesis Inactive and couch potato flies have less activity  less metabolism  less ROS and ATP production.

10. Activity and Longevity  DAM activity monitor with computer data recording  Longevity cages Google images

11. Mitochondrial H 2 O 2 Production  Mitochondria isolated from whole bodies  Hydrogen peroxide microplate assay Google images

12. ATP Production  Mitochondria isolated from whole bodies  ATP bioluminescence Google images

13. Activity Results - LD  iav and cpo: not hypoactive Fig. 1 Activity graph of three genotypes, 78 iav, 70 iav, and E023 cpo. Horizontal activity vial

14. Activity Results - LD Fig. 3 78 iav actogram Fig. 4 70 iav actogram Fig. 2 CS actogram Fig. 5 E023 cpo actogram

15. Activity Results - DD Fig. 6 CS actogramFig. 7 78 iav actogram Fig. 8 70 iav actogram Fig. 9 E023 cpo actogram

16. Activity Results - DD % rhythmicity in constant darkness:  78 iav: 50% (mean = 23.4)  70 iav: 50% (mean = 23.6)  E023 cpo: 80% (mean = 27.6)

17. Climbing Ability  iav and cpo: impaired negative geotropic movement Genotypes% climbed above 5 cm line 78 iav7.50 70 iav47.0 E023 cpo83.9 CS wild- type 89.7 iav CS

18. H 2 O 2 Production Results  iav: more H 2 O 2 than CS  cpo: less H 2 O 2 than iav Fig. 10 Rate of H 2 O 2 production for 78, 70, and E023 mutants and CS wild-type flies at ZT8 and ZT20. 820

19. ATP Production Results  iav and cpo: more ATP than CS  cpo: more ATP than iav Fig. 11 Rate of ATP production for 78, 70, and E023 mutants and CS wild-type flies at ZT8 and ZT20. 820

20. Longevity Results  iav: shorter lifespan than CS  cpo: longer lifespan than iav Fig. 12 Kaplan-Meyer survival curves for 78, 70, and E023 mutants and CS wild-type flies. TO BE CONTINUED

21. Discussion  iav compared with CS  cpo defects  Compare results with Hyperkinetic (Hk)

22. Significance  Level of beneficial physical activity  ROS production affects longevity  Promotion of healthy aging

23. Acknowledgements  Dr. Natraj Krishnan  Dr. Jadwiga Giebultowicz  Dr. Kevin Ahern  HHMI Summer Undergraduate Research Program