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

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/zns jpeg Dark

Feedback Loop From Dr. Jadwiga Giebultowicz

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

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

Activity Links to ROS Production ↑ activity = ↑ respiration = ↑ reactive oxygen species (ROS)

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

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

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

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

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

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

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

Activity Results - LD Fig iav actogram Fig iav actogram Fig. 2 CS actogram Fig. 5 E023 cpo actogram

Activity Results - DD Fig. 6 CS actogramFig iav actogram Fig iav actogram Fig. 9 E023 cpo actogram

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)

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

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 ZT

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 ZT

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

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

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

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