Kelly Crotty Dr. Tory Hagen

1. Lipoic Acid Induces Nrf2 Activation in an mTOR-dependent Manner Kelly Crotty Dr. Tory Hagen

2. Oxidative Stress Response Systems Decline with Age Young Old

3. Nrf2: A Transcription Factor in the Detoxification System • Influences the expression of over 200 genes with the Antioxidant Response Element (ARE) • These genes produce detoxification enzymes and antioxidant proteins • Levels of Nrf2 in the nucleus decline with age • The detoxification system is less effective with age

4. Lipoic Acid improves stress response Lipoic acid Supplementation • Lipoic Acid (LA) increases levels of nuclear Nrf2 Old

5. Nrf2 Is Newly Translated Cycloheximide = an inhibitor of protein synthesis

6. Nrf2 Synthesis Under Normal Conditions: Cap Dependent Translation • mTORis a kinase that senses energy and stress levels in the cell • Raptor is an adapter that brings mTOR to the substrate (4E-BP) P P P Ribosome and proteins mTOR Raptor P P 4E-BP eIF4E IRES mRNA Nrf2 Synthesis

7. Nrf2 Synthesis Under Oxidative Stress: Cap Independent Translation Ribosome and proteins P P P 4E-BP eIF4E mRNA Internal Ribosomal Entry Site Stress Synthesis of Stress Response Proteins (Nrf2)

8. Question: How does lipoic acid increase levels of Nrf2?

9. Hypothesis: Lipoic acid increases levels of phosphorylated mTOR complex, inducing cap-dependent production of Nrf2 Prediction: Cells treated with lipoic acid will have increased amounts of phosphorylated mTOR complex compared to a non-treated control.

10. Methods: Treat cells in vitro with lipoic acid Blocking Primary Antibody Secondary Antibody Development Perform SDS-PAGE to separate proteins by weight Measure levels of mTOR and Raptor by Western Blot analysis

11. mTOR activation is lost with LA treatment mTOR total mTOR in the cell increases with LA treatment mTOR No LA 4 hr. LA P-mTOR Active mTORdisappears with LA treatment P P mTOR No LA 4 hr. LA Raptor P-Raptor Total and active Raptor disappear with LA treatment Raptor No LA 4 hr. LA No LA 4 hr. LA

12. mTOR Complex is inactive • Lipoic acid treatment increases total mTOR protein, but decreases mTOR complex activity • This is counter to our hypothesis • LA decreases mTOR complex activity, so we are not seeing cap-dependent translation of Nrf2 P P P mTOR Raptor 4E-BP eIF4E

13. Implications: Switch from Cap-Dependent to Cap-Independent Translation Because lipoic acid does increase translation of Nrf2, these data suggest cap-independent translation of Nrf2. Conclusion: Under lipoic acid treatment, cells inhibit global protein synthesis and cap-independent translation is preferred

14. New Hypothesis: LA acts as a weak stress in the cell • Hormesis: the favorable response of being exposed to a small stress regularly for an extended period of time in order to have a larger capacity to respond to greater stresses • LA treatment induces a stress response in the cell • Old animals taking LA supplements respond to great stresses equally as well as young animals • LA is regimenting the cell to respond to stress

15. Next Step • Do a time course to see when mTOR complex activity returns • Probe for other proteins associated with cap-independent translation

16. Thank you to: The HHMI Summer Research Program Dr. Kevin Ahern Dr. Tory Hagen Dr. Kate Shay Judy Butler Dr. Dove Keith and the rest of the Hagen lab