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

Young Old Oxidative Stress Response Systems Decline with Age

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

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

Cycloheximide = an inhibitor of protein synthesis Nrf2 Is Newly Translated

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

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

How does lipoic acid increase levels of Nrf2? Question:

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.

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

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

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

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

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

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

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