Kelly crotty dr tory hagen
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Lipoic Acid Induces Nrf2 Activation in an mTOR-dependent Manner. Kelly Crotty Dr. Tory Hagen. Oxidative Stress Response Systems Decline with Age. Young. Old. Nrf2: A Transcription Factor in the Detoxification System.

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Kelly Crotty Dr. Tory Hagen

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Kelly crotty dr tory hagen

Lipoic Acid Induces Nrf2 Activation in an mTOR-dependent Manner

Kelly Crotty

Dr. Tory Hagen


Kelly crotty dr tory hagen

Oxidative Stress Response Systems Decline with Age

Young

Old


Kelly crotty dr tory hagen

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


Kelly crotty dr tory hagen

Lipoic Acid improves stress response

Lipoic acid

Supplementation

  • Lipoic Acid (LA) increases levels of nuclear Nrf2

Old


Kelly crotty dr tory hagen

Nrf2 Is Newly Translated

Cycloheximide = an inhibitor of protein synthesis


Kelly crotty dr tory hagen

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


Kelly crotty dr tory hagen

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)


Kelly crotty dr tory hagen

Question:

How does lipoic acid increase levels of Nrf2?


Kelly crotty dr tory hagen

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.


Kelly crotty dr tory hagen

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


Mtor activation is lost with la treatment

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


Mtor complex is inactive

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


Implications switch from cap dependent to cap independent translation

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


Kelly crotty dr tory hagen

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

Next Step

  • Do a time course to see when mTOR complex activity returns

  • Probe for other proteins associated with cap-independent translation


Kelly crotty dr tory hagen

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


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