Kelly CrottyDr. 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

mTOR Raptor

P P P

Nrf2 Synthesis

IRES

eIF4E

Ribosome and

proteins

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

mRNAeIF4E

4E-BP

Internal

Ribosomal

Entry Site

Ribosome and

proteins

Synthesis of Stress

Response Proteins

(Nrf2)Stress

PP

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

Raptor P-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

No LA 4 hr. LA No LA 4 hr. LA

Raptor

mTOR

mTOR

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

mTOR Raptor

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 ProgramDr. Kevin AhernDr. Tory HagenDr. Kate ShayJudy Butler

Dr. Dove Keith

and the rest of the Hagen lab