Transcript Enantiomers of Lipoic Acid Differentially Affect Nrf2 Activation

Lipoic Acid Induces Nrf2
Activation in an mTORdependent Manner
Kelly Crotty
Dr. Tory Hagen
Oxidative Stress Response Systems
Decline with Age
Young
Old
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
Lipoic Acid improves stress
response
• Lipoic Acid (LA)
increases levels of
nuclear Nrf2
Lipoic acid
Supplementation
Old
Nrf2 Is Newly Translated
Cycloheximide = an inhibitor of protein synthesis
Nrf2 Synthesis Under Normal Conditions:
Cap Dependent
Translation
P
P P
mTOR
P
Raptor
P
•Raptor is an adapter that
brings mTOR to the
substrate (4E-BP)
4E-BP
eIF4E
•mTOR is a kinase that
senses energy and stress
levels in the cell
IRES
mRNA
Nrf2 Synthesis
Nrf2 Synthesis Under Oxidative Stress:
Cap Independent
Translation
P
P
P
4E-BP
eIF4E
mRNA
Internal
Ribosomal
Entry Site
Synthesis of
Stress Response
Proteins
(Nrf2)
Question:
How does lipoic acid
increase levels of Nrf2?
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:
Treat cells in vitro with lipoic
acid
Perform SDS-PAGE to
separate proteins by weight
Measure levels of mTOR and
Raptor by Western Blot
analysis
Blocking
Primary
Antibody
Secondary
Antibody
Development
mTOR activation is lost with LA
treatment
mTOR
No LA
4 hr. LA
P-mTOR
No LA
4 hr. LA
Raptor
No LA
total mTOR in
the cell increases
with LA treatment
Active mTOR
disappears with
LA treatment
P-Raptor
4 hr. LA
No LA
4 hr. LA
mTOR
P
P
mTOR
Total and active
Raptor disappear
with LA treatment
Raptor
mTOR Complex is inactive
• Lipoic acid treatment increases total mTOR
protein, but decreases mTOR complex activity
• This is counter to our hypothesis
P
P
mTOR
4E-BP
eIF4E
P
Raptor
• LA decreases mTOR
complex activity, so we
are not seeing capdependent 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 capindependent 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