Molecular Mechanisms Underlying Menthol
Cigarette Addiction
Nadine Kabbani, Ph.D.
George Mason University
Nicotinic
Receptors
in Brain
Menthol cigarette smokers have greater α4β2 nAChR
up-regulation than non-menthol smokers from Brody et al., 2013
Structural and Functional Analysis of
Menthol Interaction with Nicotinic
Receptors
Nadine Kabbani: PI
• Project management and implementation
• Direct the experimental analysis
• Organize weekly meetings
• Direct future strategy
• Prepare published work
Amarda Shehu: co-PI
• Project management and implementation
• Direct the experimental analysis
• Direct future strategy
• Prepare published work
EXPERIMENTAL
Justin King
Neuroscience PhD student
• Genetic mutagenesis experiments
• Calcium imaging studies
• Functional analysis of compounds
BIO-COMPUTATIONAL
Weekly project meetings
Joint publications
Joint meeting presentations
Daniel Veltri
Bioinformatics PhD student
• Structural modeling
• Library screen
Pentameric structure of the a7 nicotinic receptor
ligands
M3 M4 M1
Extracellular region
M2
M1
M3
M4
M2
Transmembrane domains
M1
M2
Na+
Ca++
K+
M3
M2
M4
M4
M1
M2
M3
M4
M3
M1
Top-down view of the ion channel
Intracellular loops
Kabbani et al., 2013
Nicotine
8s
10s
12s
Control
6s
Bgtx
Menthol
10μm
Nic+Menthol
5
Nic
5
Soma ΔF/F
Neurite ΔF/F
**
4
3
2
1
0
Nic+Bgtx
**
4
3
2
1
0
0
5
10
Seconds
15
20
0
10
20
Seconds
30
Structural homology of menthol/propofol
binding at the nicotinic receptor
propofol
menthol
Menthol Binding to the a7 Nicotinic Receptor
Menthol Binding to 5HT3 Serotonin
Receptor
Menthol Binding to Nicotinic Receptor
Alters Receptor Function
Biochemical and proteomic analysis of
nicotinic receptors and nicotine
addiction
Menthol’s effect on nicotinic acetylcholine receptor expression and synaptic structure
in the rodent brain*
S. P. BRIDGES, J. R. KING, P. MULDOON, S. ALSHARARI, M. I. DAMAJ, N. KABBANI
SUMMARY OF
EXPERIMENTAL
DESIGN
Treatment
groups
Saline
Nicotine
Menthol
Behavior
testing
In
collaboration
with Dr.
Damaj
(VCU)
LC MS/MS
determination of
nicotine levels in
plasma in
collaboration
with Dr. Tyndale
(CAMH)
Brain
tissue
dissection
and
proteomic
analysis
*Supported by a Virginia Youth
Tobacco Program Grant Award
A mass spectrometry proteomic strategy for
identifying nicotinic receptor interactions
Proteomic Profiling
20
Figure 2. Nicotine and menthol regulate α4β2 nAChR
expression in the brain.
70%
OD (% of Control)
# *
0%
**
60%
***
40%
50%
40%
30%
20%
-20%
n
Nic
+M
en
10%
50%
D
β2
**
α4
30%
10%
-10%
β2
-50%
PFC
*
*
**
*
OD (% of Control)
**
-30%
90%
70%
0%
Me
Ni
Co
C
α4 (70)
β2 (50)
GAPDH
(36)
*
c
β2
n
α4
-40%
OD (% of Control)
OD (% of Control)
60%
Ni
c
Me
n
Nic
+
***
**
80%
*
***
***
***
B
α4 (70)
β2 (50)
GAPDH (36)
80%
20%
Me
n
Nicotine + Menthol
Co
n
α4 (70)
β2 (50)
GAPDH
(36)
Menthol
Me
n
N ic
+M
en
Ni
c
Con
A
Nicotine
***
Control
90%
Hippocampus
Striatum
70%
50%
50%
30%
10%
30%
-10%
-30%
10%
-50%
-10%
-30%
-70%
α4
β2
α4
Figure 4. An effect of menthol and nicotine
on α7 nAChR cellular localization and
trafficking in neural cells.
***
***
40
30
20
Control
Menthol
***
***
***
50
***
***
***
60
C
10
0
B
45
40
soma
neurite
terminal
10 µm
***
***
***
35
30
***
***
***
Arbitrary Fluorescence Units
A
25
20
15
10
5
0
soma
neurite
terminal
Nicotine
Nicotine+Menthol
Lab
Justin King, PhD Student
Samuel Bridges, BS student
Steven Black, MS student
Rafique Islam, PhD student
Collaborators
Amarda Shehu (GMU)
Darwin Berg (UCSD)
Imad Damaj (VCU)
Jerrel Yakel (NIEHS)
Christopher Del Negro (W&M College)
Grant Support
Jeffress Memorial Foundation
Virginia Foundation for Healthy Youth
NIAMS, NIH
Wings for Life Spinal Cord Research Foundation