Download Lester-Lect to CaltechAssociates-Nov

Three responses to chronic nicotine exposure:
Studies on
genes, proteins, drugs, cells, circuits, and behavior
Henry Lester
November, 2007
1/24
1. How does one explain nicotine addiction?
Does it matter? Won’t everyone stop smoking soon?
Smokeless tobacco?
2. Nicotine as an imperfect therapeutic drug
Best example: Parkinson’s disease
3. Cellular / molecular approaches to better therapies
2/24
Progress on smoking cessation is very slow
30
Prevalence of current smoking
among adults aged 18 years and over
United States, 1997–2006
percent
25
In 2002, individuals with a current
psychiatric disorder comprised 7%
of the US population,
20
15
but they smoked 34-46% of all
cigarettes in the US.
10
5
“Self-medication”
may be the reason
(National Health Interview Survey)
0
1996
1998
2000
2002
year
2004
2006
3/24
The nicotine video
Produced for Pfizer to explain varenicline (Chantix) to physicians
This summarizes knowledge in ~ 2004.
“ligand” is a molecule that binds to another.
“physical” addiction vs “psychological” addiction.
“Desensitization“ and “Upregulation”
receptors become “bored”
1 million
channels
Some abbreviations on future slides:
ACh, acetylcholine
nAChR, nicotinic acetylcholine receptor
DA, dopamine
nicotine
20 seconds
4/24
Focus on a4β2 receptors
Conclusions from knockout and hypersensitive mice (2005):
Activation of a4b2-containing (a4b2*) receptors by nicotine
Is necessary and sufficient for
sensitization, tolerance, reward, (but withdrawal?)
What are the mechanisms?
5/24
Dialysate DA (nM)
1. Chronic nicotine exposure causes tolerance of dopamine release
The “yoked self-administration” experiment
Yoked animal
Master animal
4.0
3.5
Yoked saline
Yoked nicotine
3.0
2.5
2.0
1.5
1.0
0.5
0.0
Saline
-40
0
Nicotine
40
80
120
160
Time (min)
Rahman, Zhang, Engleman, & Corrigall, 2004
6/24
2. Chronic nicotine exposure causes cognitive sensitization
In the human context, cognitive sensitization is epitomized by smokers’
reports that they think better when they smoke;
this anecdotal observation is confirmed by data that smokers who smoke
nicotine cigarettes (but not nicotine-free cigarettes) display several cognitive
enhancements.
In the rodent context, rats show more contextual fear conditioning if, one day
after withdrawal from chronic nicotine, they receive an acute nicotine dose;
also chronic nicotine produces better spatial working memory performance in
the radial arm maze.
7/24
3. Inverse correlation between long-term tobacco smoking and Parkinson’s disease
In identical twins discordant for both Parkinson’s disease & smoking,
the unaffected twin smoked at a significantly higher rate.
In those twins where one or both smoked,
The unaffected twin smoked 12 pack-years more.
There are good indications that nicotine itself is a protective agent.
Clinical trials of nicotine patches have given mixed results because of side effects
Beneficial results of short-term nicotine exposure:
Pain reduction.
Increased concentration: ADHD, Schizophrenia.
Alzheimer (Aricept = donepezil, a cholinesterase inhibitor; Reminyl = galantamine)
Decreased inflammation.
Antidepressant actions.
8/24
Possible mechanism 1: The “Molecular Relay Race”:
Signal transduction triggered by a ligand-gated channel
receptor
G protein
i q s t
effector
channel enzyme
nAChRs
are highly permeable to Ca2+
as well as to Na+.
intracellular
messenger
Ca2+ cAMP
kinase
phosphorylated
protein
9/24
Possible Mechanisms 2a, 2b:
a. The “Bored Receptor” (desensitized)
versus
b. The “Exuberant Receptor” (upregulated)
Chronic exposure to nicotine induces more nicotinic receptors
The “Receptor Dilemma”: How (if at all) do changed receptors contribute to . . .
nicotine addiction?
neuroprotection?
If the upregulated
receptors are
Active (“exuberant”),
If the upregulated
receptors are
Desensitized (“bored”),
If the upregulated
receptors are
active,
upregulation might cause
better synaptic
transmission and
excitation, leading to
cognitive sensitization.
this might cause
decreased synaptic
transmission and
excitation, leading to
tolerance.
excitotoxicity might
exacerbate
Parkinson’s disease.
But this does not explain
tolerance.
But this does not explain
cognitive sensitization.
If the upregulated
receptors are
desensitized, this
might be
neuroprotective. 10/24
Strategy to choose between the “bored” or “exuberant” receptors
in the response to chronic nicotine exposure
1. Generate mice with fully functional,
fluorescent a4* receptors. (Why mice?)
2. Chronically expose the mice to
nicotine (2 weeks).
3. Find the brain regions and cell types
with changed fluorescence.
4. Perform experiments on these
regions and cells to decide whether the
new receptors are “bored” or
“exuberant”.
5. Model the cellular and circuit changes
11/24
Chronic nicotine increases a4 fluorescence ~ 2-fold in hippocampus
--a brain area that provides a good model for cognition.
Alveus
Py
Or
Functional studies show:
the new receptors are
“exuberant”, not “bored”
Rad
LMol
V
200 mm
Medial Perforant
Path
Temperoammonic
Path
12/24
Midbrain dopaminergic cells (tyrosine hydroxylase stain)
Substantia nigra pars compacta (SNc, controls motion);
Ventral tegmental area (VTA, controls reward)
Substantia nigra pars reticulata (SNr, GABAergic)
14/24
a4-YFP knock-in: substantia nigra pars compacta neurons
Spectrally unmixed a4YFP
Spectrally unmixed
background autofluorescence
10 mm
10 mm
a4YFP
YFP Intensity
1500
Background
1000
Shortcut to Projections of 32-32-LS5unmix.avi.lnk
500
0
500
520
540
560
580
Wavelength (nm)
600
15/24
Substantia nigra data also support the “exuberant receptor” idea
Chronic nicotine does not change
a4 levels
in dopaminergic neurons . . .
Cumulative Percentage
100
80
60
Substantia Nigra
Pars Compacta
40
20
0
0
500
1000
1500
2000
2500
3000
α4 intensity per TH+ neuron
. . . but does upregulate
a4 levels
in GABAergic inhibitory neurons.
Cumulative Percentage
100
80
60
Substantia Nigra
Pars Reticulata
40
20
0
0
500
1000
1500
α4 intensity per GAD+ neuron
16/24
Chronic nicotine cell-specifically upregulates a4* receptors:
Basis for circuit-based tolerance in midbrain
via “exuberant inhibition”
Chronic Saline
Endogenous
ACh
VTA
LDT
Cholinergic
Dialysate DA (nM)
4.0
3.5
Upregulated a4* nAChRs
Craving
NAc
DAergic
Chronic Nicotine Tolerance
2A
1A
Endogenous ACh
Endogenous ACh
GABAergic
Yoked saline
Yoked nicotine
2B
1B
1B
Reward
Decreased Reward
3.0
2.5
2.0
1A
Plus Acute Nicotine
(1st expsoure)
1.5
1.0
0.5
0.0
Plus Acute Nicotine
(repeated exposure)
2B
2A
Saline
Nicotine
-40 -20 0 20 40 60 80 100 120140 160180
Time (min)
Rahman et al, 2004
+ acute nicotine
17/24
Hypothesis:
Circuit-based neuroprotection by chronic nicotine in substantia nigra
via
Cholinergic, Dopaminergic, and GABAergic neurons in Hindbrain & Midbrain
Upregulated a4* nAChRs
Striatum
SNc
DAergic
GABAergic neurons
may have increased
or more regular firing
in chronic nicotine. . .
PPTg
Thalamus,
superior
colliculus
Cholinergic
GABAergic
Endogenous ACh
SNr
. . . As produced by “deep brain stimulation”
in subthalamic nucleus
18/24
Conclusions from hypersensitive & fluorescent mice
When a4* nicotinic receptors are repeatedly occupied/activated
these receptors become “exuberant” in specific neurons.
This produces improved cognition via forebrain synapses,
but tolerance occurs via changes in a GABA-dopamine circuit.
How do we develop better therapeutics based on these ideas?
19/24
The nicotinic receptor’s interfacial “aromatic box” occupied by nicotine
Showing the cation-p interaction with unnatural amino acids
aY198
C2
aW149
B
aY93
A
aY190
C1
non-aW55
D
Collaboration with Dennis Dougherty, Hoag Professor of Chemistry
20/24
“Stolen” photons tell us which subunits are near each other
. . . After 24 hours in nicotine,
exuberant receptors
are assembled more tightly.
17.5
15.0
439 nm12.5
FRET Efficiency (%)
439 nm
514 nm
10.0
485 nm
Experiments like these may show us
nm
how to develop better535
therapies
for Parkinson’s Disease.
7.5
5.0
485
2.5 nm 535 nm
0.0
a4-b2XFP
a4-b2XFP
+
nicotine
21/24
The ultimate reductionist approach,
studying nAChR traffic/regulation at the single molecule level.
TIRF microscopy of nAChR geGFP in oocytes
1
3
2
2
3
1
4
4
12 μm
22/24
Caltech Bruce Cohen, Ryan Drenan, Purnima Deshpande, Carlos Fonck,
“Alpha Club” Sheri McKinney, Raad Nashmi, Qi Huang, Rigo Pantoja,
Johannes Schwarz, Cagdas Son, Andrew Tapper, Larry Wade,
Cheng Xiao
“Unnatural Amino Joanne Xiu, Nyssa Puskar, Jai Shanata, Shawna Frazier,
Acid Club” Dennis A. Dougherty
Univ of Cambridge Sarah Lummis
Univ Queensland Stephan Pless, Joseph Lynch
Univ of Colorado, Sharon Grady, Al Collins, Mike Marks, Jeremy Owens,
Boulder Tristan McClure-Begley, Paul Whiteaker
UCLA Jim Boulter, Istvan Mody, Oliver Dorigo, Arnie Berk, Max Shao,
Jack Feldman
Univ. Pennsylvania Jon Lindstrom
Rockefeller Univ Julie Miwa, Nathaniel Heintz
Institut Pasteur Uwe Maskos, Jean-Pierre Changeux
23/24
More pontifications about upregulation (“exuberance”)
Increased nAChR due to chronic nicotine exposure probably confers no
selective advantage . . . could be a thermodynamic necessity.
A substantial, regulated pool of unassembled or cytoplasmic high-sensitivity
nAChRs receptors may confer a selective advantage.
If so, the selective advantage may involve responding to circadian
rhythms in ACh levels.
If so, is there a disease caused by faulty nAChR regulation?
Autosomal dominant nocturnal frontal lobe epilepsy?
24/24
“Exuberant receptors” are a thermodynamic consequence of durg-receptor Interactions
Nicotine accumulates in cells
1 mM Nicotine+
(pKa = 7.9)
Binding eventually favors high-affinity states
+
unbound
0 mV
Free Energy
-70 mV
Bound
states
with
increasing
affinity
20 mM Nicotine+
Highest affinity
bound state
C
AC
A2C
A2O
A2D
Reaction Coordinate
+
+
Free Energy
Free
subunits
Increasingly
stable
assembled
states
+ nicotine
RLS
Covalently
stabilized
RHS
Degradation AR*HS
?
Nicotine
Increased
High-Sensitivity
Receptors
Nicotine may stabilize subunit interfaces
Reaction Coordinate
0
20
40
60
hr
25/24
“Upregulation”
Chronic exposure to nicotine causes upregulation of nicotinic receptor binding
(1983: Marks & Collins; Schwartz and Kellar);
Upregulation 1) Involves no change in receptor mRNA level;
2) Depends on subunit composition (Lindstrom, Kellar, Perry).
Shown in experiments on clonal cell lines transfected with nAChR subunits:
Nicotine seems to act as a
“pharmacological chaperone” (Lukas, Lindstrom)
or
“maturational enhancer” (Sallette & Corringer, Heinemann)
or
“Novel slow stabilizer” (Green).
Upregulation is “cell autonomous” and “receptor autonomous” (Henry).
26/24
Midbrain slice recordings: functional upregulated receptors in a circuit produce tolerance
Cheng Xiao
27/24
Substantia nigra data also support the “exuberant receptor” idea
Substantia Nigra Pars Compacta
Number of Neurons
Chronic nicotine
does not change
a4 levels in
dopaminergic neurons
...
40
30
20
10
0
0
500
1000
1500
2000
100
Cumulative Percentage
Saline (n=612)
Nicotine (n=581)
50
80
60
40
20
0
2500
0
Mean a4YFP Intensity per TH+ Neuron
500
1000
1500
2000
2500
3000
Substantia Nigra Pars Reticulata
Saline (n=256)
Nicotine (n=237)
40
30
20
10
0
100
Cumulative Percentage
50
Number of Neurons
. . . but
does upregulate
a4 levels in
GABAergic inhibitory
neurons
80
60
40
20
0
0
250
500
750
1000 1250 1500
Mean a4YFP Intensity per GAD67+ Neuron
0
500
1000
1500
28/24
Simple model for
cognitive
sensitization:
Acute Nicotine
Chronic
Saline
10 min
Acute Saline
80 min
Chronic
Saline
0.5 mV
0.5 mV
10 ms
10 ms
1.5
10 ms
Chronic Acute
Nicotine Nicotine
Saline
Nicotine
160
150
140
130
120
110
100
90
80
1 mV
0.5
0
20
10 ms
40
80
30
Stimulus Strength (mA)
120
110
100
90
100
0
20
40
40
40
Acute Saline
P < 0.001
20
10
0
Saline
Nicotine
Chronic
80
100
Chronic Nicotine
40
40

30
60
Time (min)
LTP Induction (% increase)
20
130
80
60
Acute Nicotine
10
Chronic Acute
Nicotine Saline
Saline
Saline
Time (min)
0.0
0
140
1 mM Nicotine
1.0
LTP Induction (% increase)
Slope (-mV/ms)
2.0
Nicotine
1mV
30
20
10
0
Saline
Nicotine
Chronic
LTP Induction (% increase)
1 mV
Nicotine
fEPSP Slope (%)
2.5
5 ms
fEPSP Slope (%)
chronic nicotine
+
acute nicotine
lowers the threshold
for perforant
pathway LTP
80 min
10 min
30
20
10
Acute
Saline
Mecamylamine
0
-10
-20

p < 0.001
29/24