Post on 29-Jan-2016
1/24
Three responses to chronic nicotine exposure:
Studies on
genes, proteins, drugs, cells, circuits, and behavior
November, 2007
Henry Lester
2/24
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
3/24
Prevalence of current smoking among adults aged 18 years and over
(National Health Interview Survey)
United States, 1997–2006
1996 1998 2000 2002 2004 20060
5
10
15
20
25
30
perc
ent
year
Progress on smoking cessation is very slow
“Self-medication”may be the reason
In 2002, individuals with a current psychiatric disorder comprised 7%
of the US population,
but they smoked 34-46% of all cigarettes in the US.
4/24
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”
Some abbreviations on future slides:
ACh, acetylcholine
nAChR, nicotinic acetylcholine
receptor
DA, dopamine
receptors become “bored”
nicotine20 seconds
1 millionchannels
5/24
Conclusions from knockout and hypersensitive mice (2005):
Activation of 42-containing (*receptors by nicotine
Is necessary and sufficient for
sensitization, tolerance, reward, (but withdrawal?)
Focus on 4β2 receptors
What are the mechanisms?
6/24
1. Chronic nicotine exposure causes tolerance of dopamine releaseThe “yoked self-administration” experiment
Master animal
Yoked animal
Rahman, Zhang, Engleman, & Corrigall, 2004
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Saline Nicotine
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Time (min)
Dia
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A (
nM)
7/24
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.
8/24
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.
9/24
kinase
phosphorylatedprotein
cAMPCa2+
intracellularmessenger
receptor
tsqiG protein
enzymechannel effector
nAChRs
are highly permeable to Ca2+
as well as to Na+.
Possible mechanism 1: The “Molecular Relay Race”:
Signal transduction triggered by a ligand-gated channel
10/24
Chronic exposure to nicotine induces more nicotinic receptors
Possible Mechanisms 2a, 2b:
If the upregulated receptors are Active (“exuberant”),
upregulation might cause better synaptic transmission and excitation, leading to cognitive sensitization.
But this does not explain tolerance.
If the upregulated receptors are Desensitized (“bored”),
this might cause decreased synaptic transmission and excitation, leading to tolerance.
But this does not explain cognitive sensitization.
nicotine addiction?
The “Receptor Dilemma”: How (if at all) do changed receptors contribute to . . .
If the upregulated receptors are active,
excitotoxicity might exacerbate Parkinson’s disease.
If the upregulated receptors are desensitized, this might be neuroprotective.
neuroprotection?
a. The “Bored Receptor” (desensitized)
versus b. The “Exuberant Receptor”
(upregulated)
11/24
Strategy to choose between the “bored” or “exuberant” receptors in the response to chronic nicotine exposure
1. Generate mice with fully functional, fluorescent 4* 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
12/24
200 m
Medial Perforant Path
Py Or Rad
LMol
Alveus
Temperoammonic Path
Chronic nicotine increases 4 fluorescence ~ 2-fold in hippocampus--a brain area that provides a good model for cognition.
Functional studies show: the new receptors are “exuberant”, not “bored”
V
14/24
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)
15/24
4-YFP knock-in: substantia nigra pars compacta neurons
Spectrally unmixed 4YFP Spectrally unmixed background autofluorescence
10 m 10 m
Shortcut to Projections of 32-32-LS5unmix.avi.lnk
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4YFP Background
16/24
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Substantia Nigra Pars Reticulata
. . . but does upregulate 4 levels in GABAergic inhibitory neurons.
Chronic nicotine does not change 4 levels in dopaminergic neurons . . . Substantia Nigra
Pars Compacta
Substantia nigra data also support the “exuberant receptor” idea
α4 intensity per TH+ neuron
α4 intensity per GAD+ neuron
17/24
Chronic Saline
1A
Endogenous ACh
1B
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Time (min)
Dia
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nM
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Rahman et al, 2004
2BDecreased Reward
Plus Acute Nicotine(repeated exposure)
Chronic nicotine cell-specifically upregulates 4* receptors: Basis for circuit-based tolerance in midbrain
via “exuberant inhibition”
Endogenous ACh VTA
LDT
Cholinergic
NAc
DAergic
GABAergic
Chronic Nicotine Tolerance
2A
Upregulated 4* nAChRs
Craving
Endogenous ACh
1B Reward
Plus Acute Nicotine(1st expsoure)
+ acute nicotine
1A
2A2B
18/24
. . . As produced by “deep brain stimulation” in subthalamic nucleus
GABAergic neurons may have increased or more regular firing
in chronic nicotine. . .
Hypothesis: Circuit-based neuroprotection by chronic nicotine in substantia nigra
viaCholinergic, Dopaminergic, and GABAergic neurons in Hindbrain & Midbrain
Thalamus,
superior colliculus
GABAergic
DAergicSNc
SNrEndogenous ACh
PPTg
Cholinergic
Striatum
Upregulated 4* nAChRs
19/24
When 4* 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.
Conclusions from hypersensitive & fluorescent mice
How do we develop better therapeutics based on these ideas?
20/24
The nicotinic receptor’s interfacial “aromatic box” occupied by nicotine Showing the cation- interaction with unnatural amino acids
Y198C2
Y190C1
Y93A
W149B
non-W55D
Collaboration with Dennis Dougherty, Hoag Professor of Chemistry
21/24
485 nm535 nm 485 nm535 nm
439 nm 439 nm
514 nm
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FR
ET
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icie
nc
y (%
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4-2XFP 4-2XFP+
nicotine
. . . After 24 hours in nicotine,exuberant receptors
are assembled more tightly.
“Stolen” photons tell us which subunits are near each other
Experiments like these may show us
how to develop better therapies for Parkinson’s Disease.
22/24
12 μm
1
4
3
2
1
2
3
4
The ultimate reductionist approach,studying nAChR traffic/regulation at the single molecule level.
TIRF microscopy of nAChR eGFP in oocytes
23/24
Bruce Cohen, Ryan Drenan, Purnima Deshpande, Carlos Fonck, Sheri McKinney, Raad Nashmi, Qi Huang, Rigo Pantoja, Johannes Schwarz, Cagdas Son, Andrew Tapper, Larry Wade, Cheng Xiao
Joanne Xiu, Nyssa Puskar, Jai Shanata, Shawna Frazier, Dennis A. Dougherty
Sarah Lummis
Stephan Pless, Joseph Lynch
Sharon Grady, Al Collins, Mike Marks, Jeremy Owens, Tristan McClure-Begley, Paul Whiteaker
Jim Boulter, Istvan Mody, Oliver Dorigo, Arnie Berk, Max Shao, Jack Feldman
Jon Lindstrom
Julie Miwa, Nathaniel Heintz
Uwe Maskos, Jean-Pierre Changeux
Univ Queensland
Univ of Colorado, Boulder
Caltech “Alpha Club”
UCLA
Univ. Pennsylvania
Rockefeller Univ
Institut Pasteur
“Unnatural Amino Acid Club”
Univ of Cambridge
24/24
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?
25/24
+ +
Fre
e E
nerg
y
Reaction Coordinate
Free subunits
Increasingly stable
assembled states
Nicotine may stabilize subunit interfaces
20 M Nicotine+
1 M Nicotine+
(pKa = 7.9)
0 mV-70 mV
Nicotine accumulates in cells
+Boundstates with
increasing affinity
Fre
e E
nerg
y
Reaction Coordinate
C
AC
A2C A2O
A2D
Highest affinity bound state
unbound
Binding eventually favors high-affinity states
“Exuberant receptors” are a thermodynamic consequence of durg-receptor Interactions
Nicotine
hr0 20 40 60
Incr
ease
d H
igh-
Sensi
tivit
y R
ece
pto
rs
RLS RHS
Covalently stabilized
AR*HSDegradation
+ nicotine
?
26/24
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).
“Upregulation”
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).
27/24
Midbrain slice recordings: functional upregulated receptors in a circuit produce tolerance
Cheng Xiao
28/24
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Substantia Nigra Pars Reticulata. . . but does upregulate 4 levels in GABAergic inhibitoryneurons
Chronic nicotine does not change 4 levels in dopaminergic neurons . . .
Substantia Nigra Pars Compacta
Substantia nigra data also support the “exuberant receptor” idea
29/24
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1 M Nicotine
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fEP
SP
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Acute Nicotine
10 min 80 min
1mV
10 ms
10 ms
Saline
Nicotine
0.5 mV
Acute Nicotine
Chronic10 min 80 min
Saline
Nicotine
0.5 mV
5 ms
Acute SalineChronic
Acute Saline
Acute
Simple model forcognitive
sensitization:
chronic nicotine +
acute nicotine lowers the threshold
for perforant pathway LTP