Prof. Uri Littauer Lecture: Changes in the brain during chronic exposure to nicotine Studies on genes, receptor binding, proteins, drugs, cells, circuits, and behavior February, 2009 Henry Lester 1/33
Jan 05, 2016
Prof. Uri Littauer Lecture:
Changes in the brain during chronic exposure to nicotine
Studies on
genes, receptor binding, proteins, drugs, cells, circuits, and behavior
February, 2009Henry Lester
1/33
Conclusions from hypersensitive and knockout mice (2005):
Activation of 4-containing (*receptors by nicotine
Is sufficient and necessary for
tolerance, sensitization, reward, (but withdrawal?)
Picciotto, Marubio, Maskos, Tapper . . . . Caltech, Pasteur
There are good reasons to focus on 42* nAChRs
What are the mechanisms?
(But remember that some 42* receptors contain:≥ one 5, 6, or β3 subunit)
There are excellent reasons to focus on
the nicotinic acetylcholine receptors (nAChR) themselves:
subtypes: 1-10, 1 - 4, γ, δ, ε
2/33
1. Nicotine is highly membrane-permeant. ACh is not.
Ratio unknown, probably > 1000.
2. ACh is usually hydrolyzed by acetylcholinesterase (turnover rate ~104 /s.) In
mouse, nicotine is eliminated with a half time of ~ 10 min.
Ratio: ~105
3. EC50 at muscle receptors: nicotine, ~400 μM; ACh, ~ 45 μM.
Ratio, ~10. Justified to square this because nH = 2.
Functional ratio, ~100. What causes this difference?
Nicotine and ACh act on many of the same receptors, but . . .
3/33
Binding region
Membrane region
Cytosolicregion
(incomplete)
Colored by secondary
structure
Colored by subunit(chain)
Nearly Complete Nicotinic Acetylcholine Receptor, a Well-Studied Cys-loop Receptor
~ 2200 amino acids in 5 chains
(“subunits”),
MW ~ 2.5 x 106
4/33
5/30
The AChBP interfacial “aromatic box” occupied by nicotine (Sixma, 2004)
W149BY93
A
non-W55D
Y198C2
Y190C1
(Muscle Nicotinic numbering)
Nicotine makes a stronger cation-π interaction with Trp Bat α4β2 receptors than at muscle receptors;
this partially explains α4β2 receptors’ high binding affinity for nicotine.
4 3 2 1 01
10
100
1,000
nic
otin
e E
C50
,M
Number of F-Trp atoms
Receptor muscle (~3-fold) (47-fold)
WT
WT, without cation-π
interaction
6/33
NH
HN
NH
W149
O T150
O
replace i+1 byanalogous
-hydroxy acid
NH
ONH
W149
O Tah150
O
HN+ H
N+ weakenedhydrogen bond
A BC1
C2
D
7/30
Nicotine makes a stronger H-bond to a backbone carbonyl
at α4β2 than at muscle receptors:With amide to ester substitution,
EC50 increases 20-fold vs 1.5-fold
Weaker hydrogen bond
Deleted hydrogen bond
8/30Xiu, Puskar, Shanata, Lester, Dougherty. 2009. Nature in press
Nicotine EC50 values:
Underlying the 400-fold higher nicotine sensitivity
of
neuronal vs muscle receptors:
Factor of ~16 for the cation-π interaction;
Factor of ~ 12 for H-bond;
16 x 12 = 192. We still can’t explain a factor of 400/192 ~ 2.
Muscle nAChR single component ~ 400 μM
α4β2 two components ~ 1 μM, ~200 μM
kinase
phosphorylatedprotein
cAMPCa2+
intracellularmessenger
receptor
tsqiG protein
enzymechannel effector
NMDA receptors
and
nAChRs
are highly permeable to Ca2+
as well as to Na+.
Possible molecular mechanism #1 for changes with chronic nicotine:
Signal transduction triggered by a ligand-gated channel
Brunzell, Russell, & Piccotto, 2003
9/33
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).
Possible Mechanism #2 for changes with chronic nicotine: “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, Changeux, & Corringer; Heinemann)
or
“Novel slow stabilizer” (Green).
Upregulation is “cell autonomous” and “receptor
autonomous” (Henry). 10/62
+ +
Fre
e E
nerg
y
Reaction Coordinate
Free subunits
Increasingly stable
assembled states
Nicotine stabilizes subunit interfaces
2.5 M Nicotine+
1 M Nicotine+
(pKa = 7.8)
pH 7.4
pH 7.0
Nicotine accumulates in acidic cells & organelles
+Boundstates with
increasing affinity
Fre
e E
ne
rgy
Reaction Coordinate
C
AC
A2C A2OA2D
Highest affinity bound state
unbound
Binding eventually favors high-affinity states
Nicotine
hr0 20 40 60
Incr
ease
d H
igh-
Sensi
tivit
y R
ece
pto
rs
RLS RHS
Covalently stabilized
AR*HSDegradation
+ nicotine ?
Upregulation is a thermodynamic consequence of nicotine-nAChR interactions
SePhaCHARNS“Selective Pharmacological Chaperoning of Acetylcholine Receptor Number and Stoichiometry”
11/33
12
Nucleus
TIRFM
FRET
High-resolution fluorescence microscopy to study SePhaChARNS
Golgi
ER
PMLTP / Opioids: regulation starts herePharmacological chaperoning: upregulation starts here
Förster resonance energy transfer (FRET): a test for subunit proximity
ECFPXFP =EYFP
mEYFPmVenusmCerulean mEGFP mCherry
Ligand binding M1 M2 M3 M4M3-M4 loop
M4
M3 - M4loopα4
c-myc tag XFP
4-XFP 2-XFP
HA tag XFP
FRET pairs(m = monomeric)
N C N Cβ2
-20 0 20 40 60 800
200
400
600
800
1000
1200
1400
1600
1800
2000
2200
Data: Data1_C54Model: GaussEquation: y=y0 + (A/(w*sqrt(PI/2)))*exp(-2*((x-xc)/w)^2)Weighting: y No weighting Chi^2/DoF = 288.49226R^2 = 0.9912 y0 4.19078 ±1.65095xc 12.03086 ±0.06603w 20.05847 ±0.15945A 12986.99416 ±114.84783
Nu
mb
er o
f P
ixel
s
% FRET Efficiency
2ECFP 4EYFP
FRET NFRET
13/33
Theory of FRET in pentameric receptors with αnβ(5-n) subunits
No FRET
No FRETE
1/2 1/4 1/4
E1 E2 E3 E4
1/8
1/4
1/4
1/8 1/8 1/8
50% α-CFP, 50% α-YFP
b/a =1.62; 1.62-6 = 0.055
0
20
40
60
80
0 20 40 60 80 100Distance between adjacent subunits, A
FR
ET
Eff
icie
ncy 100%(3(
100%((3
100% α3β2100% α2β3
% receptors with α3 14/33
A key experiment: changes in subunit stoichiometry caused by chronic nicotine!
Cagdas Son
0
2
4
6
8
10
12
14
16
(4CFP + YFP) : 21:1
+ Nicotine
% F
RE
T E
ffic
ienc
y
control
(CFP + YFP)1:1
+ Nicotinecontrol
Neuro2a 15/33
16
4 hour nicotine exposure causes an increase in (4)2(2)3 assembly
0 2 4 6 8 10 12 14 16 18 200
5000
10000
15000
20000
25000
30000
35000
40000
45000
50000
55000
Pix
el c
ount
NFRET (%)
0 2 4 6 8 10 12 14 16 18 200
500
1000
1500
2000
2500
3000
3500
4000
Pix
el c
ount
NFRET (%)
0 2 4 6 8 10 12 14 16 18 200
5000
10000
15000
20000
25000
30000
35000
40000
45000
50000
55000
Pix
el c
ount
NFRET (%)
WHOLE CELLNo treatment
R2 = 0.999y0 = 0 xc1 = 8.7 ± 0.06w1 = 2.22 ± 0.12A1 = 88465 ± 34150xc2 = 10 ± 0.36w2 = 2.92 ± 0.19A2 = 109476 ± 34316
GOLGINo treatment
R2 = 0.999y0 = 0 xc1 = 8.28 ± 0.07w1 = 1.9 ± 0.05A1 = 6756 ± 620xc2 = 9.72 ± 0.07w2 = 1.8 ± 0.04A2 = 5298 ± 621
WHOLE CELL+ 1 M NICOTINE 4 h
R2 = 0.998y0 = 0 xc1 = 8.5 ± 0.18w1 = 2.4 ± 0.1A1 = 130438 ± 36122xc2 = 10.1 ± 0.26w2 = 2.24 ± 0.14A2 = 64907 ± 26106
0 2 4 6 8 10 12 14 16 18 200
500
1000
1500
2000
2500
3000
3500
4000
Pix
el c
ount
NFRET (%)
GOLGI+ 1 M NICOTINE 4 h
R2 = 0.998y0 = 0 xc1 = 8.37 ± 0.02w1 = 2.33 ± 0.03A1 = 11498 ± 239xc2 = 10.21 ± 0.04w2 = 1.51 ± 0.06A2 = 1986 ± 233
PM-mCherry α4-GFPβ2 Overlay
A. Control
B. 0.1 µM nic 48 h
TotalInternal
ReflectionFluorescenceMicroscopy
(TIRFM)
17/33
18
Differential subcellular localization and dynamics of α4GFP* receptors
α4GFPβ2
α4GFPβ4 (1:1)
α4GFPβ2 overlayplasma memb. mCherry
α4GFPβ4 overlay
3 RXR per β subunit
0 RXR per β subunit
Strategy to evaluate the cell specificity of 4* upregulation in chronic nicotine
1. Generate knock-in mice with fully functional, fluorescent 4* receptors
2. Expose the mice to chronic nicotine
3. Find the brain regions and cell types with changed receptor levels
4. Perform physiological experiments on these regions and cells to verify function
5. Model the cellular and circuit changes
YFP,Leu9’Ala-YFP,CFP19/33
Cellular and subcellular specificity of SePhaChARNS
Thalamus,
superior colliculus
SNc
SNr
Striatum
Upregulation?
Transmitter Soma Term. Region / projection
Glu ?? Yes* Entorhinal cortex → dentate gyrus
ACh No No Medial habenula → Interpeduncular nucleus
DA No* Yes* Ventral tegmental area, substantia nigra pars compacta →
Striatum
GABAA Yes* Yes* SN pars reticulata, VTA → SNC, VTA
CA
DG
ECMH
IPNMedial Perforant Path
* = upregulation shown with electrophysiology
Nashmi et al J Neurosci 2007; Xiao et al, in prep.20/33
The Caltech 4 fluorescent mice . . . normal in all respects
21/33
VTA GABAergic and DA neurons have contrasting responses to nicotine in vivo
DA neuron, ~ 1700 spikes
Nicotineinjection
GABAergic neuron (5 s smoothing), ~ 8300 spikes
0.05 m V2 m s
0.05 m V2 m sF
requ
ency
, H
z
0 100 200 300 400 500 600 700
0
2
4
6
0
5
10
15
20
25
s
Fre
quen
cy,
Hz
0.1 m V
0.5 m s
0.1 mV0.5 ms
A B C D0.05 m V2 m s
0.05 mV2 ms
4*, 6*, and/or 7
4* only
V
GABAergic
DAergic
VTA
WT mouse
22/30
4-YFP knock-in: substantia nigra pars compacta neurons
Raad Nashmi
Spectrally unmixed 4YFP Spectrally unmixed background autofluorescence
10 m 10 m
Shortcut to Projections of 32-32-LS5unmix.avi.lnk
23/33
24/24
0 500 1000 1500 2000 2500 30000
20
40
60
80
100
Cu
mu
lativ
e P
erc
en
tag
e
0 500 1000 15000
20
40
60
80
100C
um
ula
tive
Pe
rce
nta
ge
Substantia Nigra Pars Reticulata(& VTA, not shown)
. . . but does upregulate 4 levels in GABAergic inhibitory neurons.
Chronic nicotine does not change 4 levels in dopaminergic neurons . . .
Substantia Nigra Pars Compacta(& VTA, not shown)
Midbrain data show cell specificity of SePhaChARNS
α4 intensity per TH+ neuron
α4 intensity per GAD+ neuron
Chronic Saline
1A
Endogenous ACh
1A
2A
1B
2B
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0 Yoked salineYoked nicotine
Saline Nicotine
-40 -20 0 20 40 60 80 100120140160180
Time (min)
Dia
lysa
te D
A (
nM
)
Rahman et al, 2004
2BDecreased Reward
Plus Acute Nicotine(repeated exposure)
Chronic nicotine cell-specifically up-regulates functional 4* receptors:
Basis for circuit-based tolerance in midbrain(Nashmi et al, 2007)
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 nicotine25/33
Midbrain slice recordings: functional upregulated receptors in a simple circuit
Cheng Xiao
In SNr of α4 knockout, these effects of chronic nicotine vanish
26/33
Chronic nicotine increases firing rate of SNr GABAergic neurons in vivo . . .
V
Cheng Xiao
. . . we’re still gathering data for DA neurons
27/33
Chronic nicotine 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 certain cognitive
enhancements (Rusted and Warburton, 1992; Rusted et al., 1995).
In the rodent context, mice show more contextual fear conditioning if, one day
after withdrawal from chronic nicotine, they receive an acute nicotine dose
(Davis et al., 2005); β* dependent
also chronic nicotine produces better spatial working memory performance in
the radial arm maze (Levin et al., 1990; Levin et al., 1996).
28/33
200 m
Medial Perforant Path
Py Or Rad
LMol
Alveus
Temperoammonic Path
Chronic nicotine increases perforant path 4 fluorescence ~ 2-fold
TV Bliss, T Lömo (1973)
Long-lasting potentiation of synaptic
transmission in the dentate area of the
anaesthetized rabbit following
stimulation of the perforant path.
J Physiol. 232:331-56.
29/33
0 10 20 30 400.0
0.5
1.0
1.5
2.0
2.5
Slo
pe (-m
V/m
s)
Stimulus Strength (A)
1 mV
10 ms
1 mV
10 ms
Saline Mecamylamine
-20
-10
0
10
20
30
40
LTP
Indu
ctio
n (%
incr
ease
)
p < 0.001
0 20 40 60 80 10080
90
100
110
120
130
140
150
160
fEP
SP
Slo
pe
(%
)
Time (min)
Chronic Acute Nicotine Nicotine Saline Nicotine
1 M Nicotine
0 20 40 60 80 10080
90
100
110
120
130
140
fEP
SP
Slo
pe (
%)
Time (min)
Chronic Acute Nicotine Saline Saline Saline
Saline Nicotine0
10
20
30
40
LTP
Indu
ctio
n (%
incr
ease
)
Chronic
Chronic Nicotine
Saline Nicotine0
10
20
30
40
LTP
Indu
ctio
n (%
incr
ease
)
P < 0.001
Chronic
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
30/33
Some changes in the brain during chronic exposure to nicotine
1. Nicotine potently activates some neuronal nAChRs (because it participates in both cation-π and H-bond interactions within the conserved aromatic box).
2. Chronic exposure to nicotine chaperones α4β2* number and stoichiometry.
3. These processes lead to cell-specific α4β2* upregulation.
4. a. Upregulation explains tolerance to chronic nicotine, via a GABAergic-DA circuit in the midbrain.b. Upregulation also explains enhanced LTP in the perforant path, via a direct presynaptic mechanism. This is a simple model for cognitive sensitization
Genes
Binding
Proteins
Cells
Circuits
Behavior
5. We do not yet understand several processes, including somatic signs of withdrawal and stress-induced nicotine use.
31/62
Understanding the changes in the brain during chronic exposure to nicotine:Translational relevance of SePhaChARNS
1. Nicotine addiction (~1 billion people, since 1540)
2. Strong inverse correlation between smoking & Parkinson’s disease (~ 50 million people, since 1819)
3. Tobacco use suppresses seizures in patients with autosomal dominant nocturnal frontal-lobe epilepsy, linked to the α4 and β2 subunits (~1000 patients, since 1994)
32/62
Bruce Cohen, Purnima Deshpande, Ryan Drenan, Carlos Fonck, Sheri McKinney, Raad Nashmi, Johannes Schwarz, Rahul Srinivasan, Cagdas Son, Andrew Tapper, Larry Wade, Cheng Xiao
Al Collins, Sharon Grady, Mike Marks, Erin Meyers, Tristan McClure-Begley, Charles Wageman, Paul Whiteaker
Merouane Bencherif, Greg Gatto, Daniel Yohannes
Jon Lindstrom
Mike McIntosh
Julie Miwa, Nathaniel Heintz
Univ of Colorado, Boulder
Caltech“Alpha Club”
Targacept
Univ. Pennsylvania
Univ. Utah
Rockefeller Univ
Dennis DoughertyKiowa Bower, Shawna Frazier, Ariel Hanek, Fraser Moss, Nyssa Puskar, Rigo Pantoja, Kristin Rule, Erik Rodriguez, Jai Shanata, Mike Torrice, Joanne Xiu
Neil Harrison, Sarah Lummis, Claire Padgett, Kerry Price, Andy Thompson
Stephan Pless, Joe Lynch
Caltech“Unnatural Club”
Uni Queensland
Univ. of Cambridge
33/33
kinase
P P. . .other proteins
bind to the phosphates . . .
Activated GPCRs are sometimes phosphorylated and endocytosed. This “downregulation” terminates signalling.
P P
But continual signalling can activate genes
During activation, the G protein leaves . . .
. . . revealingphosphorylationsites . . .
(not a synaptic vesicle)
. . . triggeringendocytosis.
34/31
FRET with fluorescent subunits tells us about nicotinic receptor assembly
4Y/
4C/2
4/2
Y/2C
6Y/
6C/2
4/2
/3Y/3
C
*
Neuro2a35/62
The nicotine video
Produced for Pfizer to explain varenicline (Chantix) to
physicians
This summarizes knowledge in ~ 2004.
“physical” addiction vs “psychological” addiction.
Desensitization and “Upregulation”
Some abbreviations on future slides:
ACh, acetylcholine
nAChR, nicotinic acetylcholine receptor
DA, dopamine
nicotine20 seconds
1 millionchannels
36/31
Chronic nicotine causes tolerance of dopamine release
Master animal
Yoked animal
Rahman, Zhang, Engleman, & Corrigall, 2004
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0 Yoked salineYoked nicotine
Saline Nicotine
-40 0 40 80 120 160
Time (min)
Dia
lysa
te D
A (
nM)
37/31