1. Modulation of Neuronal Nicotinic
α7 receptor by Genistein:
Upregulation Or PAM
M. Faridul Islam
Farley Lab
2012
2. Roadmap
• Background and our research interest
• Basics of α7
• Modulation: Allosteric, tyrosine kinase and
numerical up regulation.
• Methodology
• Result
• Discussion
3. Problem
Nicotine addiction is one of the most widespread
addiction problem but the exact role/mechanism is
poorly understood.
The effect of nicotine is very wide spread (Central
and peripheral).
To complicate further, there are many different
receptor types and subtypes and there is
differential expression in terms of neurons and
there subcellular parts ( dendrites vs soma vs
axons).
5. Differential Nicotine Action at Receptor Level
Chronic nicotine exposure induces:
•
Upregulation: as evidenced by:
Increase in nicotinic radioligand binding sites for all nAChR subtypes. Involves post-transcriptional mechanisms, because subunit
mRNA levels are not significantly changed by chronic nicotine exposure.
•
Desensitization:
Decrease in nAChR functional responsiveness to subsequent nicotinic agonist application (particularly in heterologous expression
systems).
Desensitization time frame:
A comparatively rapid in onset and rapidly reversible “desensitization” that recovers after 5 min of drug removal, and a more slow
to develop and slow to recover “persistent inactivation” functional loss requiring more than 5 min for recovery.
Receptor subtype for Upregulation and desensitization
1. α7 : Does not markedly upregulate α7* (Marks et al., 1986; Collins et al., 1994), α3β4*, or α6* nAChRs (Nguyen et al., 2003;
McCallum et al., 2006a,b; Mugnaini et al., 2006; Perry et al., 2007; Walsh et al., 2008)
But
2. α4β2 : Does effectively upregulate α4β2* nAChRs in some cell types and brain regions. Example: For instance, chronic
nicotine upregulates α4β2* nAChRs in hippocampus, cortex, striatum, hypothalamus, and midbrain, but not in interpeduncular
nucleus, medial habenula, or cerebellum (Flores et al., 1992; Marks et al., 1992; Perry et al., 1999; Nguyen et al., 2004; Nashmi et
al., 2007).
Soma Vs projections:
Does not occur for nAChRs in soma of VTA DA neurons.
6.
7. Functional correlate of upregulation
and desensitization
• Chronic nicotinic agonist-induced functional
inactivation (desensitization) and numerical
upregulation of nAChR.
• In vivo, the upregulated receptors are probably
active rather than desensitized.
• For example: upregulation contribute to the
addictive property while desinsitization might
contribute to tolerance. Upregulated receptor
state may provide better “Buzz” for each smoking
episode after a while but the desensitized state
might contribute the “Craving”.
8. Our Research Interest
Among the difference receptors of nicotine α4β2 nAChRs are comparatively well studied.
Our lab and me are more interested to a particular variety of nAchR, the α7.
We are interested on how the α7 receptors are
being modulated, upregulated and
densinsitized.
Currently we are working with Genistein,
Nicotine and A beta peptide 1-42
9. Snap Shot of α7 nAchR
• Location: Wide spread but particularly in
Stiatum, VTA/NA, Hippocampus, Neocortex.
• Subcellular location: Mostly presynaptic but
also some post synaptic. None or few in soma.
• Physiologic function: Learning and memory,
synaptic plasticity, neuroprotection,
inflammation, and presynaptic regulation of
neurotransmitter release.
15. Gating of Channel: Allosteric modulation
A.
Three negatively chagred rings around
the pore 1.external and 3. internal
may serve as prefilters and divalent
blocking sites. The central ring (2)
within the bilayer may contribute to the
selectivity filter for cations,
B.
M2 and flanking regions of each of the
five subunits.
1, 2, and 3 identify the three rings of
negative charge. The position of the
aligned serine and threonine residues
within M2, which help form the
selectivity filter.
C.
Gating
Each M2 segment is split into two
cylinders, one on top of the other.
Left: In the closed state each M2
cylinder points inward toward the
central axis of the channel. A ring of
five hydrophobic leucine residues
(large spheres, one from each M2
segment) occludes the pore.
Right: In the open state the cylinders
tilt, thus enlarging the ring of leucines.
A ring of hydrophilic threonine residues
(small spheres) may form the
selectivity filter near the inner mouth of
the channel. (Based on Unwin 1995)
16. Allosteric Modulation
• ACh receptor exhibits properties typical of allosteric
proteins:
• 1. ACh-binding site and the ion channel are spaced far
apart (35A ° ).
• 2. nAChR can undergo reversible transitions between
distinct allosteric conformations.
• 3. Positive and negative nAChR effectors can alter ACh
affinity, dose-response curve slope, and response
amplitude
17. Allosteric Modulation
•
protein
conformations
and the state
preferentially
stabilized when a
ligand is bound to
the orthosteric site
•
Binding of a
molecule at an
allosteric site
•
resting closed
state
•
L0, L1, L2 are the
isomerization
coefficients
•
The desensitized
closed state.
Binding of an allosteric
ligand can affect the
energy barrier between
transitions resulting in a
displacement of the
equilibrium between
states
PAM lower the
energy barrier
between the
resting and
active states
increase the
agonist-evoked
response
19. Different PAMs and there response
http://www.sciencedirect.com/science/article/B6T4P-4P6M5YK-3/2/63b819a2acccc122679049d0ef46e56f
20. Kinase Modulation
• Serine Threonine kinase
• Tyrosine Kinase
Abundant literature suggest Nicotinic
neuronal receptors are regulated by
Tyrosine kinase activity.
So we tested how much it contributes in α7
receptors upregulation by genistein.
23. Genistein: few words
Causes:
• Activation of PPARs
• Inhibition tyrosine kinase, mostly
of epidermal growth factor receptor EGFR
• Modulation of CFTR channel, potentiating
its opening at low concentration and
inhibiting it a higher doses.
• PAM effect
24. Key compounds we used:
•
•
•
•
•
Cyclosporine
H7
Nicotine
Genistein
A beta
34. Genistein Effect.
100 uM bath-applied genistein (an inhibitor of many PTKs) enhance
murine peak a7 nAChR currents in oocytes.
Increase in membrane capacitance (Cm), as expected if genistein’s
potentiation of peak currents was due to increased addition of
channels to the plasma membrane via exocytosis.
39. Peak to steady-state ratio of oocytes from the 2th
day VS 5th day following injection
• Peak to steady-state ratio of oocytes from
the 2th day following injection
• Control, 1 min and 24 H genistein exposed
oocyte are similar and there is no
difference.
40. The peak to steady-state ratio of oocytes from the 5th day following injection
43. Cyclosporine A and H-7
Exposure to 10 uM cyclosporine A (CsA), a potent and specific inhibitor of CaN,
increased the magnitude of a7 currents by a factor of ~2, and also
increased Cm.
The facilitatory effect of CsA upon a7 current was blocked by 100 uM H-7, a
non-specific inhibitor of serine/threonine PKs.
The combination of genistein and CsA produced a greater enhancement of the
a7 current than either agent alone; their combined effect was approximately
additive.
44.
45.
46. Nicotine
Currently working on:
How much nicotine can upregulate α7
How much nicotinic upregulation depends
on tyrosine phosphorylation or serine
threonine phosphorylation or other
potential factors such as intracellular ca++.
How can we prevent this upregulation that
can be potential as treatment of
upregulation related phenomenon etc.
1.The increased DA overflow in the accumbal shell is hypothesized to increase the ‘pleasure’ or ‘hedonic impact’derived from behaviors associated with the delivery of the drug. As a result, the probability that the individual will repeat the behavior isgreatly enhanced2.stimuli that are paired with drug delivery acquire pleasurable reinforcing properties. Once the associationbetween the delivery of nicotine and the presentation of the conditioned stimuli has been established, presentation of stimuli alone canreinforce nicotine-seeking behaviors.3the increase in DA overflow in the accumbal core elicited by a nicotineinjection greatly increases the probability that presentation of the CS will elicit nicotine-seeking behavior; thus, nicotine evokes thecompulsive drug-seeking behavior which is characteristic of addiction. Reproduced from Balfour DJK (2006) Complementary roles forthe accumbal shell and core in nicotine dependence. In: Bock G and Goode JA (eds.) Understanding Nicotine and Tobacco Addiction,Novartis Foundation Symposium 275, pp. 96–115. Chichester and New York: Wiley, with permission from Novartis Foundation.
Homeostatic model of α4β2* nAChRupregulationThe diagram shows a hypothetical scheme of a “cholinergic synapse” in the context of a neuronal circuit under five different conditions. The first three conditions represent the synapse under normal conditions (left), during acute (center) and prolonged (right) application of nicotine. Through upregulation of nAChRs, the synapse adapts to the prolonged presence of nicotine. The right two conditions illustrate possible states of the synapse following withdrawal from nicotine, with receptors either functionally upregulated (left) or downregulated (right). Any of the states that are not homeostatically balanced are potentially capable of initiating downstream changes that could underlie behavioral tolerance and sensitization.
Structural model of the nACHR from the Torpedo electric ray. Ribbon representations were prepared using coordinatesdeposited in the research collaboratory for structural bioinformatics (RCSB) protein data bank (PDB) under ID# 2BG9. The ribbonrepresentations are shown on the background of the gray-colored molecular surface. The two facing a1 subunits are colored in orangewhile the b1, g, and d subunits are shown in cyan, yellow, and green, respectively. (a) Side view, from within the membrane. The thick graylines delineate the membrane borders. (b) Top view, from the extracellular side. The M2 segments are five a helices closely organizedaround the axis of ion conduction that is at the center of the molecule and is perpendicular to the viewer. In both panels, the extrcellular endof M2 of the right a1 subunit is labeled with a black asterisk. Note that most of the cytoplasmic domain is missing due to the lack of 3-Dstructural information.
Definition:Type:Example
Comparison of the effects of three positive allosteric modulators on the agonist concentration curves recorded in oocytes expressing the human α7 nAChR. Responses to ACh test pulses (3 s), were recorded first in control and after a brief exposure (30 s) to the allosteric modulator. Peak ACh-evoked currents were measured and plotted as a function of the logarithm of the agonist concentration. Cells were held at −100 mV throughout the experiment.
PTKs can be divided in 2 classes 1. Transmembrane receptor PTKs: Insulin receptor Growth factor receptor such as Epidermal growth factor (EGF) Fibroblast growth factor (FGF) Platelet-derived growth factor (PDGF) Vascular endothelial growth factor (VEGF) and Nerve growth factor (NGF) 2. Nonreceptor PTKs (NRTKs) NRTKs are integral components of signaling cascade trigged by RTKs and other cell surface receptors such as G protein-coupled receptors. NRTKs includes Src, Faks and AbI.
Within the cell, nAChRs are synthesized in the endoplasmic reticulum as core-glycosylated high-mannose subunits. Within this compartment, a fraction of the subunits assemble into homo and hetero-pentameric complexes, the misfolded proteins being degraded by the proteasome machinery. For the Torpedo receptor, assembly likely proceeds through a sequential pathway, from αβγ trimers, to αβγδ tetramers and αβγδα pentamers. Only pentameric complexes exit from the endoplasmic reticulum compartment to reach the cell surface. This latter stage involves targeting of the receptors to relevant subcellular compartments, for instance post-synaptic, pre-synaptic or somatic for neurons. At the neuromuscular junction, nicotinic acetylcholine receptors are located mainly in the postsynaptic membrane under the motor nerve terminal. This distribution results from a differential transcriptional regulation of nAChR genes at the level of the sub-junctional nuclei as well as from their targeting through a local secretory pathway. Local immobilisation of AChR molecules involve a large assembly of cytoskeletal proteins, including the 43K-rapsyn protein that is thought to directly cross-link nAChR molecules.Current model for nicotine upregulation of α4β2 nAChRs. a Schematic of a cell indicatingmajor steps in the lifecycle of a nAChR. Nicotine accumulates within the cell.Within the endoplasmicreticulum, nicotine binds to nAChR subunits to facilitate assembly, or binds at the interfaceof an αβ subunit pair to enhance maturation of a pentamericnAChR (Sallette et al. 2004, 2005).The strong influence of nicotine on maturation of the β2 subunit might also favour a change innAChR stoichiometry, from (α4)3(β2)2 to (α4)2(β2)3 (Moroni et al. 2006). These actions couldresult in an increase in the membrane insertion of competent nAChRs. The possibility of an additionalaction of nicotine to impede nAChR turnover or degradation is indicated by the dottedline. b Binding of nicotine to the extracellular domain of unassembled nAChR subunits facilitatesassembly. c Binding of nicotine at an αβ interface facilitates maturation of a pentamericnAChR.Items b and c adapted from Nashmi and Lester (2007)
Oocyte Harvest and Injection3-4 lobes from the ovaries of adult Xenopuslaevis were removed and defolliculated with 45 – 60 min of 2mg/ml collagenase type 1A (Sigma).Oocytes were incubated in ND-96 PK at 18 °C.1 day after harvest, oocytes were injected with 13-20 µL of 0.1 µg/µL mouse α7 mRNA in Ca2+-free ND-96 Oocytes were then incubated at in ND-96 PK at 18 °C for 4-5 days following injection.
Electrophysiological recordings were conducted using two-electrode voltage-clamp:An oocyte was placed in 2.0 mL ND-96The oocyte was then impaled with two glass microelectrodes containing 3.0 M KCl.The oocyte’s resting membrane potential was recorded, then current was passed though one of the electrodes to hold the voltage constant at 60 mV.For all genistein incubations, 100 µM of genistein was added to the recording bath before introducing the cell.
Figure 2. Average Ach-evoked currents of oocytes 5 days following injection, +/- SEM. The asterisk (*) indicates that both 1 min. as well as 24 hour Genistein incubations produced significant facilitation compared to controls (p<0.05), as determined by a two-tailed t-test performed without assuming unequal variance.
Figure 1. Average Ach-evoked currents of oocytes 2 days following injection, +/- SEM. The asterisk (*) indicates that both 1 min. as well as 24 hour Genistein incubations produced significant facilitation compared to controls (p<0.05), as determined by a two-tailed t-test performed without assuming unequal variance.
Table 1. (a) The results of a set of t-tests from the Ach-evoked currents on the second day following injection. Levene’s test for equality of variances was first conducted to see if an equal variance assumption was justified. In all but one case, indicated by the double asterisks (**), the variance between groups is unequal at the 95% confidence interval, thus in all but this case, unequal variance was assumed. As indicated by the single asterisk (*), both 1 min and 24 h incubation with genistein produced significantly greater currents than controls. However, average Ach-evoked response between 1 min and 24 h genistein incubations, although qualitatively different, is not quite statistically significant at the 95% confidence interval. (b) The results of a set of t-tests from the Ach-evoked currents on the fifth day following injection. According to Levene’s test, none of the variances are equal. The t-tests recapitulate the trend observed in the 2 day data with both 1 min and 24 h incubation with genistein produced significantly greater currents than controls (*). However, average Ach-evoked response between 1 min and 24 h genistein incubations, although qualitatively different, is not quite statistically significant at the 95% confidence interval.
Figure 3. The peak to steady-state ratio of oocytes from the 5th day following injection. The asterisks (*) indicate that the difference compared to controls is significant (p<0.05) using a 2-tailed t-test, without assuming equal variance. For untreated oocytes (controls), the holding current returned to baseline within 6 sec of Ach application. Both 1 min and 24 h incubated genisteinoocytes, however, displayed much slower desensitization kinetics. The peak to steady-state ratios from the 2nd day show the opposite trend, with the ratio for each data point closely approximating the peak current (data not shown), indicating that the critical factor is the day of treatment and recording rather than the exposure time in determining genistein’s effects on kinetics.
Figure 1. Representative traces of the departure from baseline for untreated occytes as well as two genistein conditions tested on the 2nd post-injection.
