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Long-term treatment with nicotine or selective α7 nicotinic acetylcholine receptor (nAChR) agonists increases the number of α7 nAChRs and this up-regulation may be involved in the mechanism underlying the sustained procognitive effect of these compounds. Here, we investigate the influence of type I and II α7 nAChR positive allosteric modulators (PAMs) on agonist-induced α7 nAChR up-regulation. We show that the type II PAMs, PNU-120596 (10 μM) or TQS (1 and 10 μM), inhibit up-regulation, as measured by protein levels, induced by the α7 nAChR agonist A-582941 (10 nM or 10 μM), in SH-EP1 cells stably expressing human α7 nAChR, whereas the type I PAMs AVL-3288 or NS1738 do not. Contrarily, neither type I nor II PAMs affect 10 μM nicotine-induced receptor up-regulation, suggesting that nicotine and A-582941 induce up-regulation through different mechanisms. We further show in vivo that 3 mg/kg PNU-120596 inhibits up-regulation of the α7 nAChR induced by 10 mg/kg A-582941, as measured by [125I]-bungarotoxin autoradiography, whereas 1 mg/kg AVL-3288 does not. Given that type II PAMs decrease desensitization of the receptor, whereas type I PAMs do not, these results suggest that receptor desensitization is involved in A-582941-induced up-regulation. Our results are the first to show an in vivo difference between type I and II α7 nAChR PAMs, and demonstrate an agonist-dependent effect of type II PAMs occurring on a much longer time scale than previously appreciated. Furthermore, our data suggest that nicotine and A-582941 induce up-regulation through different mechanisms, and that this confers differential sensitivity to the effects of α7 nAChR PAMs. These results may have implications for the clinical development of α7 nAChR PAMs.
The α7 nicotinic acetylcholine receptor (nAChR) is a pentameric cation-selective ligand-gated ion channel that is widely expressed in the brain (Dani and Bertrand 2007; Albuquerque et al. 2009). Numerous studies have demonstrated that α7 nAChR agonists improve a wide range of cognitive parameters in animal models (Hajos 2009; Thomsen et al. 2010). Clinical studies have also demonstrated cognitive improvement in healthy volunteers and patients with schizophrenia, although somewhat restricted to effects on attention and working memory domains (Kitagawa et al. 2003; Olincy et al. 2006; Freedman et al. 2008). Consequently, α7 nAChR ligands are currently being developed for the treatment of cognitive deficits in diseases such as schizophrenia and Alzheimer’s disease (Wallace and Porter 2011).
Positive allosteric modulators (PAMs) of the α7 nAChR have no intrinsic effect on channel activation, but increase the effectiveness of an agonist. They are normally divided into two types depending on whether they decrease receptor desensitization (type II) or not (type I) (Grønlien et al. 2007). It has been argued that allosteric modulation may be preferable over direct agonism for therapeutic purposes because this merely modulates the effects of endogenous transmitters, rather than activating the receptor per se, thus restricting the drug effects to areas where acetylcholine is being released (Faghih et al. 2008), but the lack of activation by the PAMs alone may diminish their effect in patients with decreased levels of endogenous activation. As there are no published clinical results with α7 nAChR PAMs, it is not clear at the moment, whether α7 nAChR agonists or PAM are preferable for clinical use. But from the limited data on the behavioral effects of α7 nAChR PAMs in animals, they seem to produce similar acute effects as α7 nAChR agonists, including improvements of pre-pulse inhibition and auditory gating as well as short- and long-term memory (Hurst et al., 2005; Ng et al. 2007; Timmermann et al. 2007; Dunlop et al. 2009; Dinklo et al. 2011). There are currently no behavioral data demonstrating in vivo differences between the effects of type I and II α7 nAChR PAMs.
It is well known that nicotine and selective α7 nAChR agonists increase binding of the α7 nAChR antagonist [125I]-bungarotoxin (BTX) in the rodent brain after systemic administration (Marks et al. 1985, 1986a; Rasmussen and Perry 2006; Christensen et al. 2010). Several mechanisms may be involved in agonist-induced up-regulation of nAChRs, such as decreased cell surface turnover, increased receptor trafficking to the surface, increased subunit maturation and assembly, decreased subunit degradation, and conformational changes at the membrane leading to increased ligand affinity (reviewed in (Lester et al. 2009; Govind et al. 2009)). However, the mechanisms underlying agonist-induced up-regulation of the α7 nAChR are not well understood.
Underscoring the potential behavioral effects of nAChR up-regulation, it has been shown that increased [125I]-BTX binding and enhanced novel object recognition in mice occur in parallel 4–48 h after administration of the specific α7 nAChR agonist AZD0328 (Werkheiser et al. 2011), and that up-regulation of [3H]-nicotine binding correlates directly with performance in the Morris water maze task several days after nicotine administration (Abdulla et al. 1996). These data suggest that increased nAChR levels may partly underlie the cognitive improvements seen with nAChR agonists. We have recently demonstrated that α7 nAChR up-regulation does not occur with administration of α7 nAChR PAMs in the rat brain, demonstrating a fundamental in vivo difference between agonists and PAMs (Christensen et al. 2010).
Here, we use an SH-EP1 cell line transfected with the α7 nAChR coupled to yellow fluorescent protein and ex vivo [125I]-BTX binding to show that type II α7 nAChR PAMs inhibit up-regulation of the α7 nAChR by the α7 nAChR agonist A-582941, whereas type I α7 nAChR PAMs do not affect A-582941-induced up-regulation. Neither the type I or II PAMs affect α7 nAChR up-regulation by nicotine. These results demonstrate a fundamental difference between type I and II PAMs that may affect the long-term behavioral effects of these compounds. Furthermore, the results suggest that different agonists may mediate up-regulation of the α7 nAChR through different mechanisms.
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The main findings in this article are that type II α7 nAChR PAMs inhibit A-582941-induced up-regulation of α7 nAChR levels in vitro and in vivo, whereas type I PAMs do not. Furthermore, nicotine-induced up-regulation of α7 nAChR levels in vitro is not affected by type I or II PAMs, suggesting that A-582941 and nicotine increase α7 nAChR levels through different mechanisms.
Both A-582941 and nicotine produced a dose-dependent increase in α7 subunit protein expression in SH-EP1 cells transfected with the α7 nAChR coupled to enhanced yellow-fluorescent protein. Nicotine-induced up-regulation of BTX binding in cell cultures is a well-known phenomenon (Molinari et al. 1998; Kawai and Berg 2001), and it has previously been shown that nicotine increases BTX binding in SH-EP1 cells transfected with the human α7 subunit by ∼80% (Peng et al. 1999). Our data suggest that at least part of the nicotine-induced increase in BTX binding is because of increased protein expression of the α7 subunit. A-582941 was ∼1000-fold more potent than nicotine and slightly more efficacious. The increased potency of A-582941 over nicotine may be because of a higher affinity and potency of A-582941 for the α7 nAChR. Thus, the Ki for A-582941 binding to rat and human brain tissue is 10.8 and 16.7 nM, respectively (Bitner et al. 2007), compared to a Ki of ∼400–8900 nM for nicotine (Marks et al. 1986b; Gotti et al. 1997; Haustein and Groneberg 2009). And, A-582941 activates rat and human α7 nAChRs expressed in Xenopus oocytes with an EC50 of 2.45 and 4.26 μM, respectively (Bitner et al. 2007), compared with an EC50 of ∼18–91 μM for nicotine (Peng et al. 1994b; Briggs et al. 1995; Haustein and Groneberg 2009).
The α7 nAChR antagonist MLA inhibited the up-regulation of α7 protein levels by A-582941 or nicotine. This confirms that binding of the agonist to the orthosteric site of the α7 nAChR is required for up-regulation induced by either compound. MLA has previously been shown to increase BTX binding in SH-SY5Y and transfected HEK293 cells (Molinari et al. 1998; Ridley et al. 2001), but MLA alone had no effect on α7 subunit protein expression in SH-EP1 cells. Although these effects may vary between cell types, one explanation is that MLA affects nAChR levels, as reflected in BTX binding, downstream of protein synthesis.
Neither the type I PAMs, AVL-3288 and NS1738, nor the type II PAMs, PNU-120596 and TQS, affected α7 subunit protein expression in SH-EP1 cells, at doses well above their EC50 for increasing acetylcholine-evoked currents (0.7, 3.4, 0.21, and 5.5 μM, respectively) (Hurst et al., 2005; Grønlien et al. 2007; Ng et al. 2007; Timmermann et al. 2007). The lack of effect of type I or II PAMs corresponds with the lack of effect on in vivo brain BTX binding, previously reported after repeated administration of PNU-120596 or NS1738 (Christensen et al. 2010). This provides further support for the notion that allosteric modulation of the α7 nAChR does not affect the basal levels of the receptor, contrary to what is seen with α7 nAChR agonists.
Importantly, the type II PAMs PNU-120596 and TQS dose-dependently inhibited the increase in α7 subunit protein expression induced by both 10 nM and 10 μM A-582941 in SH-EP1 cells. The inhibition with PNU-120596 or TQS occurred at concentrations which correspond well with those needed to affect agonist current responses at the α7 nAChR (Grønlien et al. 2007). This provides further support that the effect of A-582941 on α7 protein expression occurs directly at the α7 nAChR. Up-regulation of the α7 subunit may depend on specific conformational states, such as desensitized states induced by the ligand. In line with this, it has been shown that receptor desensitization initiates nicotine-induced up-regulation of α4β2 nAChRs (Fenster et al. 1999). Binding of allosteric modulators affects receptor conformations by favoring certain states, and this may diminish the propensity of receptors to enter desensitized states (Williams et al. 2011). Thus, the type II α7 nAChR PAMs, PNU-120596 and TQS, greatly diminish agonist-induced desensitization of the α7 nAChR, and can even revert desensitized receptors into a conducing state (Hurst et al., 2005; Grønlien et al. 2007). It may therefore be the effect on receptor desensitization that enables PNU120596 and TQS to inhibit A-582941-induced up-regulation of the α7 nAChR, whereas the type I α7 nAChR PAMs, AVL-3288 and NS1738, which do not affect receptor desensitization, did not affect the A-582941-induced increase in α7 protein expression in SH-EP1 cells. However, it is also possible that the different PAM compounds bind to different allosteric sites on the α7 nAChR (Thomsen and Mikkelsen 2012), which may confer different modulation of A-582941-mediated effects. Finally, metabotropic signaling through the α7 nAChR has previously been described (Chernyavsky et al. 2009, 2010), and we cannot exclude the possibility that agonists induce receptor up-regulation by activating intracellular cascades through metabotropic signaling, similar to what is observed with, e.g., the regulation of NMDA receptors through metabotropic glutamate receptors (Benquet et al. 2002).
As an extension of the in vitro data, we demonstrated that 3 mg/kg PNU-120596 inhibited the A-582941-induced increase in BTX binding in the frontal cortex and hippocampus in vivo, whereas 1 mg/kg AVL-3288 did not. These data are the first to demonstrate an in vivo difference between type I and II PAMs, and they suggest that type II PAMs have activity-dependent effects that occur on a much longer time scale (i.e. several hours) than previously reported using electrophysiology. Furthermore, as the endogenous agonist, acetylcholine, might also induce up-regulation of nAChRs, and given the potential involvement of receptor up-regulation in the prolonged effect of nAChR ligands, these data allow us to speculate that repeated treatment with type II versus I PAMs may have very different cognitive effects, particularly in situations with high levels of endogenous or experimental agonists. It should be noted, however, that these data are based on single-point BTX measurements, so although we have previously shown with saturation curves that α7 nAChR agonists increase α7 nAChR numbers without affecting ligand affinity, we cannot exclude that α7 nAChR PAMs reduce BTX binding by reducing ligand affinity. In addition, although we have chosen behaviorally active doses of both PAMs (Hurst et al., 2005; Ng et al. 2007), different in vivo kinetics of the two PAMs might influence the results. Thus, administration of 10 mg/kg PNU-120596 results in an approximate brain level of 15 ng/mL, corresponding to 0.048 μM, which declines with a half life of 7.9 h (McLean et al. 2011). Administration of 0.3 mg/mL AVL-3288 results in brain levels of 1 μM after 10 min, which declines to 0.3 μM after 90 min (Ng et al. 2007). So, although PNU-120596 reaches a lower concentration than AVL-3288, the prolonged presence of the compound in the brain may facilitate its effect on an A-582941-induced increase in BTX binding.
The correspondence between our results from in vitro protein data and in vivo BTX-binding data for A-582941 and PNU-120596 suggests that at least part of the observed increase in BTX binding is because of increased α7 subunit protein expression. However, it has been demonstrated in vitro and in vivo that the majority of α7 nAChRs exist as an intracellular pool (Fabian-Fine et al. 2001; Zhong et al. 2008; Mielke and Mealing 2009; Murray et al. 2009). Therefore, an increase in BTX binding might readily occur without increased protein synthesis, and it is possible that other factors, such as increased transport of α7 nAChRs to the cell surface occur in parallel with increased protein synthesis. Vice versa, increases in α7 protein may not necessarily lead to increased BTX binding. Thus, a single administration of 1 mg/kg nicotine did not affect BTX binding in the frontal cortex in vivo. It is well documented that prolonged exposure to nicotine increases nAChR levels, including the α7 nAChR (Marks et al. 1985, 1986a), whereas to our knowledge, there are no reports on the effects of acute nicotine administration on α7 nAChR levels. This effect of nicotine might therefore require prolonged exposure. This illustrates another difference between selective experimental α7 nAChR agonists and nicotine, regarding the mechanism of α7 nAChR up-regulation. This difference may be related to different affinities for the α7 nAChR, as the experimental agonists that demonstrate α7 nAChR up-regulation after acute administration all have higher affinities for the α7 nAChR than nicotine (Christensen et al. 2010). However, although we have previously shown, A-582941-induced up-regulation of the α7 nicotinic receptor to be evident several days after administration (Christensen et al. 2010), we cannot exclude the possibility that the shorter half-life of nicotine (∼45 min) (Matta et al. 2007) compared to A-582941 (∼2 h) in rats (Tietje et al. 2008) precludes us from observing an effect of nicotine.
PNU-120596 has previously been shown to reduce cell viability in α7 nAChR-expressing SH-SY5Y cells, because of the prolonged opening of the α7 nAChR, characteristic of type II PAMs, leading to Ca2+-mediated toxicity (Ng et al. 2007). Another study, however, found no effect of PNU-120596 on cell viability in PC12 cells or rat primary cortical neurons (Hu et al. 2009). We measured LDH release, as a measure of cytotoxicity, and found no effect of any of the α7 nAChR ligands used in our studies. Therefore, the reduced expression of α7 in SH-EP1 cells co-incubated with A-582941 and PNU-120596 or TQS is not because of cytotoxicity of the compounds.
Contrary to the effects on A-582941-induced up-regulation, neither type I nor II α7 nAChR PAMs affected nicotine-induced α7 up-regulation in vitro. This indicates a mechanistic difference in how A-582941 and nicotine induce α7 up-regulation. It has been suggested that nicotine induces up-regulation of high-affinity nAChRs primarily by acting as a molecular chaperone, promoting assembly and maturation of nAChRs, in the endoplasmic reticulum (Kuryatov et al. 2005; Lester et al. 2009). Because of its unique chemical properties, nicotine readily crosses the cellular membrane and is enriched in intracellular organelles such as the endoplasmic reticulum (Lester et al. 2009). It is therefore possible that nicotine primarily affects intracellular receptors, and that these receptors are not as accessible to A-582941 or the α7 nAChR PAMs used in our studies. However, one study has indicated that the degree of up-regulation of high-affinity nAChRs that occurs with nicotine in transfected HEK cells cannot be accounted for merely by increased maturation of already existing subunits (Vallejo et al. 2005), and recent studies have shown that nicotine-induced increases in α4β2*-binding sites can be explained by increased α4 and β2 protein levels (Moretti et al. 2010; Marks et al. 2011). Conformational changes leading to increased ligand binding, and reduced receptor turnover has also been shown to affect ligand binding after nicotine treatment (Peng et al. 1994a; Kuryatov et al. 2005; Vallejo et al. 2005). Furthermore, it has been shown that repeated nicotine-induced up-regulation of α7 nAChRs, but not high-affinity nAChRs, requires protein synthesis and glycosylation in cortical cultures (Kawai and Berg 2001). Proteasomal activity is also an important regulator of α7 nAChR levels (Christianson and Green 2004), and repeated nicotine-mediated inhibition of proteasomal activity has been shown to mediate α7 nAChR up-regulation (Rezvani et al. 2007). In summary, nicotine may affect α7 nAChR levels through several mechanisms, and it is currently not known whether other nAChR agonists, such as A-582941, exhibit the same properties. However, our data with PAM inhibition suggest that A-582941 is dependent on receptor-specific conformations, such as desensitized states, to increase α7 nAChR levels whereas nicotine may not be.
Our data demonstrate a fundamental difference between the effects of α7 nAChR agonists and type I and II PAMs on receptor up-regulation that is evident both in vitro at the level of protein expression in single cells, and in vivo at the level of assembled α7 nAChRs in several brain regions. As nAChR up-regulation may underlie the ability of nAChR agonists to produce, particularly long-lasting, cognitive effects (Abdulla et al. 1996; Briggs et al. 1997; Buccafusco et al. 2005; Werkheiser et al. 2011), it is pertinent to investigate the potential behavioral consequences of this differential effect on agonist-induced up-regulation of the α7 nAChR. Therefore, further studies are warranted to determine whether type II PAMs may inhibit the long-lasting cognitive effects of α7 nAChR agonists.
It is currently not known to what degree endogenous cholinergic signaling affects nAChR levels or on what time scale. However, if up-regulation does occur in response to increased acetylcholine or choline levels, e.g. during performance, then inhibiting this up-regulation may interfere with long-term cognitive performance. Finally, our results highlight the importance of the arousal state of the animals when performing experiments with PAMs, as they are dependent on endogenous signaling for their effect. Thus, both the acute and long-term effects of PAM administration may be very different in non-performing animals with low levels of endogenous neurotransmitter, and in aroused animals with high levels of endogenous neurotransmitter.