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New Insights on Neuronal Nicotinic Acetylcholine Receptors as Targets for Pain and Inflammation: A Focus on α7 nAChRs

Background Nicotine and nicotinic acetylcholine receptors (nAChRs) have been explored for the past three decades as targets for pain control. The aim of this review is to introduce readers particularly to α7 nAChRs in a perspective of pain and its modulation. Methods Developments for α7 nAChR modulators and recent animal studies related to pain are reviewed. Results Accumulating evidences suggest that selective ligands for α7 nAChRs hold promise in the treatment of chronic pain conditions as they lack many of side effects associated with other nicotinic receptor types. Conclusion This review provides the reader recent insights on α7 nAChRs from structure and function to the latest findings on the pharmacology and therapeutic targeting of these receptors for the treatment of pain and inflammation.


INTRODUCTION
Nicotine and other ligands that act on nicotinic acetylcholine receptors (nAChR) have been explored for the past three decades as a strategy for pain control. These receptors are widely expressed throughout the central and peripheral nervous system as well as on immune cells. Despite encouraging results with many selective α4β2* agonists in animal models of pain, human studies showed a narrow therapeutic window between analgesic efficacy and toxicity associated with the use of these agonists as analgesics (For a recent chapter see [1]). However, several recent developments have potentially opened new windows of opportunity in the use of nicotinic agents for analgesia. α7 nAChR agonists have shown to be beneficial for central nervous system disorders characterized by cognitive deficits, such as Alzheimer's disease and schizophrenia [2]. In addition, accumulating evidences suggest that agonists and modulators of nicotinic receptors that contain the α7 subunit hold promise for the treatment of chronic inflammatory pain conditions. *Address correspondence to this author at the Department of Pharmacology and Toxicology, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, VA 23298-0613; Tel/Fax: +1-804-828-9256; E-mail: dbagdas@vcu.edu

Alpha7 Nicotinic Acetylcholine Receptor Structure and Modulation
The nAChRs are pentameric assemblies of subunits configured around a central ion channel pore that can be gated by the binding of ligands (orthosteric agonists) to sites in the N-terminal extracellular domains at the interface between subunits. The first nAChR to be studied and isolated were those of the neuromuscular junction and the homologous electric organ of the Torpedo ray. These receptors evolved to rapidly and efficiently activate when stimulated with a large increase in acetylcholine (ACh) concentration [3]. These muscle-type receptors were found to have four different types of subunits, referred to as α1, β1, γ and δ, with two α1 subunits per receptor. For the most part, each subunit had a relatively well-conserved structure, with three transmembrane domains following the N-terminal extracellular domain, a more variable intracellular domain [4], followed by a fourth transmembrane domain. The α1 subunits contained a pair of disulfide-linked vicinal cysteines, and all of the subunits contained a disulfide-constrained loop in a region later shown to be at the interface between the extracellular and transmembrane domains. This hallmark "Cys-loop" was ultimately found in all nAChR subunits subsequently cloned, as well as in homologous receptors activated by GABA, gly-cine, or serotonin, identifying the Cys-loop superfamily of ligand-gated ion channels (LGIC).
The family of nAChR subunits was ultimately expanded to include one additional alternative muscle subunit, epsilon, and twelve additional subunits cloned from neuronal tissues. The neuronal subunits were classified as alpha subunits (α2 -α10, although α8 is found only in birds) if they contained vicinal cysteines homologous to those in α1, or as β subunits (β2 -β4) if they did not. Each subunit has a unique pattern of expression associated with diverse functions for nAChR in different tissues and brain structures [5]. Most nAChR subtypes are heteromers containing both α and non-alpha subunits with two orthosteric agonist-binding sites contributed by α subunits in each receptor, as in the muscle-type receptor. In the rodent brain the predominant nAChRs contain combinations of α4 and β2 subunits, and these receptors were shown to bind radiolabeled ACh or nicotine with high affinity. It was also shown that a second class of nAChR is present in brain that did not bind ACh or nicotine with high affinity but did bind the neuromuscular receptor blocker αbungarotoxin [6]. These were subsequently shown to be associated with the expression of the α7 subunit, which could form receptors without the co-expression of a beta subunit [7].
Homomeric α7 nAChR lacks the specializations for synaptic function that are present in muscle-type and ganglionic nAChR [3]. The absence of non-alpha subunits modifies the agonist binding site, reducing high-affinity agonist binding to desensitized states and increasing sensitivity to the ubiquitous ACh precursor choline. In regard to functioning as a homopentamer, α7 nAChR is like the proton-gated channels Gloeobacter ligand-gated ion channel (GLIC) [8] and Erwinia ligand-gated ion channel (ELIC) [9] found in bacteria, which are believed to be homologs of the vertebrate Cysloop LGIC.
The unique cellular and subcellular α7 expression patterns indicate special roles for this receptor subtype. This unique pattern includes wide spread expression in nonneuronal cells, including cells of the immune system where α7 has been uniquely implicated in regulating the cholinergic anti-inflammatory pathway. Additionally, due to its ability to be activated by choline in addition to ACh and its rapid concentration-dependent desensitization of ion channel currents, α7 nAChR will respond in a very different way from other nAChR to endogenous cholinergic signals, including paracrine signals in peripheral tissues.
Open α7 ion channels have high calcium permeability [7]. While the high calcium permeability of NMDA-type glutamate receptors is responsible for their key role in synaptic plasticity, it has been implicated to lead to the potential for excitotoxic activation, in the case of α7. This feature is offset by the fact that normally the open probability of the α7 receptor channel is extremely low compared to other nAChR [10,11]. Changes in intracellular calcium concentration subsequent to α7 stimulation are typically more due to release of calcium from intracellular stores rather than calcium influx through the α7 channels [12], suggesting a metabotropic-like function for α7 nAChR. This may especially be the case for non-neuronal cells, where no α7-mediated ionic currents can be detected [13][14][15].
The identification of cholinergic anti-inflammatory activity mediated by α7 nAChR in cells of the immune system [16][17][18] has drawn attention to the likelihood that ligandinduced conformational changes of α7 receptors are global and apparently encompass changes in signaling associated with the receptor's interactome [19] and potentially with Gprotein-mediated signals [20]. While the prokaryotic Cysloop receptor homologs lack any intracellular domains, the vertebrate nAChR subunits show remarkable diversity and specialization in their intracellular domains, and the unique intracellular domain of α7 receptors has been especially well conserved throughout the evolution of vertebrates [4].
As noted above, the unique configuration of the α7 orthosteric agonist binding between pairs of identical, rather than specialized subunits, allows for these receptors to be effectively targeted by multiple classes of selective agonists [21]. The presence of five potential agonist-binding sites per receptor also permits multiple types of ligand-induced conformational states based on the level of binding site occupancy. Data suggest that only relatively low levels of binding site occupancy effectively promote channel activation (albeit with low probability) [22-24] and that higher levels of agonist occupancy preferentially induce nonconducting (i.e. desensitized) states that are far more stable than the open channel state. The existence of these unique nonconducting states has been confirmed by the activity of α7-selective positive allosteric modulators (PAMs), which, at least in the case of efficacious type II PAMs like PNU-120596 [25], can destabilize the nonconducting conformations and couple them to unique open-channel states [11,26,27]. The identification of diverse classes of α7-selective PAMs [25] opened up a new avenue for α7-directed drug development [28] and ultimately led to the identification of another novel class of α7-channel activators, ago-PAMs, including 4-BP-TQS [29], its active isomer GAT107 [30], and more recently, the structurally unrelated B-973 [31], that function both as PAMs and allosteric agonists. The allosteric agonism has been hypothesized to be associated with a specific allosteric activation site in the extracellular domain [32].
Agonists, PAMs and ago-PAMs are all capable, to varying degrees, of activating α7 ion channel currents, although for a PAM that activation is conditional on the coincident presence of an agonist. All of these currents can be antagonized by both competitive and non-competitive antagonists, for example MLA and mecamylamine, respectively. However, the conducting states activated in the presence of a PAM differ subtly in their sensitivity to specific antagonists [27] than those activated by orthosteric agonist in the absence of a PAM. Additionally, molecules have been identifies which specifically antagonize the effects of PAMs with little or no effects on activation by ordinary orthosteric agonists [33]. Conversely, silent agonists [34], which produce channel activation in the presence of a PAM, behave as very low efficacy partial agonists in the absence of a PAM.
While the primary focus for the use of α7-PAMs has been as tools to overcome the intrinsic limitations to α7 channel activation, they have also been used to investigate the induction of nonconducting conformational states of the receptor that may play important roles in the metabotropic functions of α7 receptors. As noted above, while the openchannel state of α7 is very unstable, the nonconducting states that can be converted to conducting states by the activity of PAMs are far more stable. This has allowed the use of PAMs to identify "silent agonists", ligands that bind to a site that overlaps the agonist binding site [34], do not produce significant activation when applied alone, but will activate currents in the presence of a PAM. NS6740 was the first silent agonist introduced in the literature. It was reported to be an α7 ligand that required a PAM to produce channel activation. NS6740 was later shown to be very effective at inducing stable nonconducting states of the receptor and also to be very effective at reducing the behavioral manifestations of pain in several animal models. While NS6740 was ineffective in an animal model of cognition, it was subsequently shown that both NS6740 and GTS-21, a low-efficacy α7selective partial agonist, were effective modulators of immune signaling in microglia [35]. Interestingly, the α7 ago-PAM GAT107, which also induces stable nonconducting states, had a profile similar to that of NS6740 in the same pain models [36]. The long-term effects of NS6740 on AChevoked responses are desensitizing [37], while those of GAT107 are potentiating [30], suggesting that the antiinflammatory activity reducing the manifestations of pain may be specifically associated with the nonconducting states of the receptor.
Both RIC-3, and a newly identified accessory protein NACHO [61] have been shown to be crucial regulators of α7 functional expression on both cellular and subcellular levels. While the only known function for NACHO has been as a regulator of α7, RIC-3 has been more broadly implications as a regulator of other cys-loop receptors [61][62][63][64][65].

Anti-inflammatory Signaling of alpha7 Nicotinic Acetylcholine Receptors
α7 nAChR endogenous ligands, ACh and choline, are closely associated with controlling immune cell functions, attenuation of pro-inflammatory cytokine production and inhibition of the inflammatory process via activation of α7 nAChRs [15,66]. This neurophysiological mechanism decreases inflammation by reducing cytokine synthesis via release of ACh in organs of the reticulo-endothelial system, such as the lungs, spleen, liver, kidneys and gastrointestinal tract [67]. It has been revealed that α7 nAChRs are implicated in modulating tumor necrosis factor (TNF-α), interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-18 (IL-18), high mobility group box 1 (HMGB1) and some other proinflammatory cytokines without affecting the antiinflammatory cytokine interleukin-10 (IL-10) [39,66,[68][69][70]. Indeed, a critical role for α7 nAChR as a peripheral component in cholinergic anti-inflammatory pathway has been demonstrated using α7 subunit knockout (KO) mice [17]. Intraplantar complete Freund's adjuvant or inactivated and dried mycobacteria, which induces chronic inflammation and inflammatory pain, injection increased more edema, hyperalgesia and allodynia in the α7 KO mice compared with the wild-type (WT) littermates [71]. ACh and nicotine pretreatment inhibited lipopolysaccharide (LPS)-induced TNF-α release in murine-derived microglial cells through α7 nAChR activation [60]. Another study also showed that pretreatment with ACh inhibited LPS-induced matrix metalloproteinase 9 (MMP-9) production and macrophage migration in vitro [72]. It has been reported that activation of these receptors by an agonist attenuated TNF-α and IL-1β levels in human whole blood activated by exposure to endotoxin [50] and microglial α7 nAChR activation reduced TNF-α release but not IL-1β [58]. Choline has also been shown to modulate TNF-α release via α7 nAChR-mediated signaling [73]. CDPcholine is an endogenously synthesized nucleotide which rapidly metabolizes to choline and cytidine/uridine. Consistently, exogenous administration of CDP-choline results in elevations in plasma and tissue levels of choline [74,75]. When CDP-choline, also a source for the synthesis of the cholinergic neurotransmitter ACh, was locally administered, it reduced the production of the TNF-α, reduced edema and reversed the mechanical hyperalgesia through α7 nAChR, suggesting that the local application of α7 nAChR activators may provide a tool to reduce the local inflammation and pain [76]. Nuclear translocation of nuclear factor-κB (NF-κB) is the main factor of immune cell activation and proinflammatory cytokines expression [77]. It has been shown that α7 nAChR activation decreases NF-κB activity and reduces inflammatory response [17,51]. It has been suggested that different mechanisms effect α7 nAChR activation on NF-κB activity [51,78]. One possible mechanism is the recruitment of Janus kinase-2 (Jak2) to the α7 nAChR and activation of Jak2, thereby initiating the anti-inflammatory signal transducer and activator of transcription 3 (STAT3) and suppressor of cytokine signaling 3 (SOCS3) signaling cascade. Vagus nerve stimulation has been shown to attenuate macrophage activation through activating the Jak2-STAT3 signaling pathway and STAT3 phosphorylation, a potential negative regulator of inflammatory responses, depends on α7 nAChR activation [78,79]. In addition, α7 nAChR activation leads to suppression of the phosphorylation of I kappa B (I-κB) which results in inhibition of transcriptional activity of NF-κB and inhibition of proinflammatory mediators [51].
Another possible mechanism may result from an interaction between α7 nAChRs and toll-like receptor 4 (TLR4) through myeloid differentiation factor 88 (MyD88) and NF-κB. MyD88, a key intracellular reactive protein for TLR4, could activate I-κB kinases (IKK) and induce NF-κB translocation [80]. Nicotine has been shown to decrease the expression of MyD88 and to interfere NF-κB transcription in an α7 nAChR antagonist α-bungarotoxin dependent manner. Consequently, nicotine reduced TNF-α expression through α7 nAChR/MyD88/NF-κB pathway [81]. Many other downstream pathways may involve α7 nAChRs and TLR4 interaction, such as overexpression of IL-1 receptor-associated kinase M (IRAK-M), a negative regulator of TLR4, in macrophages resulted in decreased TNF-α production through a Jak2-STAT3 signaling pathway by α7 nAChR activation [82]. HMGB1, a cytokine mediator of inflammation, has been reported to upregulate α7 nAChR expression [83]. In addition, vagus nerve stimulation or cholinergic receptor agonists have been suggested to inhibit the activation of NACHT, LRR, PYD domains-containing protein 3 (NLRP3) inflammasome, a key modulator of innate immunity, whereas genetic deletion of α7 nAChR enhances inflammasome activation. Mitochondrial α7 nAChR activation suppressed the release of mitochondrial DNA, an NLRP3 ligand, as a result IL-1β and HMGB1 expressions were suppressed [84]. It has also been shown that α7 nAChR gene expression was increased in septic patients' peripheral blood mononuclear cells and these patients responded dynamically to the inflammatory challenge; besides a group of patients with low levels of α7 expression had a defective response to infection and showed worst clinical outcome with respect to inflammation [85]. This is also suggestive that there might be bilateral interactions between α7 nAChR and inflammatory mediators. With regard to these results, α7 gene expression level in septic patients might be used to assess cholinergic anti-inflammatory pathway activity as a clinical marker. Furthermore, ligands for α7 nAChRs might open new strategies to monitor and cure inflammatory diseases such as ulcerative colitis, sepsis, acute pancreatitis and asthma. Therefore, these receptors present an alternative therapeutic strategy for modulation of inflammation-based syndromes.

Effects of alpha7 Nicotinic Acetylcholine Receptors Agonists and Modulators in Rodents Pain Models
Animal studies have shown that α7 nAChR agonists such as choline, compound B [(R)-N-(1-azabicyclo[2.2.2]oct-3yl)(5-(2-pyridyl)thiopene-2-carboxamide)], JN403, PHA-543613 and AR-R17779 exhibit anti-inflammatory effects in various inflammatory pain models in rodents [86][87][88][89][90]. In addition, they were found to be active in chronic neuropathic pain models [91][92][93]. For example, repeated administration of a selective α7 agonist decreases allodynia in a chronic neuropathic pain model in the rat and reverses signs of neuroinflammation and neurodegeneration (macrophage infiltration, decreases in axon compactness and diameter with a significant loss of myelin sheaths) [93]. Not limited to direct α7 nAChR agonists, CDP-choline which is an intermediate in the biosynthesis of phosphatidylcholine and leads to an increase in choline levels, has been reported as effective to modulate nerve injury-and chemotherapy-induced neuropathic pain through α7 nAChRs [94,95]. The partial agonist GTS-21 has been reported to have antinociceptive properties in a mouse model of postoperative pain [50]. Stimulation of cholinergic anti-inflammatory pathways resulted in attenuation of neuroinflammation in a tibial fracture model of rats through α7 nAChR [96]. Choline has been also reported to attenuate a model of postoperative pain in mice [97]. Choline and nicotine have been shown to enhance inhibitory synaptic transmission in the dorsal horn neurons of spinals cords of rats [98]. However, there are some limitations to the development α7 nAChR agonists for the treatment of pain, such as receptor selectivity issues (cross-reactivity with 5-HT3 receptors, which have high homology with α7 nAChRs, and possible related adverse effects), overactivation and possible desensitization of the receptor. In addition, while α7 nAChR agonists have shown beneficial effects in chronic pain models in some studies, this effect was not consistently seen in others [99]. Efficacy of various α7 nAChRs ligands in various animal models of pain is shown in Table 1. Therefore, alternative approaches to overcome these limitations associated with direct nAChR agonists were developed in the last decade.
In addition to agonists, α7 nAChRs PAMs also provide a unique strategy for rational drug design and discovery. As discussed above, PAMs for α7 nAChRs increase the potency and/or maximal efficacy of endogenous (ACh and choline) or exogenous agonists for the α7 nAChRs without directly activating α7 nAChRs. Recently, several structurally distinct and selective α7 nAChR PAMs were identified and classified as type I and type II based on their electrophysiological properties [100,101]. Type I PAMs increase agonist response with little or no effect on desensitization of α7 nAChRs, whereas type II PAMs increase agonist response and slow down the apparent desensitization profile of the agonist response [28]. It has been suggested that PAMs may modulate conformational states for both channel activity and ion channel-independent signaling [27]. Importantly, it has been reported that in contrast to α7 agonists, α7 nAChR PAMs do not induce upregulation of α7 nAChR expression in the brain in vivo [102].
Both PAM types have been recently tested in vivo for their efficacy in animal models of inflammatory and neuropathic pain. Type II but not type I PAMs were shown to be efficacious in these models [92,103]. This is different from rodent models of cognition and memory, where both type I and type II PAMs for the α7 nAChRs showed cognitive enhancement. There are several examples of antinociceptive, antiallodynic and antihyperalgesic activities of type II PAMs. PNU-120596, an α7 nAChR type II PAM, has well described activity in tonic, inflammatory and chronic pain models in rodents [92,103,104]. Recently, another α7 nAChR type II PAM, 2,4,2',5'-tetrahydroxychalcone, has been found active against complete Freund's adjuvant-induced inflammatory pain in rats [105]. In addition, 3-furan-2-yl-Np-tolyl-acrylamide, an α7 nAChR type II PAM agent reduces neuropathic and inflammatory pain outcomes in mice in a dose and time dependent manner [92,106]. Different mechanisms may underlie PAM-induced anti-inflammatory effects.  PNU120596 reduced TNF-α and IL-6 within the hind paw edema in a rat model of the carrageenan test [104]. The activation of spinal extracellular signal-regulated kinase-1/2 (ERK1/2) pathways are the likely one possible post receptor signaling mechanism for the antinociceptive effect of PNU-120596 in the formalin test [92]. Although, α7 nAChR modulation attenuates inflammatory and neuropathic pain, they are mostly lack of efficacy for acute pain [36,37,103]. Additional studies to fully clarify the analgesic-like properties of α7 nAChR are necessary in animal models of chronic pain.
An alternative therapeutic approach involves allosteric agonist and allosteric modulators called ago-PAMs. While PAMs are thought to bind to a distinct binding site from the orthosteric site and thus lack intrinsic agonist activation, recent studies have reported that some molecules show dual activity as allosteric modulators and allosteric agonists [33,107]. GAT107 is a potent α7 nAChR type II PAM with intrinsic allosteric agonist activities suggesting it is an ago-PAM for α7 nAChRs [107]. In several mouse models of chronic inflammatory and neuropathic pain, GAT107 attenuated pain behavior and showed anti-inflammatory effects. Furthermore, intrathecal, but not intraplantar, injections of GAT107 reversed nociception in the complete Freund's adjuvant-induced inflammatory pain model, suggesting a spinal mechanism of action. Immunohistochemical evaluation of the lumbar spinal cord dorsal horn of mice treated with complete Freund's adjuvant showed an increase in the expression of astrocyte-specific glial fibrillary acidic protein (GFAP) and phosphorylated p38-mitogen-activated-kinase (p-p38MAPK). Treatment with intrathecal GAT107 robustly attenuated these inflammatory changes. These findings suggest that α7 ago-PAMs represent a novel and effective pharmacological strategy for treating inflammatory and neuropathic pain in mice [36].
An additional new class of selective ligand has been identified for α7 nAChR. While direct α7 nAChR agonists bind to the receptor on the orthosteric binding site to activate the channel, ligands called "silent agonists" also bind the orthosteric binding site of the receptor, but preferentially induce a nonconducting state associated with desensitization that can none the less modulate signal transduction [108,109]. Recent studies showed that α7 nAChR-selective silent agonism may provide a novel strategy for pain management. NS6740, α7 silent agonist, induces nonconducting states of the receptor. NS6740 modulates the inflammatory responses of microglia cells in vitro and it is effective in both chronic constriction nerve injury-induced neuropathic pain and formalin model of tonic inflammatory pain [37]. Another α7 nAChR silent agonist PMP-072 has also been shown to have anti-inflammatory effects in collagen-induced arthritis in mice [109]. The behavioral and pharmacological profile of PMP-072 and NS6740 in these models are consistent with the induction of nonconducting conformational states of the receptor which confirmed in vitro studies. Indeed, data obtained from α7 nAChR silent agonist R-47 (another name of compound PMP-072) support the hypothesis that the antiinflammatory effects of silent agonism was mediated by a signal transduction pathway that was independent of ion current [110]. α7 nAChRs are downregulated in dorsal root ganglion and spinal cord of rats by oxaliplatin treatment [111] suggesting that α7 nAChRs might have modulatory role in chemotherapy-induced neuropathic pain. In support of this hypothesis, another study showed that α7 nAChR activation protected neurons from oxaliplatin-induced toxicity through a mechanism related to the neuroprotective functions of astrocytes [112]. Ligands that favor non-conducting states are favorable compared to α7 full agonists that enhanced tumor growth in small cell lung cancer cell lines [113], and in mouse model of lung cancer [114]. Because of these considerations, α7 agonists have limited potential for the treatment of chemotherapy-induced neuropathic pain. Targeting alternative conformational states of α7 nAChRs, such as silent agonism, may provide a better therapeutic approach for this common type of neuropathic pain. The recent characterization of α7 nAChR silent agonists has created new opportunities for targeting these receptors as analgesic agents for cancer and chemotherapy-induced peripheral neuropathy. It is very possible that α7 silent agonists may be more efficacious to prevent or attenuate the chemotherapy-induced neuropathic pain. Further pre-clinical behavioral studies will clarify the effects of silent agonists potentially influencing their future clinical development.
Pain has been described as a multi-dimensional state composed of sensory, affective, and cognitive components [115][116][117]. As such, pain states that require clinical intervention are often accompanied by changes in affective behaviors that have complex interplay with the nociceptive aspects of the pain response [118][119][120][121]. A positive feature of α7 nAChR ligands is that in contrast to opioids, they appear to improve pain related affective behaviors in preclinical models. The conditioned place aversion model [122] was used to test the activities of several α7 nAChR. The type II PAM 3-furan-2yl-N-p-tolyl-acrylamide, silent agonist NS6740 and ago-PAM GAT107 were effective to reverse negative affective behaviors associated with visceral pain induced by acetic acid in mice [36,37,106]. These findings suggest that α7 nAChR modulation may serve an efficacious strategy to treat both the sensory and affective components of pain.

CONCLUSION
In summary, many of immune cells and non-immune neuronal cells have been reported to express α7 nAChR subtype. This receptor subtype affords a critical role on the initiation, maintenance and modulation inflammation in addition to direct neuronal signaling. Exploring different pathways to the activation and/or modulation of α7 nAChRs and determining downstream signaling pathways will provide data critical to develop beneficial α7 nAChR ligands for translational research on pain and inflammation.

CONFLICT OF INTEREST
The authors declare no conflict of interest, financial or otherwise. [10] Williams, D.