1521-0081/67/4/1025–1073$25.00 http://dx.doi.org/10.1124/pr.113.008581 PHARMACOLOGICAL REVIEWS Pharmacol Rev 67:1025–1073, October 2015 Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics

ASSOCIATE EDITOR: TIMOTHY A. ESBENSHADE Therapeutic Potential of a7 Nicotinic Receptors

Daniel Bertrand, Chih-Hung L. Lee, Dorothy Flood, Fabrice Marger, and Diana Donnelly-Roberts HiQScreen Sàrl, Geneva, Switzerland (D.B., F.M.); AbbVie Inc., North Chicago, Illinois (C-H.L.L., D.D-R.); and FORUM Pharmaceuticals Inc., Waltham, Massachusetts (D.F.)

Abstract...... 1025 I. Introduction...... 1026 II. From Gene to Function ...... 1028 A. The CHRNA7 Gene and Its Variants...... 1028 Downloaded from B. Functional Properties of a7 Nicotinic Acetylcholine Receptors ...... 1030 C. The a7 Nicotinic as a Prototypical Allosteric Protein ...... 1034 D. The a7 Nicotinic Acetylcholine Receptor Heteromers ...... 1035 E. Effects of Chronic Exposure of a7 Nicotinic Acetylcholine Receptors ...... 1036

F. Modulation of a7 Nicotinic Acetylcholine Receptor Signaling...... 1037 pharmrev.aspetjournals.org G. Expression and Function of a7 Nicotinic Acetylcholine Receptors in Brain ...... 1038 III. Ligands Active at a7 Nicotinic Acetylcholine Receptors...... 1039 A. Structure-Activity Relationship of a7 Nicotinic Acetylcholine Receptor ...... 1039 1. The Clinical Trials...... 1042 B. From the Crystal Structure to Developments in Structure-Activity Relationship ...... 1043 C. Target Engagement and Overview of the Development of Biomarkers...... 1044 D. The Chemical Strategy for a7 Nicotinic Acetylcholine Receptor Positive Allosteric

Modulators ...... 1044 at Universite Laval on October 1, 2015 IV. The Relevance of a7 Nicotinic Acetylcholine Receptors in Diseases ...... 1047 A. Alzheimer’s Disease ...... 1047 B. ...... 1050 C. Autism ...... 1052 D. Microdeletion Syndrome ...... 1053 E. Parkinson’s Disease ...... 1054 F. Nonneuronal Related Diseases...... 1056 1. Cancer...... 1056 G. a7 Nicotinic Acetylcholine Receptors and the Immune Connection ...... 1057 1. Sepsis...... 1058 2. Inflammatory Bowel Disease, Focus on Ulcerative Colitis...... 1058 3. a7 Nicotinic Acetylcholine Receptors and Osteoarthritis...... 1059 H. a7 Nicotinic Acetylcholine Receptors and Cardiac Function ...... 1060 I. The Role of a7 Nicotinic Acetylcholine Receptors in Renal Function...... 1060 V. Conclusions ...... 1062 Acknowledgments ...... 1063 References ...... 1063

Abstract——Progress in the fields of neuroscience and nicotinic acetylcholine receptors (nAChRs) has high- molecular biology has identified the forebrain choliner- lighted, in particular, the role of a7nAChRsinthese gic system as being important in many higher order brain higher order brain functions as evidenced by their functions. Further analysis of the genes encoding the peculiar physiologic and pharmacological properties. As

Address correspondence to: Daniel Bertrand, HiQScreen Sàrl, 6, rte de Compois, 1222 Vésenaz, Geneva, Switzerland. E-mail: daniel. [email protected] dx.doi.org/10.1124/pr.113.008581.

1025 1026 Bertrand et al. this receptor has gained the attention of scientists from affecting a7 nAChRs into therapeutics has to date only academia and industry, our knowledge of its roles in progressed to the stage of testing in clinical trials. various brain and bodily functions has increased im- Notably, however, most recent human genetic and bio- mensely. We have also seen the development of small chemical studies are further underscoring the crucial molecules that have further refined our understanding of role of a7 nAChRs and associated genes in multiple organ the roles of a7 nAChRs, and these molecules have begun systems and disease states. The aim of this review is to to be tested in clinical trials for several indications. discuss our current knowledge of a7 nAChRs and their Although a large body of data has confirmed a role of a7 relevance as a target in specific functional systems and nAChRs in cognition, the translation of small molecules disease states.

I. Introduction 1988; Gopalakrishnan et al., 1995). Several genes encoding for nAChRs were identified from different invertebrate Although is the most widely used “recrea- and vertebrate species. Genes encoding for the neuronal tional drug” for more than 400 years, our understanding nAChRs were identified and subsequently classified as of its effects on the brain remains limited. With the CHRNA and CHRNB (CHRNA2–10 and CHNRB2–4)and introduction in 1929 of electroencephalography, nicotine their chromosomal localizations were identified in the exposure was found to cause modifications of brain human genome, as illustrated by the human karyotype electrical activity but provided no insights into the – – possible mechanisms underlying these modifications shown in Fig. 1A. CHRNA2 10, encoding the a2 10 (reviewed in Yamamoto and Domino, 1965). In the first subunits of neuronal nAChRs, were defined as sharing intracellular recordings conducted in brain slices, nico- homology with the gene encoding for the a1nAChR tine exposure failed to demonstrate significant activity; subunit of the neuromuscular junction and, more specifi- thus it was concluded that nicotine did not mimic the cally, in the extracellular domain with the presence of two – acetylcholine (ACh). Histologic investigations adjacent cysteines corresponding to amino acids 192 193 carried out at approximately the same time revealed, in the Torpedo marmorata sequence, from which the a1 however, surprising results illustrating that the toxin nAChR subunit was cloned (Devillers-Thiery et al., 1979). from the snake Bungarus multicintus (a-, In the case of the adult form of the neuromuscular junction a-Btx), which displays a high affinity for muscle nAChRs, nAChR, ACh binds at the interface between a and d or a abundantly labeled specific regions in brain slices and and « subunits (Pedersen and Cohen, 1990; Changeux and that this labeling was displaced by incubation with Edelstein, 1994; Karlin and Akabas, 1995). Subsequently, nicotine (Clarke et al., 1985). Two possibilities could genes encoding for neuronal nAChRs were isolated simul- explain the discrepancy between electrophysiological taneously in avians (chick) and in the rat and were initially and histologic studies, either a nonspecific binding of divided into a and b subunits according to the presence of a-Btx with no functional significance occurred or the two adjacent cysteines equivalent to those observed in the absence of a functional signal detected by intracellular a1 subunit of the muscle receptors. Functional receptors recording resulted in a false negative finding. were observed only with coexpression of a and b subunits, Cloning of the neuromuscular junction nAChRs marked suggesting that the a subunit harbors the principal a turning point for the investigation of ligand-gated ion component for the ACh binding site, whereas the b subunit channels and offered the possibilities for investigation of wasconsideredasaconstitutive subunit. However, since related proteins at the proteomic and genomic levels. In these initial studies, it has been shown that both the a and addition, the availability of complementary DNA and the b subunits contribute to the formation of the ligand- mRNA allowed reconstitution of functional proteins in binding domain (LBD) in the neuronal nAChRs in which host systems such as Xenopus laevis oocytes or cell lines ACh binds at the interface between an a and an adjacent (Miledi et al., 1982; Barnard et al., 1982; Ballivet et al., subunit.

ABBREVIATIONS: ACh, acetylcholine; AChBP, acetylcholine binding protein; AKI, acute kidney injury; AD, Alzheimer’s disease; AMPA, a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; APP, amyloid precursor protein; ASD, autism spectrum disorder; Ab, amyloid-b; BACE1, b-site amyloid precursor protein-cleaving enzyme 1; BD, bipolar disorder; bp, base pair; BP, break point; a-Btx, a-bungarotoxin; [11C]CHIBA-1001, 1,4-diazabicyclo-[3.2.2]nonane analog 4-[11C]methylphenyl 2,5-diazabicyclo[3.2.2]noane-2- carbocylate; CD, Crohn’s disease; CKD, chronic kidney disease; CNS, central nervous system; CREB, cAMP response element-binding protein; DBA, Dilute Brown Non-Agouti;

DHbE, di-hydro-beta-erythroidine; DLB, with Lewy bodies; DSS, dextran sulfate sodium; EC50, half activation concentration; ERK1/2, extracellular signal-regulated kinase; FISH, fluorescence in situ hybridization; GABA, g-aminobutyric acid; hERG, human ether à go-go-related gene; HMGB1, high-mobility group box 1; HO-1, heme-oxygenase 1; HRV, heart rate variability; 5-HT, 5-hydroxytryptamine; I/R, ischemia-reperfusion; IBD, inflammatory bowel disease; JAK2, janus kinase 2; LBD, ligand binding domain; LPS, lipopolysaccharide; LTP, long-term potentiation; MCCB, MATRICS Consensus Cognitive Battery; MCI, mild cognitive impairment; MLA, ; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; nAChRs, nicotinic acetylcholine receptors; NF-kb, nuclear factor kappa-light-chain-enhancer of activated B cells; NMDA, N-methyl-D-aspartate; NOR, novel object recognition; OA, ostheoarthritis; PAM, positive allosteric modulator; PD, Parkinson’s disease; PET, positron emission tomography; PK, pharmacokinetic; S9-GSKb, serine 9 on glycogen synthase kinase 3b; SANS, Scale for the Assessment of Negative Symptoms; SAR, structure-activity relationship; SNP, single nucleotide polymorphism; SPECT, single photon positron emission tomography; STAT3, signal transducer and activator of transcription 3; TMD, transmembrane domain; TNBS, 2,4,6-trinitrobenzenesulfonic acid; TNFa, tumor necrosis factor alpha; UC, ulcerative colitis. Therapeutic Potential of a7 nAChRs 1027

Fig. 1. The CHRNA7 gene, its duplication and microdeletion. (A) Human karyotype with the localization of CHRNA7 on chromosome 15. (B) Schematic representation of CHRNA7 and its duplication CHRFAM7A. (C) Karyotype of a person showing a microdeletion on chromosome 15 labeled with fluorescence in situ hybridization (FISH) with a probe specific for CHRNA7 and a control probe reveals the presence of only one copy of the CHRNA7 gene. (D) FISH analysis of another patient reveals a higher signal (see inset) indicative of the triplication of CHRNA7 and illustrates the variability in the chromosomal region 15q13.3. FISH images were kindly provided by Dr. C. Schaaf (see also Schaaf, 2014; Soler-Alfonso et al., 2014).

First identified in avian and, almost simultaneously, nAChR subunit was the first to yield functional recep- in rat, the CHRNA7 gene encoding for the a7 nAChR tors when expressed in isolation, indicating that they subunit, which exhibits unique structural features, can form functional homomers (Couturier et al., 1990; initially proved difficult to express in a reconstituted Séguéla et al., 1993). Confirmation that a-Btx labeling system (Couturier et al., 1990; Séguéla et al., 1993). It corresponded to a7 nAChR expression was later was, however, immediately recognized that the a7 obtained with a7 nAChR knockout mice that lacked nAChR subunit shared a sufficient degree of structural a-Btx labeling (Orr-Urtreger et al., 1997). similarity with the neuromuscular junction a1 nAChR This initial and determinant step marked a turning subunit, to which a-Btx also binds, and thus the a7 point in our understanding of nAChRs and opened new nAChRs could be the high affinity a-Btx binding sites in opportunities and challenges to discover the contribution the brain (Couturier et al., 1990). Moreover, the a7 and relevance of the various nAChR subtypes in the 1028 Bertrand et al. brain and, more generally, in other organ systems. In schizophrenia, and given the high level of expression of this review, we shall discuss the current knowledge of a7 nAChRs in brain regions known to contribute to the functions of a7 nAChRs and examine the develop- auditory processing, it was hypothesized that stimula- ment of new ligands designed to address therapeutic tion of these receptors might improve the patient’s needs for Alzheimer’s disease (AD), cognitive impair- condition. In healthy volunteers, sensory gating to audi- ment in schizophrenia, or other diseases. tory cues (two brief tones or clicks) is characterized by an attenuation of the evoked potential response to the II. From Gene to Function second stimulus. In patients with schizophrenia, atten- uation of the evoked potential response to the second A. The CHRNA7 Gene and Its Variants stimulus is reduced (Freedman et al., 1996). As early as As previously mentioned, cloning of the a7 nAChR 1998, experiments conducted in animal models revealed subunit from chick (Couturier et al., 1990) and rat that a7 nAChR agonists were able to normalize this (Séguéla et al., 1993) was achieved in the early 1990s, sensory gating deficit by restoring attenuation to the followed shortly by the cloning of the human CHRNA7 second stimulus (Stevens et al., 1998). Reinforcing the gene (Doucette-Stamm et al., 1993) and determination hypothesis of a critical role of a7 nAChRs in ameliorating of its chromosomal location (Fig. 1) (Chini et al., 1994). the sensory processing deficits in schizophrenia, these Sequence alignment of CHRNA7 with the genes for the experiments stimulated further studies that have led to other known nAChR subunits revealed certain similar- the discovery of multiple properties of a7nAChRsand ities, but also differences. Comparisons across species the related protein products. revealed that CHRNA7 was conserved throughout At about the same time, genetic studies unveiled evolution and that its ancestor gene could be traced to possible splice variants of CHRNA7 and a partially approximately one million years ago (Le Novère and duplicated gene located upstream in close proximity of Changeux, 1995). The consensus protein structure has CHRNA7, coined dup a7 (Chini et al., 1994; Gault et al., the typical signature of nAChRs with four putative 1998). Although the relevance of splice variants in a7 transmembrane domains (TMD I–IV) and a large cyto- nAChR function still needs to be fully characterized, plasmic domain between TMD III and IV, resulting in further efforts have been dedicated to more detailed a mature protein of ;55–60 kDa (Seto et al., 1981; analysis of the duplicate gene, which corresponds to exons Whiting et al., 1987; Gotti et al., 1991). The intracellular 5-10 of CHRNA7 and is located 1.6 Mb centromeric to domain of the a7 nAChR was subsequently reported to CHRNA7, which is now referred to as CHRFAM7A (for play important physiologic roles, including receptor a recent detailed review, see Sinkus et al., 2015). The targeting or phosphorylation. Mapped in humans to corresponding protein, dup a7, is widely expressed, but in chromosome 15, CHRNA7 encodes a mature protein of lower levels than a7 nAChRs throughout the body but 479 amino acids and displays multiple unusual fea- elevated in peripheral leukocytes (Villiger et al., 2002; tures, including putative regions that resemble known Benfante et al., 2011, Costantini et al., 2015). Although so transcription factors. far no specific function has been directly associated with CHRNA7 mRNA was shown to be widely expressed at CHRFAM7A, it was proposed that it might regulate a7 low levels in post mortem human brain, but with highest nAChR activities. Coexpression experiments conducted in expression in hippocampus and thalamus. Thalamic a heterologous expression system confirmed this hypoth- sensory nuclei to include the lateral and medial genicu- esis and revealed that dup a7 subunits when coexpressed late nuclei had high expression levels of CHRNA7 can assemble with a7 nAChR subunits to alter the mRNA. The medial geniculate nucleus is the relay center functionality of these receptors (Araud et al., 2011; de between the inferior colliculus and the auditory cortex, Lucas-Cerrillo et al., 2011). Despite the limitation in- and these regions have been implicated in processing of herent in heterologous expression systems, these results sensory information (Breese et al., 1997). As expression indicate that the dup a7 subunit, and its high level of of CHRNA7 transcripts revealed regions matching the variation in the human population, might play a role in a-Btx binding site comparable to the a1 nAChR subunit, modulating a7 nAChR expression and/or function. A this confirmed the initial observation that a7nAChR further complexity observed in CHRFAM7A is the pres- subunits yield a receptor that binds with high affinity to ence of a 2-base pair (bp) deletion that has been associated this snake toxin. CHNRA7 transcripts were also highly with the sensory gating deficit in a subpopulation of expressed in the prefrontal cortex and other brain schizophrenia patients (Sinkus et al., 2009). regions contributing to sensory and information process- Further genetic analysis showed that the a7 nAChR ing (Musso et al., 2007; Parikh et al., 2010; Wallace and protein contains sequences corresponding to Sp1, AP-2, Bertrand, 2013b). The reduction of CHNRA7 transcripts Egr-1, and CREB transcription factors that might play observed in schizophrenia patients prompted the idea a role in its regulation (Gault et al., 1998). Variants in the that this receptor might play a determinant role in this promoter region of CHRNA7 were also identified and pathology (Freedman et al., 1995). From the clinical associated with differential expression levels of a7 observation, it was noted that sensory gating is altered in nAChR protein. Because these variants were associated Therapeutic Potential of a7 nAChRs 1029 with symptoms of schizophrenia or the sensory gating missing is the detection of the CHRFAM7A transcript, deficit, it was proposed that dysregulation of a7 nAChR but as noted in a recent review the similarity of this gene expression might be at the origin of the disease. Most with CHRNA7 renders difficult the query in genome-wide recent genetic data have highlighted that the region of association studies. Nonetheless, CHRFAM7A was re- chromosome 15 that encompasses CHRNA7 is one of the cently proposed to be associated with several cognitive most unstable within the human genome (Szafranski deficits (Sinkus et al., 2015). et al., 2010; Schaaf, 2014). As described by these authors, Data presented in Table 1 illustrate a series of break points referred to as BP1–BP6 are observed in this CHRNA7 single nucleotide polymorphisms (SNPs) that region of the chromosome. Paracentric inversion between have been shown to positively correlate with various BP4 and BP5 is frequent in the population and was disorders. A significant association of SNP rs8028396 proposed to be associated with clinical features including with smoking, as well as with smoking in schizophrenia, mental retardation and seizures (Chini et al., 1994; Sharp in a non-Hispanic Caucasian population was observed et al., 2008). In turn, these break points can predispose to (Stephens et al., 2012; Neri et al., 2012). SNP rs3087454 nonallelic homologous recombination, resulting in re- was first shown to be significantly associated to schizo- current reciprocal BP4–BP5 microdeletions and micro- phrenia (Stephens et al., 2009) and also influenced duplications. Copy number variants of CHRNA7 have differences in synaptic activity, as detected by functional been reported, leading to multiple classes of microdupli- magnetic resonance imaging, between patients with cations varying in size from 350 kb to 1.6 Mb (Schaaf, schizophrenia and control subjects performing an audi- 2014). The microdeletions are now recognized as chromo- tory oddball task (Tregellas et al., 2010). A Korean some 15q13.3 deletion syndrome (OMIM #612001). Be- population-based study on the development of either cause they are rather prevalent and often accompanied schizophrenia or bipolar disorder (BD) demonstrated that by multiple neurodevelopmental and intellectual disabil- SNPs rs2337506, rs6494223, and rs12916879 exhibited ities, it was recommended that testing for such micro- only a marginal association with BD (Joo et al., 2010) and deletions should be conducted in individuals showing that SNP rs6494223 was associated with a protective unexplained intellectual disability, seizures, and/or mild effect in BD (Ancin et al., 2010) and impaired attention in dysmorphic features (Deutsch et al., 2011). Psychiatric patients with euthymic bipolar disorder (Ancin et al., disorders have been associated with this microdeletion 2011). Additionally, in two separate studies in patients syndrome, which is characterized by a 2-Mb deletion in with AD in Northern Ireland, SNP rs6494233 was sig- 15q13.3 and shown to be inherited in 75% of patients with nificantly associated with delusional symptoms (Carson this syndrome (Masurel-Paulet et al., 2010). Although et al., 2008a), whereas SNPs rs1514246, rs2337506, and microdeletions in chromosome 15q13.3 generally encom- rs8027814 were significantly associated with a reduced pass several genes and therefore render difficult the risk of AD (Carson et al., 2008b). There have been interpretation of the pathologic mechanisms, the identi- a number of other genetic association studies between fication in a smaller number of patients with homozygous CHRNA7 SNPs and various disease states, which all microdeletion of CHRNA7 is starting to shed new light on the pathology associated with the absence of a7 nAChRs (Shinawi et al., 2009; Hoppman-Chaney et al., 2013; Le TABLE 1 Pichon et al., 2013). Identification of the missing CHRNA7 SNPs with positive disease correlation CHRNA7 can be performed using fluorescence in situ Summary of the SNPs reported for CHRNA7 showing a positive correlation to a disease hybridization (FISH) analysis as illustrated in Fig. 1. Additional relevance for the role of a7 nAChRs was SNP Disease Author recently identified with the finding of triplicate expres- rs8028396 Smoking and smoking in Neri et al., 2012; Stephens schizophrenics et al., 2012 sion of CHRNA7 that is associated with both impairment (Caucasian & non- in cognition and neuropsychiatric phenotypes in a three- Hispanics) rs3087454 Perinatal P50 inhibition; Stephens et al., 2009; generation pedigree (Soler-Alfonso et al., 2014). These Schizophrenia and Tregellas et al., 2010; data illustrate that imbalance of the expression levels of improvement in Ross et al., 2013 a7 nAChRs with either a reduced expression or an functional magnetic resonance imaging excessive expression is associated with neurologic im- (Caucasian & non- pairment and underscore the need for tight regulation of Hispanics) rs6494233 Delusional symptoms in AD Carson et al., 2008a CHNRA7 expression (Liao et al., 2011; Cubells et al., rs1514246 2011; Mikhail et al., 2011). Chromatin analysis of rs2337506 Protective with respect to Carson et al., 2008b 15q11.2-13.3 has revealed the CHRNA7 transcript to be risk of AD rs8027814 significantly diminished in the cerebral cortex of patients rs2337506 with autism and Rett syndrome (Yasui et al., 2011) as well rs883473 Ancin et al., 2011; rs6494223 Marginal significance to Ancin et al., 2010; as bipolar disorder, further supporting the hypothesis bipolar disorder that reduced a7 nAChR expression may result in abnor- rs904952 Joo et al., 2010 mal brain development and function. However, what is rs12916879 1030 Bertrand et al. failed to show any significant association (Iwata et al., a number of clinical disorders, all with a component of 2007; Joo et al., 2010; Neri et al., 2012; Cabranes et al., cognitive dysfunction, and suggests the importance of 2013). a 7 nAChRs in cognitive performance. Recently, patent WO 2013/057687 demonstrated the potential value that genetic analysis can afford by B. Functional Properties of a7 Nicotinic identifying responders of a7 nAChR agonist ther- Acetylcholine Receptors apy (Arias et al., 2013). Genetic variations in 15q24, The most prominent functional features of a7 possessing certain “indicative SNPs,” have been un- nAChRs expressed in heterologous systems are 1) their expectedly accurate in predicting responsiveness of high sensitivity to a-Btx and methyllycaconitine (MLA); a patient with cognitive impairment to treatment with 2) their ability to form functional channels in the an a7 nAChR agonist. These “indicative SNPs” reside in absence of other nAChR subunits; 3) their fast desensi- genomic coordinates of either CHRNA5 or CHRNA3, tizing channels (millisecond time scale) upon prolonged encoding for the a5ora3 nAChR subunit, respectively. application of agonist; 4) their enhanced permeability to In particular, patients could be more responsive to an calcium (Ca2+) ions; and 5) their strong inward rectifi- a7 nAChR agonist if they carried CHRNA5 SNP cation (Couturier et al., 1990; Séguéla et al., 1993; rs55854698-T or SNP rs16969968 and/or a SNP forming Bertrand et al., 1993; Peng et al., 1994; Puchacz et al., a haplotype with them, or the CHRNA3 SNP rs6495308 1994). Ca2+ permeability of homomeric a7 nAChRs was or SNP rs1051730, or even a haplotype forming with measured to be at least fourfold higher than that either of these SNPs. Thus before treatment, a genotyp- reported for N-methyl-D-aspartate (NMDA) glutamate ing analysis of a patient afflicted with cognitive impair- receptors (Mayer and Westbrook, 1987) in various ment that includes SNP rs55854698, for example, species (avian, rat, or human) (Séguéla et al., 1993; might predict whether treatment with an a7 nAChR Bertrand et al., 1993; Fucile, 2004; Gilbert et al., 2009). ligand will be efficacious in that particular patient. To The investigation of these unique functional features further expand, patients that were homozygous for became more feasible upon the development of the CHRNA5 SNP rs55853698T showed a significant re- stable expression of human a7 nAChR subunits in sponse to an administered a7 nAChR agonist versus a human cell line, HEK293, which has been shown to placebo on visual learning and memory parameters be devoid of endogenous nAChR subtype expression (P , 0.015). These data further illustrate the complexity (Gopalakrishnan et al., 1995). These studies revealed of the genetic relationship between a7 nAChR SNPs that stably expressed homomeric human a7 nAChR and SNPs for other nAChR subunits but also offer the subunits had an affinity for a-Btx binding with compa- promises for determining patients carrying influential rable currents and fast kinetics to those expressed in SNPs to design the best treatment regimen for them. Xenopus oocytes (Peng et al., 1994). Further studies in Although these results highlight the complexity in this stable cell line, using voltage-clamp or fast ratio- genetic analysis of CHRNA7 and of the 15q13.3 chro- metric intracellular imaging (Delbono et al., 1997), were mosomal locus, the complexity is further increased with able to confirm that homomeric a7 nAChRs, unlike the the presence of CHRFAM7A. However, these complex- heteromeric nAChR subtypes, exhibit a higher Ca2+ ities clearly point to a determinant role of a7 nAChRs in permeability relative to monovalent cations and to human brain function and that altered expression of a7 support previous work in Xenopus oocytes (Bertrand nAChRs is associated with cognitive impairment and et al., 1993; Séguéla et al., 1993; Sands et al., 1993) in neuropsychological deficits. SNP analyses are leading which the intracellular Ca2+ rises within 10 ms with to the potential of personalized medicine for patients a mean peak concentration of 356 nM, the importance of treated with a7 nAChR ligands, once these ligands which will be discussed later. Mutagenesis studies become approved therapies. revealed that the Ca2+ permeability of a7 nAChRs is Results from the genetic analysis conducted so far controlled by distinct amino acids strategically posi- indicate that the chromosome region harboring the gene tioned at each end of the pore forming the channel, encoding for the a7 nAChR subunit presents an unusual which are unique to this nAChR subtype (Galzi et al., complexity with 1) a duplication of exons 5–10 of 1992; Bertrand et al., 1993). CHRNA7 as the CHRFAM7A gene that encodes for Determination of the concentration-response curves the a7 dup protein; 2) important variability of the to ACh and nicotine revealed that a7 nAChRs displayed chromosome in this region due to homologous recombi- a higher sensitivity for nicotine than for ACh and that nation resulting in inversions, microdeletions, and both agonists caused a fast and rapid desensitization of microduplications; and 3) variations in the promoter the receptors. The half activation concentration (EC50) region of CHRNA7. All of these modifications appear to of a7 nAChRs by ACh is about 200 mM, and at this affect expression levels of a7 nAChR protein, suggesting concentration, the response consists of a large inward the importance of tightly regulated expression for the current, with a maximum amplitude at about 10 ms normal function of a7 nAChRs. Furthermore, dysregu- (Puchacz et al., 1994; Gopalakrishnan et al., 1995). lation of a7 nAChR expression has been linked to Intracellular recordings conducted in brain slices Therapeutic Potential of a7 nAChRs 1031 confirmed that native a7 nAChRs exhibited comparable suggesting that inward rectification is a relevant prop- physiologic and pharmacological properties as receptors erty that has been maintained throughout evolution expressed in heterologous systems (Alkondon et al., (Forster and Bertrand, 1995). Although the relevance of 1996). Moreover, these native receptors were selectively these properties of a7 nAChRs to drug discovery has not blocked by low concentrations of a-Btx and MLA. The been thoroughly evaluated, it should be pointed out that rapid desensitization has often been proposed to repre- they represent additional and yet unexplored possibil- sent a protective mechanism, preventing excessive Ca2+ ities for a7 nAChR modulation. influx into the cell that could prove cytotoxic. Evidence Given their capacity to reconstitute functional homo- for such a hypothesis was provided by the observation meric channels, the a7 nAChRs have been a workhorse that mutations in the second TMD, which reduce for structure-function studies of four TMD proteins receptor desensitization, are associated with increased because it has been sufficient to mutate a single amino cell death and are lethal in an animal model (Orr- acid to influence the entire complex in the LBD or Urtreger et al., 2000; Lukas et al., 2001). An interesting channel domain. These studies also pointed out numer- parallel can be drawn with the a-amino-3-hydroxy-5- ous features of a7 nAChRs and confirmed that a small methyl-4-isoxazolepropionic acid (AMPA) receptors, pocket localized at the border between two adjacent which have fast activation and desensitization compa- subunits constituted the orthosteric LBD (Galzi et al., rable with those of the a7 nAChRs (Traynelis et al., 1991). Since then, many studies have attempted to 2010). AMPA receptors are further subdivided into Ca2+ resolve the three-dimensional structures of a7 nAChRs permeable and Ca2+ impermeable, and it was shown and how the LBD is formed (Andersen et al., 2013). A that Ca2+-permeable receptors play a pivotal role in further confirmation of the clear structural determi- synaptic plasticity, triggered by the increase of the nants, with a distinct extracellular domain and TMD, intracellular Ca2+ (Liu and Cull-Candy, 2000; Malinow was obtained using chimeras between receptors from and Malenka, 2002). Similar to AMPA receptors, different families. Namely, based on sequence align- a growing body of evidence has revealed that a7 ments it was proposed that homologies between the nAChRs also contribute to the modulation of synaptic serotoninergic 5-HT3A receptor and a7 nAChR were and cellular functions and influence the release of sufficient to allow the creation of a chimera containing other neurotransmitters (Dani and Bertrand, 2007; the extracellular domain of the a7 nAChR and the Albuquerque et al., 2009). Diverse studies have revealed channel domain of the 5-HT3A receptor that maintained the contribution of a7 nAChRs in fast neurotransmis- the functionality of a ligand-gated ion channel (Eiselé sion, especially in the hippocampus where these recep- et al., 1993). This chimeric construct yielded functional tors might participate in memory processes (Hefft et al., receptors that displayed the pharmacological profile of 1999; Grybko et al., 2010, 2011). Contributions of a7 an a7 nAChR and the channel properties of a 5-HT3A nAChRs to neuronal network functions are not re- receptor (Eiselé et al., 1993). Marking a turning point in stricted to the hippocampus and cerebral cortex but our understanding of the functional domains governing are also observed in the cerebellum, where these the properties of four TMD receptors, this approach was receptors were shown, both in vitro and in vivo, to cause successfully used in many studies to identify defined a switch from long-term depression to long-term poten- properties, such as the binding site for allosteric tiation (LTP) (Prestori et al., 2013). These studies modulators of the a7 nAChR (Bertrand et al., 2008). indicate that ACh in the extracellular space reached These data also suggested that the N-terminal domain sufficient concentrations to activate a7 nAChRs and of the a7 nAChR, which is in the extracellular space, can that, despite its short response time, this receptor can be thought of as an independent structure that is contribute to brain signaling. functionally coupled to the channel domain. Structural The strong inward rectification, which is a hallmark analysis suggested that this domain, referred to as the of all neuronal nAChR subtypes, implied that a7 LBD, resulted from the assembly of b-sheets in a barrel- nAChRs can contribute to signal processing only if the like manner. Confirmation of this hypothesis was cell’s membrane potential is below 240 mV. Site- obtained with the discovery of the acetylcholine binding directed mutagenesis conducted in the second TMD protein (AChBP) (Brejc et al., 2001). Initially identified that lines the channel pore has shown that rectification in Lymnea stagnalis, this water-soluble protein forms is associated with the glutamate residue at the opening a high-affinity complex that binds ACh, thereby con- of the channel and that mutation to an aspartic acid was tributing to the homeostasis of the ligand in the in- sufficient to abolish rectification (Forster and Bertrand, vertebrate nervous system (Smit et al., 2001). Sharing 1995). The inward rectification was associated with the a high degree of sequence homology with the N-terminal blockade caused by intracellular magnesium in a mech- domain of nAChRs, this protein was amenable to anism that strikingly resembled that observed for crystallization and provided the first atomic resolution NMDA receptors (Nowak et al., 1984; Kupper et al., image of the LBD (Brejc et al., 2001). Since this initial 1998). Finally, it is important to note the high degree of work, crystallization of AChBP from several inverte- conservation of the glutamate residue across species, brates, including Aplysia californica and Capitella 1032 Bertrand et al. teleta, with different ligands, has been used as a tool to between an a7 nAChR subunit and its adjacent subunit, direct more efficient drug development (Hansen et al., whichisapointthatmustbe considered when assessing 2005; Smit et al., 2006; Gao et al., 2006; Rucktooa et al., the properties of a7 subunit-containing heteromeric 2009; Sander et al., 2010; Nemecz and Taylor, 2011; nAChRs. It recently became evident that it is necessary Kombo et al., 2012; Billen et al., 2012). Despite these to evaluate ligand binding and the accompanied small interesting results, important differences remained changes in the conformation of the LBD, as well as the between a ligand bound to AChBP versus to a7 nAChRs, properties of the channel domain and its interaction with as shown in studies that attempted to link the develop- the N-terminal domain, to understand the activation of ment of new a7 nAChR ligands based on predictions the receptor and to evaluate the properties of the channel from AChBP binding (Ulens et al., 2009; Akdemir et al., domain and its interaction with the N-terminal domain. 2011). Using site-directed mutagenesis, efforts were In early years of research in the field, a novel and dedicated to modifying the structure of the AChBP to powerful approach to examine the domain properties of mimic as much as possible the human a7 nAChR the channel was the utilization of site-directed mutagen- extracellular domain while maintaining protein solu- esis, which allowed study of the physiologic consequences bility (Nemecz and Taylor, 2011). This work fully of the substitution of single or multiple amino acid confirmed that the multiple interactions were made residues. Mutations conducted in the channel domains between a ligand and the interface of two adjacent a7 confirmed the relevance of the amino acids residues that nAChR subunits. Crystal structures obtained for differ- are pointing outward toward the ionic pore as critical for ent ligands bound to AChBP led to the hypothesis that ionic selectivity. Although it would go beyond the scope of the N-terminal domain of the a7 nAChR subunit under- this work to review in detail the multitude of mutations goes conformational changes upon binding of a full that were designed and tested, it is of relevance to mention agonist to include the closure of the loop-C, which the mutation, initially called L247T with reference to the contains the two adjacent cysteines in the a nAChR chick amino acid sequence, and subsequently called L99T subunits, whereas partial agonists and antagonists with reference to its position in the channel domain maintained the loop-C in a more open conformation (Bertrand et al., 1992). The exchange of this leucine, which (Hibbs et al., 2009; Brams et al., 2011a). is in the upper third of the channel domain, for a shorter Providing a valuable insight into the N-terminal amino acid caused three major effects: 1) a profound domain of a7 nAChRs, crystal structure data were modification of receptor desensitization; 2) an increase of heavily used to dissect the structure-function relationship ;200-fold in sensitivity to ACh; and 3) a change in the between ligand binding and channel activity. Comple- pharmacological profile of some compounds, with compet- mented by experiments using site-directed mutagenesis itive antagonists becoming agonists. Although at first and/or sulfhydryl reagents to modify accessibility to the surprising, these three properties are closely related and cysteines in the LBD, data further highlighted the role of can be explained by considering that the mutation key amino acid residues that are thought to contribute changed the gating properties of the channel in the to the pharmacophore (Papke et al., 2011a). Computer desensitized state. Independent of the scientific interest modeling and docking of molecules based on known in understanding the different transition states occurring structures rapidly expanded the number of molecules upon activation and desensitization of a7 nAChRs, this that demonstrated selectivity for a7 nAChRs, as illus- mutation offered a window into the binding properties of trated in the medicinal chemistry literature (Li et al., a7 nAChRs in a desensitized state (Revah et al., 1991; 2010; Ghiron et al., 2010; Schrimpf et al., 2012; Kombo Bertrand et al., 1992). The observation that competitive et al., 2012; Zanaletti et al., 2012). Computer modeling inhibitors of neuronal nAChRs, such as dihydro-beta- further pointed to the role of fractional hydrophobicity/ erythroidine (DHbE) or d-tubocurarine become agonists hydrophilicity of the LBD and the contribution of the in L99Tmutanta7 nAChRs indicated that these molecules cation-p interaction of the ligand’s basic nitrogen atom can bind with high affinity and selectivity, but because with 149 of the LBD (Kombo and Bencherif, they stabilized a desensitized state, they act as competi- 2013). The LBD interaction with a ligand is, however, tive inhibitors. By demonstrating without ambiguity the probably more complex, as revealed by studies conducted existence of multiple states showing selective pharmaco- using nonnatural amino acids that point to an unusual logical profiles, this mutant offers an additional advan- interaction of with a7 nAChRs and the tage. Namely, the L99Tmutanta7nAChRallowedthe absence of a cation-p interaction with a tryptophan determination of which state a given compound was residue (Van Arnam et al., 2013). This study further stabilizing and its affinity. Finally, by providing a func- revealed that the leucine at position 119 and tryptophan tional window on the desensitized states, the L99Tmutant at position 55 are important for the binding of large a7 nAChR provided insight into the discrepancy between molecules such as or varenicline but are binding experiments yielding high affinities for a given dispensable for binding of smaller molecules such as agonist and low levels of activity revealed by electrophys- ACh. Consistent with the initialobservations,allstudies iology. To understand these differences and how the L99T confirmed that ligands are binding at the interface mutant a7 nAChR could permit the resolution of this Therapeutic Potential of a7 nAChRs 1033 conundrum, it should be recalled that binding experi- contain consensus phosphorylation sites (Charpantier ments are generally conducted at equilibrium, that is, et al., 2005). Based on this observation it was proposed with sustained exposure to the ligand of interest, whereas that compounds known to alter phosphorylation might functional assays are monitoring the receptor activation modify the properties of a7 nAChRs. Indeed, exposure using brief agonist test pulses. Because a7nAChRsare to the general kinase inhibitor genistein caused a desensitized by sustained exposure to agonists (see Fig. concentration-dependent, substantial increase in the 2), it becomes evident that binding and functional experi- current evoked by ACh test pulses. Effects of genistein or ments are challenging two different states of a7nAChRs, specific Src-family kinase inhibitors were observed both with the desensitized state showing a higher affinity for on reconstituted a7 nAChRs expressed in Xenopus compounds than the active state. The availability of the oocytes or cell lines as well as on native receptors and L99Tmutanta7 nAChR or equivalent mutants permitted confirmed that activity of a7 nAChRs can be modulated investigations of how compounds are activating and by phosphorylation (Charpantier et al., 2005). More desensitizing a7 nAChRs in an unprecedented manner. recent experiments confirmed the relevance of T cell As indicated above, the a7 nAChR contains a long receptors in the phosphorylation and modulation of a7 amino acid sequence between TMD III and TMD IV that nAChRs in the brain (Komal et al., 2014). Activation of faces the intracellular cytoplasm and has been shown to T cell receptors, expressed in neocortical neurons,

Fig. 2. Activation and desensitization of the a7 nAChRs. (A) Activation of a7 nAChRs by brief pulses of nicotine at increasing concentrations compared with a saturating ACh test pulse. Plot of the peak inward current, as a function of the logarithm of the nicotine concentration, yields a typical concentration-response curve as shown by s (C). Data were normalized versus the current evoked by 1.28 mM ACh. (B) Sustained exposure to nicotine inhibits the current evoked by a brief ACh test pulse (100 mM). Plot of the peak ACh-evoked current as a function of the logarithm of nicotine concentration yields the inhibition curve shown by the d (C). Inhibition was scaled to 50% of the maximal response corresponding to the fraction of current evoked by 100 mM ACh. Continuous curves through the data points are the best fits obtained with a Hill equation (D. Bertrand, personal communication). 1034 Bertrand et al. caused a reduction in the firing rate of layer I interneu- favor the stabilization in a nonactive state are defined as rons of frontal cortex. The prominent role of a7 nAChRs negative allosteric modulators. One important conse- in the frontal cortex and their modulation by phosphor- quence of allosteric modulation is the alteration on the ylation opens new strategic avenues in drug discovery. receptor’s pharmacological profile. Namely, exposure to In the absence of a crystal structure of the entire a7 a PAM might change the profile of an agonist from partial nAChR, comparison with other known and related to full agonist, as illustrated by the influence of ivermec- structures and computer modeling represented the best tin on the concentration-response relationship of a7 alternative to gain further insight into a7 nAChR nAChRs to 1,1-dimethyl-4-phenylpiperazinium (Krause function. The discovery of four TMD-related channels et al., 1998). Conversely, exposure to a negative allosteric in bacteria that offer the possibility of crystallization, modulator is expected to reduce activity and of marked an advance in our understanding (Bocquet compounds, as exemplified in engineered a7 nAChRs et al., 2007; Hilf and Dutzler, 2009; Bocquet et al., containing the point mutation M253L in which ivermec- 2009). Largely confirming previous hypotheses, these tin becomes a negative allosteric modulator (Collins and crystal structures pointed out the critical role of the Millar, 2010). interface between the LBD and the channel domain a7 nAChRs display typical characteristics of alloste- and, more specifically, of the short segment delimited by ric proteins; that is, they are activated or stabilized in TMD II and TMD III (Lee et al., 2009; Bouzat, 2012; the active state by orthosteric ligands such as ACh, and Calimet et al., 2013). these orthosteric ligands can be allosterically modu- Expression studies of a7 nAChRs in recombinant lated by other molecules. The first example of a7 systems revealed that expression of this nAChR subunit nAChR allosteric modulation was provided by the alone results in functional homomeric receptors, which finding that increased the receptor’s appar- display a fast activation, rapid desensitization, and are ent affinity and amplitude of the current evoked by highly permeable to Ca2+. Structure-function analysis ACh (Krause et al., 1998). Several molecules active at confirmed that a7 nAChRs result from the assembly of a7 nAChRs have since then been identified and divided five subunits with the orthosteric LBD formed at the in two groups. Type-I modulators are compounds such interface between adjacent subunits. The pore domain as 5-hydroxy indole (Zwart et al., 2002) or NS-1738 lies in the center of the protein assembly and is lined by [1-(5-chloro-2-hydroxy-phenyl)-3-(2-chloro-5-trifluorome- the second TMD. Site-directed mutagenesis and forma- thylphenyl)-urea] (Timmermann et al., 2007), which tion of chimeras with portions of the a7 nAChR protein increased the sensitivity of the receptor but do not mod- have been used to demonstrate how a7 nAChR function ify its response time course and Type-II modulators can be modulated by compounds acting in the extracel- such as PNU-120596 [1-(5-chloro-2,4-dimethoxy- lular or transmembrane domain and by phosphoryla- phenyl)-3-(5-methyl-isoxazol-3-yl)-urea] (Hurst et al., tion in the intracellular domain. 2005), which modulated a7 nAChR activity and re- duced or abolished desensitization. Use of chimeric C. The a7 Nicotinic Acetylcholine Receptor as receptors comprised of the extracellular domain of the a Prototypical Allosteric Protein a7 nAChR and TMD and C-terminal domain of the Introduced in the 1960s, the term allosteric model 5-HT3A receptor allowed examination of the critical (Monod et al., 1963) was proposed to describe the fact protein determinants required for modulation by that proteins can have different conformations and that Type-I and Type-II PAMs (Bertrand et al., 2008). binding of a ligand can preferentially stabilize a given Conservation of the activity of the Type-I modulator state. Initially developed to describe enzymatic reac- NS-1738 at the a7-5-HT3 receptor chimera indicated tions, this concept was soon extended to signal trans- that this compound must bind in the extracellular duction proteins such as metabotropic receptors or domain of the receptor, whereas the loss of activity of ligand-gated ion channels (Changeux and Edelstein, PNU-120596 suggested that this Type-II modulator 2005). Moreover, the model accounted for the fact that probably interacts with the a7 nAChR TMD or in- allosteric proteins comprise the main, or orthosteric, tracellular domain (Bertrand et al., 2008). Since these binding site and possibly other, or allosteric, sites where initial studies, effects of Type-I and Type-II a7 nAChR molecules interact. The term allosteric was coined from PAMs have been widely studied in various models of the Greek etymology, with “allos” meaning other and cognitive impairment and (refer to “stereos” indicating the object. In such a model, mole- Table 4). cules that bind to allosteric sites can influence the Functional properties of the a7 nAChRs can be protein transitions and thereby favor or inhibit modulated by molecules that bind at a site different the stabilization in the active state (Bertrand and from the LBD. These molecules, also called allosteric Gopalakrishnan, 2007). modulators, can have positive or negative actions Molecules that favor stabilization in the active state depending on the state that is preferentially stabilized. increased the protein activity and are called positive PAMs were further subdivided in Type-I and Type-II allosteric modulators (PAMs), whereas molecules that depending on their effects on receptor desensitization. Therapeutic Potential of a7 nAChRs 1035

D. The a7 Nicotinic Acetylcholine Receptor Heteromers The possible incorporation of other nAChR subunits Although most experiments, including those using types with a7 nAChR subunits into the pentameric immunoprecipitation, point to the existence of homo- complex raises questions about the formation of the meric a7 nAChRs, the expression of multiple genes LBD. Recalling that ACh binds at the interface between encoding for the neuronal nAChRs exist in the same cell. two adjacent a7 nAChR subunits, novel and distinct For example, it is known that SH-SY5Y cells express a3, subunit sites might be formed at the interface of a7 and a5, b2, b4, and at a low level a7 nAChR subunits but that other nAChR subunits. To tackle this question, it was overexpression of a7 nAChR subunits yielded robust shown that a7 and b2 nAChR subunits can assemble currents attributable to a7 nAChRs alone that were quite into functional heteromers that have pharmacological properties resembling a7 nAChR homomers but with distinct from those observed in naive SH-SY5Y cells lower maximal amplitudes of agonist-evoked currents (Lukas et al., 1993; Puchacz et al., 1994). Since then, (Murray et al., 2012). Evidence for heteromeric assem- several studies have attempted to determine if a7 nAChR bly included colocalization of tagged subunits using subunits assemble purely as homomers or if, in some laser scanning confocal microscopy, fluorescence För- cases, a7 nAChR subunits could assemble with other ster resonance energy transfer, and total internal re- nAChR subunits. Experiments were conducted to iden- flection fluorescence microscopy. Electrophysiological tify neurons that did not express other nAChR subunits, evaluation demonstrated no significant difference be- and recently it was proposed that the hypothalamic tween a7b2 nAChR heteromers and a7 nAChR homo- histaminergic tuberomammillary neurons expressed a7 mers in the pharmacological sensitivity to agonists. A nAChR subunits but no detectable levels of mRNA significant difference was, however, found with the encoding for other nAChR subunits (Tischkau et al., competitive antagonist DHbE. In agreement with this 2014). These data suggested that bona fide a7nAChR observation, the use of cysteine mutants indicated that homomers expressed in neurons show similar properties this ligand does not bind in a functionally productive to those observed in overexpressed cell systems. manner at the interface between a7 and b2 nAChR Alternative strategies pursued by other laboratories subunits (Murray et al., 2012), suggesting that only the attempted to resolve the question of possible heteromeri- a7-a7 nAChR subunit interface is able to activate the zation of a7 nAChR subunits by coexpression with other a7b2 nAChR heteromers and produce the ACh-evoked nAChR subunits types. As cells expressing a7 nAChR response. This result supports the observation that subunits often also expressed b4 subunits, experiments of a7b2 nAChR heteromers show reduced maximal ampli- coexpression of a7 nAChR subunits with other nAChR tudes of inward currents. Recently, a more in-depth subunits (a2, a3, a4, and b4) were conducted by immu- interrogation of the unique pharmacology of the nopurification and Western blot analysis (Criado et al., rat a7b2 nAChR heteromers expressed in Xenopus 2012). The results indicated that b4 nAChR subunits can oocytes showed that nonselective a7 nAChR agonists coassemble with a7 nAChR subunits but that the phar- such as varenicline, nicotine, and became less macological properties of the resulting receptors showed efficacious in a7b2 nAChR heteromers and selective no significant differences in the response time course or agonists of b2 subunit-containing nAChRs such as pharmacological profile from homomeric a7 nAChRs, ABT-089 ([2-methyl-3-(2-(S)-pyrrolidinylmethoxy)pyridine except for a minor difference in the sensitivity to cytisine dihydrochloride salt]), sazetidine-A, and , (Criado et al., 2012). Additional studies examined the which demonstrated no agonist activity, suggesting localization of a7withb2 nAChR subunits in brain stem that the incorporation of a b2 nAChR subunit did not and hippocampus and suggested that coassembly of these impose b2-like pharmacology on a7b2 nAChR hetero- subunits have a developmental role and influence the oc- mers (Zwart et al., 2014). Selective a7 nAChR agonists currence of sudden infant death syndrome (Machaalani such as PNU-282987 (N-[(3R)-1-azabicyclo[2.2.2]oct-3- et al., 2011; Machaalani et al., 2010). A word of caution yl]-4-chlorobenzamide hydrochloride);, SSR-108911 1,4- should, however, be given, because these studies were diazabicyclo(3.2.2)nonane-4-carboxylic acid, 4-bromophenyl conducted with antibodies and it is known that such ester, TC-5619 (N-[2-(pyridin-3-ylmethyl)-1-azabicyclo[2.2.2] labeling might be inaccurate (Moser et al., 2007; Jones oct-3-yl]-1-benzofuran-2-carboxamide), and and Wonnacott, 2005). However, additional studies have (EVP-6124; [(R)-7-chloro-N-quinuclidin-3-yl)benzo[b] found that neurons in the basal forebrain and hippocam- thiophene-2-carboxamide]) exhibited comparable po- pus expressed a7b 2 nAChRs and that this expression tencies with heteromeric a7b2 nAChRs as seen with resulted in a heteromeric nAChR that is functionally and homomeric a7 nAChRs but had a significantly reduced pharmacologically distinct from homomeric a7 nAChRs, efficacy. Interestingly, there was no decrease in the with slower desensitization rates and increased sensitiv- efficacy of ACh with a7b2 nAChRs compared with a7 ity to DHbE, a selective antagonist of b2-containing nAChRs; and the loss of efficacy for varenicline with a7b2 nAChRs (Liu et al., 2012; Liu et al., 2009). Furthermore, nAChRs was less than for cytisine and the selective a7 these publications discussed a greater sensitivity of a7b2 nAChRs agonists. Although these results are not the final nAChRs than a7 nAChRs to the amyloid-b (Ab)peptide. word on the pharmacology and function of heteromeric 1036 Bertrand et al. a7 nAChRs, they clearly demonstrate that differential (Rezvani et al., 2007). In addition, these studies com- expression in the brain must be taken into account and plement past efforts demonstrating that ultra-low that this diversity might offer additional strategies in the exposure to a7 nAChR partial agonists result in sus- design of active compounds. tained efficacy in models of working memory because of Although the a7 nAChRs were initially thought to be upregulation of cell surface a7 nAChRs (Werkheiser expressed only as homopentamers, more recent evi- et al., 2011) or because of reduced turnover from dence points to the possibility of heteromeric assembly inhibition of proteasomal activity (Rezvani et al., 2007). of a7 subunits with another nAChR subunit such as b2, Evidence obtained from animal models suggest that b4, or dup a7 (discussed in section II.A). This further exposure to these a7 nAChR agonists, GTS-21 (3-(2,4- complexity must be taken into account when examining dimethoxybenzylidene)anabaseine) (Stevens et al., 2010), the role of a7-containing nAChRs in brain function. ABT-107 (5-(6-[(3R)-1-azabicyclo[2,2,2]oct-3-yloxy] pyridazin-3-yl)-1H-indole) (Bitner et al., 2010), RG3487 E. Effects of Chronic Exposure of a7 Nicotinic (N-[(3S)-1-azabicyclo[2.2.2]oct-3-yl]-1H-indazole-3- Acetylcholine Receptors carboxamide hydrochloride) (Wallace et al., 2011), and One important property of the a7 nAChR to take into encenicline (van Goethem et al., 2015), caused no tachy- consideration in the concept of drug design is receptor phylaxis or receptor upregulation. Maintained efficacy and desensitization. Namely, although brief exposures to absence of tachyphylaxis of a7 nAChR agonists has also agonists activate the receptors, sustained exposures to been observed in Phase 1 and Phase 2 clinical trials, agonists are known to inhibit their subsequent re- suggesting that a7 nAChR agonists may be good candi- sponses by desensitization. Moreover, as illustrated in dates for long-term treatment of cognitive disorders (Olincy Fig. 2, concentrations causing inhibition are of orders of et al., 2006; Freedman et al., 2008; Preskorn et al., 2014; magnitude lower than those evoking a response, with Keefe et al., 2015). On closer examination of both the limited or no overlap between the desensitization and preclinical and clinical studies, it is apparent that effica- activation curves. From this observation, it follows that cious concentrations of a7 nAChR agonists are extremely sustained administration of an a7 nAChR agonist is low, below those required to activate a 7 nAChRs alone and unlikely to evoke a sustained response of the receptors. below those that desensitize a7 nAChRs. The finding that This raises the question whether ligands produce an at low concentrations, a7 nAChR agonists can increase the efficacious effect by preferentially activating or inhibit- amplitude of ACh responses at a7nAChRsexpressedin ing a7 nAChRs. Moreover, in clinical settings, longer cells has suggested that a7 nAChR agonists in vivo may and cumulative exposures need to be examined to similarly be acting in conjunction with the native ligand assess if the therapeutic effects of a molecule do not ACh.Invitro,theeffectofa7 nAChR agonists on enhancing cause tachyphylaxis, the loss of effect of a . the activity of ACh has been termed "priming" (Quik et al., The observation of an increase in ligand binding in 1997; Papke et al., 2011b) and has also been observed with smokers’ brains revealed another puzzling property of high-affinity a7 nAChR agonists (Wallace et al., 2011; nAChRs (Perry et al., 1999). Subsequently, the term Prickaerts et al., 2012). Although the underlying mecha- receptor upregulation was coined to describe the appar- nisms of priming have not been fully revealed, a suggested ent increase in receptor numbers, and further exami- mechanism has been described for the neuromuscular nation revealed that this process is not identical for all junction nAChR, in which priming may represent the nAChR subtypes. Moreover, upregulation of hetero- stabilization of receptor conformations by ACh that are meric receptors such as the a4b2 nAChR depends upon more easily activated by subsequent exposure to another multiple factors (Govind et al., 2012). A fast increase in molecule of ACh (Mukhtasimova et al., 2009). Alterna- a-Btx binding was observed upon nicotine exposure, tively, an a7 nAChR agonist may act to synchronize better suggesting the incorporation of additional a7 nAChRs receptor activation upon exposure to ACh. Additional work into the membrane in chick ganglia (Berg et al., 2006). is required to describe better priming in vitro and to Similarly, it was concluded that potentiation of the a7 establish whether similar mechanisms are in effect in vivo. nAChR-mediated current observed in Xenopus oocytes Collectively, these data point out the need for additional and rat hippocampal interneurons after treatment with analyses before reaching a conclusion on the complexity of the kinase inhibitor genistein was associated mechanisms underlying the maintenance of receptor den- with an increase in receptor number (Cho et al., 2005). sity in the membrane. An alternative conclusion was, however, presented in Sustained exposure of a7 nAChRs to an agonist such other experiments, suggesting that genistein might act as nicotine causes a profound desensitization that can by an allosteric mechanism rather than due to an block the response to ACh. Sustained exposures to an a7 increase in receptor numbers (Charpantier et al., nAChR agonist can, however, modify the fraction of 2005). Nicotine exposure caused an increase in a-Btx receptor available. Although upregulation, that is an binding in mouse prefrontal cortex that was attributed increase in the receptor number, was observed for some to reduced proteasome-dependent degradation due to molecules and experimental conditions in vivo, another the accumulation of ubiquitinated a7 nAChR subunits possible mechanism by which a7 nAChR agonists are Therapeutic Potential of a7 nAChRs 1037 efficacious in vivo may be by priming of the receptor. induction of HO-1 and reduction of reactive oxygen This second mechanism may explain the absence of species (Parada et al., 2013). This study supported the tachyphylaxis observed with different molecules ren- role of a7 nAChRs expressed on microglia and their dering them attractive therapeutics. role in neuroprotection as a result of excitotoxic insults. The direct role of a7 nAChRs in neuroprotection and F. Modulation of a7 Nicotinic Acetylcholine more importantly in the ability to modulate cognition Receptor Signaling came about with the development of selective a7 nAChR The high Ca2+ permeability of the a7nAChRis agonists, demonstrating efficacy in a battery of preclinical suggestive of a role downstream in events such as in- cognitive measurements (Wallace and Porter, 2011). tracellular signaling, neurite outgrowth, synaptic trans- These selective a7 nAChR ligands will be discussed in mission, modulation of neutrophic factors, and excitotoxic detail in section III; however, one such preclinical study processes (Role and Berg, 1996; Albuquerque et al., 1997). involved the exploration of A-582941, a selective a7 The neuroprotective effect of ligands acting at a7nAChRs nAChR agonist shown to be efficacious in several behav- was supported by various studies (de Fiebre et al., 1995; ioral assays such as rat social recognition, mouse in- Donnelly-Roberts et al., 1996; Meyer et al., 1998b). The hibitory avoidance, and monkey delayed matching to mode of entry, but more importantly, the level of in- sample, that evaluated short-term recognition, long-term tracellular Ca2+ achieved upon activation of a7nAChRs memory consolidation, and working memory, respec- is pivotal to the outcome of downstream signaling events. tively, as well as normalizing sensory gating deficits in Studies conducted in a stably transfected HEK293 cell line DBA/2 mice (Bitner et al., 2007). It was further shown expressing a7 nAChRs revealed that stimulation elicited that the signaling pathways involved in cognitive func- a significant increase in intracellular Ca2+ (;350 nM) tion [i.e., extracellular signal-regulated kinase (ERK1/2) (Delbono et al., 1997). Experiments conducted with the and cAMP response element-binding protein (CREB)] Type-II a7 nAChR PAM PNU-120596 caused an increase were phosphorylated by activation of a7 nAChRs. It was in intracellular Ca2+ concentration but was not cytotoxic in previously shown that phosphorylated ERK1/2 has a key either PC12 cells or neurons from rat primary cultures (Hu role in activating CREB, a transcription factor involved in et al., 2009). According to the Ca2+ set-point theory for cell long-term memory formation (Adams and Sweatt, 2002). survival (Franklin and Johnson, 1992), activation of a7 Further studies examined A-582941 to demonstrate that nAChRs yielded an intracellular Ca2+ concentration in- this a7 nAChR agonist induced an increase in phosphor- crease in the range of 200–400 nM, which permitted cell ylation of serine 9 on glycogen synthase kinase 3b (S9- survival without the aid fromtrophicfactors.Thus,in GSKb), which was not observed in a7 nAChR knockout contrast to glutamate receptors, in which prolonged mice (Bitner et al., 2009). GSKb has been shown to be activation was shown to provoke cell death, activation of a predominant kinase in AD, involved in tau hyper- a7 nAChRs should be neuroprotective. This, as well as the phosphorylation (Grimes and Jope, 2001). Additionally, importance of Ca2+ homeostasis to neuroprotection, was A-582941 continuously infused subcutaneously to a subsequently shown in diverse preparations using specific steady-state exposure in Tg2576 mice, an AD model of ligands of a7 nAChRs (Verkhratsky and Toescu, 1998; amyloid precursor protein overexpression, significantly Donnelly-Roberts and Brioni, 1999) through the coupling of increased phosphorylation of S9-GSKb in hippocampus Ca2+ regulation to second messenger pathways (Berg and and in a double transgenic AD mouse (TAPP, tau P301L Conroy, 2002). Neuroprotective effects caused by exposure X amyloid precursor protein) decreased phosphorylation to SEN12333 (5-morpholin-4-yl-pentanoic acid (4-pyridin- of tau (Bitner et al., 2009). Increased phosphorylation of 3-yl-phenyl)-amide), a specific ligand of a7nAChRs,were ERK1/2, CREB, and S9-GSKb and decreased phosphor- also exemplified in a quisqualate-lesioned animal model ylation of tau were replicated with another a7 nAChR (Roncarati et al., 2009). Stimulation of a7nAChRsbythe agonist of another chemotype (ABT-107) (Bitner et al., selective agonist PNU-282987 protected retinal neurons 2010). In addition, acute administration of A-582941 also from the cytotoxic effect of glutamate, illustrating the com- dose dependently amplified both c-Fos and Arc, cytoskel- mon mechanisms of neuroprotection against various cyto- etal proteins, in regions of brain involved in attention toxic insults via regulation of Ca2+ homeostasis (Iwamoto and working memory, although only in juvenile but not et al., 2013). In addition, posttreatment of SH-SY5Y cells adult rats (Thomsen et al., 2008). The data collectively with PNU-282987 reversed oxidative damage from reactive support the hypothesis that brain exposure to a7nAChR oxygen species production as a result of treatment with agonists, shown to demonstrate cognitive enhancement, a combination of rotenone and oligomycin A, which inhibit may be acting through these modulatory signaling path- mitochondrial complexes I and V, respectively, by induc- ways via an increase in protective Ca2+ concentrations tion of heme oxygenase 1 (HO-1), a nuclear factor and, furthermore. that through GSKb inhibition, this (erythroid-derived 2)-like 2-regulated phase II enzyme neuroprotection may also have the potential to be disease (Parada et al., 2010). This group recently extended these modifying. studies to demonstrate that activation of a7 nAChRs In vitro studies using PC12 cells have also shown elicited neuroprotection in a model of brain ischemia via that several a7 nAChR agonists, in the presence of the 1038 Bertrand et al. a7 nAChR PAM, PNU-120596, all increased ERK1/2 are highly expressed presynaptically or perisynaptically, phosphorylation, as well as phosphorylation of upstream suggesting that these receptors might be involved in kinases, p38 MAP kinase and mitogen-activated protein a critical role in modulation of the release of other kinase kinase1/2, that have been linked to synaptic neurotransmitters (Fabian-Fine et al., 2001; Jones and plasticity and cognition (Gubbins et al., 2010). Other Wonnacott, 2004). The presynaptic role of a7nAChRs studies using SH-SY5Y cells have also shown that PNU- was confirmed by the observation that their stimulation 282987 can rescue cells from apoptosis induced by rote- induced the release of other neurotransmitters (Girod none and oligomycin A in a dose-dependent manner when et al., 2000; Rousseau et al., 2005; Zhu et al., 2005; Biton applied after the insult (Parada et al., 2010). It was further et al., 2007; Livingstone et al., 2009; Sydserff et al., 2009; demonstrated through use of specific kinase inhibitors Parikh et al., 2010; Huang et al., 2014a,b; Koranda et al., that this rescue was activating HO-1 via the JAK1/Akt 2014). pathway to afford neuroprotection. A slightly different picture emerges when examining Neuroprotective effects caused by a7 nAChR agonists the electrophysiological properties of neurons using offer a promising possibility of disease modification, in intracellular recordings and probing the response to addition to symptomatic treatment, for patients with specific compounds. Recordings performed cognitive deficits associated with neurodegeneration. in hippocampal slices revealed the importance of a7 Neuroprotection was shown to result from different nAChRs on inhibitory interneurons, as illustrated by mechanisms including the increase of intracellular Ca2+ the sensitivity to different cholinergic agonists and and/or stimulation of second messenger cascades. Of- allosteric modulators (Buhler and Dunwiddie, 2001; fering new therapeutic avenues, neuroprotection Christophe et al., 2002; Hurst et al., 2005; Charpantier remains one of the important challenges in the field of et al., 2005). Projections from the basal forebrain a7 nAChRs. innervate the cortex, and release of ACh was found to stimulate a7 and non-a7 nAChRs that are preferen- G. Expression and Function of a7 Nicotinic tially expressed on interneurons of the different cortical Acetylcholine Receptors in Brain layers (Arroyo et al., 2012). MLA blocked fast neuro- To comprehend the role of a7 nAChRs in central transmission indicative of the a7 nAChR contribution, nervous system (CNS) function, it is necessary to first whereas slow, non-a7 nAChR transmission was blocked examine which areas of the brain express them. As by DHbE, with some cells showing a dual fast and slow described above, the most reliable labeling strategies are component (Arroyo et al., 2012; Bennett et al., 2012). the use of a7 nAChR-specific radiolabeled ligands such Cholinergic modulation of interneuronal activity was as a-Btx, MLA, A-585539, CHIBA-1001, AZ11637326, shown to participate in signal processing and the A-582941, A-844606, NS14492, or NS14490 (Han et al., formation of LTP when the presynaptic cell was stim- 2003; Tribollet et al., 2004; Anderson et al., 2008; ulated and postsynaptic cell was hyperpolarized Hashimoto et al., 2008; Toyohara et al., 2010; Ettrup (Griguoli et al., 2013). The initial evidence of a modulatory et al., 2011; Maier et al., 2011; Rötering et al., 2014). An role for a7 nAChRs in synaptic activity was obtained in alternative approach is to detect a7 nAChR expression rat hippocampal CA1 pyramidal neurons by examining levels by measuring the levels of CHRNA7 mRNA, as spontaneous activity and the effect of stimulation of the was performed in monkey brain (Han et al., 2000) and in Schaffer collaterals (Alkondon et al., 1998, 2000; Frazier post mortem human brain (Breese et al., 1997). Alto- et al., 1998; Hefft et al., 1999). gether, these data from mRNA expression and a-Btx Figure 3 illustrates the localization and laminar binding indicate that a7 nAChRs are widely expressed in localization of a7 nAChR expression in the cerebral the brain and are at especially high densities in hippo- cortex obtained by detailed electrophysiological record- campus, cerebral cortex, lateral and medial geniculate ings and pharmacological testing (Poorthuis et al., 2013; nuclei, reticular thalamic nucleus, basilar pontine nuclei, Arroyo et al., 2014). The fast and slow activity caused by diagonal band of Broca, nucleus basalis of Meynert, ACh release observed in different neuronal types indi- inferior olive, posterior hypothalamus, mammillary bod- cates that these cells must be expressing different ies, dorsal nucleus of the vagus, and cerebellum (Breese nAChR subtypes that might be colocalized or expressed et al., 1997). In vivo imaging using positron emission in distinct subcellular areas such as post- and/or peri- tomography (PET) tracers, such as CHIBA-1001, con- synaptically. The imbalance of fast and slow cholinergic firmed a similar distribution of a7 nAChRs (Tanibuchi signals might result in different functional outcomes. For et al., 2010). Although these studies provided a general example, when considering the effects of an acetylcho- distribution of a7 nAChRs, a fine cellular and subcellular linesterase inhibitor, increased half-life of ACh in the localization of these receptors is required to examine synaptic cleft and its vicinity is expected to result in their possible physiologic role. Successful subcellular a larger effect at the non-a7 nAChR component of the localization of a7 nAChRs was obtained using fluores- ACh-evoked response to cause a weighting toward tonic cent a-Btx or immunogold with an anti-a7nAChR ACh activity. Exposure to an a7 nAChR antagonist such antibody and revealed that in hippocampus, a7 nAChRs as MLA should result in diminished a7 nAChR activity Therapeutic Potential of a7 nAChRs 1039

III. Ligands Active at a7 Nicotinic Acetylcholine Receptors

A. Structure-Activity Relationship of a7 Nicotinic Acetylcholine Receptor Agonists Over the past two decades, medicinal chemistry has remarkably expanded the development of compounds acting via a7 nAChR agonism by agonists and PAMs. Extensive basic and clinical research studies were conducted and yielded numerous active and selective molecules that are reviewed in this section. The major attention will be kept on developing the various chemo- types that were brought up to clinical trials and are summarized in Table 2. The majority of a7 nAChR agonists described to date are comprised of the quinu- clidine moiety, which encompasses structures such as the spirooxazolidinones, and quinuclidine carbamates, amides, and ethers. GTS-21 (DMXBA), one of the first reported ligands differing from nicotine to show binding specificity for the a7 nAChR, was described as a func- Fig. 3. Distribution of a7 nAChRs in the cerebral cortex. Schematic representation of the cell types and their laminar localization in the tionally selective in comparison with cerebral cortex. a7 nAChR channels are inserted to indicate their laminar ACh (Hunter et al., 1994; Briggs et al., 1995; Meyer distribution. Cells expressing a7 nAChRs are indicated in dark gray with light gray areas not expressing a7 nAChRs. (Drawn from Arroyo et al., et al., 1998a). This compound is not solely selective for 2014; Bloem et al., 2014). a7 nAChRs and also binds to a4b2 nAChRs, but with a lower affinity. Over the years, GTS-21 has been characterized extensively both in vitro and in vivo and and a similar imbalance between the fast and slow is one of the first molecules that was advanced into early transients. In contrast, an a7 nAChR agonist could phase clinical trials. However, newer ligands, stemming induce a weighting favoring fast versus slow transients from scaffolds, such as the azaadamantane (ABT-126) as long as the agonist was not present at desensitizing and quinuclidine (encenicline, TC-5619, MEM3454/ concentrations. These data point to the necessity of RG3487, and AQW051 [(R)-3-(6-p-tolyl-pyridin-3-yloxy)- establishing a better understanding of the respective 1-aza-bicyclo(2.2.2)octane]), have expanded the chemical contribution of different nAChRs subtypes at the cellu- space also to include additional azabicyclic tertiary lar and circuit levels, especially when considering amine templates. compounds like acetylcholinesterase inhibitors that The most explored class of ligands for a7 nAChR is have effects on multiple nAChR subtypes and nAChR the quinuclidine amine-based moiety. AR-R17779 subtype-selective compounds that can similarly alter (–)-spiro[1-azabicyclo[2.2.2]octane-3,59-oxazolidin-29- the balance of nAChR subtype activity. one], a spirooxazolidinone, from the group at Astra- Expression of a7 nAChRs is not restricted to neurons Zeneca (London, United Kingdom) was another early but was also shown both histologically and functionally molecule in this chemical space (Mullen et al., 2000). to be on glial cell types (Vélez-Fort et al., 2009), Several research groups followed with expansion on this including astrocytes (Sharma and Vijayaraghavan, particular series to result in structurally diverse and 2001), oligodendrocytes, and microglia (Shytle et al., selective compounds that were active preclinically. 2004). Stimulation of the a7 nAChRs on glia caused However, the challenges of these initial compounds in many physiologically relevant activities that need to be this series remained, which involved cross reactivity taken into account when evaluating effects of mole- with the 5-HT3 receptors and limited penetration into cules targeting a7 nAChRs. the CNS. Astra-Zeneca further developed the structure- Widely expressed in the brain, a7 nAChRs were activity relationship (SAR) in the spirooxazolidinone shown to be localized pre- and postsynaptically. Pre- series with identification of AZD0328 [(29R)-spiro-[1- synaptic receptors were shown to modulate the release azabicyclo[2.2.2]octane-3,29(39H)-furo[2,3-b]pyridine] of neurotransmitter at both excitatory (glutamate) or D-tartrate], a spirofuropyridine, that has been charac- inhibitory (GABA) neurons. Postsynaptic and extra- terized in preclinical models (Sydserff et al., 2009). synaptic a7 nAChRs can also modulate neuronal AZD0328 was the first spirooxazolidine analog with activity and participate in neurotransmission. In addi- good selectivity and potency for a7 nAChRs (Ki = 3 nM) tion, a7 nAChRs were also shown to be expressed by and showed favorable pharmacokinetic (PK) properties glial cells, but their role on these cell types remains to sufficient for advancement into clinical studies. This be clarified. compound acted as a partial agonist at rat and human 1040 Bertrand et al.

TABLE 2 a7 nAChR agonists entered clinical trials and in discovery phase

Development Compounds Therapeutic Indications Status Company Reference Alzheimer’s disease Phase 2 Comentis Briggs et al., 1995; Azuma Cognitive deficiency in schizophrenia et al., 1996, 1999; Mahnir Parkinson’s disease et al., 1998; Li et al., 1999; Smoking cessation Adams et al., 2000; Crutcher, 2000; Bruchfeld et al., 2010; Loram et al., 2010; Vukelic et al., 2013

GTS-21 Alzheimer’s disease Phase 2 Papke et al., 2005; Ishikawa Cognitive deficiency in schizophrenia and Hashimoto, 2011; Pain Stegemann et al., 2013

Tropisetron Alzheimer’s disease Phase 1 Pfizer Bodnar et al., 2005; Hajós Cognitive deficiency in schizophrenia et al., 2005

PNU-282987 Alzheimer’s disease Discovery Sanofi Biton et al., 2007; Pichat et al., Cognitive deficiency in schizophrenia 2007; O’Donnell et al., 2009

SSR-180711 Alzheimer’s disease Phase 2 AbbVie Gault et al., 2015 Cognitive deficiency in schizophrenia Pain

ABT-126

Alzheimer’s disease Phase 2 and 3 Forum Prickaerts et al., 2012; Barbier Cognitive deficiency in schizophrenia et al., 2015; Huang et al., Smoking cessation 2014a; Preskorn et al., 2014; Keefe et al., 2015

Encenicline (EVP-6124) Alzheimer’s disease Phase 2 Memory/Roche Rezvani et al., 2009; Wallace Cognitive deficiency in schizophrenia et al., 2011; Huang et al., 2014b; Umbricht et al., 2014

MEM3454/RG3487 Alzheimer’s disease Phase 2 Targacept Hauser et al., 2009; Mazurov Cognitive deficiency in schizophrenia et al., 2012; Lieberman et al., 2013; Walling et al., 2015

TC-5619

(continued) Therapeutic Potential of a7 nAChRs 1041

TABLE 2—Continued

Development Compounds Therapeutic Indications Status Company Reference Alzheimer’s disease Discovery Novartis Feuerbach et al., 2007, 2009; Cognitive deficiency in schizophrenia Enz et al., 2009; Jiang et al., Pain 2009; Arias et al., 2013

JN403 Alzheimer’s disease Discovery Weyth Ghiron et al., 2010; Marquis Cognitive deficiency in schizophrenia et al., 2011

SEN34625/WYE-103914 Alzheimer’s disease Phase 2 Novartis Di Paolo et al., 2014; Cognitive deficiency in schizophrenia Feuerbach et al., 2015 L-DOPA induced dyskinesias

AQW051

a7 nAChRs and displayed an EC50 of 338 nM and an demonstrated partial agonist activity at a7nAChRs(Ki= efficacy of 64.7%; however, this compound also activated 10 nM) and antagonist activity at 5-HT3 receptors (IC50 5-HT3A receptors with an EC50 of 474 nM but an efficacy , 10 nM) (Prickaerts et al., 2012). Encenicline demon- of only 12% (Sydserff et al., 2009). In vivo, this strated preclinical procognitive effects in rat NOR, as compound exhibited activity in a variety of preclinical well as efficacy in Phase 1 and Phase 2 trials in normal models including novel object recognition (NOR) in volunteers and patients with schizophrenia (Barbier mouse, reversal of short-term memory deficits in et al., 2015; Keefe et al., 2015), and is now in Phase 3 fimbria-fornix-lesioned rats, and improvement in work- trials for treatment of cognitive impairment in schizo- ing memory in the spatial delayed response task in phrenia and Alzheimer’s disease. Like the amide series rhesus monkey (Sydserff et al., 2009; Castner et al., from FORUM, MEM3454 (RG3487) also exhibited an- 2011; Werkheiser et al., 2011). tagonism at the 5-HT3 receptor and produced procogni- Quinuiclidine and ring expansion analogs have been tive effects in rat NOR and in aged mouse and aged rat extended by modification of functional linkers such as in a water maze (Wallace et al., 2011). As expected for an heteroaryls, amides, and carbamates. Pharmacia/Pfizer a7 nAChR agonist, RG3487 increased and (Groton, CT) developed and reported on one of the earlier ACh release in the rat hippocampus and prefrontal cortex compounds in this series with PNU-282987, a quinucli- (Huang et al., 2014b). Similarly, encenicline increased dine benzamide (Bodnar et al., 2005). PNU-282987 dopamine and ACh, as well as glutamate release in the showed both selectivity and potency as an a7nAChR prefrontal cortex (Huang et al., 2014a). Tested in clinical agonist (Ki = 29 nM), retaining full agonist efficacy trials in schizophrenia patients, RG3487 showed no relative to nicotine in functional assays (Hajós et al., significant improvement of the cognitive deficit associ- 2005). PNU-282987 has been thoroughly characterized ated with schizophrenia, but patients with moderate both in vitro and in vivo, demonstrating the restoration negative symptoms exhibited a significant improvement of P50 gating deficits in rodents, and has served as a tool in their symptoms (Umbricht et al., 2014). Novartis molecule to advance basic research efforts (Hajós et al., (Basel, Switzerland) also recently disclosed a quinucli- 2005; Vicens et al., 2010; McLean et al., 2011). However, dine ether a7 nAChR agonist, AQW051 (Di Paolo et al., a major drawback of this compound was the interaction 2014; Feuerbach et al., 2015). In vitro assessment of with the human ether à go-go-related gene (hERG) AQW051 at a7 nAChRs expressed in Xenopus oocytes channel, which could represent a major cardiovascular revealed that this compound displayed an EC50 of 7.5 mM risk (Walker et al., 2006). Thus, to improve the selectivity but acted as a partial agonist, evoking only 75% of the and to diminish functional activity at the hERG channel, ACh-evoked current. This compound inhibited the 5-HT3 an analog of PNU-282987 was designed that demon- receptors with an IC50 of 19 mM, demonstrating a better strated improved absorption, distribution, metabolism, a7 nAChR/5-HT3 selectivity profile than RG3487 and and excretion properties, reduced hERG activity, as well encenicline (Feuerbach et al., 2015). This compound also as an adequate therapeutic index. This analog, PHA- demonstrated a sufficient PK profile, with rapid CNS 543613 (N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]furo[2,3-c] permeability, and activity in exploration in the rat social pyridine-5-carboxamide) (Ki =9nM)(Ackeretal., recognition model and an improved mouse sensory 2008), advanced into Phase 1 clinical studies. Recently, gating profile (DBA/2). Another amide quinuclidine encenicline, a potent quinuclidine amide analog re- compound (Targacept’s [Winston-Salem, NC] TC-5619) ported by FORUM (formerly EnVivo, Waltham, MA), demonstrated potency and selectivity for a7nAChRs(net 1042 Bertrand et al. current EC50 = 33 nM), with full efficacy relative to ACh micromolar range. Compounds with a quinuclidine in Xenopus oocytes (Hauser et al., 2009). TC-5619 (a7 moiety were extensively researched and yielded the nAChR Ki = 1 nM) exhibited superior in vitro selectivity discovery of several molecules that have progressed into against human 5-HT3 receptors (IC50 . 10 mM). This clinical trials. compound also exhibited adequate in vivo properties, 1. The Clinical Trials. To test the utility of a7 including PK profile and rapid CNS permeability as well nAChR agonists for enhancing cognitive function and being active in rat NOR and in mouse social exploration ultimately to assess the utility of a7 nAChR agonists in (Table 2). treating cognitive deficits, several compounds such as Additionally, structural diversity began to emerge in GTS-21, RG3487, encenicline, TC-5619, ABT-126, and the literature from this series within the amine portion AQW051 have advanced into clinical trials (see Table 2). of a7 nAChR agonists. However, some of this diversity Despite strong preclinical evidence supporting procog- also led to azabicyclic amines that exhibited activity as nitive effects, clinical results thus far have been limited. ligands for other nAChR subtypes such as the a4b2 The paucity of results may be attributable to confound- nAChR. Several alternate amines were recently pub- ing issues that include 1) properties of compounds that lished with a7 nAChR activity and include the diazabi- may not exhibit adequate receptor selectivity and cyclononanes such as SSR180711 (Biton et al., 2007; functional profile; 2) inadequate PK, particularly, CNS Pichat et al., 2007), the octahydropyrrolo[3,4-c]pyrrole A- exposure to achieve necessary receptor occupancy and 582941 (Tietje et al., 2008), and the (3R,5R)-1-azabicyclo- pharmacodynamic effects; and 3) design [3.2.1]oct-3-yl-carboxamide PHA-709829 (Acker et al., issues. For example, the GTS-21 Phase 2 trial failed to 2008) and ABT-107 (Bitner et al., 2010; Malysz et al., show significant effects on cognition. Specifically, per- 2010). The selective partial agonist SSR180711A is a 1,4- formance on the six domains of the MATRICS Consen- diazabicyclo[3.2.2]nonane carbamate derivative (Ki = sus Cognitive Battery (MCCB) did not differ between 50 nM, EC50 = 800 nM) active in NOR, Morris water maze, GTS-21 and placebo, although a significant effect of and an MK-801-induced memory deficit model (Pichat GTS-21 treatment was observed on the Scale for the et al., 2007). Researchers at AbbVie (formerly Abbott, Assessment of Negative Symptoms (SANS) total score North Chicago, IL) described A-582941, a novel biaryl (Freedman et al., 2008). However, it should be noted diamine a7 nAChR agonist (Buccafusco et al., 2007; that GTS-21 is not a prototypical a7 nAChR agonist. Tietje et al., 2008). A-582941 exhibited good affinity for GTS-21 has higher affinity at a4b2 nAChRs, where it is the human a7 nAChRs (Ki = 16.7 nM), with partial a functional antagonist compared with its interaction agonist efficacy but with a favorable PK profile and CNS with a7 nAChRs (Ki = 650 nM at rat and 2000 nM at penetration. In addition, A-582941 improved cognitive human) with only weak agonist efficacy (6% efficacy performance in assays, such as the monkey delayed relative to ACh) (Briggs et al., 1997). Thus, in the dose matching-to-sample task and rat social recognition range used in the clinic, GTS-21 is more likely to (Buccafusco et al., 2007) and mouse inhibitory avoidance, interact with a4b2 than a7 nAChRs, and it is mis- and normalized sensory gating deficits induced by MLA leading to conclude that the lack of clinical benefit can in rats (Tietje et al., 2008). Overall, the large number of be attributed to the a7 nAChR pharmacology. Sub- potent and selective a7 nAChR agonists that have been sequently, a number of a7 nAChR agonists/5-HT3 re- synthesized provide insight into the in vivo pharmacol- ceptor antagonists have been advanced to the clinic. For ogy and absorption, distribution, metabolism, and excre- example, RG3487 has relatively equal affinities at a7 tion properties, which has been instrumental in nAChRs (Ki = 6 nM) and 5-HT3 receptors (Ki = 2 nM) and advancement of compounds into clinical trials over the showed improvement in episodic secondary memory in years. However, biomarkers that can monitor target a healthy volunteer study, although the cognitive engagement have not been fully developed to accompany enhancing effects were not confirmed using the MCCB these clinical trials. Early work in this area has sug- composite score in a double-blind, placebo-controlled gested that endophenotypes associated with schizophre- Phase 2 study in schizophrenia (Wallace and Porter, nia, such as event-related electroencephalographic 2011; Umbricht et al., 2014). Although the lack of deficits in mismatch negativity, P300 (Preskorn et al., efficacy in this clinical trial might have different origins, 2014) and P50 (Olincy et al., 2006), are improved by a7 one additional complexity relies on the methodology nAChR agonists and that these endophenotypes might and assessment scale used. In this respect it should be serve as biomarkers of target engagement in future recalled that few studies share the same measurement clinical studies. scales and that so far there is no agreement on the best Functional expression of a7 nAChRs in recombinant method to be used for a given symptom domain in systems has highlighted the similarities and differences schizophrenia. Interestingly, a common trait emerges, between the a7 and other nAChR subtypes. These with some of these studies demonstrating an improve- findings stimulated the search for a7 nAChR-specific ment on the SANS (Umbricht et al., 2014; Keefe et al., ligands and led to the discovery of families of molecules 2015); although Walling et al. (2015) showed no benefit showing selective a7 nAChR agonist activity in the of TC-5619 on the SANS. Indicative of the fact that a7 Therapeutic Potential of a7 nAChRs 1043 nAChR agonists have not only reached the brain and of expression of a7 nAChRs in the brain and the have also modified the SANS scale, improvement in relevance of cholinergic function to cognition suggest negative symptoms could be used as a kind of bio- that a7 nAChR selective compounds may still become marker. Moreover, as negative symptoms are very therapeutics for cognitive impairment. The success or important in the day-to-day functioning of schizo- failure of the encenicline Phase 3 studies will likely phrenic patients, a drug that could reduce them would influence the likelihood that other a7 nAChR agonists still be beneficial, even if there is no improvement in the are developed in the near future, regardless of how cognitive symptoms. compelling the preclinical evidence is for the a7 nAChR More recently, positive signals of efficacy have been as a target for treating cognitive impairment. reported in schizophrenia with other a7 nAChR/5-HT3 re- B. From the Crystal Structure to Developments in ceptor ligands. , an a7 nAChR agonist/5-HT3 Structure-Activity Relationship (Ki =6.9nMfora7 nAChRs and Ki = 5.3 nM for 5-HT3 receptors), which is primarily used The discovery of the water-soluble AChBP, which as an antiemetic, was reported to improve sustained allows the preparation of high resolution crystal struc- attention and to significantly improve sensory gating in tures, initially reported by Sixma and collaborators a randomized, double-blind, placebo-controlled study in (Brejc et al., 2001; Sixma and Smit, 2003), marked schizophrenia (Shiina et al., 2010). More recently, FORUM a turning point in SAR research of compounds targeted reported an improvement in cognitive deficits with at the a7 nAChR. Since then, other proteins sharing encenicline, another a7 nAChR agonist/5-HT3 receptor similar structure and functions have been identified in antagonist using the CogState test battery, with trends invertebrates and provide additional and valuable in- for improvement on the MCCB composite score, as well formation about the ACh LBD (Hansen et al., 2005; as improvement in the SANS in a Phase 2, double-blind, Hibbs et al., 2009; Sander et al., 2010). The crystal placebo-controlled clinical trial in schizophrenia that structure of AChBP bound to different molecules included smokers and nonsmokers (Keefe et al., 2015). brought new insights to the understanding of protein- The importance of trial design was underscored in this ligand interactions and suggested that the LBD differ- study by the secondary analysis in which the consistency entially changes conformation upon binding of an in time of day of cognitive testing was critical to agonist or an antagonist (Hibbs et al., 2009; Brams demonstrating efficacy with the MCCB (Hufford et al., et al., 2011a). Crystal structures have been obtained for 2014). TC-5619, an a7 nAChR agonist without 5-HT3 epibatidine bound a7 nAChR/AChBP chimeras, with receptor antagonist activity, was found to improve three additional Aplysia californica AChBP mutants cognitive dysfunction as assessed by the Groton Maze showing further structural features with a variety of Learning Task of the CogState Schizophrenia Battery nicotinic ligands. These constructs may provide realistic and to reduce negative symptoms in a 12-week study in templates for structure-aided drug design. Homology schizophrenia (smokers and nonsmokers) (Lieberman models of this type, coupled with docking studies and et al., 2013). However, a second Phase 2 trial of negative regional analysis, have become a tool for the rational symptoms and cognition failed to find any benefit of design of new, selective nAChR ligands. Sequence TC-5619 in schizophrenia (Walling et al., 2015). Collec- analysis of AChBPs revealed that these proteins share tively, these recent clinical results with compounds that about 20226% overall sequence identity to the nAChR exhibited improved receptor selectivity and enhanced extracellular domains, but homologies within the PK profiles further validated the approach of activating orthosteric LBD are higher. a7 nAChRs as a therapeutic target. Studies comparing results obtained using the AChBP Clinical trials conducted with a7 nAChR agonists with functional assays for a7 and a4b2 nAChRs showed promising results in most of the Phase 1 studies, revealed some limitations, because some molecules with compounds demonstrating safety and efficacy in known to bind with high affinity to AChBP had no normal healthy volunteers. Cardiovascular events, such activity on receptors expressed in heterologous systems as reported for PNU-282987, marked, however, a halt of (Ulens et al., 2009). Nonetheless, AChBP provides studies for compounds from this class. Paucity of effects a useful template for the identification of novel mole- or lack of sufficient selectivity between a7 nAChRs and cules that were subsequently reported to have high 5-HT3 receptors might be the cause of other discontin- affinity for the 5-HT3 receptor and a7 nAChR (Akdemir ued compounds in clinical development. In the absence et al., 2011; Akdemir et al., 2012; Armishaw et al., 2009; of clear reports, it is difficult to discuss the limited Bourne et al., 2010; Brams et al., 2011b). success of a7 nAChR agonists in clinical studies report- Recent publications of the GABAA and 5-HT3 receptor ed so far. However, it should be noted that Phase 3 structures illustrate the feasibility of crystallization of clinical studies of encenicline, an a7 nAChR agonist/5- members of the cys-loop family of ligand-gated ion HT3 antagonist, are currently in progress, with two channels and suggest that the three-dimensional struc- trials for Alzheimer’s disease as well as two trials for ture of the a7 nAChR might be resolved in the near cognitive impairment in schizophrenia. The high degree future (Hassaine et al., 2014; Miller and Aricescu, 1044 Bertrand et al.

2014). Paving the way to SAR discoveries, the pro- unlikely. Recently, a novel series of octahydropyrrolo gressive refinement of the three-dimensional protein [3,4-c]pyrrole moieties were described by AbbVie (for- structure at the molecular level is expected to expand merly Abbott) as ligands for nAChRs (Briggs et al., 2008; our understanding of the relationship between ligand Tietje et al., 2008). Two octahydropyrrolo[3,4-c]pyrrole binding and functional activity of a7 nAChRs. derivatives were characterized in the literature to be Although crystal structure of a full a7 nAChR has not selective a7 nAChRs agonists adequate for labeling with been achieved, progress made with related proteins 11C, 2-methyl-5-[6-phenylpyridazine-3-yl]octahydropyrrolo such as the 5-HT3 receptor or AChBP has brought our [3,4-c]pyrrole (A-582941) and 2-(5-methyl-hexahydro- understanding of the nAChRs one step further. Crys- pyrrolo[3,4-c]pyrrol-2-yl)-xanthene-9-one (A-844606) tallization of the a7 nAChR is expected to shine a new (Toyohara et al., 2010). These compounds possessed light on ligand-protein interactions and is expected to required properties of potency and selectivity indis- open the discovery to new and more specific molecules. pensable for PET. A-582941 displaced specifically the radioligand [3H]A-585539 binding to a7nAChR C. Target Engagement and Overview of the membranes from both rat brain and human frontal Development of Biomarkers cortex with Ki values of 10.8 and 17 nM, respectively Research in the nicotinic field provided, over the years, (Anderson et al., 2008). A-582941 competed with the extensivedatatosupportthata7 nAChRs play a patho- specific binding of [3H]MLA to rat brain membranes physiological role in several psychiatric and neurologic with a Ki of 88 nM and exhibited much lower affinity disorders, such as schizophrenia,AD,anxiety,depression, for the a4b2 nAChR subtype, as measured using 3 drug addiction, and autism. Altogether, this propelled a7 [ H]cytisine binding to rat brain membranes (Ki . nAChRs as an attractive target for the design of selective 100,000 nM). In addition, A-582941 (10 mM) did not molecules desired for a number of therapeutic indications. exhibit any significant affinity for 78 other targets in Moreover, it has been demonstrated in post mortem a Cerep panel analysis, with the sole exception of 5-HT3 human brain samples that a7 nAChRs levels are altered receptors, in which [3H]-BRL 43694 (granisetron) bind- in schizophrenia and AD patients. This finding generated ing was displaced. The Ki value of A-582941 for 5-HT3 interest in studies to image and assess alterations in a7 receptors was 150 nM, which translates into a 15-fold nAChRs levels in living brains of patients with such higher Ki than for a7 nAChRs (Tietje et al., 2008). neuropsychiatric disorders. In this respect, it would also Over the past decade, there has been a considerable be relevant to measure in the intact brain and possibly in effort to expand the development of a7 nAChR ligands, neuropsychiatric patients, the receptor occupancy of with more than 20 compounds radiolabeled for PET and potential therapeutic a7 nAChR drugs that would provide SPECT, but previous efforts by several research groups an assessment of target engagement. A number of radio- to develop a clinically viable a7 nAChR tracer for PET or ligands were synthesized by different laboratories to SPECT have proven unsuccessful. None of these radio- undertake such a measurement and to provide quantita- ligands had sufficiently high specific binding at a7 tively the distribution of a7 nAChRs in the human brain nAChRs in vivo. Even [11C]CHIBA-1001, the recent through the utilization of PET tracer and single photon PET radioligand for human subjects, exhibited a low a7 emission computed tomography (SPECT) (see Table 3). nAChR binding affinity and poor in vivo selectivity. However, challenges ensued with such radioligand devel- In vivo imaging of the a7 nAChR distribution, opment due to lack of lead chemical structures that possibly up to the subcellular level, represents one of provided properties of high affinity and strategically the indispensable steps toward a better understanding attached functional groups to be labeled with PET and of these receptors in brain function. Development of SPECT radioisotopes. It is important to stress that radioligands, specific for a7 nAChRs and amenable to chemistry of ligands suitable for PET or SPECT imaging PET or SPECT studies is an enormous challenge that requires the possibility of rapidly inserting the desired was brought one step further with molecules that were radioactive group in the last step of synthesis, which examined in primates. Indicative of positive outcomes, seriously increases the challenge in the design of new these pioneering studies are opening additional strate- molecules. In 2008, the 1,4-diazabicyclo-[3.2.2]nonane gies to evaluate the contribution of the cholinergic analog 4-[11C]methylphenyl 2,5-diazabicyclo[3.2.2]noane- system in brain function. 2- carbocylate ([11C]CHIBA-1001) was developed by CHIBA University (Tokyo, Japan), and its selective D. The Chemical Strategy for a7 Nicotinic uptake was confirmed in the conscious monkey brain Acetylcholine Receptor Positive Allosteric Modulators by PET (Hashimoto et al., 2008). In recent years, medicinal chemistry has evolved to To date, [11C]CHIBA-1001 has been the sole PET include the synthesis of allosteric modulators active at ligand accessible for clinical trials to monitor in the a7 nAChRs with a focus on the identification of novel human brain a7 nAChR images. Although [11C]CHIBA- chemical entities. In this review, we will highlight such 1001 exhibited adequate properties as an a7 nAChR ligands as illustrated in Table 4, which are those most imaging tool, its development for human studies remains extensively characterized in the literature and have Therapeutic Potential of a7 nAChRs 1045

TABLE 3 a7 nAChR PET ligands

Compounds Species Tested Company Reference Rodent Monkey and Human Chiba Hashimoto et al., 2008; Sakata et al., 2011; Yin et al., 2013

[11C]-CHIBA-1001 Rodent and Pig Neurosearch Ettrup et al., 2011

[11C]-NS-14492 Rodent AbbVie Horti et al., 2013

[11C]-A-833834 Rodent Astra-Zeneca Gordon et al., 2010; Maier et al., 2011

[18F]-AZ11637326 Rodent and Human Johns Hopkins Wong et al., 2014

[18F]-ASEM

served as tools to make a significant advancement in the was acting by potentiating a7 nAChRs, as confirmed by a7 nAChR field. These compounds embody a new series inhibition of the effects by MLA (Callahan et al., 2013). of ligands that modulate the activity of a7 nAChRs and More recently, the SAR within this chemical series thus provide a potentially new therapeutic opportunity was determined to be narrow, but the series contained to treat a7 nAChR-associated cognitive deficits in compounds with improved potency, physicochemical schizophrenia. Initial work in the area of allosteric properties, and PK when tested in vivo. A biaryl urea modulation of a7 nAChRs was accomplished using series was identified by the group at NeuroSearch ivermectin or 5-hydroxyindole (Krause et al., 1998; (Copenhagen, Denmark), with NS1738 as an example Zwart et al., 2002). The disclosure of the selective a7 of such a compound from the series (Timmermann et al., nAChR Type-II PAM PNU-120596 provided the essen- 2007). NS1738 was also reported to enhance agonist tial selective tool to exploit the mechanistic potential of potency, as well as the efficacy. Although this compound such ligands (Hurst et al., 2005). Using an engineered has limited CNS penetration, it exhibited the ability to variant of the human a7 nAChR and ACh-evoked rescue scopolamine-induced deficits in acquisition of inward currents in hippocampal interneurons, PNU- a water maze learning task in rats and enhance 120596 increased agonist-evoked Ca2+ flux (Hurst et al., performance in rat social recognition (Thomsen et al., 2005). This compound also suppressed desensitization 2011). Interestingly, a team at the University of when tested in vitro and exhibited robust activity in the California-Irvine synthesized another molecule that is in vivo -induced P50 gating deficit model. a kind of chimera between compounds active at the Experiments conducted in aged rodents and nonhuman GABAA receptor and at the a7 nAChR, by taking primates showed that low doses of PNU-120596 aug- advantage of sequence homologies between these recep- mented the effects of the acetylcholinesterase inhibitor tors. From a library screen of modulators of the GABAA on learning and memory (Callahan et al., receptor, the group identified a class of compounds 2013). The positive effects were observed with low doses highlighted by compound 6 (XY4083) (Ng et al., 2007). of PNU-120596 that were otherwise ineffective when XY4083 exhibited properties of an a7 nAChR PAM but, applied alone and indicated that this drug combination unlike PNU-120596, did not significantly prolong the 1046 Bertrand et al.

TABLE 4 a7 nAChR positive allosteric modulators

Modulator Compounds Therapeutic Indications Type Company Reference Alzheimer’s disease I XYTis Ng et al., 2007

XY4083 Alzheimer’s disease II Pfizer Hurst et al., 2005; Barron et al., 2009; Cognitive deficiency in schizophrenia Young et al., 2008; Young and Geyer, 2013

PNU-120596 Alzheimer’s disease I Neurosearch Timmermann et al., 2007; Thomsen and Cognitive deficiency in schizophrenia Mikkelsen, 2012

NS-1738 Alzheimer’s disease II AbbVie Malysz et al., 2009, 2010 Cognitive deficiency in schizophrenia

A-867744 Alzheimer’s disease II Eli Lilly Broad et al., 2006 Cognitive deficiency in schizophrenia

LY-1078733 Alzheimer’s disease Discovery Roche Sahdeo et al., 2014

RO5126946 response time course. This compound reversed sensory in the series related to LY-2087101 exhibited an enhance- gating deficits in rodents and improved working mem- ment in potency and maximal efficacy at both a7 and ory. Eli Lilly (Indianapolis, IN) and Johnson and a4b2 nAChRs. JNJ1930942 (2-[[4-fluoro-3-(trifluoro- Johnson (New Brunswick, NJ) identified from high methyl)phenyl]amino]-4-(4-pyridinyl)-5-thiazolemetha- throughput screenings a series of thiazole derivatives nol) was reported to be selective for a7 nAChRs (Dinklo as a7 nAChR PAMs. LY-2087101, a (2-amino-5-keto) et al., 2011). This compound enhanced the peak agonist- thiazole compound (Broad et al., 2006), was derived evoked current amplitude, and similar to PNU-120596, from that series and exhibited activity at the various was classified as a Type-II PAM with slowed desensi- brain subtypes of nAChRs and thus was not selective tization kinetics. JNJ1930942 improved sensory gating for a7 nAChRs (Young et al., 2008). Other compounds of auditory evoked potentials in DBA/2 mice. More Therapeutic Potential of a7 nAChRs 1047 recently, Roche (Basel, Switzerland) described another brain (Agulhon et al., 1999). Improper function of these PAM selective for a7 nAChRs, RO5126946, or 5-chloro-N- receptors might therefore alter the organization of the [(1S,3R)-2,2-dimethyl-3-(4-sulfamoyl-phenyl)-cyclopropyl]- brain. Although our knowledge of the relationship 2-methoxy-benzamide), which is also classified as a Type-II between a7 nAChRs and brain development is rather PAManddisplayedeffectsbothinvitroandinvivo limited, developments in imaging technologies, com- (Sahdeo et al., 2014). It was concluded and reported that bined with genetic associations and clinical phenotypes subtlesubstitutionsinthischemicalseriestranslateto are paving the way to future research, as illustrated by profound effects on selectivity among the homomeric and the computer analysis of multiple genes contributing to heteromeric nAChR subtypes. schizophrenia (Gilman et al., 2012). The finding that a7 nAChR activity can be modulated The aim of this section is to review the most by different molecules suggested that PAMs represent prominent correlations found between CHRNA7 and an additional therapeutic possibility. The identification neurologic and psychiatric diseases. In the view of the of Type-I and Type-II PAMs that were classified determinant properties of a7 nAChRs and their impli- according to their effects on receptor desensitization cation in brain development, it is probable that addi- unveiled the broad possibilities offered by allosteric tional diseases associated with this receptor will be modulation of a7 nAChRs. Although efforts in the discovered as progress is made in small patient cohorts search for additional PAMs are still in their infancy, it or even in the case of pedigree studies (see Table 5). is clear that this strategy will be further exploited in the near future. A. Alzheimer’s Disease In the United States alone, AD has become a devas- tating neurologic disorder affecting over 5 million IV. The Relevance of a7 Nicotinic Acetylcholine patients and is the major form of dementia in the aging Receptors in Diseases population. First identified in 1901 in a 51-year-old Normal brain function relies on precise connectivity patient showing presenile dementia, this case allowed and equilibrium between neuronal activities from mul- Dr. Alois Alzheimer to study the evolution of a disease tiple sources. Although there are numerous examples of that initiated with loss of short-term memory and was single amino acid mutations causing altered brain followed by progressive loss of cognitive function. function, the profound modifications caused by muta- Autopsy and histologic observations of this patient’s tion in the AMPA receptor regulatory protein g2 (or brain revealed a peculiar formation of amyloid plaques stargazin) are particularly noteworthy (Osten and and neurofibrillary tangles that were subsequently Stern-Bach, 2006). Mutant stargazin alters trafficking used as the post mortem diagnostic criteria of the of AMPA receptors and leads to a complex phenotype, disease (Simchowicz, 1911). which includes changes in AMPA receptor function, Initiating with mild cognitive impairment (MCI) and aberrant pyramidal cell orientation, and epilepsy. Dur- progressing to loss of short-term memory, the symptoms ing development, as cells are progressively migrating of AD disable patients in their day-to-day functioning and differentiating, neurotransmitters are synthesized and pose an enormous burden on patients’ relatives and and released to aid in the establishment of neuronal caregivers. It is therefore of no surprise that the finding connections. Expression of receptors is therefore one of of a possible relationship between histologic evidence the primary steps taking place early in development, and cognitive decline triggered immediate attention. and CHRNA7 mRNA is found early in several nuclei Since these initial studies, replication of the clinical and that receive sensory information in the human fetal histologic observations were made by other laboratories

TABLE 5 a7 nAChR-associated diseases

Disease Reference Auditory dating deficits in families with schizophrenia Freedman et al., 1996; Leonard et al., 2002; Gault et al., and smokers with schizophrenia 2003; Mexal et al., 2010; Flomen et al., 2013 Triplication in three generation pedigree of cognitive Soler-Alfonso et al., 2014 impairment and neuropsychiatric phenotype Inherited phenotypic trait of schizophrenia Adler et al., 1998 Neuropsychiatric disorders Riley et al., 2002 Epilepsy Elmslie et al., 1997; Rozycka et al., 2013 AD susceptibility; MCI risk and conversion to AD Barabash et al., 2009; Heinzen et al., 2010; Swaminathan et al., 2011, 2012a,b AD, Dementia with DLB or Pick’s Feher et al., 2009 Benign rolendic epilepsy Neubauer et al., 1998 Autism and Rett Syndrome Allen-Brady et al., 2010; Yasui et al., 2011 Developmental Delay, Mental Retardation and/or ASD Mikhail et al., 2011 Microdeletion Syndrome Masurel-Paulet et al., 2010; Szafranski et al., 2010 1048 Bertrand et al.

(Cras et al., 1991; Bloom, 2014). Additional observations to the treatment of patients suffering from MCI and reported, however, that the presence of amyloid plaque age-related decline with aceytlcholinesterase inhibitors and neurofibrillary tangles could also be observed in (see for review Tan et al., 2014). In view of the parallel brains of elderly patients without the necessary associ- reductions in ACh and nicotine binding sites in cerebral ation with dementia (Bloom, 2014). Casting doubt on the cortex, experiments were conducted to determine if causality between histologic markers and the clinical nicotine could be a suitable treatment to restore phenotype, these studies indicate that additional factors cognitive function. Exposure to nicotine alleviated probably need to be taken into account to fully explain deficits in attention and informational processing AD (Bloom, 2014). Since then, genetic studies were associated with AD (Wesnes and Warburton, 1984; conducted on large populations in an attempt to find Sahakian et al., 1989; Newhouse et al., 2012). The possible associated genes, as well as protective factors positive effects of nicotine treatment observed in these that would prevent or delay the onset of the AD (Bloom, studies were initially attributed to nicotine interacting 2014; Rosenblum, 2014). Genome-wide association stud- with the high-affinity a4b2 nAChRs. However, given ies revealed that mutations in amyloid precursor pro- the lack of robustness of a4b2 nAChR agonists in tein, presenilin 1, and presenilin 2 are accountable for improving cognition, attention was refocused on other the rare early-onset autosomal dominant forms of AD, nAChR subtypes and more specifically on a7 nAChRs. fueling the hypothesis that amyloid is a key causal Additional correlation between these receptors and AD factor in AD (Armstrong, 2013). The apolipoprotein E was provided by the observation of a reduced expression gene «4 allele is associated with the more common late- of a7 nAChR labeling in post mortem AD cortex onset and complex forms of AD (reviewed in Kim et al., (Burghaus et al., 2000). 2014a). As these studies have progressed, more genes To further examine the putative relationship between have been identified to be putatively associated with a7 nAChRs and AD, it is important to recall key findings AD, forcing the field to revisit the amyloid hypothesis as obtained in different models. Following the hypothesis the primary the etiology of AD. that the increase of Ab1-42 might be one of the de- Despite a clear genetic basis for the presenile or early- terminant factors causing AD, experiments were con- onset form of AD, a genetic signature explaining the key ducted to determine whether this peptide interacted factors responsible for triggering senile or late-onset AD with a7 nAChRs and the consequences of this interac- and its progression is less obvious, with several genes tion. As a first step, studies examined if Ab1-42 peptide implicated in modifying the risk of late-onset AD. In could bind to nAChRs and more specifically to a7 addition to the hallmark pathologies of AD, other nAChRs. Some studies report a direct interaction evidence suggests that the cognitive deficits are associ- between Ab1-42 and a7 nAChRs from mouse and human, ated with reduced cholinergic function (Whitehouse suggesting a binding interaction (Wang et al., 2000, et al., 1983; Whitehouse and Kalaria, 1995; Whitehouse, 2009). An absence of a correlation between a7 nAChR 1998). In light of the negative correlation between binding and the presence of Ab plaques was also smoking and Parkinson’s disease (PD) and the relevance reported in other studies (Ikonomovic et al., 2009). of the cholinergic system in prefrontal cortical activity, it Moreover, it was suggested that Ab might disrupt the was hypothesized that a similar correlation might exist membrane structure and indirectly affect a7 nAChR between AD and smoking. Careful histologic examina- expression and packing of lipids (Small et al., 2007). tion of brains from AD patients and age-matched controls Appropriate caution must however be maintained, revealed no clear evidence for a link between nicotine because false positives might be generated when eval- intake and AD (Ulrich et al., 1997). Because the study uating protein expression using commercially available revealed a possible protective effect of nicotine on antibodies (Herber et al., 2004; Jones and Wonnacott, neurofibrillary changes, the authors concluded that 2005). Experiments conducted with competitive antag- nicotine might have an influence on the structural onists such as a-Btx or MLA have found opposing alterations of AD. results: an increase in expression of a7 nAChRs thought Nonetheless, the progressive neuronal loss in the to compensate for the loss of ACh in AD (Counts et al., cholinergic basal forebrain and prefrontal cortex was 2007; Liu et al., 2013) and reduced expression of a7 correlated with reduced cholinergic activity and a re- nAChRs in other studies (Burghaus et al., 2000). A duction in [3H]acetylcholine and [3H]nicotine binding in lower level of a7 nAChR expression on neurons, at the AD (Whitehouse and Au, 1986). Studies of the effects of same time as expression was higher on astrocytes, was blockade of the cholinergic system in young healthy also reported in sporadic and familial AD (Yu et al., subjects further supported the relationship between 2005). Although determination of the level of expression cognitive function and the cholinergic system (Bartus and possible affinity between the Ab1-42 and a7 nAChRs et al., 1982). In view of these observations, it was constitutes the first step toward the understanding of proposed that use of a compound that would block a possible correlation between Ab1-42 and cholinergic acetylcholinesterase activity might be beneficial in re- functions, these studies rely on cross-sectional analyses ducing the cognitive deficit in AD, which eventually led and are therefore lacking the indispensable longitudinal Therapeutic Potential of a7 nAChRs 1049 aspect that would be requiredtoanalyzethecorrelation nAChRs and inhibits their function, as measured by between these parameters with the evolution of the Ca2+ influx in CHO-K1 cells expressing a7nAChRs(Ju disease in individual patients. Functional experiments et al., 2014). Unlike other Ab mutations, such as the aimed at examining the effects of Ab1-42 on the activities E22Q, which gives rise to a highly distinct phenotype of the a7 nAChRs, together with pharmacological in- with amyloid angiopathy leading to recurrent hemor- tervention with a7 nAChR agonists, provide an alter- rhage, the E22G mutation causes only cognitive deficits native to evaluate the role of the cholinergic system in (Nilsberth et al., 2001). Although differences observed AD. Initially, patch-clamp experiments conducted on between distinct Ab protein products highlight the rat hippocampal neurons in culture showed that expo- complexity of interactions of these amyloid peptides, sure to rat Ab1-42 inhibited in a dose-dependent manner results obtained with a7 nAChRs suggest a plausible the current evoked by ACh and that this inhibition was specific interaction that is therapeutically worthwhile noncompetitive (Liu et al., 2001). One difficulty in pursuing. functional studies concerns the sequence and oligomer- Additionally, a7 nAChR stimulation could poten- ization of the Ab used. Namely, species, sequence and tially play a role in rescuing presynaptic deficits in AD peptide length (e.g., Ab1-40 versus Ab1-42, etc.) differ- as a result of decreasing levels of b-site amyloid ences have been reported and, more importantly, Ab precursor protein-cleaving enzyme 1 (BACE1) (Vassar oligomerization is difficult to control and probably et al., 2009; Kandalepas and Vassar, 2014; Yan and represents a crucial variable that cannot be analyzed Vassar, 2014). In 2010, an intriguing study examined when comparing effects reported from different labora- the connection between activation of a7 nAChRs in tories. This is illustrated by considering opposite results BACE1 knockout mice, showing restoration of pair- obtained in two laboratories reporting 1) activation of pulsed facilitation from mossy fiber-to-CA3 synapses, a7 nAChRs by exposure to 10 pM Ab1-42 (Dineley et al., which is reflective of deficits in presynaptic release 2002; Tong et al., 2011) and 2) absence of effect reported (Wang et al., 2010a). Second, activation of a7 nAChRs for apparently comparable experimental conditions also restored LTP deficits at these terminals in BACE1 (Small et al., 2007). Use of chimeric approaches and in- knockout mice. Both of these restorations by a7nAChR tracellular calcium measurements suggest that Ab1-42 agonists, in this case by nicotine and PNU-282987, interacts with the tyrosine residue Tyr-188 of a7 were selective because pretreatment with 100 nM nAChRs (Tong et al., 2011). Evidence for the effects of a-Btx blocked the agonist actions (Wang et al., 2010a). Ab on a7 nAChRs in animal models was obtained using One mechanistic insight afforded by this study was that different approaches. For example, it was shown that this a7 nAChR-induced restoration in BACE1 knockout intraventricular injection of Ab1-42 inhibited the pressor mice was due to one of the hallmark characteristics of a7 response of the heart rate caused by infusion (Li nAChR stimulation, which is elevation of protective and Buccafusco, 2004). Further agreement for an in- levels of intracellular Ca2+ and the concomitant in- teraction between a7 nAChRs and Ab was obtained in crease in glutamate release from synaptic endings that studies relying on selective a7 nAChR agonists (Wang affected downstream intracellular signaling cascades et al., 2010b; Chen et al., 2010; Inestrosa et al., 2013). A such as the ERK pathway (Dickinson et al., 2008). These further complexity might reside in the structural dif- studies clearly suggest that the combination of an a7 ferences between homomeric a7 and heteromeric a7b2 nAChR agonist with a BACE1 inhibitor may be an nAChRs, as shown by intracellular recording in brain attractive approach to tackling the complexity in treat- slices and from nAChRs expressed in Xenopus oocytes ing AD. (Liu et al., 2009, 2012). The apparent contradictory In view of the unique procognitive properties of a7 observation of an exacerbation of AD effects in a7 nAChR agonists and their neuroprotective activity in nAChR knockout mice (Hernandez et al., 2010) versus different models, it is reasonable to speculate that improvement observed in another model (Dziewczapolski stimulation of a7 nAChRs could be disease modifying et al., 2009) can be reconciled when considering the rather than solely compensate for cognitive decline. stage and conditions. More specifically, if it is thought Thought of as the “missing link” between the histopath- that stimulation of a7 nAChRs is neuroprotective, then ological hallmarks of amyloid or phosphorylated tau it is understandable that inhibition of these receptors and the clinical manifestation of AD, a7 nAChRs will exacerbate Ab neurotoxicity, but inhibition of a7 constitute an important area of research (reviewed in nAChRs may also cause impairment of cognitive func- Bencherif and Lippiello, 2010; Parri et al., 2011). tion. A balance between neuroprotection and reduction The safety and efficacy of the a7 selective agonist in cholinergic neurotransmission must therefore be ABT-126 observed in a Phase 2 clinical trial conducted taken into account when considering the effects and on subjects with mild-to-moderate AD dementia was requires long-term studies. recently published (Gault et al., 2015). Although this Experiments conducted with Ab40 that contains the study failed to demonstrate statistically significant mutation E22G corresponding to the so-called “Artic improvements with doses up to 25 mg; exposure- Ab” variant revealed that this polypeptide binds to a7 response analysis suggested that additional data with 1050 Bertrand et al. higher concentrations should be performed either as been developed, the hope is that a7 nAChR agonists mono or add-on therapy. may fill this role, if only partially as a treatment to There are also other forms of dementia that may substantially slow disease progression, if not halt it benefit therapeutically from an a7 nAChR agonist. MCI entirely. Current Phase 3 clinical trials conducted with is clinically defined as a patient having problems with the a7 nAChR agonist/5-HT3 antagonist encenicline are memory with or without cognitive deficits that interfere hoped to mark a turning point in AD treatment. While with daily function (Petersen et al., 2001). Unfortu- time will tell if encenicline can be successfully mar- nately, 10–15% of these patients annually will convert keted, it is likely that a7 nAChRs represent a promising to dementia, particularly to AD (Petersen, 2009). A target for the treatment of cognitive impairment in AD genetic study suggested that the CHRNA7 might act as and may also alter the course of the disease. a modifier gene in patients with MCI in protection from conversion to AD (Barabash et al., 2009). A recent study B. Schizophrenia demonstrated that in a 6-month study, transdermal Affecting about 1% of the population or more than 50 nicotine improved measures of attention, memory, and million people worldwide, schizophrenia is a life-long mental processing in nonsmoking MCI patients, al- severely disabling mental disorder characterized by though not in the global impression clinical rating scale deficits in thought processes, perception, and emotional (Newhouse et al., 2012). responsiveness. Schizophrenia onset typically occurs in Dementia with Lewy bodies (DLB) is the second most early adulthood and symptoms such as cognitive im- prevalent form of dementia, and in DLB the psychiatric pairment and reduced attention might be indicative of symptoms, mainly visual hallucinations, emerge earlier altered brain function prior to the first psychotic episode than in AD. Thus these patients use more resources to and diagnosis. The role of a7 nAChRs in this disease was cope with the disease and as a consequence early recently thoroughly reviewed and supported by a biology intervention may prove beneficial in quality of life for that correlates with the pathophysiology of the disease these patients and their caregivers (Mollenhauer et al., and the involvement of cholinergic nuclei and, more 2010). Interestingly, visual hallucinations and delu- specifically, a7 nAChRs (Wallace and Bertrand, 2013a; sional misidentification associated with DLB were Freedman, 2014). From its revised and expanded char- correlated with lower a-Btx binding in the temporal acterization by the Swiss psychiatrist Paul Eugen cortex, indicative of a7 nAChR localization, and not Bleuler (1857–1939), who coined the term schizophre- with [3H]-epibatidine binding, indicative of a4b2 nia, it was recognized that basic symptoms included nAChR localization (Rei et al., 2000; Court et al., “negative symptoms” with disorganized speech, think- 2001). As nAChRs are also strongly expressed in the ing, affective incongruence, and withdrawal from reality visual system, including the retina, the lateral genicu- and “positive symptoms” with tactile, auditory, late nucleus and visual cortex, it would be of value to visual, and gustatory hallucinations and delusions know if the density of receptors is also modified in other (Pearlson and Ford, 2014). Additionally, cognitive im- brain areas. As listed in Table 5, the CHRFAM7A gene pairment in multiple domains is a core feature of without the 2-bp deletion has been shown to be signif- schizophrenia and accounts for much of the continued icantly correlated with AD (P = 0.011), DLB (P = 0.001), disability and poor functional outcomes in patients after and Pick’s disease (P , 0.0001) compared with healthy management of the positive symptoms (Green et al., controls (Swaminathan et al., 2011, 2012a). Patients 2004; Nuechterlein et al., 2011). Although impressive with DLB or PD with dementia share the same profile of progress in treating positive symptoms has been made dopaminergic and cholinergic deficits with widespread with the discovery of typical and atypical antipsy- reductions in choline acetyltransferase-positive neu- chotics, it is now recognized that these molecules are rons in DLB and PD with dementia compared with PD ineffective at treating negative and cognitive symptoms alone (Rei et al., 2000; Fujishiro et al., 2006). If lower (Kay and Singh, 1989; Chue and Lalonde, 2014; Keefe, functional a7 nAChRs are present in these other forms 2014). Schizophrenia, as with many other brain disor- of dementia, then the therapeutic use of a potent a7 ders, is certainly multifactorial and includes environ- nAChR agonist could be potentially beneficial as well. mental etiologies; however, as shown by recent twin Numerous observations point to a critical role of the studies, there are genetic factors predisposing for the cholinergic system in AD, as well as in other dementing development of schizophrenia (Cardno and Owen, illnesses. This is best illustrated by the fact that today 2014). The need for better molecules that would be acetylcholinesterase inhibitors represent one of the few efficacious for negative and cognitive symptoms is tools available for the treatment of dementia. Develop- therefore tremendous and should be readily evaluated. ment of a7 nAChR agonists and the observation that Without entering into a detailed review of all the these molecules not only can be procognitive but also elements linking a7 nAChRs and schizophrenia, it is neuroprotective has strengthened the need to pursue worthwhile recalling that a large percentage of patients research into their utility in the treatment of AD. are smokers and that their cigarette consumption is Because no disease-modifying treatment of AD has yet about twice that of smokers in the general population. Therapeutic Potential of a7 nAChRs 1051

This prompted the idea that nicotine intake from The importance of the cholinergic system was largely smoking might constitute an attempt to self-medicate documented in animal models that supported the de- and that nicotine could reduce some of the symptoms. terminant role of a7 nAChRs by studying the effects of Post mortem examination of brain tissue revealed a re- molecules specific for this receptor subtype. Exposure to duction of a-Btx binding sites up to 50% in schizo- a7 nAChR agonists or PAMs was found to restore phrenic patients (Freedman et al., 1995; Marutle et al., cognitive impairments caused by pharmacological 2001). Subsequent analysis of smoking versus non- treatments. Consistent results obtained in different smoking schizophrenics confirmed the reduced a7 animal species ranging from rodent to nonhuman nAChR expression at the cell surface but concluded primates further strengthened the role of a7 nAChRs that smoking might cause some compensation for this in cholinergic system functioning. Preclinical studies deficit (Leonard et al., 2000). Genetic studies conducted have demonstrated the efficacy of several a7 nAChR by different laboratories indicated that mutations in the agonists in an N40 sensory gating model (Hashimoto CHRNA7 gene, its promotor region, or CHRFAM7A are et al., 2005; Simosky et al., 2008; Wildeboer-Andrud and associated with schizophrenia (Riley et al., 2000; Raux Stevens, 2011), in particular ABT-107 (Radek et al., et al., 2002; Martin et al., 2007; Sinkus et al., 2009; 2012). Other molecules such as TC-5619, SSR-180711, Stephens et al., 2009; Bakanidze et al., 2013). A survey A-582941, or encenicline also demonstrated efficacy in of the literature on schizophrenia and genetic associa- cognitive models (Pichat et al., 2007; Tietje et al., 2008; tion revealed that several studies failed to detect any Mazurov et al., 2012; Prickaerts et al., 2012). Although association with CHRNA7 or CHRFAM7A, which is application of an a7 nAChR agonist alone was ineffec- indicative of the rarity of these mutations in the tive in a DBA/2 mouse model of sensorimotor gating schizophrenic population, the complexity of schizophre- (prepulse inhibition), it increased the effects of haloper- nia, and the need for a better classification of the disease idol or (Kohlhaas et al., 2012). TC-5619 (Petrovsky et al., 2009). Because schizophrenia is showed a statistically significant effect on executive probably multifactorial, influenced by both environ- function based on measurements for the Groton Maze mental factors and genetics, with many other genes Learning Task but not on other cognitive domains identified as linked to the disease, it would not be (Lieberman et al., 2013). However, secondary measure- surprising that absence of an association would be ments for negative symptoms demonstrated efficacy in observed in certain patient cohorts. TC-5619-treated patients with schizophrenia, and these Because the first manifestations of the disease often effects were more pronounced in smokers than non- occur in late adolescence, it would be of interest to smokers (Lieberman et al., 2013). However, a second identify biomarkers that could allow detection and Phase 2 trial with TC-5619 failed to demonstrate any possible treatment of the disease before the appearance benefit in alleviating either negative or cognitive symp- of overt clinical symptoms. Interestingly, studies con- toms (Walling et al., 2015). The logical next steps for ducted in children have shown that delay in P50 proof-of-concept were obtained in early clinical studies inhibition is associated with attention problems during using a7 nAChR agonists (Olincy et al., 2006; Tregellas maturation (Ross et al., 2010). In view of the fact that et al., 2011; Barbier et al., 2015; Preskorn et al., 2014). amniotic choline activates fetal a7 nAChRs and facili- Although a more detailed discussion of these clinical tates the development of central inhibition, it was trials is presented in section III, it is important to note hypothesized that choline supplementation during that studies of the 5-HT3 receptor antagonist tropise- pregnancy might improve child development (Ross tron, which also acts as an agonist at a7 nAChRs, et al., 2010). Randomized, placebo-controlled clinical caused a normalization of the P50 response in schizo- trials conducted on more than 100 volunteers revealed phrenic patients (Shiina et al., 2010; Zhang et al., 2012). that 76% of the children treated with phosphatidylcho- The complex modifications observed in behavior and line during development showed suppression of the P50 multiple higher cognitive functions are, however, un- response at the 5th postnatal week, a marker of central likely to be caused by the modification of the a7nAChR inhibition, versus 43% in placebo-treated infants. Al- pathway alone but might implicate some interdepen- though the difference progressively diminished be- dence with other neurotransmitter systems. Contribu- tween treated and placebo groups by the 13th week, tion of the glutamatergic system and more specifically perinatal choline treatment might help in the develop- the hypofunction of the NMDA receptor is one of the ment of central inhibition (Ross et al., 2013). In hypotheses that is preponderantly discussed in the addition, studies examining children with autism spec- field of schizophrenia (Veerman et al., 2014). This was trum disorder (ASD) have shown a reduction in the supported by the observation that exposure of healthy latency to evoke a P50 response, as well as a reduction subjects to NMDA antagonists such as causes in prepulse inhibition to a startle response, similar to schizophrenic-like symptoms (Adler et al., 1999). A that observed in schizophrenia, and suggests a role for possible cross-interaction between NMDA receptor and a7 nAChR agonist therapy for this condition (Deutsch a7 nAChR neurotransmission would therefore repre- et al., 2010). sent a plausible mechanism that would explain results 1052 Bertrand et al. observed with these two classes of receptors. Speaking Huang et al., 2014a). The recent hypothesis that ket- in favor of a cross-interaction, the following facts must amine might be used for the treatment of depression be considered. First, important evidence comes from indicates that in view of the a7 nAChR/NMDA receptor the pharmacological interactions observed in rodent interaction, additional targets might be envisaged to and monkey in which impairment caused by ketamine treat a wide range of psychologic afflictions. or MK-801 exposure could be improved by the a7 The high prevalence of schizophrenia in the popula- nAChR agonist GTS-21 (Cannon et al., 2013; Callahan tion and lifelong debilitation associated with the disease et al., 2014). Similarly, in a mouse model, tropisetron calls for the development of new molecules that could improved the cognitive deficits caused by phenyclidine help patients suffering from this disease. Progressive (Hashimoto et al., 2006). The a7 nAChR agonists enceni- cognitive deficits as well as negative symptoms ob- cline and TC-5619 have also been shown to reverse served in patients with schizophrenia suggest that -induced deficits that mimic negative-like drugs targeting the cholinergic system and more spe- symptoms of schizophrenia in mice (Pedersen et al., 2014). cifically the a7 nAChRs should be appropriate for the Deficits induced by the open channel blocker MK-801 can treatment of this disease. Current Phase 3 clinical trials be reversed by a7 nAChR agonists as well as by a7 are expected to provide additional information about nAChR PAMs (Jones et al., 2014). The use of a specific a7 the utility of a7 nAChR agonists for treatment of both nAChR agonist is probably necessary, as nicotine, a non- cognitive impairment and negative symptoms in schizo- selective ligand, could not restore the ketamine-induced phrenia. Parallel developments brought by refined deficits in humans (D’Souza et al., 2012). Interestingly, genetic analysis of the chromosome 15 sequence in the however, nicotine was able to alleviate ketamine-induced CHRNA7 region are expected to provide additional sensory and memory impairment and improve attention information about the association between schizophre- in subjects displaying a predisposition for auditory nia or cognitive deficits and genetic modifications. hallucinations/delusions (Knott et al., 2012). The reports Together, these data will be valuable in determining of a possible pharmacological cross-reactivity between the the contribution of a7 nAChRs or a7 dup in cognitive NMDA receptors and a7 nAChRs call, however, for a word impairments and the population of patients that might of caution in the analysis of currently available data. benefit from treatment with a7 nAChR agonists. Namely, it was shown that , ketamine, and MK-801 can interact with a7nAChRs,whichrequires C. Autism a precise quantification of the concentration used in the Autism spectrum disorders (ASDs), which include experimental conditions, before reaching a definitive autistic disorder, Asperger’s disorder, and pervasive conclusion (Maskell et al., 2003; Alkondon et al., 2011; developmental disorder, are defined by socialization Banerjee et al., 2012; Moaddel et al., 2013). The impor- and communication deficits and a need for preservation tance of the functional interactions between NMDA of “sameness.” Initially considered in 1943 as the receptors and a7 nAChRs was further defined by studies manifestation of a single affliction, autism and schizo- illustrating that chronic inactivation of a7nAChRs phrenia became classified as two separate diseases, markedly increased NMDA receptors at the cell surface although they shared several clinical features, such as (Lin et al., 2010). Differences in the distribution of NMDA social withdrawal, communication impairment, and receptors from synaptic to extrasynaptic and a change in poor eye contact (Barneveld et al., 2011). With a preva- the pharmacological sensitivity to D-serine were also lence of up to 2/1000 individuals, autism is about four observed in a7nAChRknockoutmice(Linetal.,2014). times more frequent in men than women and shows up to Direct interaction between a7 nAChRs and NMDA 90% genetic inheritability (Casey et al., 2012). Despite receptors was recently proposed with a protein-protein evidence for genetic transmission, genome-wide analysis interaction that could be disrupted by the use of small performed on large cohorts has failed to identify associ- peptide fragments (Li et al., 2012, 2013). These data need, ations with specific genes or mutations, suggesting that however, to be replicated and extended to understand the variants exert only a weak effect on ASD (Anney et al., putative mechanism of direct receptor interaction. 2012). An alternative genetic analysis conducted on more The large body of literature documenting the effects than a thousand patients was aimed at elucidating why of NMDA receptors on memory and other intrinsic a disease that is highly heritable cannot be associated brain functions indicate that a recognized interaction with a given gene or set of genes (Casey et al., 2012). In between these glutamate receptors and a7nAChRs rare cases, autism is associated with agents that cause further supports the fundamental role of a7 nAChRs in birth defects, but the impressive increase in autism of cognition. Development of compounds acting at the a7 about 30% between 2012 and 2014 suggests that envi- nAChR might therefore also serve to alleviate NMDA ronmental causes might be at the origin of ASD (Neggers, receptor dysfunction, as hypothesized in schizophre- 2014). Similar observations were made in a meta- nia. Indeed, glutamate release from prefrontal cortex analysis of results from an Asian population (Feng was elevated after treatment with a7 nAChR agonists et al., 2013). Questions about recent increases in ASD in rats and nonhuman primates (Yang et al., 2013; have been raised because this might reflect a difference Therapeutic Potential of a7 nAChRs 1053 in diagnostic practice and government-subsidized finan- Another challenging hypothesis has been suggested cial incentives for named diagnoses more than a real that autism, schizophrenia, and attention deficit disor- increase in the frequency of ASD. der are part of a single neurologic disorder correspond- With regard to the cholinergic system, neuropatho- ing to a spectrum of imbalances in neurotransmission logical abnormalities in the basal forebrain cholinergic (Lippiello, 2006). Because a7 nAChRs modulate a num- nuclei originally indicated that dysfunction of cholin- ber of neurotransmitter systems in brain regions af- ergic neurotransmission may be involved in the etiol- fected in ASD, therapeutically targeting this receptor ogy of autism (Ray et al., 2005). Neurochemical studies may be beneficial. have shown a loss of a7 nAChRs in thalamus (specif- Although the linkage between autism and a genetic ically the paraventricular nucleus and nucleus cause remained elusive for a long time, the newest reuniens) in autistic versus control brains, suggesting findings reported for the CHRNA7 locus on chromosome a role in the relay of information from the periphery to 15 provide a stimulating hypothesis. Complementing cerebral cortex and in modulation of cortical outputs other observations of cholinergic and a7 nAChR deficits (Lee et al., 2002; Martin-Ruiz et al., 2004). Data from in autism, these genetic studies are pointing to a de- this small sample suggest that cholinergic dysfunction terminant role of a7 nAChRs in cognitive impairment in the thalamus of persons with autism may contribute associated with autism. Further confirmation for the to disrupted regulation of sensory input to the cerebral correlation between a7 nAChRs and autism will be cortex and disturbance of the processing of emotions provided by progress in genome-wide association studies and cognition. A 15q11.2-13.3 chromatin analysis and will determine whether future development of new revealed alterations in epigenetic regulation resulting treatments for ASD will include a7nAChRagonists. in reduced transcription of CHRNA7 in Rett syndrome and autism brains (Yasui et al., 2011). This study also D. Microdeletion Syndrome revealed an association between ASD and genomic The orphan disease of 15q13.3 microdeletion syn- instability at chromosome 15q13.3 due to recurrent drome (see Table 6) was first described in 2008 (Sharp microdeletions, also resulting in a reduced level of et al., 2008), and since then, using whole genomic CHRNA7 transcripts in human frontal cortex in ASD. microarray analysis (International Schizophrenia Con- CHRNA7 transcripts were high in normal subjects less sortium, 2008; Stefansson et al., 2008; Masurel-Paulet than 1 year of age and declined until about 20 years of et al., 2010), over 150 individuals worldwide with age. Interestingly, the postnatal decline in CHRNA7 a frequency of 1/30,000–1/40,000 have been identified levels around 1 to 2 years of age corresponds to the onset that carry these deletions. Several studies have been of autistic symptoms (Yasui et al., 2011). Modifications conducted that demonstrate an association between of the cholinergic system are expected to affect multiple these microdeletions and neurodevelopmental pheno- brain areas including the cerebellum. The abnormal types such as bipolar depression, epilepsy, schizophre- anatomy of the cerebellum in ASD, together with nia (Stefansson et al., 2008), and autism, as well as cerebellar motor and cognitive deficits, suggests that developmental delays, mental retardation, and variable this structure might play an important role in ASD facial and digital dysmorphisms (Sharp et al., 2008; (Fatemi et al., 2012). Interestingly, recent studies Lowther et al., 2015). As described in detail in section II, conducted in vitro and in vivo showed a determinant the area of this microdeletion in 15q13.3 contains the role of a7 nAChRs in the gating of LTP versus long-term previously mentioned break points of BP4 and BP5 that depression at cerebellar mossy fiber-granule cell syn- flank the CHRNA7 gene (OMIM #118511) (Deutsch apses to regulate plasticity and behavioral adaptation et al., 2011). It was also recently suggested that (Prestori et al., 2013). a haploinsufficiency of CHRNA7 is causative for the

TABLE 6 Microdeletions of chromosome 15q13.3

Disease Reference Epilepsy Taske et al., 2002; Sharp et al., 2008; Stefansson et al., 2008; Helbig et al., 2009; Dibbens et al., 2009 Schizophrenia Stefannson et al., 2008 Psychiatric Disorders Masurel-Paulet et al., 2010 Autism Spectrum Disorder Sharp et al., 2008; Ben-Shachar et al., 2009; Pagnamenta et al., 2009; Miller et al., 2009; Deutsch et al., 2011; Mikhail et al., 2011 Intellectual Disability Sharp et al., 2008; Ben-Shachar et al., 2009 Dysmorphic features of face and digits and severe Liao et al., 2011 neurodevelopmental features Recurrent rage outbursts Cubells et al., 2011 Microdeletion Syndrome Masurel-Paulet et al., 2010; Hoppman-Chaney et al., 2013; Le Pichon et al., 2013; Schaaf, 2014 1054 Bertrand et al. various phenotypes connected to the 15q13.3 micro- E. Parkinson’s Disease deletion syndrome (Szafranski et al., 2010). In a review Parkinson’s disease (PD) is a neurodegenerative of the literature on genetic data derived from screening disease that is characterized by hypokinetic rigid samples with genomic arrays, it was concluded that the syndrome or paralysis agitans because of the tremor clinical presentations of 15q13.3 microdeletion syn- observed in the extremities (Lees, 2007). Historically drome were due to haploinsufficiency of the CHRNA7 recognized, this disease began to gain the attention gene and that there is a logical rationale to test a7 of clinicians only at the turn of the 19th century with nAChR agonists and PAMs for potential therapeutic the description by James Parkinson in his essay on the efficacy (Deutsch et al., 2011; Schaaf, 2014). Another Shaking Palsy published in 1817 to provide one of review of patient samples and literature suggested that the earliest descriptions of the clinical symptoms of CHRNA7 could potentially be a susceptibility factor for a disorder now known as Parkinson’s disease. Since both juvenile myoclonic epilepsy and childhood centro- then, it was shown that PD is the consequence of temporal spikes and epilepsy (Masurel-Paulet et al., degeneration in the ventrolateral tier of the substan- 2010). It was suggested that genetic screening and tia nigra pars compacta (Brigo et al., 2014; Grosset identification of the 15q13.3 microdeletions could result et al., 2014; Niccolini et al., 2014; Stoessl et al., 2014). in early intervention with special education and speech The observation that exposure to 1-methyl-4-phenyl- therapy, instead of waiting for symptoms to fully 1,2,3,6-tetrahydropyridine (MPTP) causes clinical manifest (Deutsch et al., 2011). For example, a patient symptoms that resemble PD suggested that lesions suffering from 15q13.3 deletion syndrome and also caused by this toxic agent could be used for animal presenting with recurrent episodes of aggressive rage models of PD (Javitch et al., 1984). However, this outbursts showed a dramatic decline in such events molecule does not show the same toxicity in human upon treatment with , an a7 PAM and acetylcholinesterase inhibitor, combined with behavior- and rodents, and the model of choice retained for rats al management (Cubells et al., 2011) to suggest the and mice is the local injection of 6-hydroxy-dopamine potential of this approach. into the brain regions containing dopaminergic cell It was further suggested that clinical intervention bodies, axons, or synaptic terminals (Perese et al., could also theoretically be possible with first, the 1989). identification of patients expressing this microdeletion, Degeneration of dopaminergic neurons causes multiple followed by the administration of an a7 nAChR agonist symptoms including hyposomnia, visual hallucinations, alone or in combination therapy with an a7 nAChR olfactory dysfunction, anxiety, cognitive disorders, and PAM in cases such as these in which there are decreased motor control deficits (Lees, 2007). As for most neurode- functional a7 nAChRs (Deutsch et al., 2011; Schaaf, generative diseases, prevalence increases with age and 2014). A preclinical proof-of-concept study for testing most recent epidemiologic studies indicate a prevalence of the efficacy of a7 nAChR agonists and PAMs in 15q13.3 PDofabout0.5%attheageof65years(WrightWillis microdeletion syndrome became feasible with the re- et al., 2010; Schneider and Obeso, 2015). Genome-wide cent development of a mouse model that captures the association studies of large PD cohorts revealed that main characteristics of the human syndrome, such as several genes might contribute to PD, with an important schizophrenia-like auditory processing deficits, in- role for a-synuclein, and that mutations in the Parkin creased seizure susceptibility, and increased body gene are associated with an early age of onset of PD weight (Fejgin et al., 2014). This model may provide (Ramanan and Saykin, 2013). a way to test compounds preclinically for their ability In the extrapyramidal system there is a balance to ameliorate symptoms in microdeletion syndrome between the dopaminergic projection from the substan- and establish whether clinical trials with a7 nAChR tia nigra pars compacta onto the striatum and the agonists or PAMs are warranted for microdeletion corticostriatal inputs that impacts the striatum’s pro- syndrome. jection to the thalamus and the feedback loop from the The recently discovered 15q13.3 microdeletion syn- thalamus back to the cortex. Dysfunction of the dopa- drome is marking a new step in the understanding of minergic projection from the substantia nigra directly the role of a7 nAChRs and cholinergic function in impacts upon the function of the striatum and therefore neurodevelopmental disorders. The combination of the on the cortical interactions with different brain nuclei. development of better analysis of allelic expression with The substantia nigra is an important player in partic- appropriate cognitive assessment will provide the in- ular for control of eye movement, motor planning, dispensable tools to identify the syndrome early and in reward seeking, or learning. Moreover, as the thalamus time for effective therapeutic intervention. The devel- can be considered as a relay between the sensory systems opment of an animal model will aid in the determination and the cortex, indirect impairment of the thalamus of whether patients with 15q13.3 microdeletion syn- caused by the dysfunction of the substantia nigra alters drome may be candidates for a7 nAChR agonist or PAM sensory perceptions (Müller et al., 2013; Barter et al., treatment. 2014). Therapeutic Potential of a7 nAChRs 1055

The observation that PD is associated with a specific (Court et al., 2000). Analysis of the cortical distribution of degeneration of dopaminergic neurons suggested that nAChRs, conducted with Western blots, revealed a re- adding exogenous dopamine might counterbalance the duced expression of both the a4b2 and a7 nAChRs in PD dysfunction of the substantia nigra. Successful introduc- patients (Burghaus et al., 2003), suggesting that a re- tion in the 1960s of levodopa, a brain permeable precursor duction of nAChR-mediated neurotransmission might of dopamine, marked a turning point in the treatment of aggravate the cognitive deficits associated with PD. In PD (Katzenschlager and Lees, 2002). Long-term treat- addition, impairment of cholinergic neurotransmission ment of PD with levodopa, however, includes significant in the premotor and motor areas could contribute to the side effects with motor dyskinesias, visual hallucinations, bradykinesia (slowness of movement) or other movement- etc. Statistical analysis of PD cohorts showed, however, associated functions. Simulation of a7 nAChRs might no toxicity associated with levodopa treatments or the therefore produce neuroprotective effects and represent acceleration of symptoms (Simuni and Stern, 1999). an important strategy to minimize the cognitive decline or Alternative treatments include deep brain electrical or impaired motor functions associated with PD. magnetic stimulation, which are thought to stimulate the Further analysis of the distribution of a7 nAChRs in remaining neurons in the substantia nigra (Sanghera human brain revealed an additional important expres- et al., 2004; Chang et al., 2011). Although pharmacolog- sion of these receptors in cerebellum, both post mortem ical treatments and stimulation represent a significant and in vivo (Court et al., 2000; Toyohara et al., 2009). In advance for patients, these therapies fail to change the view of the determinant role of the cerebellum in motor course or the progression of the disease. coordination as well as in cognitive processes, it will be Based on the observation of a lower prevalence of PD in of value to review if, and how, a7 nAChRs are altered in smokers, it was proposed that nicotine may exert a neuro- the cerebellum of PD patients. protective and possibly beneficial effect (Nefzger et al., As indicated above, long-term treatment with levo- 1968; Gorell et al., 1999; Quik, 2004). Experimental dopa causes multiple side effects including dyskinesia. evidence in animal models suggesting that nicotine in- Stimulation of nicotinic receptors reduced PD- creased production of neurotrophic factors further sup- associated dyskinesia by interaction with multiple re- ported this hypothesis (Maggio et al., 1998). Albeit other ceptor subtypes including a7 nAChRs (Quik et al., 2013; factors, including consumption, were proposed to Zhang et al., 2013, 2014). In agreement with these be associated with a reduction of PD prevalence, the observations, although nicotine reduced apomorphine- protective effects of nicotine were replicated in many induced rotational behavior in hemiparkinsonian rats studies. Furthermore, a reduction of the olfactory im- injected unilaterally with 6-hydroxydopamine, rota- pairment in PD was observed in PD-affected smokers tional behavior worsened during exposure to the nico- (Lucassen et al., 2014). tinic blocker (Han and Wang, 2007). Although the mechanisms by which nicotine could Different doses of nicotine, capable of producing activa- reduce the susceptibility to PD may involve different tion or desensitization, indicated that the rotational nAChRs subtypes, we shall focus the discussion on the behavior was only affected by desensitizing doses of possible role of a7 nAChRs. Examination of a-Btx binding nicotine, not by nAChR activation. The specific a7 in the monkey substantia nigra revealed moderate nAChR agonist ABT-107 at 0.03–1 mg/kg decreased labeling, indicative of a7 nAChR expression (Han et al., levodopa-induced dyskinesias in parkinsonian monkeys 2003; Kulak and Schneider, 2004). In monkeys, low doses (Zhang et al., 2014). Similar effects were reported for of MPTP that did not cause motor symptoms resulted in another selective a7 nAChR agonist, AQW051 (15 mg/kg) an increase in a-Btx binding, whereas a change in a-Btx (Di Paolo et al., 2014), indicating that this therapeu- binding was not observed in animals treated with an tic strategy can apply to higher mammals and may be acute high dose of MPTP or long-term escalating doses of beneficial in humans. It remains, however, to be de- MPTP, in which PD-like motor symptoms were present termined whether activating or desensitizing concen- (Kulak and Schneider, 2004). This was interpreted as trations of a selective a7 nAChR agonist will be required reflecting a compensatory elevation of a7 nAChR expres- in humans, because ABT-107 was efficacious at activat- sion in response to the low doses of MPTP and could ing and desensitizing doses and AQW051 at only desen- participate in minimizing the dysfunction caused by this sitizing doses in the MPTP monkey model (Bitner et al., molecule. Interestingly in the same study, the low doses 2010; Feuerbach et al., 2015). If desensitizing concen- of MPTP caused cognitive impairments, and brain trations are required, the effects on dyskinesia of regions involved in cognition (e.g., hippocampus, pre- a7 nAChR agonists may be achieved at the expense of frontal cortex and cingulate gyrus) did not show increases improvements in cognition. in a-Btx binding. PD is characterized first by movement disorders In contrast to the monkey, a-Btx binding in human that are thought to be caused initially by the neuro- revealed a high level of labeling in the substantia nigra degeneration of the dopaminergic neurons in the pars compacta, indicating that progressive atrophy of this substantia nigra. PD is also accompanied by choliner- brain area could affect a7 nAChR neurotransmission gic deficits and cognitive impairment in some patients. 1056 Bertrand et al.

The negative association between PD and smoking the main features that characterize cancer and the provided the first clues for the possible contribution of development of tumors. It is generally accepted that nAChRs in this disease. Most recent evidence is now cancer begins by a modification of DNA that triggers pointing to the role of a7 nAChRs in treating levodopa- abnormal cell division. Furthermore, when, in normal induced dyskinesias and cognitive impairment. conditions, cells begin aberrant division they undergo apoptosis and cancer cells are not stopped and can F. Nonneuronal Related Diseases initiate further divisions. Importantly, as the tumor grows, its general metabolism increases, requiring more The discoveries that the neuromuscular junction is oxygen and nutrients, and is accompanied by a neo- a chemical synapse and that these synaptic events are vascularization to support its metabolic requirements. mediated by nAChRs suggested that such neurotransmit- Cells that initiate migration and will ultimately cause ter receptors were restricted to the nervous system. metastases are thought to be changing fate and, However, expression of mRNAs in different tissues sur- temporarily, returning to a more embryonic cell type prisingly revealed that genes encoding for ligand-gated ion with the capacity of migration through lymphatic and channels are not exclusively expressed in neurons but can blood vessels to other organs. also be expressed in many other cell types. As an example, Smoking-associated lung cancers with more than NMDA receptors are also expressed in red blood cells about 200,000 new cases per year in the United States (Makhro et al., 2013). However, when considering biologic alone and more than 150,000 associated deaths per year, questions from a broader perspective, it became obvious are the most obvious cancers linked to nicotine. Lung that cells from different tissues might exploit the vast cancers are subdivided into non-small cell lung cancers repertoire of membrane proteins encoded in the genome. and small cell lung cancers, according to histologic In this respect, it is interesting to note that nAChRs are observations (DeSantis et al., 2014). The devastating link widely expressed throughout the body and that a7 between products and cancer results from the nAChRs are thought to play a role in many physiologic association of carcinogenic compounds such as nitros- functions outside the CNS. In this section we shall discuss amines N9-nitrosonornicotine and 4-(methylnitrosamino)- evidence for CHRNA7 expression in different tissues, the 1-(3-pyridyl)-1-butanone together with nicotine (Hecht, possible role(s) of a7 nAChRs in nonneuronal cells, and 2003; West et al., 2003). Although there is still debate their validity as a therapeutic target for nonneuronal about the carcinogenicity of nicotine itself, recent evidence related diseases. points to a possible effect of nicotine on long-term mini- 1. Cancer. Cancer is characterized by abnormal cell organ cultures (Ginzkey et al., 2014). Additional evidence division and proliferation and, sometimes, by migration for nicotine-induced mutations was provided by the recent of a small number of cells to form metastases (http:// analysis of nicotine and oxidative stress in lung cancer www.cancer.gov). The main categories of cancer (Bavarva et al., 2014). There is good agreement, however, include: onthefactthatnicotinepromotescellsurvivaland division, which are key factors for tumor growth and can Carcinoma - cancer that originates in the skin or in precipitate development of lung cancer in susceptible tissues that line or cover internal organs, (i.e., cells individuals (Davis et al., 2009; Schaal and Chellappan, of epithelial origin). Numerous subtypes of carci- 2014). Furthermore, the antiapoptotic activity of nicotine, noma exist to include adenocarcinoma, basal cell which is beneficial in other conditions like neurodegener- carcinoma, squamous cell carcinoma, and transi- ative diseases, is considered to be deleterious in lung tional cell carcinoma. cancer (Zeidler et al., 2007; Egleton et al., 2008; Schuller, Sarcoma - cancer that originates in bone, cartilage, 2012). An additional and determining factor in tumor fat, muscle, blood vessels, or other connective or formation associated with nicotine is its angiogenic supportive tissues (i.e., cells of mesenchymal activity of promoting neovascularization (Cooke and origin). Ghebremariam, 2008; Dasgupta and Chellappan, 2006; Leukemia - cancer that originates in blood-forming Arias et al., 2009; Lee and Cooke, 2012). A susceptibility tissues such as the bone marrow and causes large locus for lung cancer and nAChRs was found on chromo- numbers of abnormal blood cells to be produced some 15 but mapped to 15q25, which corresponds to the and enter the blood. position of the CHRNA5 gene encoding for the a5nAChR Lymphoma and myeloma - cancers that origi- subunit (Hung et al., 2008). Expression of CHRNA7 was, nate in the cells of the immune system. however, observed in human lung, indicating that a7 Central nervous system cancers – cancers that nAChRs might contribute to the regulation of lung originate in the tissues of the brain and spinal epithelia (Plummer et al., 2005). cord and are neuronal or glial in origin. Data pointing to the determinant role of nicotine in Whereas it would be beyond the scope of this work to the development of lung cancer have prompted numer- review all forms of cancer, their origins, outcomes, and ous studies using culture models and shown that pro- the possible role of nAChRs, it is important to delineate liferation could be reduced by use of nAChR antagonists Therapeutic Potential of a7 nAChRs 1057

(see for example Tsurutani et al., 2005; Improgo et al., mechanism that is critical for survival. The innate 2013). In view of the important role of a7 nAChRs, both immune system is activated during infection and injuries in terms of their expression levels and their relevance by initiating the release of proinflammatory cytokines for downstream biologic pathways to which they are such as interleukin-6 and tumor necrosis factor alpha associated, it was proposed that specific a7 nAChR (TNFa) to control infection and promote tissue repair antagonists such as toxins might be of use in cancer (Medzhitov, 2008). However, a balance must be main- treatment (Alama et al., 2011; Pillai and Chellappan, tained, because an excessive release could lead to a more 2012; Brown et al., 2012). severe inflammatory state and spread of inflammation Because angiogenesis is indispensable for tumor (Nathan, 2002). Neural reflex circuits that detect periph- growth, it is of interest to examine by which mechanism eral inflammation provide regulatory feedback via spe- stimulation of a7 nAChRs could lead to the develop- cific nerve signals to suppress the innate immune ment of neovascularization. Critical features of a7 response (Olofsson et al., 2012). The autonomic nervous nAChRs include their unique Ca2+ permeability and system has been shown to regulate this cytokine pro- wide range of expression in neuronal and nonneuronal duction via neural pathways involving the vagus nerve, cells including vascular endothelial cells. Biochemical which provides parasympathetic innervation to most pathway analysis revealed that several regulatory organ systems, with ACh being the principle neurotrans- elements, including adhesion factors, and stimulation mitter connecting the nervous system to the immune of vascular endothelial growth factor are modified by system. Collectively, these connections form the “cholin- nicotine and can be blocked by a-Btx, confirming the ergic anti-inflammatory pathway” (Wang et al., 2003). A determinant role of a7 nAChRs in these regulatory detailed review of the neural circuits involved in the processes (Heeschen et al., 2002; Hoshino et al., 2005; reflex control over the innate immune response has been Arias et al., 2009; Alamanda et al., 2012). Proangio- published elsewhere (Tracey, 2009). genesis mediated by a7 nAChRs was also observed in About 10 years ago, it was found, through use of models of age-related macular degeneration, which is antisense and pull-down experiments, that the cholin- one of the major ophthalmologic risks associated with ergic control of inflammation depended on the a7 nicotine consumption (Dom et al., 2011). nAChR subunit (Wang et al., 2003). Experiments con- Nicotine is associated with several forms of cancer in ducted with a7 nAChR knockout mice confirmed the which the common mechanisms of cell migration, tumor essential role of these receptors in the cholinergic anti- growth, etc., observed in lung cancer are replicated. For inflammatory pathway, with these mice exhibiting example, nicotine stimulated cell proliferation and in- higher TNFa serum levels over wild-type mice 2 hours creased cell survival in colon cancers, and these effects post lipopolysaccharide (LPS) injection (Wang et al., were inhibited by a-Btx (Cucina et al., 2012; Dinicola 2003). The vagus nerve has been shown to be a vital et al., 2013). In pancreatic cancers, nicotine was also component in this pathway, involving transduction of shown to stimulate the production of membrane-bound vagal signals via the celiac and superior mesenteric mucin, a protein known to induce tumor initiation and ganglia to result in release of in the development by modulating cellular growth, differenti- spleen (Vida et al., 2011) and in inhibition of proin- ation, transformation, adhesion, invasion, and immune flammatory cytokines through a7 nAChR signaling on surveillance (Kunigal et al., 2012; Momi et al., 2013). macrophages (Wang et al., 2003; Sun et al., 2013). Additional insights in the biochemical pathways Additional studies involving experimental models of implicated in nicotine-associated cancer are discussed local and systemic inflammation used electrical stimu- in a recent review (Grando, 2014). lation of the vagus nerve or cholinergic drugs acting via Although smoking-associated cancer is well acknowl- a7 nAChRs to modulate inflammation and showed that edged, the direct contribution of nicotine as a carcinogenic stimulation of a7 nAChRs results in blockade of endo- compound remains rather tenuous. Nonetheless, differ- thelial cell activation and the subsequent recruitment of ential expression of nAChRs was reported in several leukocytes during inflammation (Saeed et al., 2005). forms of cancer with notably overexpression of the a7 Stimulation of a7 nAChRs in a murine macrophage nAChRs. Because inhibition of these receptors was shown cell line by the agonists nicotine GTS-21 and choline to reduce tumor growth, neovascularization, and metas- have all been shown to inhibit LPS-induced TNFa and tasis, an a7 nAChR antagonist might broaden the high-mobility group box 1 (HMGB1) protein release to therapeutic possibilities to fight some forms of cancer. improve survival in experimental sepsis (Wang et al., 2004), which supports the role of a7 nAChR agonists as G. a7 Nicotinic Acetylcholine Receptors and the anti-inflammatory agents. It was initially postulated Immune Connection and later confirmed that in macrophages and other Inflammation caused by excessive cytokine produc- inflammatory-related cells a7 nAChRs interact with tion is the culprit in many diseases, leading to both janus kinase 2 (JAK2) to stimulate and phosphorylate human morbidity and mortality; however, at the same the anti-inflammatory signal transducer and activator time inflammation is an important part of the defense of transcription 3 (STAT3), a key component of the 1058 Bertrand et al.

STAT3-NF-kB cascade, which dimerizes to decrease receptors and a7 nAChRs (Deutschman and Tracey, 2014). proinflammatory cytokines, such as TNFa, HMGB1, This reaction in sepsis is extreme such that an anti-TNFa and interleukin (de Jonge and Ulloa, 2007; Marrero and antibody approach has met with limited success due to Bencherif, 2009). It was also demonstrated that stimu- the fact that this disease involves multiple cytokines lation of the vagus nerve attenuates inflammation in and requires a treatment that would have a broad effect. macrophages of STAT3 knockout mice (de Jonge and As mentioned in the introduction of this section, Ulloa, 2007). Recent data have further expanded this a selective a7 nAChR agonist, acting through the mechanism to demonstrate that a7 nAChR activation cholinergic anti-inflammatory pathway, could be more significantly induced microRNA, miR-124, a potential efficient at inhibiting production of proinflammatory modulator in the anti-inflammatory response (Sun cytokines by preventing the phosphorylation of STAT3 et al., 2013). and in turn preventing activation of the NF-kB pathway Outside the CNS, a7 nAChRs are thought to have (Peña et al., 2010). It has been shown that administra- a pivotal role in peripheral immune cells via the tion of nicotine to mice after cecal ligation and puncture cholinergic anti-inflammatory pathway, with the poten- or LPS treatment significantly reduced serum levels of tial for therapeutic intervention in several inflamma- HMGB1 (Huston and Tracey, 2011) by suppressing Toll- tory diseases such as endotoxemia (Rosas-Ballina et al., like receptor expression through activation of the P13K/ 2008), rheumatoid arthritis (van Maanen et al., 2009, Akt pathway (Kim et al., 2014b). It has been reported 2010), artherosclerosis (Bencherif et al., 2011), and that HMGB1 has a deleterious effect on epithelial and inflammatory bowel disease (Sandborn, 1999). The a7 endothelial barriers, causing a failure at these sites of nAChR subunit is widely expressed in immune cells protection and making the events of sepsis result in including monocytes (Matsunaga et al., 2001), macro- organ as well as mitochondrial dysfunction by depleting phages (Rosas-Ballina et al., 2008), B and T cells (Fujii cells of ATP needed to support survival (Deutschman et al., 2008), and dendritic cells (Aicher et al., 2003). and Tracey, 2014). A recent paper demonstrated how a7 More recently, a7 nAChR subunit expression has been nAChR signaling inhibits the events of inflammasome found within the celiac and superior mesenteric ganglia activation, as well as the release of mitochondrial DNA, and splenic nerve fibers, key components of the vagus through the use of a selective agonist or vagus nerve nerve signaling pathway to the spleen (Downs et al., stimulation, all of which pointed to a direct role of a7 2014). nAChRs expressed on mitochondria in this cascade of Stimulation of the vagus nerve was found to induce events (Lu et al., 2014). Further evidence was provided multiple effects, including a reduction of inflammation. in a7 nAChR knockout mice where there was a failure of Results accumulated over the past 10 years point to inhibition of inflammasome activation with vagal nerve a role of a7 nAChRs in the regulation of inflammatory stimulation (Lu et al., 2014). Collective understanding pathways, and it was proposed that ACh released of these studies and events may eventually lead to during vagus nerve stimulation mediates its effects a more efficacious method of treatment of the compli- through stimulation of this nAChR subtype. As vagus cations of sepsis. nerve stimulation effects were mimicked by a7 nAChR Defined as a systemic response to inflammation, agonists, this opens new strategies for the treatment of sepsis is expected to benefit from the findings that inflammation with a7 nAChR agonists. inflammation may be downregulated through the cho- 1. Sepsis. Severe sepsis is the third leading cause of linergic anti-inflammatory pathway. This pathway can death in most developed countries and accounts for be activated by stimulation of the vagus nerve and the .9% of overall deaths in the United States alone (Ulloa, response is mediated through a7 nAChRs. Further 2005). Sepsis has been defined as systemic inflamma- study of the role of a7 nAChR activation in sepsis will tion in response to an infection, whereas "severe sepsis" yield a better understanding of mechanisms to curb refers to organ dysfunction observed during systemic inflammasome activation and perturbation of mito- inflammation with or without an associated infection chondrial activity. (Ulloa and Tracey, 2005). To date there are no approved 2. Inflammatory Bowel Disease, Focus on Ulcerative drugs specific for the treatment of sepsis, a costly Colitis. Inflammatory bowel disease (IBD), which illness that is poorly understood (Deutschman and encompasses Crohn’s disease (CD) and ulcerative colitis Tracey, 2014). It is known that severe sepsis involves (UC), affects approximately 3.6 million patients in the a flood of cytokine production, including TNFa, United States and Europe combined (Loftus, 2004). interleukin-1b, interleukin-10, interferon gamma, and Unlike CD, UC is a disease of nonsmokers, exhibiting HMGB1 (Bencherif, 2009; Bencherif et al., 2011), in a fivefold increased risk, whereas smoking decreased which HMGB1 is a late lethal mediator of sepsis (Kim the risk of disease and had a favorable effect on disease et al., 2014b). This flood of cytokines involves the course and severity (Harries et al., 1982). Conventional participation of intracellular and extracellular “danger treatments include corticosteroids and aminosalicy- signals” that activate inflammasomes to mediate the lates but only 60–80% of IBD sufferers achieve release of these cytokines and involves both P2X7 remission with these drugs, however, not without Therapeutic Potential of a7 nAChRs 1059 complications due to unwanted side effects. UC has It will be interesting if a selective a7 nAChR agonist been associated with altered STAT3 expression and can attenuate the severity of colitis and reduce the phosphorylation, and it is possible that an a7 nAChR unwanted side effects of other therapies, as well as agonist can reverse that alteration in the STAT3 clarify the mixed results that have been associated with pathway (Wang et al., 2004). Thus, therapeutic in- transdermal nicotine. It will also be of significance if tervention with new small molecule a7 nAChR agonists the human-specific CHRFAM7A gene, shown to be has potential that would be greatly needed. expressed in human intestinal epithelial cells (Dang It has been shown that nicotine, given by transdermal et al., 2015), plays a mechanistic role in regulating a7 or enema delivery, attenuated disease in colitis models nAChR activity in IBD. induced by 2,4,6-trinitrobenzenesulfonic acid (TNBS) or Inflammatory bowel disease (IBD) affects a growing dextran sulfate sodium (DSS), as well as in clinical number of people throughout the world. Although the trials, with improvements in histologic and global etiology of IBD in most cases remains elusive, the need for clinical scores of colitis (Pullan et al., 1994). However, additional treatment strategies that could, at least, several clinical trials using nicotine have led to mixed attenuate the effects is rapidly increasing, because results because of the limitations of associated side current therapies are not effective in all patients and effects (McGilligan et al., 2007). In a TNBS-induced have unwanted side effects. The observation that stimu- colitis model, anabaseine (GTS-21), an a7 nAChR lation of the vagus nerve can attenuate the symptoms, agonist, showed less tissue damage, less myeloperox- together with results from animal models, suggests that idase activity, less TNFa production in the colon, and activation of a7 nAChRs might prove beneficial for the inhibited NF-kB activation (Bai et al., 2007). Other treatment of IBD. studies have shown that acetylcholinesterase inhibitors 3. a7 Nicotinic Acetylcholine Receptors and effectively modulate the acute colonic inflammation in Osteoarthritis. Smoking-associated side effects reveal colitis possibly by an increase in the cholinergic tone of a plethora of activity of nicotine on different organ the colon (Miceli and Jacobson, 2003). Vagal nerve systems. For example, smoking is thought to affect bone stimulation via ACh also ameliorates disease activity healing (Truntzer et al., 2015; Miller, 2014) and repre- (van der Zanden et al., 2009). In this same study, it has sents a detrimental factor when considering dental been shown that vagotomized a7 nAChR knockout mice implants (Baig and Rajan, 2007; Heitz-Mayfield and display more severe colitis than wild-type mice. More Huynh-Ba, 2009). In other studies it is suggested that recently, nicotine suppressed hyperexcitability of co- nicotine might reduce or prevent osteoarthritis (OA) lonic dorsal root ganglion neurons in DSS-induced (Gullahorn et al., 2005), a finding that is supported by colitis, which was attenuated by the a7 nAChR antag- analysis of total knee replacement surgery in large onist MLA (Abdrakhmanova et al., 2010). In the same Chinese populations where there was a 51% reduction study, nicotine failed to suppress hyperexcitability of in the number of smokers versus nonsmokers requiring dorsal root ganglion neurons in a7 nAChR knockout surgery (Leung et al., 2014). However, a less clear image mice. In a 2011 study, nicotine attenuated oxazolone- emerges from other studies, indicating no positive induced colitis (a model of UC) but worsened TNBS- reduction in OA associated with smoking (Wilder induced colitis (a model of CD), and these opposing et al., 2003). Because it was shown that nicotine exerts effects were dependent on the differential expression of both positive and negative modulatory effects as a func- a7 nAChRs on CD4+ T cells in the colon, with more tion of its concentration (Rothem et al., 2009), differ- than two-thirds of CD4+ T cells expressing a7nAChRs ences in conclusions from the various studies might in the UC model versus less than 3% of CD4+ Tcells depend upon the amount of circulating nicotine in the in the CD model (Galitovskiy et al., 2011). This study patient’sblood. also associated the improvement in the UC model with Inflammation is an important factor in OA, and the an immunosuppressive effect of increasing CD25+/ role of cholinergic modulation of the inflammatory Foxp3+ regulatory T cells and reducing Th17 cells pathway provides insight into the understanding of versus in the CD model in which the Th17 pro- the complex mechanisms underlying OA. Examination inflammatory cells were increased. More recently, it of the inflamed synovium from OA patients revealed was shown that the dose and route of administration of expression of a7 nAChRs predominantly in the intimal nicotine was critical to the protective outcome in the lining in vivo, as well as in fibroblast-like synoviocytes DSS-induced colitis model, with administration of low in vitro (van Maanen et al., 2009). Nicotine treatment (12.5–50 mg/ml), but not high, doses of oral nicotine in decreased infiltration of inflammatory cells into the DSS-treated mice having a significant effect on de- synovial tissue and bone erosion in a DBA/1 mouse creasing disease severity, histologic scores of damage model of collagen-induced arthritis (van Maanen et al., and TNFa levels in colon tissue (AlSharari et al., 2013). 2010). As a complement to these observations, a7 These data are consistent with a role for activation nAChR knockout mice present an exacerbated response rather than desensitization of a7 nAChRs in the DSS- in the collagen-induced arthritis model (van Maanen induced colitis model. et al., 2010). The presence of choline acetyltransferase 1060 Bertrand et al. mRNA and marked expression of a7 nAChRs was build-up and total cholesterol and triglyceride levels reported in the pannus of the knee joint, supporting as well as lowering blood pressure in these mice. This the hypothesis that this nAChR subtype is involved in group plans to study this compound using a7 nAChR/ OA (Forsgren, 2012). apolipoprotein E double knockout mice to establish a more Because nicotine interacts with many nAChRs sub- definitive connection between a7 nAChRs and atheroscle- types, it is important to examine the effects of selective rosis. Chronic hypertension has also been shown to a7 nAChR compounds including agonists and PAMs. contribute to end-organ damage and a study tested if a7 The attenuation of cytokine release in stimulated whole nAChR dysfunctional signaling is also involved. In this blood cells isolated from patients with OA by nicotine or study, a7nAChRknockoutmicewereusedinatwo- GTS-21 supports the hypothesis of a predominant role kidney one-clip hypertension model, which caused the of a7 nAChRs in the inflammatory response (Bruchfeld release of proinflammatory cytokines and more severe et al., 2010). A similar conclusion was reached from organ damage than in wild-type mice (Li et al., 2011). In experiments conducted with the a7 nAChR agonist addition, the a7 nAChR agonist PNU-282987 was used to AR-R17779, which attenuated the elevation in TNFa in chronically treat spontaneously hypertensive rats and the blood and synovial tissue, delayed the onset of shown to attenuate the inflammatory effects induced in disease, and was protective against joint destruction this hypertension model (Li et al., 2011). (van Maanen et al., 2009). In contrast, the role of a7 nAChRs in heart rate Joint afflictions are a serious cause of morbidity, with variability (HRV) was examined in endotoxemic rats effects lasting for decades. Improving OA would have because HRV, a reflection of the strength of vagus nerve broad consequences for the general population and signaling, has been used as a noninvasive measure of more specifically for the elderly. Given the strong link sepsis (Mazloom et al., 2013). In this study, the a7 between inflammation and OA, it is readily understood nAChR agonist PHA-543613 was unable to prevent the that molecules acting on inflammatory pathways will be reduction in HRV that occurs in endotoxemic rats but beneficial for this disease. Preliminary data obtained was able to modulate the effect of LPS on body with a7 nAChR agonist tool compounds suggest that temperature, supporting past research that demon- stimulation of these receptors represents a novel strat- strates a tonic role for a7 nAChRs in systemic in- egy to treat OA that needs to be followed up with more flammation (Andersson and Tracey, 2012; Mazloom potent a7 nAChR agonists displaying better drug-like et al., 2013). However, pretreatment with the a7 nAChR properties. antagonist, MLA, was able to further reduce HRV and induce a febrile response in endotoxemic rats but had no H. a7 Nicotinic Acetylcholine Receptors and effect on either measure in naive rats. Future analysis Cardiac Function with selective a7 nAChR agonists or a7 nAChR knock- Atherosclerosis is an additional inflammatory condi- out mice will continue to provide insight into the role of tion that is a major contributor to cardiovascular a7 nAChRs in heart rate dynamics during sepsis. disease and often leads to sudden cardiac arrest or Cardiovascular diseases are recognized to be one of myocardial infarction. In the progression of atheroscle- the major factors causing premature death, with ath- rosis, inflammatory cells are engaged to release a host of erosclerosis being one of multiple contributory factors. proinflammatory cytokines, chemokines, adhesion mol- Atherosclerosis is often closely related to inflammation ecules, and growth factors (Libby, 2002). a7 nAChRs are and would therefore represent another possible target expressed on rat atrium and localized to the endothelial for a7 nAChR specific compounds. layer (Mazloom et al., 2013) and in the intracardiac ganglion (Cuevas and Adams, 1994; Cuevas and Adams, I. The Role of a7 Nicotinic Acetylcholine Receptors in 1996). As previously mentioned, the a7 nAChRs are also Renal Function expressed on immune cells and are key mediators in the Data from the Centers for Disease Control and Pre- cholinergic anti-inflammatory pathway in which a7 vention reveal that more than 20 million Americans nAChRs become activated via release of ACh from the may have kidney disease, with the risk increasing each vagus nerve to attenuate the inflammatory response year. Kidney disease falls into several categories but the that occurs by activation of the JAK2-STAT3 and focus here will be on the two main forms. The sudden suppression of the NF-kB pathways (Wang et al., loss of kidney function is referred to as acute kidney 2004). In a recent study using infusion of angiotensin II injury (AKI), also known as acute renal failure, which to induce atherosclerosis in apolipoprotein E knockout can occur due to traumatic injury, damage from shock or mice, it was demonstrated that AR-R17779, a selec- sepsis, damage from drugs or toxins, obstruction, or tive a7 nAChR agonist, attenuated atherosclerosis as sudden reduction in blood flow. When kidney damage well as abdominal aortic aneurysms via decreased and decreased function last longer than 3 months, it is interleukin-6 and interleukin-1b gene expression in referred to as chronic kidney disease (CKD) and is aortic tissue (Hashimoto et al., 2014). In addition, a devastating illness that has recently increased at an AR-R17779 also decreased atherosclerotic plaque alarming rate and now affects over 13% of the Therapeutic Potential of a7 nAChRs 1061 population (Couser et al., 2011), caused primarily by cells and renal dysfunction), was also investigated with diabetes (type 1 and 2) and high blood pressure. The use of a7 nAChR agonists for efficacy in preclinical involvement of a7 nAChRs in renal disease was shown models. Pretreatment with nicotine or GTS-21 attenu- in a study that found nicotine pretreatment protected ated acute tubular injury and renal dysfunction in the mice from renal dysfunction in a dose-dependent manner I/R model in rats but was not significantly protective if and that this protection was absent in a7nAChR the agonists were administered 2 hours after the onset knockout mice (Sadis et al., 2007). Subsequent studies of the injury (Yeboah et al., 2008). This study also showed that a7 nAChRs are strongly expressed in kidney demonstrated that functional a7 nAChRs were detected cortex with the highest protein levels in the proximal in rat tubular epithelial cells, which are also involved in tubules compared with the distal tubules (Rezonzew the inflammatory response of this AKI model, and et al., 2012). Flow cytometry on human kidney cells and suggested a local cholinergic effect. More recently, an cell lines demonstrated the expression of a7, a4, and b2 innovative approach demonstrated that an ultrasound nAChR subunits, with a7nAChRbeinghigherthanthe treatment 24 hours before I/R also prevented renal other two subunits in the proximal tubule epithelial cell injury in mice (Gigliotti et al., 2013). It was further line (HK-2) and renal glomerular endothelial cells shown that cytisine, an a7 nAChR agonist, also trig- (Chatterjee et al., 2012). Collectively, these studies place gered this protective effect. The protective effect of a7 nAChRs in areas affected by renal disease. ultrasound pretreatment was abrogated in a7 nAChR Severe sepsis and septic shock are major contributing knockout mice or by a-Btx treatment in wild-type mice, factors leading to AKI, because the kidney is the main suggesting involvement of the splenic “cholinergic anti- target of the proinflammatory assault. AKI is also inflammatory pathway” and activation of a7 nAChRs in a major issue for many intensive care patients, account- causing the reduction in the inflammation in this injury ing for more than 50% of patients affected with AKI model. Splenectomy and adoptive transfer showed that (Zarjou and Agarwal, 2011), and to date there is no the spleen and CD4+ T cells mediated the protective effective treatment. Sepsis-induced AKI has a patho- effects of ultrasound pretreatment. This study revealed genesis that involves multiple pathways and requires the importance of an intact spleen for the efficacy of a7 a therapeutic approach that would attack this compli- nAChR agonists, because activation of adrenergic cated cascade of events. An a7 nAChR agonist ap- receptors on the spleen activated CD4+ T cells to proach, involving the cholinergic anti-inflammatory stimulate the production of ACh, triggering the activa- pathway, may be beneficial. In an LPS-induced AKI tion of a7 nAChRs and the accompanying anti- sepsis model, nicotine and GTS-21 attenuated kidney inflammatory response (Gigliotti et al., 2013). This injury with a reduction in serum TNFa levels as well as study clearly demonstrated the role of a7 nAChRs in in kidney levels of TNFa, chemokine CCL2, and chemo- AKI, as well as the importance of the spleen versus kine CXCL10. Both nicotine and GTS-21 also improved a direct effect on the kidney. renal function as measured by blood-urea-nitrogen Cigarette smoking has been shown to be a factor levels and leukocyte infiltration (Chatterjee et al., associated with high risk for several chronic diseases, 2012). Mice with LPS-induced AKI exhibited signifi- with CKD being no exception, because cigarette smoking cantly enhanced renal proteasome activity compared has contributed to the progression of this disease (Orth, with saline control mice, but treatment with nicotine or 2000, 2002). In the well-validated 5/6 nephrectomized GTS-21 attenuated both the ATP-dependent and in- model of human CKD disease, rats exhibited increased dependent renal proteasome activity induced by LPS proteinuria and increased oxidative stress, and chronic (Chatterjee et al., 2012). It was concluded by these nicotine pretreatment of the rats further exacerbated the studies that proteasome inhibition preserves the in- proteinuria and oxidative stress (Rezonzew et al., 2012). hibitor of kappa B alpha-NF-kB complex and that a7 Pretreatment with the a7 nAChR antagonist MLA nAChR agonists regulate proteasome activity, which significantly improved the glomerular injury score as supports previous studies demonstrating that a7 well as reduced oxidative stress. This result is in nAChR agonism regulates protein turnover by partial contrast to the beneficial effects of a7 nAChR agonists inhibition of proteosome activity (Rezvani et al., 2007). in AKI, and these opposing effects in AKI versus CKD In addition, many kidney diseases have been associated models may parallel the contrasting effects of nicotinic with altered STAT3 expression and phosphorylation agonists in UC and CD models of IBD. However, more (Liu et al., 2014) and it is possible that a7 nAChR research is needed in this area with the use of safer and agonists may reverse that alteration, because they have more selective agonists than nicotine. In addition, been shown to be neuroprotective through the JAK2/ studies need to be performed across species, age, and STAT3 and NF-kB pathways (de Jonge and Ulloa, 2007; sex to ensure consistent results and to account for the Marrero and Bencherif, 2009). sex differences noted in both preclinical (Kang et al., Ischemia-reperfusion (I/R) injury, another model of 2014) as well as clinical studies demonstrating that sex human AKI with the characteristic signs of kidney plays a role in the prediction of renal decline (Halbesma inflammation (i.e., infiltration of circulating immune et al., 2008). From preclinical studies, the sex difference 1062 Bertrand et al. resulted in greater renovascular vasodilation in female CHRFAM7 on the same chromosome arm that encodes rat kidney tissue with the suggestion that it could be the dup a7 protein. Most recent work using different facilitated by estrogen having a direct effect on a7 techniques combined with FISH examination have nAChR downstream signaling (El-Mas et al., 2011). surprisingly revealed an unforeseen degree of variation Overall, the studies mentioned in this section high- at this gene locus in the human population. Moreover light the recent interest in a7 nAChRs and kidney- a correlation emerges between these variants in chro- related diseases. The association between smoking and mosome 15 and cognitive performance in patients with CKD is illustrative of the presence of a7 nAChRs in the a variety of neurologic and psychiatric disorders. De- kidney and their relevance in renal function. Prelimi- letion of CHRNA7 observed in some patients correlates nary data from animal models indicate, however, that with severe cognitive impairment. These genetic find- by reducing inflammation, a7 nAChR agonists should ings further support the hypothesis of the involvement be beneficial for certain kidney diseases, most notably of a7 nAChRs in cognition that was initially suggested AKI. On the contrary, it was found that treatment with by studies with a7 nAChR agonists and PAMs. a7 nAChR antagonists showed beneficial outcomes in To exploit the promise offered by selectively targeting other renal conditions such as CKD. Refined character- a7 nAChRs, efforts were dedicated to the identification ization and classification of the kidney disease and of selective a7 nAChR agonists to serve as new phar- design of adequate treatment regimens with a7 nAChR macological tools and as therapeutic interventions. agonists or antagonists may allow novel approaches for These studies conducted by several laboratories yielded treating renal disorders. a panoply of small molecules displaying exquisite binding selectivity for a7 nAChRs. After the identifica- tion of PNU-282987, chemical structures containing V. Conclusions a quinuclidine moiety represent the majority of com- Our understanding of the nervous system has been pounds, showing sufficient discrimination of a7 versus advanced by DNA cloning and sequencing that has other nAChR subtypes. The close structural homology allowed the discovery of entire families of genes encod- among the ligand-gated ion channels offered an addi- ing for neurotransmitter receptors. The nAChRs are tional challenge because many molecules displaying a good example of the diversity existing within a single agonistic properties at the a7 nAChR acted as antago- receptor family. Given its structure and particular nists of the 5-HT3 receptor. Nonetheless, molecules such physiologic properties, which include a high Ca2+ as RG3487 and encenicline (EVP-6124) displayed ade- permeability, the a7 nAChR has been the center of quate profiles to be introduced into clinical trials. Some attention of many laboratories, opening up speculation success was achieved in reducing activity at the 5-HT3 about its physiologic role in cognitive processes and its receptor with a7 nAChR agonists such as TC-5619, contribution in neurologic and psychiatric diseases. AQW051, and ABT-126, which were also studied in Structure-function studies conducted at the homo- clinical trials through Phase 2. meric a7 nAChRs concluded that a functional complex is Because a7 nAChR agonists were primarily targeted composed of five subunits arranged around an axis of as cognitive enhancers, novel chemical compounds were pseudosymmetry with the ion channel in the center. mainly tested in AD or schizophrenic patients. One of Each subunit spans the membrane four times, with the the difficulties encountered in clinical studies relates to N and C termini facing the extracellular domain. The the cognitive tests employed to examine the efficacy of channel pore is made by the assembly of the five a7 nAChR agonists and the multiplicity of available identical a7 subunits in a barrel-like manner and lined tests (e.g., ADAS-Cog 13, Cog-State, RBANS, MCCB, by the second TMD. Specific cationic selectivity of the a7 etc.) and the lack of consensus to date on the most nAChR is provided by the specificity of the amino acids effective testing instruments. Nonetheless, progress of the second TMD that are pointing toward the ionic made with the a7 nAChR agonist encenicline, which is pore. The LBD, where ACh binds, lies at the interface now in Phase 3 clinical trials for AD and schizophrenia, between two adjacent a7 subunits, and, given its illustrates the continued hope that use of an a7 nAChR homomeric structure, the a7 nAChRs display five agonist will be beneficial in the treatment of cognitive identical LBDs. Recent studies, however, indicate that deficits in these disorders. in some conditions (in vivo and in vitro), a7 subunits can Development of new molecules acting at a7 nAChRs assemble with another subunit, with heteromeric a7 is not limited to direct acting agonists and antagonists, nAChRs containing b2ora7 dup subunits, which have but data from the literature illustrate the feasibility of been described. These heteromeric nAChRs may display identifying allosteric modulators. Binding at a site different physiologic or pharmacological characteristics. distinct from the ACh orthosteric site, these compounds Recent detailed genetic studies have demonstrated are able to modulate receptor function by potentiating the complex nature of the chromosome localization of or inhibiting the response to a ligand and, in some cases, CHRNA7, the gene encoding for the a7 nAChR, and the to reduce or suppress agonist-induced desensitization. presence in human of a duplication of exons 5-10 in Preclinical studies have already shown the potential of Therapeutic Potential of a7 nAChRs 1063 these molecules as cognitive enhancers, and it is likely Alamanda V, Singh S, Lawrence NJ, and Chellappan SP (2012) Nicotine-mediated induction of E-selectin in aortic endothelial cells requires Src kinase and E2F1 that new and even more active compounds will be transcriptional activity. Biochem Biophys Res Commun 418:56–61. 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