Paul Whiteaker

Functional Nicotinic Acetylcholine Receptors Containing α6 Subunits Are on GABAergic Neuronal Boutons Adherent to Ventral Tegmental Area Dopamine Neurons

Diverse nicotinic acetylcholine receptor (nAChR) subtypes containing different subunit combinations can be placed on nerve terminals or soma/dendrites in the ventral tegmental area (VTA). nAChR α6 subunit message is abundant in the VTA, but α6*–nAChR cellular localization, function, pharmacology, and roles in cholinergic modulation of dopaminergic (DA) neurons within the VTA are not well understood. Here, we report evidence for α6β2*–nAChR expression on GABA neuronal boutons terminating on VTA DA neurons. α-Conotoxin (α-Ctx) MII labeling coupled with immunocytochemical staining localizes putative α6*–nAChRs to presynaptic GABAergic boutons on acutely dissociated, rat VTA DA neurons. Functionally, acetylcholine (ACh) induces increases in the frequency of bicuculline-, picrotoxin-, and 4-aminopyridine-sensitive miniature IPSCs (mIPSCs) mediated by GABAA receptors. These increases are abolished by α6*–nAChR-selective α-Ctx MII or α-Ctx PIA (1 nm) but not by α7 (10 nm methyllycaconitine) or α4* (1 μm dihydro-β-erythroidine)–nAChR-selective antagonists. ACh also fails to increase mIPSC frequency in VTA DA neurons prepared from nAChR β2 knock-out mice. Moreover, ACh induces an α-Ctx PIA-sensitive elevation in intraterminal Ca2+ in synaptosomes prepared from the rat VTA. Subchronic exposure to 500 nm nicotine reduces ACh-induced GABA release onto the VTA DA neurons, as does 10 d of systemic nicotine exposure. Collectively, these results indicate that α6β2*–nAChRs are located on presynaptic GABAergic boutons within the VTA and modulate GABA release onto DA neurons. These presynaptic α6β2*–nAChRs likely play important roles in nicotinic modulation of DA neuronal activity.

Attenuation of CNS inflammatory responses by nicotine involves α7 and non-α7 nicotinic receptors.

A considerable number of in vivo studies have demonstrated that the cholinergic system can dampen the peripheral immune response, and it is thought that the α7-nicotinic acetylcholine receptor (nAChR) subtype is a key mediator of this process. The goal of the present study was to determine if nicotine modulates immunological mechanisms known to be involved in the development of experimental autoimmune encephalomyelitis (EAE), a mouse model for CNS autoimmune disease, via α7-nAChRs. Here we show that nicotine exposure attenuates EAE severity and that this effect is largely abolished in nAChR α7 subunit knock-out mice. However, nicotine exposure partially retains the ability to reduce lymphocyte infiltration into the CNS, inhibit auto-reactive T cell proliferation and helper T cell cytokine production, down-regulate co-stimulatory protein expression on myeloid cells, and increase the differentiation and recruitment of regulatory T cells, even in the absence of α7-nAChRs. Diverse effects of nicotine on effector and regulatory T cells, as well as antigen-presenting cells, may be linked to differential expression patterns of nAChR subunits across these cell types. Taken together, our data show that although α7-nAChRs indeed seem to play an important role in nicotine-conferred reduction of the CNS inflammatory response and protection against EAE, other nAChR subtypes also are involved in the anti-inflammatory properties of the cholinergic system.

Synthesis and Characterization of 125I-α-Conotoxin ArIB[V11L;V16A], a Selective α7 Nicotinic Acetylcholine Receptor Antagonist

The α7 nicotinic acetylcholine receptors (nAChRs) are widely expressed both in the central nervous system (CNS) and periphery. In the CNS, 125I-α-bungarotoxin is commonly used to identify α7 nAChRs specifically. However, α-bungarotoxin also interacts potently with α1* and α9α10 nAChRs, two receptor subtypes in peripheral tissues that are colocalized with the α7 subtype. [3H]Methyllycaconitine is also frequently used as an α7-selective antagonist, but it has significant affinity for α6* and α9α10 nAChR subtypes. In this study, we have developed a highly α7-selective α-conotoxin radioligand by iodination of a naturally occurring histidine. Both mono- and diiodo derivatives were generated and purified (specific activities were 2200 and 4400 Ci mmol-1, respectively). The properties of the mono- and diiodo derivatives were very similar to each other, but the diiodo was less stable. For monoidodo peptide, saturation binding to mouse hippocampal membranes demonstrated a Kd value of 1.15 ± 0.13 nM, similar to that of 125I-α-bungarotoxin in the same preparations (0.52 ± 0.16 nM). Association and dissociation kinetics were relatively rapid (kobs for association at 1 nM was 0.027 ± 0.007 min-1; koff = 0.020 ± 0.001 min-1). Selectivity was confirmed with autoradiography using α7-null mutant tissue: specific binding was abolished in all regions of α7-/- brains, whereas wild-type mice expressed high levels of labeling and low nonspecific binding. 125I-α-conotoxin ArIB[V11L; V16A] should prove useful where α7 nAChRs are coexpressed with other subtypes that are also labeled by existing ligands. Furthermore, true equilibrium binding experiments could be performed on α7 nAChRs, something that is impossible with 125I-α-bungarotoxin.

αConotoxin Arenatus IB[V11L,V16D] Is a Potent and Selective Antagonist at Rat and Human Native α7 Nicotinic Acetylcholine Receptors

A recently developed α-conotoxin, α-conotoxin Arenatus IB-[V11L,V16D] (α-CtxArIB[V11L,V16D]), is a potent and selective competitive antagonist at rat recombinant α7 nicotinic acetylcholine receptors (nAChRs), making it an attractive probe for this receptor subtype. α7 nAChRs are potential therapeutic targets that are widely expressed in both neuronal and non-neuronal tissues, where they are implicated in a variety of functions. In this study, we evaluate this toxin at rat and human native nAChRs. Functional α7 nAChR responses were evoked by choline plus the allosteric potentiator PNU-120596 [1-(5-chloro-2,4-dimethoxy-phenyl)-3-(5-methyl-isoxazol-3-yl)-urea] in rat PC12 cells and human SH-SY5Y cells loaded with calcium indicators. α-CtxArIB[V11L,V16D] specifically inhibited α7 nAChR-mediated increases in Ca2+ in PC12 cells. Responses to other stimuli, 5-I-A-85380 [5-iodo-3-(2(S)-azetidinylmethoxy)pyridine dihydrochloride], nicotine, or KCl, that did not activate α7 nAChRs were unaffected. Human α7 nAChRs were also sensitive to α-CtxArIB[V11L,V16D]; acetylcholine-evoked currents in Xenopus laevis oocytes expressing human α7 nAChRs were inhibited by α-CtxArIB[V11L,V16D] (IC50, 3.4 nM) in a slowly reversible manner, with full recovery taking 15 min. This is consistent with the time course of recovery from blockade of rat α7 nAChRs in PC12 cells. α-CtxArIB[V11L,V16D] inhibited human native α7 nAChRs in SHSY5Y cells, activated by either choline or AR-R17779 [(2)-spiro[1-azabicyclo[2.2.2]octane-3,59-oxazolidin]-29-one] plus PNU-120596. Rat brain α7 nAChRs contribute to dopamine release from striatal minces; α-CtxArIB[V11L,V16D] (300 nM) selectively inhibited choline-evoked dopamine release without affecting responses evoked by nicotine that activates heteromeric nAChRs. This study establishes that α-CtxArIB[V11L,V16D] selectively inhibits human and rat native α7 nAChRs with comparable potency, making this a potentially useful antagonist for investigating α7 nAChR functions.

Heteromeric α7β2 Nicotinic Acetylcholine Receptors in the Brain

The α7 nicotinic acetylcholine receptor (α7 nAChR) is highly expressed in the brain, where it maintains various neuronal functions including (but not limited to) learning and memory. In addition, the protein expression levels of α7 nAChRs are altered in various brain disorders. The classic rule governing α7 nAChR assembly in the mammalian brain was that it was assembled from five α7 subunits to form a homomeric receptor pentamer. However, emerging evidence demonstrates the presence of heteromeric α7 nAChRs in heterologously expressed systems and naturally in brain neurons, where α7 subunits are co-assembled with β2 subunits to form a novel type of α7β2 nAChR. Interestingly, the α7β2 nAChR exhibits distinctive function and pharmacology from traditional homomeric α7 nAChRs. We review recent advances in probing the distribution, function, pharmacology, pathophysiology, and stoichiometry of the heteromeric α7β2 nAChR, which have provided new insights into the understanding of a novel target of cholinergic signaling.

Differential modulation of EAE by α9*- and β2*-nicotinic acetylcholine receptors.

Nicotine is a potent inhibitor of the immune response and is protective against experimental autoimmune encephalomyelitis (EAE). Initial studies suggested that the cholinergic system modulates inflammation via the α7-nicotinic acetylcholine receptor (nAChR) subtype. We recently have shown that effector T cells and myeloid cells constitutively express mRNAs encoding nAChR α9 and β2 subunits and found evidence for immune system roles for non-α7-nAChRs. In the present study, we assessed the effects of nAChR α9 or β2 subunit gene deletion on EAE onset and severity, with or without nicotine treatment. We report again that disease onset is delayed and severity is attenuated in nicotine-treated, wild-type mice, an effect that also is observed in α9 subunit knock-out (KO) mice irrespective of nicotine treatment. On the other hand, β2 KO mice fail to recover from peak measures of disease severity regardless of nicotine treatment, despite retaining sensitivity to nicotine’s attenuation of disease severity. Prior to disease onset, we found significantly less reactive oxygen species production in the CNS of β2 KO mice, elevated proportions of CNS myeloid cells but decreased ratios of CNS macrophages/microglia in α9 or β2 KO mice, and some changes in iNOS, TNF-α and IL-1β mRNA levels in α9 KO and/or β2 KO mice. Our data thus suggest that β2*- and α9*-nAChRs, in addition to α7-nAChRs, play different roles in endogenous and nicotine-dependent modulation of immune functions and could be exploited as therapeutic targets to modulate inflammation and autoimmunity.Nicotine is a potent inhibitor of the immune response and is protective against experimental autoimmune encephalomyelitis (EAE). Initial studies suggested that the cholinergic system modulates inflammation via the α7‐nicotinic acetylcholine receptor (nAChR) subtype. We recently have shown that effector T cells and myeloid cells constitutively express mRNAs encoding nAChR α9 and β2 subunits and found evidence for immune system roles for non‐α7‐nAChRs. In the present study, we assessed the effects of nAChR α9 or β2 subunit gene deletion on EAE onset and severity, with or without nicotine treatment. We report again that disease onset is delayed and severity is attenuated in nicotine‐treated, wild‐type mice, an effect that also is observed in α9 subunit knock‐out (KO) mice irrespective of nicotine treatment. On the other hand, β2 KO mice fail to recover from peak measures of disease severity regardless of nicotine treatment, despite retaining sensitivity to nicotines attenuation of disease severity. Prior to disease onset, we found significantly less reactive oxygen species production in the central nervous system (CNS) of β2 KO mice, elevated proportions of CNS myeloid cells but decreased ratios of CNS macrophages/microglia in α9 or β2 KO mice, and some changes in iNOS, TNF‐α and IL‐1β mRNA levels in α9 KO and/or β2 KO mice. Our data thus suggest that β2*‐ and α9*‐nAChRs, in addition to α7‐nAChRs, have different roles in endogenous and nicotine‐dependent modulation of immune functions and could be exploited as therapeutic targets to modulate inflammation and autoimmunity.

Nicotinic Receptor β2 Determines NK Cell-Dependent Metastasis in a Murine Model of Metastatic Lung Cancer

Cigarette smoke exposure markedly compromises the ability of the immune system to protect against invading pathogens and tumorigenesis. Nicotine is a psychoactive component of tobacco products that acts as does the natural neurotransmitter, acetylcholine, on nicotinic receptors (nAChRs). Here we demonstrate that natural killer (NK) cells strongly express nAChR β2. Nicotine exposure impairs the ability of NK cells to kill target cells and release cytokines, a process that is largely abrogated by nAChR β2 deficiency. Further, nicotinic suppression of NF-κB-induced transcriptional activity in NK cells is dependent on nAChR β2. This nAChR subtype also plays a large role in the NK cell-mediated control of melanoma lung metastasis, in a murine lung metastasis model exposed to nicotine. Our findings suggest nAChR β2 as a prominent pathway for nicotine induced impairment of NK cell functions which contributes to the occurrence of smoking-related pathologies.

A component of Premarin ® enhances multiple cognitive functions and influences nicotinic receptor expression

In women, ovarian hormone loss at menopause has been related to cognitive decline, and some studies suggest that estrogen-containing hormone therapy (HT) can mitigate these effects. Recently, the Womens Health Initiative study found that conjugated equine estrogens, the most commonly prescribed HT, do not benefit cognition. Isolated components of conjugated equine estrogens (tradename Premarin(®)) have been evaluated in vitro, with delta(8,9)-dehydroestrone (∆(8)E1) and equilin showing the strongest neuroprotective profiles. It has not been evaluated whether ∆(8)E1 or equilin impact cognition or the cholinergic system, which is affected by other estrogens and known to modulate cognition. Here, in middle-aged, ovariectomized rats, we evaluated the effects of ∆(8)E1 and equilin treatments on a cognitive battery and cholinergic nicotinic receptors (nAChR). Specifically, we used (125)I-labeled epibatidine binding to assay brain nicotinic receptor containing 4α and 2β subunits (α4β2-nAChR), since this nicotinic receptor subtype has been shown previously to be sensitive to other estrogens. ∆(8)E1 enhanced spatial working, recent and reference memory. ∆(8)E1 also decreased hippocampal and entorhinal cortex α4β2-nAChR expression, which was related to spatial reference memory performance. Equilin treatment did not affect spatial memory or rat α4β2-nAChR expression, and neither estrogen impacted (86)Rb(+) efflux, indicating lack of direct action on human α4β2 nAChR function. Both estrogens influenced vaginal smear profiles, uterine weights, and serum luteinizing hormone levels, analogous to classic estrogens. The findings indicate that specific isolated Premarin(®) components differ in their ability to affect cognition and nAChR expression. Taken with the works of others showing ∆(8)E1-induced benefits on several dimensions of health-related concerns associated with menopause, this body of research identifies ∆(8)E1 as a new avenue to be investigated as a potential component of HT that may benefit brain health and function during aging.

Nicotinic acetylcholine receptor α7 subunits with a C2 cytoplasmic loop yellow fluorescent protein insertion form functional receptors

Aim:Several nicotinic acetylcholine receptor (nAChR) subunits have been engineered as fluorescent protein (FP) fusions and exploited to illuminate features of nAChRs. The aim of this work was to create a FP fusion in the nAChR α7 subunit without compromising formation of functional receptors.Methods:A gene construct was generated to introduce yellow fluorescent protein (YFP), in frame, into the otherwise unaltered, large, second cytoplamsic loop between the third and fourth transmembrane domains of the mouse nAChR α7 subunit (α7Y). SH-EP1 cells were transfected with mouse nAChR wild type α7 subunits (α7) or with α7Y subunits, alone or with the chaperone protein, hRIC-3. Receptor function was assessed using whole-cell current recording. Receptor expression was measured with 125I-labeled α-bungarotoxin (I-Bgt) binding, laser scanning confocal microscopy, and total internal reflectance fluorescence (TIRF) microscopy.Results:Whole-cell currents revealed that α7Y nAChRs and α7 nAChRs were functional with comparable EC50 values for the α7 nAChR-selective agonist, choline, and IC50 values for the α7 nAChR-selective antagonist, methyllycaconitine. I-Bgt binding was detected only after co-expression with hRIC-3. Confocal microscopy revealed that α7Y had primarily intracellular rather than surface expression. TIRF microscopy confirmed that little α7Y localized to the plasma membrane, typical of α7 nAChRs.Conclusion:nAChRs composed as homooligomers of α7Y subunits containing cytoplasmic loop YFP have functional, ligand binding, and trafficking characteristics similar to those of α7 nAChRs. α7Y nAChRs may be used to elucidate properties of α7 nAChRs and to identify and develop novel probes for these receptors, perhaps in high-throughput fashion.

Chemistry, Pharmacology, and Behavioral Studies Identify Chiral Cyclopropanes as Selective α4β2-Nicotinic Acetylcholine Receptor Partial Agonists Exhibiting an Antidepressant Profile. Part II

A 3-pyridyl ether scaffold bearing a cyclopropane-containing side chain was recently identified in our efforts to create novel antidepressants that act as partial agonists at α4β2-nicotinic acetylcholine receptors. In this study, a systematic structure-activity relationship investigation was carried out on both the azetidine moiety present in compound 3 and its right-hand side chain, thereby discovering a variety of novel nicotinic ligands that retain bioactivity and feature improved chemical stability. The most promising compounds, 24, 26, and 30, demonstrated comparable or enhanced pharmacological profiles compared to the parent compound 4, and the N-methylpyrrolidine analogue 26 also exhibited robust antidepressant-like efficacy in the mouse forced swim test. The favorable ADMET profile and chemical stability of 26 further indicate this compound to be a promising lead as a drug candidate warranting further advancement down the drug discovery pipeline.