Activation Of Alpha7 Receptors Research Articles

Target identification and validation of the alpha7 nicotinic acetylcholine receptor as a potential therapeutic target in retinal disease.

The role of acetylcholine (ACh) in visual processing in the mammalian retina has been the focus of research for many decades. Pioneering work on the localization of ACh discovered that the neurotransmitter is synthesized and stored in a distinct subpopulation of amacrine (starburst) cells. It has been shown that ACh release is regulated to a low resting "tonic" level, much like what is observed at the neuromuscular junction (NMJ). If there were a dysfunction in the tonic release of ACh, might post-synaptic changes render the targets of ACh [i.e., retinal ganglion cells (RGCs)] vulnerable to disease? During my time at Pharmacia & Upjohn (PNU), selective nicotinic ACh receptor (nAChR) agonists (e.g., PNU-282987) were developed as a possible therapy for central nervous system (CNS) diseases. As RGCs are the main targets of neurodegeneration in glaucoma, could the activation of this target provide neuroprotection? In response to this question, experiments to identify alpha7 nAChRs in the retina (i.e., target ID studies) followed by "proof-of-concept" experiments were conducted. Target ID studies included binding studies with retinal homogenates, [125I]-alpha-bungarotoxin (α-BTX) autoradiography, and fluorescently tagged α-BTX binding in retinal slices. Imaging studies of intracellular calcium dynamics in the retinal slice were conducted. Reverse transcription-polymerase chain reaction (RT-PCR) analysis with alpha7 nAChR knockout mice using the "laser-capture microdissection" technique, in situ hybridization studies, and RT-PCR analysis of the human retina were conducted. Collectively, these experiments confirmed the presence of alpha7 nAChRs on specific cells in the retina. "Proof-of-concept" neuroprotection studies demonstrated that PNU-282987 provided significant protection for RGCs. This protection was dose dependent and was blocked with selective antagonists. More recently, evidence for the generation of new RGCs has been reported with PNU-282987 in rodents. Interestingly, the appearance of new RGCs is more pronounced with eye-drop application than with intravitreal injection. One could postulate that this reflects the neurogenic activation of alpha7 receptors on the retinal pigment epithelium (RPE) (eye drops) vs. a neuroprotective effect on RGCs (injections). In conclusion, there does appear to be a cholinergic retinal "tone" associated with RGCs that could be utilized as a neuroprotective therapy. However, a distinct cholinergic neurogenic mechanism also appears to exist in the outer retina that could possibly be exploited to generate new RGCs lost through various disease processes.

Ultra-low exposure to alpha-7 nicotinic acetylcholine receptor partial agonists elicits an improvement in cognition that corresponds with an increase in alpha-7 receptor expression in rodents: implications for low dose clinical efficacy

Αlpha-7 neuronal nicotinic receptors (NNRs) are considered targets for cognitive enhancement in schizophrenia and Alzheimer's disease. AZD0328 is an alpha-7 NNR partial agonist that enhances cognition in rodents and nonhuman primates at sub-microgram to microgram doses. We hypothesized that increased expression of the alpha-7 receptor contributes to this beneficial activity at low doses and tested this by examining the effect of AZD0328 using in vivo and ex vivo binding, RT-PCR and cognitive function in rodents. AZD0328 (0.00178 mg/kg) was subcutaneously administered to mice 4, 24, 48 and 72 hours prior to testing in novel object recognition and produced a significant increase in cognition at 4, 24 and 48 h post-dosing. In vivo binding was examined in rat brain using [(3)H]AZ11637326 and there was a dose-dependent reduction in receptor binding at higher doses of AZD0328 (0.001-3 mg/kg), and a second alpha-7 partial agonist, SSR180711 (0.01-30 mg/kg). Lower doses of both compounds (0.0001 mg/kg) produced a significant increase in binding of [(3)H]AZ11637326. Ex vivo binding using [(125)I]-α-bungarotoxin, showed a significant increase in receptor number (B(max.)) in the frontal cortex or hippocampus with no significant effect on receptor affinity (K(d)) 2 h post administration of AZD0328. [(3)H]AZ11637326 administered 1.5 h following AZD0328 produced a significant increase in specific binding in rat brain regions. We found that the effect on receptor number was long-lasting, with [(125)I]-α-bungarotoxin binding increased in rats given AZD0328 for 2-48 h, but this was not accompanied by increased mRNA synthesis. SSR180711 produced a similar increase in B(max.) and specific binding with no effect on K(d). Therefore, trace dose of alpha-7 partial agonists has rapid onset and produces a profound, sustained effect on novel object recognition in mice that corresponds by dose to an increase in receptor number in rat brain. These findings provide an explanation for the acute and sustained benefit of alpha-7 receptor activation in working memory in nonhuman primates and guidance for drug development initiatives and treatment regimens for nicotinic partial agonists.

Gene regulation of α4β2 nicotinic receptors: microarray analysis of nicotine‐induced receptor up‐regulation and anti‐inflammatory effects

alpha4beta2 Nicotinic acetylcholine receptors play an important role in the reward pathways for nicotine. We investigated whether receptor up-regulation of alpha4beta2 nicotinic acetylcholine receptors involves expression changes for non-receptor genes. In a microarray analysis, 10 muM nicotine altered expression of 41 genes at 0.25, 1, 8 and 24 h in halpha4beta2 SH-EP1 cells. The maximum number of gene changes occurred at 8 h, around the initial increase in (3)[H]-cytisine binding. Quantitative RT-PCR corroborated gene induction of endoplasmic reticulum proteins CRELD2, PDIA6, and HERPUD1, and suppression of the pro-inflammatory cytokines IL-1beta and IL-6. Nicotine suppresses IL-1beta and IL-6 expression at least in part by inhibiting NFkappaB activation. Antagonists dihydro-beta-erythroidine and mecamylamine blocked these nicotine-induced changes showing that receptor activation is required. Antagonists alone or in combination with nicotine suppressed CRELD2 message while increasing alpha4beta2 binding. Additionally, small interfering RNA knockdown of CRELD2 increased basal alpha4beta2 receptor expression, and antagonists decreased CRELD2 expression even in the absence of alpha4beta2 receptors. These data suggest that endoplasmic reticulum proteins such as CRELD2 can regulate alpha4beta2 expression, and may explain antagonist actions in nicotine-induced receptor up-regulation. Further, the unexpected finding that nicotine suppresses inflammatory cytokines suggests that nicotinic alpha4beta2 receptor activation promotes anti-inflammatory effects similar to alpha7 receptor activation.

Multiple Pharmacophores for the Selective Activation of Nicotinic α7-Type Acetylcholine Receptors

The activation of heteromeric and homomeric nicotinic acetylcholine receptors was studied in Xenopus laevis oocytes to identify key structures of putative agonist molecules associated with the selective activation of homomeric alpha7 receptors. We observed that selectivity between alpha7 and alpha4beta2 was more readily obtained than selectivity between alpha7 and alpha3beta4. Based on structural comparisons of previously characterized selective and nonselective agonists, we hypothesize at least three chemical motifs exist that, when present in molecules containing an appropriate cationic center, could be associated with the selective activation of alpha7 receptors. We identify the three distinct structural motifs based on prototypical drugs as the choline motif, the tropane motif, and the benzylidene motif. The choline motif involves the location of an oxygen-containing polar group such as a hydroxyl or carbonyl separated by two carbons from the charged nitrogen. The tropane motif provides alpha7-selectivity based on the addition of multiple small hydrophobic groups positioned away from the cationic center in specific orientations. We show that this motif can convert the nonselective agonists quinuclidine and ethyltrimethyl-ammonium to the alpha7-selective analogs methyl-quinuclidine and diethyldimethyl-ammonium, respectively. We have shown previously that the benzylidene group of 3-2,4, dimethoxy-benzylidene anabaseine (GTS-21) converts anabaseine into an alpha7-selective agonist. The benzylidene motif was also applied to quinuclidine to generate another distinct family of alpha7-selective agonists. Our results provide insight for the further development of nicotinic therapeutics and will be useful to direct future experiments with protein structure-based modeling and site-directed mutagenesis.

P4-399: Essential factors for the selective activation of nicotinic alpha7-type receptors, potential therapeutic targets for Alzheimer’s disease

Several observations have tied alpha7 nicotinic acetylcholine receptors (nAChR) to both the etiology and potential therapy for Alzheimer's disease. Beta–amyloid peptides bind to alpha7 nAChR, and treatment of cells expressing alpha7 nAChR with alpha7–selective agonists can prevent cytotoxic effects of the amyloid peptides. Likewise, treatment with alpha7–selective drugs can reverse learning and memory deficits in animals with experimentally–induced cholinergic hypofunction. We have developed a model for the key features of alpha7–selective agonists based on systematic analysis of numerous drug classes with varying structure but similar selectivity for alpha7. In each class, there is a core agonist structure which can activate multiple nAChR subtypes. This core structure may be structurally related to tropane, quinuclidine, anabaseine, nicotine, or similar aromatic or alkyl groups with an essential nitrogen that will be charged at physiological pH. Selectivity for alpha7 activation arises when hydrophobic groups are added to such a core agonist structure, provided that the orientation of the hydrophobic additions align with corresponding hydrophobic pockets in the receptor. We show that while alpha7 selectivity can be achieved with substitutions as small as a single methyl group, large hydrophobic substitutions (e.g. indoles or benzylidene groups) typically enhance potency for alpha7 receptor activation. We have probed the significance of hydrophobic domains within the receptor binding site and show that a tyrosine to phenylalanine mutation which decreases the potency of the endogenous agonist ACh 200–fold does not affect the potency or relative efficacy of the benzylidene anabaseine 4OH–GTS–21. By creating a new site for hydrophobic interaction within the non–alpha7 nAChR alpha4beta2, we make a receptor which previously did not respond to 4OH–GTS–21 respond four to five–fold better to 4OH–GTS–21 than to ACh. The observation that the specific mutation which relaxes the constraint for the activation of alpha4beta2 receptors by benzylidene anabaseines does not increase the effectiveness of all alpha7–selective agonists provides additional insight into how hydrophobic side groups of the various alpha7–selective agonists are oriented in the agonist binding site.

Activation of α7 nicotinic acetylcholine receptor by nicotine selectively up-regulates cyclooxygenase-2 and prostaglandin E2 in rat microglial cultures

BackgroundNicotinic acetylcholine (Ach) receptors are ligand-gated pentameric ion channels whose main function is to transmit signals for the neurotransmitter Ach in peripheral and central nervous system. However, the α7 nicotinic receptor has been recently found in several non-neuronal cells and described as an important regulator of cellular function. Nicotine and ACh have been recently reported to inhibit tumor necrosis factor-α (TNF-α) production in human macrophages as well as in mouse microglial cultures. In the present study, we investigated whether the stimulation of α7 nicotinic receptor by the specific agonist nicotine could affect the functional state of activated microglia by promoting and/or inhibiting the release of other important pro-inflammatory and lipid mediator such as prostaglandin E2.MethodsExpression of α7 nicotinic receptor in rat microglial cell was examined by RT-PCR, immunofluorescence staining and Western blot. The functional effects of α7 receptor activation were analyzed in resting or lipopolysaccharide (LPS) stimulated microglial cells pre-treated with nicotine. Culture media were assayed for the levels of tumor necrosis factor, interleukin-1β, nitric oxide, interleukin-10 and prostaglandin E2. Total RNA was assayed by RT-PCR for the expression of COX-2 mRNA.ResultsRat microglial cells express α7 nicotinic receptor, and its activation by nicotine dose-dependently reduces the LPS-induced release of TNF-α, but has little or no effect on nitric oxide, interleukin-10 and interleukin-1β. By contrast, nicotine enhances the expression of cyclooxygenase-2 and the synthesis of one of its major products, prostaglandin E2.ConclusionsSince prostaglandin E2 modulates several macrophage and lymphocyte functions, which are instrumental for inflammatory resolution, our study further supports the existence of a brain cholinergic anti-inflammatory pathway mediated by α7 nicotinic receptor that could be potentially exploited for novel treatments of several neuropathologies in which local inflammation, sustained by activated microglia, plays a crucial role.

P2-045 Neuroprotective mechanisms underlying nicotinic alpha7 receptor activation in brain

P2-045 Neuroprotective mechanisms underlying nicotinic alpha7 receptor activation in brain

Nicotinic receptor stimulation protects neurons against beta-amyloid toxicity.

beta-Amyloid (A beta), a major constituent of senile plaques in Alzheimer's disease (AD), is thought to contribute to the neurodegeneration. We examined the effects of nicotinic receptor agonists on A beta cytotoxicity in cultured rat cortical neurons. The number of viable neurons decreased significantly when cultures were exposed to synthetic A beta peptides (25-35). Concomitant administration of nicotine with A beta markedly reduced the number of dead cells. This nicotine-induced neuroprotection was dependent on the concentration. When hexamethonium or mecamylamine, nicotinic antagonist, was added, neuroprotective effect of nicotine was blocked, which indicates that effect of nicotine was mediated by nicotinic receptors. In addition, a selective alpha7-receptor antagonist, alpha-bungarotoxin (alpha-BTX), blocked the neuroprotective effect of nicotine. Furthermore, incubation with 3-(2,4)-dimethoxybenzylidene anabaseine (DMXB), a selective alpha7-receptor agonist, protected against A beta-induced neuronal death. These results suggest that alpha7-receptor activation plays an important role in neuroprotection against A beta cytotoxicity. This study suggests that nicotinic receptor stimulation, especially alpha7-receptor activation, may be able to protect neurons from degeneration induced by A beta and may have effects that counter the progress of AD.