Broad-spectrum efficacy across cognitive domains by alpha7 nicotinic acetylcholine receptor agonism correlates with activation of ERK1/2 and CREB phosphorylation pathways

Abstract

The alpha7 nicotinic acetylcholine receptor (nAChR) plays an important role in cognitive processes and may represent a drug target for treating cognitive deficits in neurodegenerative and psychiatric disorders. In the present study, we used a novel alpha7 nAChR-selective agonist, 2-methyl-5-(6-phenyl-pyridazin-3-yl)-octahydro-pyrrolo[3,4-c]pyrrole (A-582941) to interrogate cognitive efficacy, as well as examine potential cellular mechanisms of cognition. Exhibiting high affinity to native rat (Ki = 10.8 nM) and human (Ki = 16.7 nM) alpha7 nAChRs, A-582941 enhanced cognitive performance in behavioral assays including the monkey delayed matching-to-sample, rat social recognition, and mouse inhibitory avoidance models that capture domains of working memory, short-term recognition memory, and long-term memory consolidation, respectively. In addition, A-582941 normalized sensory gating deficits induced by the alpha7 nAChR antagonist methyllycaconitine in rats, and in DBA/2 mice that exhibit a natural sensory gating deficit. Examination of signaling pathways known to be involved in cognitive function revealed that alpha7 nAChR agonism increased extracellular-signal regulated kinase 1/2 (ERK1/2) phosphorylation in PC12 cells. Furthermore, increases in ERK1/2 and cAMP response element-binding protein (CREB) phosphorylation were observed in mouse cingulate cortex and/or hippocampus after acute A-582941 administration producing plasma concentrations in the range of alpha7 binding affinities and behavioral efficacious doses. The MEK inhibitor SL327 completely blocked alpha7 agonist-evoked ERK1/2 phosphorylation. Our results demonstrate that alpha7 nAChR agonism can lead to broad-spectrum efficacy in animal models at doses that enhance ERK1/2 and CREB phosphorylation/activation and may represent a mechanism that offers potential to improve cognitive deficits associated with neurodegenerative and psychiatric diseases, such as Alzheimer's disease and schizophrenia.

Figure 1.

In vitro pharmacological properties of A-582941. A, Structure of A-582941. B, Displacement of [³H]A-585539 and [³H]cytisine binding to rat brain membranes by A-582941. C, Current traces showing activation of human α7 nAChRs expressed in Xenopus oocytes by ACh (top) and A-582941 (bottom). After preincubation with methyllycaconitine (MLA; 10 nm, 5 min), current responses to both A-582941 and ACh were abolished. Responses returned to control levels on washout. D, Concentration–response data (mean ± SEM) are shown for A-582941 as agonist at human and rat α7 nAChR expressed in Xenopus oocytes. The EC₅₀ and percentage maximal efficacy values are summarized in Results.

Figure 2.

Efficacy profile in short-term memory models. A, Improvements in DTMS task performance using a titration version by adult rhesus monkeys after administration of A-582941 (3, 10, and 30 nmol/kg, i.m.). Task accuracy and the maximal delay interval titrated derived from the titrating version of the DMTS task is plotted as a function of dose (*p < 0.05 vs vehicle-treated controls; n = 6 per treatment). B, A-582941 (0.01, 0.1, and 1.0 μmol/kg) was evaluated in the rat social recognition test, a model of short-term recognition memory. Adult rats, administered A-582941 intraperitoneally immediately after initial juvenile exposure (T1), produced a significant decrease in the duration of subsequent juvenile interaction (T2) 2 h later. Whereas vehicle-treated rats exhibited a T2:T1 ratio >0.9, indicating loss of memory, rats treated with 0.1 and 1.0 μmol/kg A-582941 displayed a significantly lower ratio (*p < 0.05 vs vehicle-treated controls; n = 8 per treatment). Data are represented as mean ± SEM.

Figure 3.

Efficacy profile in a long-term memory consolidation model. A, A-582941 (0.01, 0.1, and 1.0 μmol/kg) improved memory in the mouse inhibitory avoidance test, a model of long-term memory consolidation. Administered intraperitoneally 30 min before the training (shock) trial, A-582941 increased crossover latency during the test trial (no shock) 24 h later, an index of index of memory enhancement (*p < 0.05 vs vehicle-treated control; n = 8–10 per treatment). B, Mecamylamine (1.5, 5, and 15 μmol/kg, i.p.) pretreatment (15 min) blocked inhibitory avoidance memory enhancement produced by A-582941 (1.0 μmol/kg, i.p.) (*p < 0.05 vs vehicle-treated control; ⁺ p < 0.05 vs A-582941-alone-treated; n = 8–10 per treatment). C, However, selective α4β2 nAChR antagonism by DHβE (0.3, 1.0, and 3.0 μmol/kg) did not block inhibitory avoidance memory enhancement produced by A-582941 (1.0 μmol/kg, i.p.) (*p < 0.05 vs vehicle-treated control; n = 8–10 per treatment). Data are represented as mean ± SEM.

Figure 4.

Efficacy profile in sensory gating/preattention. A, A-582941 improves N40 auditory-evoked sensory gating in rats in a model of α7-mediated preattention. A-582941 (10 μmol/kg, i.p.) reversed the N40 auditory-evoked sensory gating deficit in rats produced by intracerebroventricular administration of the α7 nAChR anatgonist MLA (30 μg). Whereas MLA alone increases the T:C ratio, an index of impaired sensory gating, pretreatment with A-582941 completely reverses the N40 deficit, as evidenced by a reduced T:C ratio (*p < 0.05 vs vehicle-treated control; ⁺ p < 0.05 vs MLA-treated control; n = 6 per treatment). B, A-582941 improved N40 auditory-evoked sensory gating in DBA/2 mice, a genetic model of impaired sensory gating. Specifically, A-582941 improved N40 auditory-evoked gating as evidenced by the reduced T:C ratio (*p < 0.05 vs vehicle-treated control; n = 16–40 per treatment) C, Improved N40 sensory gating was observed after 5 d of daily dosing in DBA/2 mice, indicating a lack of tolerance development (*p < 0.05 vs vehicle-treated control; n = 23 per treatment). Data are represented as mean ± SEM.

Figure 5.

Effects on ERK1/2 phosphorylation in vitro and in vivo. A, A-582941 increased phosphorylation of ERK1/2 in an MLA-sensitive manner after incubation in PC12 cells. B, The EC₅₀ value of A-582941 to increase ERK phosphorylation is 95 nm (95% c.i., 81–111 nm; n = 3). Compared with α7 nAChR responses measured electrophysiologically in oocytes, these EC₅₀ values are lower and apparent efficacy values are higher because of the inclusion of an α7-selective modulator in the ERK1/2 assay. C, A-582941 (0.01, 0.1, and 1.0 μmol/kg) increased ERK1/2 phosphorylation in mouse cingulate cortex (left) and hippocampus (right) at behaviorally efficacious doses (15–20 min after injection; *p < 0.05 vs vehicle-treated control; n = 6 per treatment). Scale bars, 150 μm.

Figure 6.

Effects on CREB phosphorylation in vivo. A, B, A-582941 (0.01, 0.1, and 1.0 μmol/kg) increased phosphorylation of the transcription factor CREB in mouse cingulate cortex at behaviorally efficacious doses (15–20 min after injection; *p < 0.05; **p < 0.01 vs vehicle-treated control; n = 6 per treatment). Data are represented as mean ± SEM. Scale bar, 150 μm.

Figure 7.

Influence of MEK inhibition on ERK1/2 and CREB phosphorylation in vivo. A, MEK inhibitor SL327 (300 μmol/kg, i.p.) pretreatment (15 min) blocked A-582941 (1.0)-induced and basal (tonic) ERK1/2 phosphorylation in mouse cingulate cortex (*p < 0.05 vs nontreated control; ⁺ p < 0.05 vs A-582941 alone; n = 6 per treatment). B, MEK inhibitor SL327 (300 μmol/kg, i.p.) pretreatment (15 min) only produced a partial nonsignificant attenuation of A-582941-induced CREB phosphorylation, suggesting that other non-MAPK pathways may be involved in α7 agonism-mediated CREB phosphorylation (*p < 0.01 vs vehicle-treated controls). Data are represented as mean ± SEM.