Augmentation of cognitive function by NS9283, a stoichiometry-dependent positive allosteric modulator of α2- and α4-containing nicotinic acetylcholine receptors

Abstract

Background and purpose: Positive allosteric modulation of α4β2 nicotinic acetylcholine (nACh) receptors could add a new dimension to the pharmacology and therapeutic approach to these receptors. The novel modulator NS9283 was therefore tested extensively.

Experimental approach: Effects of NS9283 were evaluated in vitro using fluorescence-based Ca(2+) imaging and electrophysiological voltage clamp experiments in Xenopus oocytes, mammalian cells and thalamocortical neurons. In vivo the compound was tested in models covering a range of cognitive domains in mice and rats.

Key results: NS9283 was shown to increase agonist-evoked response amplitude of (α4)(3) (β2)(2) nACh receptors in electrophysiology paradigms. (α2)(3) (β2)(2) , (α2)(3) (β4)(2) and (α4)(3) (β4)(2) were modulated to comparable extents, but no effects were detected at α3-containing or any 2α : 3β stoichiometry nACh receptors. Native nACh receptors in thalamocortical neurons similarly displayed DHβE-sensitive currents that were receptive to modulation. NS9283 had favourable effects on sensory information processing, as shown by reversal of PCP-disrupted pre-pulse inhibition. NS9283 further improved performance in a rat model of episodic memory (social recognition), a rat model of sustained attention (five-choice serial reaction time task) and a rat model of reference memory (Morris water maze). Importantly, the effects in the Morris water maze could be fully reversed with mecamylamine, a blocker of nACh receptors.

Conclusions and implications: These results provide compelling evidence that positive allosteric modulators acting at the (α4)(3) (β2)(2) nACh receptors can augment activity across a broad range of cognitive domains, and that α4β2 nACh receptor allosteric modulation therefore constitutes a promising therapeutic approach to symptomatic treatment of cognitive impairment.

Figure 1

Allosteric modulation of α4β2 nACh receptors by NS9283. (A) Chemical structure of 3-[3-(3-pyridyl)-1,2,4-oxadiazol-5-yl]benzonitrile (NS9283). (B) NS9283 fails to activate α4β2 nACh receptors but increases the amplitude of nicotine-evoked Ca²⁺-responses. NS9283 or control buffer was added to HEK293-hα4β2 cells after a 20 s stabilization period, and a ∼EC₂₀ concentration of nicotine was added after 200 s. Response amplitudes were background subtracted and normalized to the effect of a saturating nicotine concentration (100 µM). The traces shown represent average responses from 16 wells for each experiment. (C) Concentration–response relationship for NS9283 allosteric modulation. Nicotine-induced (0.5 µM) Ca²⁺-responses (baseline subtracted and normalized; presented as mean ± SEM of four experiments) were fitted to the Hill equation by non-linear regression. (D) Representative current traces of ACh and NS9283+ACh in two-electrode voltage-clamp experiments in Xenopus laevis oocytes. The oocyte was injected with α4 and β2 nACh receptor subunits in a 4:1 ratio. Application of ACh or NS9283 mixed with ACh is indicated by a bar above each trace. NS9283 concentrations were increased in half log unit increments starting with lowest concentration (10 nM) in application ‘1’ and ending with highest concentration (31.6 µM) in application ‘8’. (E) Concentration–response relationships for NS9283 modulation of ACh-evoked currents in X. laevis oocytes. Oocytes were injected with α4 and β2 nACh receptor subunits in a 1:4 or 4:1 ratio to obtain (α4)₂(β2)₃ or (α4)₃(β2)₂ receptors, respectively. Peak current amplitudes were baseline subtracted and normalized with respect to the current induced by ACh (1 µM for (α4)₂(β2)₃, 10 µM for (α4)₃(β2)₂), and are presented as mean ± SEM of 11–12 experiments. Data points for (α4)₃(β2)₂ were fitted to the Hill equation by non-linear regression, and the result is seen in Table 1. (F) Representative current traces from whole-cell patch-clamp experiments on human α4β2 nACh receptors transiently expressed in GH4C1 cells. Cells were clamped at −60 mV and stimulated with ACh (100 µM) in the absence, presence and after wash-out of NS9283 (10 µM), respectively.

Figure 2

Allosteric modulation of α4β2 nACh receptors by NS9283 is conferred through increases in functional agonist potency. (A) NS9283 concentration-dependently leftward shifts the nicotine concentration–response (C–R) relationship for activating Ca²⁺-flux. Nicotine C-R relationships in HEK293-hα4β2 cells are shown under control conditions and at three concentrations of NS9283. Data points, baseline subtracted and normalized to the maximal nicotine (100 µM) effect, are presented as mean ± SEM of n= 4–6 experiments and are fitted to the Hill equation by non-linear regression. (B) Leftward shift in agonist C-R fully accounts for NS9283-induced modulation. The magnitude of the nicotine C–R leftward shift was quantified as the difference in log EC₅₀ with and without NS9283 (ΔpEC₅₀-value) and plotted as a function of NS9283 concentration. These C–R data for NS9283 were fitted well by the Hill equation. (C) NS9283 leftward-shifts the C-R relationship of ACh at the (α4)₃(β2)₂ nAChR in X. laevis oocytes. Oocytes were injected with α4 and β2 nAChR receptor subunits in a 4:1 ratio, and C–R relationships for ACh were determined in the absence and presence of NS9283 (31.6 µM) as data set pairs; that is, two 5-point ACh C–R relationships obtained for each oocyte, the first without and the second with NS9283. Data points, baseline subtracted and normalized to the maximal value obtained by fitting each oocyte ACh C-R to the Hill equation by non-linear regression, are presented as mean ± SEM of n= 12 experiments; and these were then fitted to the Hill equation by non-linear regression. (D) NS9283 does not alter the C–R relationship of ACh at (α4)₂(β2)₃ nAChR in X. laevis oocytes. Oocytes were injected with α4 and β2 nACh receptor subunits in a 1:4 ratio, and ACh C–R relationships were determined as described above. Data points were treated as described in (C) and are presented as mean ± SEM of n= 15 experiments.

Figure 3

NS9283 modulates ACh-evoked currents in thalamocortical (TC) neurons. (A) Representative current trace of whole-cell patch clamp recording of a TC neuron in the presence of Cs (140 mM in the pipette), kynurenic acid and GABAzine. The experiment was initiated after the cell reached a steady-state holding current level. ACh (100 µM) was bath-applied and following current stabilization NS9283 (10 µM) and finally DHβE (10 µM) was further applied as indicated by the horizontal lines. (B) Representative current trace for an experiment similar to that in (A) except for the order of ACh and NS9283 applications. (C) NS9283 positively modulates ACh-evoked currents in TC neurons. Maximal change in current (ΔI_max) evoked by application of ACh (left), ACh+NS9283 (middle) or ACh + NS9283+DHβE (right) versus control current are presented as mean ± SD of six experiments. (D) NS9283 does not by itself evoke currents in TC neurons. Maximal current (ΔI_max) evoked by NS9283 (left) or NS9283 + ACh (right) are presented as mean ± SD of four experiments.

Figure 4

NS9283 improves performance in rat social recognition. Groups of nine adult male Sprague–Dawley rats were administered NS9283 (0.075, 0.75 and 7.5 mg·kg⁻¹, i.p.) or nicotine (0.1 mg·kg⁻¹, i.p.) immediately following initial juvenile exposure (T1), and changes in duration of subsequent juvenile interaction (T2) were assessed 2 h later. Vehicle control animals did not display recollection of the first encounter, as suggested by a recognition index of ∼1 (left bar). However, NS9283 significantly reduced the recognition index in a dose-dependent manner (middle bars), suggesting an improvement in recognition memory, equivalent to that observed for nicotine (right bar). Data are shown as mean ± SEM (n= 9) and significance levels *: P < 0.05, **: P < 0.01 and ***: P < 0.001.

Figure 5

NS9283 and nicotine improves attention performance in the rat five-choice serial reaction time task. Fifteen water-deprived male Lister hooded rats were trained to discriminate a brief visual cue randomly presented in one out of five nose-poke units and upon correct responding they received a water reward. ITI was set very high (15 s) in these experiments in order to challenge attentional processes maximally. (A1–A9) NS9283 enhance attentional functions by increasing vigilance. NS9283 (0.3, 1 and 3 mg·kg⁻¹, i.p., −30 min) significantly and dose-dependently affected errors of omissions (A2), completed trials (A9), perseverant responses (A7) and head entries (A8) but had no effects on any of the latencies (A4–A6). Minor improvements were seen on correct responses (A1) and premature responding (A3) was increased albeit with reverse dose-dependency. (B1–B9) Nicotine enhances attentional functions by increasing sustained attention in conjunction with mild stimulant effects. Nicotine (0.03, 0.1 and 0.3 mg·kg⁻¹, s.c., −10 min) significantly and dose-dependently affected correct responses (B1), premature responding (B3), head entries (B8) correct latency (B4) and magazine latency (B6). Errors of omissions (B2), incorrect latency (B5), perseverant responses (B7) and completed trials (B9) were not significantly affected by the compound. For ease of reading the y-axis titles are placed within the graphs. Results are presented as mean ± SEM (n= 15) and significance levels *: P < 0.05, **: P < 0.01, ***: P < 0.001.

Figure 6

NS9283 improvements in the Morris water maze task were mediated by nACh receptors as evidenced by target engagement studies with mecamylamine. Adult rat acquisition of a visuo-spatial learning task in the Morris water maze was monitored by video over a 4 day period. Task acquisition was impaired considerably by a sub-chronic PCP (1.5 mg·kg⁻¹ s.c., −30 min) administration all 4 days, as evidenced by the significant difference in distance, % trial platform not found and time in thigmotaxis between the vehicle and PCP groups at day 1. (A1–A4) Water maze improvements by NS9283 could be reversed with mecamylamine. NS9283 (0.3 mg·kg⁻¹, p.o., −30 min) produced a significant alleviation of the PCP-deficits and improved task acquisition to nearly control levels at days 3 and 4 depending on behavioural parameter. This effect of NS9283 was completely abolished by co-administration of the non-selective nACh receptor antagonist mecamylamine (3 mg·kg⁻¹, s.c., −30 min). A significant effect on swimming speed was only seen in two of the groups at days 3 and 4 and was generally considered minor. (B1–B4) Mecamylamine does not impair MWM performance or interact with PCP-induced deficits. Effects of mecamylamine (3 mg·kg⁻¹, s.c., −30 min) treatment alone did not differ from the vehicle group at any behavioural parameter at any of the 4 days. Likewise, mecamylamine administration did not significantly affect the main impairments seen with PCP except for an increase in swimming speed at days 1 and 4 (P < 0.05). Results are presented as mean ± SEM (n= 7–8). Significance levels indicated in the graphs were versus the PCP group for A1–A4 (full line pattern) and versus vehicle for B1–B4 (full line pattern) and *: P < 0.05, **: P < 0.01, ***: P < 0.001.