Type I and type II positive allosteric modulators of α7 nicotinic acetylcholine receptors induce antidepressant-like activity in mice by a mechanism involving receptor potentiation but not neurotransmitter reuptake inhibition. Correlation with mTOR intracellular pathway activation
Introduction
Alpha7 (α7) nicotinic acetylcholine receptors (nAChRs) are widely distributed throughout functionally important brain regions, including hippocampus, ventral tegmental area, nucleus accumbens, locus coeruleus, prefrontal cortex (PFC), and dorsal raphe nucleus, where they regulate the release of different neurotransmitters (Gotti et al., 2006). The malfunctioning of α7 nAChRs might be involved in the process of depression since α7 knockout mice showed depression-like phenotype (Zhang et al., 2016) and might regulate depression-like behavior under stressful conditions (Mineur et al., 2018).
Previous studies showed that non-selective (e.g. nicotine) (Arias et al., 2018; Vazquez-Palacios et al., 2004), α7-selective agonists (e.g. SSR180711) (Andreasen et al., 2012; Marcus et al., 2016), and α7-selective antagonists [e.g. methyllycaconitine (MLA)] (Andreasen et al., 2009; Mineur et al., 2018) induce antidepressant-like effects in animal models. Moreover, α7-selective agonists (i.e. PNU-282987) increased the antidepressant activity of serotonin selective reuptake inhibitors (e.g. citalopram) (Andreasen et al., 2011), whereas mecamylamine, a non-selective noncompetitive antagonist, improved antidepressant activity in wild-type but not α7 or β2 knockout mice (Rabenstein et al., 2006). Pre-clinical studies on α7-selective positive allosteric modulators (α7-PAMs) showed that acute administration of PAM-2 (Arias et al., 2015; Targowska-Duda et al., 2014) or NS-1738 (Marcus et al., 2016) failed to induce antidepressant-like activity in naïve mice, whereas chronic administration of PAM-2 was able to achieve such activity, and acute administration of PNU-120596 prevented depression-like behavior in lipopolysaccharide-treated mice (Alzarea and Rahman, 2018; Alzarea and Rahman, 2019). Compounds that potentiate the physiological activity of the endogenous neurotransmitter ACh without directly activating and/or desensitizing the receptor such as α7-PAMs would have an important clinical advantage respect to nicotinic agonists, which can over-/sub-stimulate the receptor as well as induce receptor desensitization and up-regulation, increasing consequently the extent of side effects and tolerance compared to α7-PAMs.
Since the antidepressant-like activity of nicotinic agonists (e.g. nicotine) seem to be related to their desensitizing rather than activating properties (Mineur and Picciotto, 2010), we want to compare the behavioral activity of type II PAMs (PAM-2 and PNU-120596), which potentiate agonist-evoked α7 nAChR currents, delay the desensitization process, and even reactivate desensitized receptors, with that elicited by NS-1738, a classical type I PAM that potentiates α7 nAChRs without modifying the desensitization process (Arias et al., 2011; Targowska-Duda et al., 2014; Targowska-Duda et al., 2019; Andersen et al., 2016). In this regard, forced swim (FST) and tail suspension (TST) tests, both widely used to determine the effectiveness of antidepressant-like drugs, were performed in naïve mice. Both tests are based on the concept that a short-term inescapable stress condition may result in increased animal hopelessness. This strategy might represent the psychological equivalent of a phenomenon called “entrapment” that has been associated with clinical depression in humans, in which behavioral passivity releases the individual from actively coping with the stressful situation (Cryan and Mombereau (2004). A stricter assessment of the FST, however, has recently concluded that it actually measures stress-coping strategy and that effective antidepressants actively promote this strategy (Commons et al., 2017). Since previous studies with α7-PAMs supported the notion that antidepressant-like activity is dependent on treatment length, we conducted behavioral tests under same animal and experimental conditions to compare the behavioral activity of PAM-2, PNU-120596, and NS-1738 after acute (30 min), subchronic (7 days), and chronic (14 days) treatments. To evaluate the potential involvement of α7 nAChRs in the behavioral activity of α7-PAMs, mice were pretreated with MLA followed by treatment with either PNU-120596 or PAM-2. To determine potential drug interactions, a subeffective dose of PAM-2 was co-administered with a subeffective dose of either DMXBA, a potent α7-agonist, or the antidepressant bupropion (BP).
The PI3K/AKT/mTOR (mammalian target of rapamycin) intracellular pathway is dysregulated in depression (Abelaira et al., 2014), supporting the concept that this signaling mechanism underlies the activity of structurally and functionally different antidepressants (Park et al., 2014; Chandran et al., 2013; Li et al., 2010). Thus, to assess whether PAM-2 activates the mTOR and/or ERK1/2 (extracellular signal-regulated protein kinases 1 and 2) pathways, phosphorylation levels of each component were measured in hippocampus and PFC from mice chronically treated with PAM-2. Since dopamine (hDAT), serotonin (hSERT), and noradrenaline (hNET) transporters are therapeutically relevant in the context of depression treatment (Kristensen et al., 2011), the interaction of type I and type II PAMs, including PAM-2 and its analogs PAM-3 and PAM-4 (Arias et al., 2011), with these human neurotransmitter reuptake systems was also evaluated.
In the present work, we showed that both type I and type II PAMs induce antidepressant-like activity in mice by a mechanism involving α7 potentiation but ruling out either α7 desensitization or neurotransmitter transporter blockade. PAM-2 also activated the mTOR intracellular signaling cascade, which might be correlated with the observed behavioral activity of α7-PAMs. These results support the view that these modulators might be clinically used to ameliorate depression and other stress disorders.
Section snippets
Drugs
[3H]Dopamine (39.4 Ci/mmol) and [3H]5-HT 27.9 Ci/mmol) were purchased from Perkin Elmer (Waltham, MA, USA). NS-1738 and PNU-120596 were purchased from Tocris Bioscience (Ellisville, MO, USA). Tween 80, methyllycaconitine (MLA), bupropion hydrochloride (BP), (2,4)-dimethoxybenzylidene anabaseine dihydrochloride (DMXBA, also called GTS-21), dopamine, serotonin, nomifensine maleate, paroxetine hydrochloride hemihydrate, nortriptyline hydrochloride, and Ro 41-0960 were obtained from Sigma-Aldrich
Dose- and treatment-dependence of antidepressant-like activity of α7-PAMs
To determine whether type I and type II α7-PAMs induce antidepressant-like activity, FSTs were performed on mice injected with the following PAMs and doses: PAM-2 (0.5 and 1.0 mg/kg), PNU-120596 (1.0 and 10.0 mg/kg), and NS-1738 (1.0 and 10.0 mg/kg). To assess treatment length dependence, FSTs were performed after acute (30 min following drug injection), or after subchronic (7 days) and chronic (14 days) treatments. The results indicated that PAM-2 (Fig. 1A), PNU-120596 (Fig. 1B), and NS-1738 (
Discussion
The main objective of this study is to determine whether PAMs that enhance the activity of α7 nAChRs by decreasing (i.e., type II PAMs such as PNU-120596 and PAM-2) or not (i.e., type I PAMs such as NS-1738) the desensitization process induce antidepressant-like activity in mice after acute, subchronic, and chronic treatments. To assess whether other targets might be related to this behavioral activity, we also determined the effect of α7-PAMs at human neurotransmitter transporters and
Role of the funding source
The funding sources mentioned in the Acknowledgments had no further role in the study design, the collection, analysis and interpretation of data, the writing of the report and the decision to submit the paper for publication.
Contributors
KTD and HRA designed the study; KTD wrote the first draft of the manuscript; managed the literature - searches and analyses; KTD, AW, CJL performed the experiments and undertook the statistical analysis; contributed to writing of the manuscript; BB, AM performed the experiments; KJ, GB, MM: revised the manuscript; DM and MNR synthesized studied compounds; HRA: critically revised and edited the manuscript. All authors contributed to and have approved the final version of the manuscript.
Conflict of interest
The authors declare no conflict of interest.
Acknowledgement
This work was supported by grants from the Polish National Science Center (SONATA funding, UMO-2013/09/D/NZ7/04549) [to K.T-D. (PI) and H.R.A. (Co-PI)], the Independent Research Fund Denmark (4183-00581) and the Novo Nordisk Foundation (NNF17OC0028582) [to C.J.L.], and by an OVPR Pilot/Seed Grant (Oklahoma State University Center for Health Sciences) (to H.R.A).
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Abbreviations: nAChR, nicotinic acetylcholine receptor; MLA, methyllycaconitine; PAM, positive allosteric modulator; PAM-2, 3-furan-2-yl-N-p-tolyl-acrylamide; PAM-3, 3-furan-2-yl-N-o-tolylacrylamide, PAM-4, 3-furan-2-yl-N-phenylacrylamide; NS-1738, N-(5-Chloro-2-hydroxyphenyl)-N'-[2-chloro-5-(trifluoromethyl)phenyl]urea; PNU-120596, N-(5-Chloro-2,4-dimethoxyphenyl)-N-(5-methyl-3-isoxazolyl)-urea; DMXBA, 3-(2,4-dimethoxybenzylidene) anabaseine; MLA, methyllycaconitine; hDAT, human dopamine transporter; hSERT, human serotonin transporter; hNET, human noradrenaline transporter; IC50, ligand concentration that produces 50% inhibition; Ki, inhibition constant; nH, Hill coefficient; PFC, prefrontal cortex; mTOR, mammalian target of rapamycin; ERK1/2, extracellular signal-regulated protein kinases 1 and 2; GSK-3β, glycogen synthase kinase 3β.