Elsevier

Progress in Neurobiology

Volume 197, February 2021, 101898
Progress in Neurobiology

Alterations in nicotinic receptor alpha5 subunit gene differentially impact early and later stages of cocaine addiction: a translational study in transgenic rats and patients

https://doi.org/10.1016/j.pneurobio.2020.101898Get rights and content

Highlights

  • α5SNP rats exhibit impairment in the acquisition of cocaine self-administration.

  • α5 knock-out rats exhibit higher cocaine-induced reinstatement of nicotine seeking.

  • Early phase of cocaine addiction is impaired in human patients carrying the α5SNP.

  • The rs950776 in CHRNB4 is associated with faster relapse to cocaine use in patients.

Abstract

Cocaine addiction is a chronic and relapsing disorder with an important genetic component. Human candidate gene association studies showed that the single nucleotide polymorphism (SNP) rs16969968 in the α5 subunit (α5SNP) of nicotinic acetylcholine receptors (nAChRs), previously associated with increased tobacco dependence, was linked to a lower prevalence of cocaine use disorder (CUD). Three additional SNPs in the α5 subunit, previously shown to modify α5 mRNA levels, were also associated with CUD, suggesting an important role of the subunit in this pathology. To investigate the link between this subunit and CUD, we submitted rats knockout for the α5 subunit gene (α5KO), or carrying the α5SNP, to cocaine self-administration (SA) and showed that the acquisition of cocaine-SA was impaired in α5SNP rats while α5KO rats exhibited enhanced cocaine-induced relapse associated with altered neuronal activity in the nucleus accumbens. In addition, we observed in a human cohort of patients with CUD that the α5SNP was associated with a slower transition from first cocaine use to CUD. We also identified a novel SNP in the β4 nAChR subunit, part of the same gene cluster in the human genome and potentially altering CHRNA5 expression, associated with shorter time to relapse to cocaine use in patients.

In conclusion, the α5SNP is protective against CUD by influencing early stages of cocaine exposure while CHRNA5 expression levels may represent a biomarker for the risk to relapse to cocaine use. Drugs modulating α5 containing nAChR activity may thus represent a novel therapeutic strategy against CUD.

Introduction

Cocaine use disorder (CUD) is a clinically devastating neuropsychiatric disease without any approved pharmacotherapy (Degenhardt et al., 2011). Relapse to cocaine seeking after a phase of abstinence is the main impediment in the treatment of CUD, with 78 % of patients relapsing within three months after discharge from hospital detoxification (Lima et al., 2019). In such pathological cocaine users, relapse can be precipitated by exposure to cocaine-associated cues, stress, or to cocaine itself (Childress et al., 1993; Jaffe et al., 1989; O’Brien, 1998; Sinha, 2001). Relapse prevention is thus a key target for clinicians in the treatment of substance use disorders in general since it has been associated with the chronicization and the burden of these disorders (Hendershot et al., 2011). Similarly, reinstatement of cocaine-seeking can be elicited in laboratory animals after extinction of cocaine self-administration (SA), by exposure to a conditioned stimulus (CS) previously associated with cocaine intake, stress or a cocaine-priming injection (Crombag and Shaham, 2002; de Wit and Stewart, 1981; Epstein et al., 2006; Fuchs et al., 1998).

Cocaine acts primarily by inhibiting monoamine transporters, such as the dopamine transporter (DAT), leading to increased extracellular dopamine concentration in the nucleus accumbens (NAcc), an effect considered essential for its reinforcing property. However, cocaine is also an inhibitor of nicotinic acetylcholine receptors (nAChRs) (Damaj et al., 1999; Francis et al., 2000), an action that can alter dopamine release in the NAcc (Acevedo-Rodriguez et al., 2014). nAChRs are pentameric ligand-gated ion channels composed of α (α2– α7, α9, α10) and β (β2– β4) subunits that co-assemble according to various combinations with distinct brain localizations and functional properties (Changeux, 2010). nAChRs have been investigated in preclinical models of cocaine addiction. For example, the nAChR antagonist mecamylamine reduces cocaine-SA and prevents cocaine-SA escalation in rodents (Blokhina et al., 2005; Hansen and Mark, 2007; Levin et al., 2000). Additionally, 18-MC, an antagonist of the α3β4 containing (*) nAChRs, decreases cocaine-SA in rats (Glick et al., 1996; Pace et al., 2004), and mice knockout (KO) for the β2 nAChR subunit exhibit decreased cocaine-induced conditioned place preference (Zachariou et al., 2001). Interestingly, mecamylamine also reduces craving for cocaine in CUD subjects (Reid et al., 1999).

Several independent large-scale human genome-wide association studies (GWAS) identified variants in a region on human chromosome 15 encoding the α3, α5 and β4 nAChR subunits that increase the risk for nicotine dependence (Bierut et al., 2007, 2008; Sherva et al., 2008). One of these variants, the non-synonymous coding single nucleotide polymorphism (SNP) rs16969968 found in exon 5 of the α5 gene (α5SNP) that causes an amino acid change D398N in the second intracellular loop of the α5 subunit, has been recently introduced into the genome of the rat, resulting in increased nicotine-SA at high doses and relapse (Forget et al., 2018). This α5SNP is very frequent in the general population and may induce a partial loss of function of the corresponding pentamer (Bierut et al., 2008; Frahm et al., 2011; Kuryatov et al., 2011) without altering the overall expression of the subunit, at least in rats (Forget et al., 2018). It does not modify the electrophysiological function of the receptor when expressed in concatemer in Xenopus oocytes (Prevost et al., 2020), and it lies away from agonists’ binding site, suggesting that it affects other features of the receptor, but its precise mechanism of action is currently unknown (Maskos, 2020). Interestingly, in candidate gene association studies, the α5SNP has also been associated with a lower prevalence of CUD in humans (Grucza et al., 2008; Sherva et al., 2010). In addition, three other SNPs in the α5 nAChR subunit, linked to altered levels of α5 mRNA in humans, have also been associated with CUD (Sherva et al., 2010; Wang et al., 2009). These genetic studies suggest an involvement of the α5 nAChR subunit in cocaine dependence, although the precise mechanisms and stages of CUD implicated have never been further assessed so far.

In fact, multiple behaviors related to CUD may be impacted by alterations in α5*nAChR density or mutations in the α5 nAChR subunit gene, that now need to be identified through longitudinal studies to understand how they contribute to the risk for this pathology. For this, the first goal of the present study was to clarify the role of α5*nAChRs and the impact of the α5SNP, found to confer protection for CUD in humans, on the different stages of an intravenous (IV) cocaine-SA procedure, from first cocaine intake to relapse to cocaine seeking after SA extinction, using transgenic rat lines previously generated in the laboratory (Forget et al., 2018). At the molecular level, since cocaine has been shown to inhibit nAChR-mediated currents in midbrain dopaminergic neurons (Acevedo-Rodriguez et al., 2014), we hypothesized that the association between α5*nAChRs and CUD may be linked to an implication of the α5 subunit in nAChR response to cocaine. Thus, the second objective of the study was to characterize the influence of the α5 subunit on nAChR functional response to cocaine in vitro. Finally, for translational purposes, we took advantage of the information generated by the SA longitudinal study implemented in our preclinical models to i) clarify, in humans, the association of the α5SNP with specific CUD phenotypes, and ii) seek for novel polymorphisms in the CHRNA5/A3/B4 gene cluster, linked to α5 expression levels, in association with these CUD phenotypes.

Section snippets

Experimental model and subject details

Male rats experimentally naive at the start of the study and initially weighing 250–275 g were used for all behavioral experiments. All rats were individually housed in a temperature-controlled environment on a 12-h reverse light/dark cycle (lights off from 08:00 h to 20:00 h) at the start of the experiments and were mildly food restricted (20 g/day/rat).

Wild-type (WT) rats, homozygous α5KO rats and homozygous rats constitutively carrying the rs16969968 SNP (α5SNP rats), on Long Evans

Acquisition of cocaine-SA, dose-response curve, saline substitution and motivation for cocaine-SA in α5SNP, α5KO and WT rats

α5SNP, α5KO and WT rats acquired the food training similarly (continuous reinforcement, no associated cues; Fig. 1A), suggesting an absence of learning or motor deficit in α5SNP and α5KO rats.

Rats were then submitted to chronic IV cocaine-SA. The three groups increased similarly their lever pressing behavior when the ratio progressively increased (FR-1 then FR-2 then FR-3 then FR-4 and finally FR-5; Fig. 1B). However, there was a significant interaction between sessions and genotypes on the

Phenotypes of α5SNP and α5KO rats in cocaine-SA and relapse

We first observed that the presence the α5SNP did not prevent rats from acquiring cocaine-SA, and did not alter the dose-response curve for cocaine-SA. Yet, contrary to WTs, α5SNP rats did not adapt their AL pressing to maintain constant amounts of cocaine intake when the ratio schedule of reinforcement was increased (Fig. 1C and D). This seems in line with previous indications of a protective effect of this polymorphism on cocaine dependence in humans (Grucza et al., 2008; Sherva et al., 2010

Declaration of Competing Interest

RI has received honoraria and support to attend congresses from Indivior and honoraria and consulting fees from Lundbeck. FV has received an honorarium from Lundbeck for a single lecture. FB has received honoraria or research or educational conference grants from Bristol-Myers Squibb, Otsuka, Eli Lilly and Co., Servier, Sanofi Aventis, Lundbeck, AstraZeneca, the European Space Agency and has received peer review research funding from French Ministry of research, Assistance Publique – Hôpitaux

Acknowledgements

Funding: This work was supported by the Institut Pasteur, Centre National de la Recherche ScientifiqueUMR 3571, Fondation pour la Recherche Médicale (SPF20140129365, DPA20140629803andEQU201903007822), Agence Nationale de la Recherche (ANR), Neuroscience "SNP-NIC" and BLANC, LABEX BIO-PSY, FP7 ERANET programme NICO-GENE grant agreement convention ANR no 2010-NEUR-004-01, European Commission FP7 RTD Project HEALTH-2009-Neurocyp.08-202088 grant 242167, French National Cancer Institute grant

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