Differential sensitivity of human neurons carrying μ opioid receptor (MOR) N40D variants in response to ethanol

Highlights

• There is a limited understanding of the precise cellular and molecular mechanisms underlying AUDs in humans.

• A functional polymorphism of the mu-opioid receptor (MOR) N40D may alter the risk of developing AUDs.

• iPS and induced neuronal (iN) cell technology provide unique opportunities to model AUDs in a human context.

• Alcohol application reveals that the MOR genotype confers differential sensitivity to synaptic output for AD-iNs.

Abstract

The acute and chronic effects of alcohol on the brain and behavior are linked to alterations in inhibitory synaptic transmission. Alcohol's most consistent effect at the synaptic level is probably a facilitation of γ-aminobutyric acid (GABA) release, as seen from several rodent studies. The impact of alcohol on GABAergic neurotransmission in human neurons is unknown, due to a lack of a suitable experimental model. Human neurons can also be used to model effects of genetic variants linked with alcohol use disorders (AUDs). The A118G single nucleotide polymorphism (SNP rs1799971) of the OPRM1 gene encoding the N40D (D40 minor allele) mu-opioid receptor (MOR) variant has been linked with individuals who have an AUD. However, while N40D is clearly associated with other drugs of abuse, involvement with AUDs is controversial. In this study, we employed Ascl1-and Dlx2-induced inhibitory neuronal cells (AD-iNs) generated from human iPS cell lines carrying N40D variants, and investigated the impact of ethanol acutely and chronically on GABAergic synaptic transmission. We found that N40 AD-iNs display a stronger facilitation (versus D40) of spontaneous and miniature inhibitory postsynaptic current frequency in response to acute ethanol application. Quantitative immunocytochemistry of Synapsin 1⁺ synaptic puncta revealed a similar synapse number between N40 and D40 iNs, suggesting an ethanol modulation of presynaptic GABA release without affecting synapse density. Interestingly, D40 iNs exposed to chronic intermittent ethanol application caused a significant increase in mIPSC frequency, with only a modest enhancement observed in N40 iNs. These data suggest that the MOR genotype may confer differential sensitivity to synaptic output, which depends on ethanol exposure time and concentration for AD-iNs and may help explain alcohol dependence in individuals who carry the MOR D40 SNPs. Furthermore, this study supports the use of human neuronal cells carrying risk-associated genetic variants linked to disease, as in vitro models to assay the synaptic actions of alcohol on human neuronal cells.

Introduction

Alcohol use disorders (AUDs), which include alcohol abuse and dependence, are among the most common types of life-threatening neuropsychiatric disorders, impacting ~15% of the population (Sridhar, 2012; Grant et al., 2017; World Health Organization, 2019). Despite the high prevalence of AUDs, the precise molecular mechanism(s) that lead to the development of dependence and tolerance remain poorly understood (Lewohl et al., 2000; Hanchar et al., 2006). One limitation to our current mechanistic understanding of AUDs is the lack of an appropriate human model system that permits functional studies.

Despite the lack of understanding of alcohol's impact on human neurons, rodent studies have provided us with invaluable information. Ethanol, also known as ethyl alcohol, produces its clearest presynaptic effects at GABAergic synapses in several brain regions in rodents (Lovinger, 2018). Specifically, early electrophysiological studies established that ethanol consistently and reproducibly potentiated GABA-mediated inhibitory transmission, and this was described as one of the clearest synaptic effects of the drug (Wan, Berton, Madamba, Francesconi, & Siggins, 1996; Weiner, Gu, & Dunwiddie, 1997). This effect was observed at GABAergic synapses in many brain regions, including the hippocampus (Ariwodola & Weiner, 2004; Sanna et al., 2004), basolateral amygdala (Butler, Chapell, & Weiner, 2014; Karkhanis, Alexander, McCool, Weiner, & Jones, 2015; Talani & Lovinger, 2015), central amygdala (Bajo, Cruz, Siggins, Messing, & Roberto, 2008), cerebellum (Kelm, Criswell, & Breese, 2008), dorsal striatum (Wilcox et al., 2014), nucleus accumbens (Hyytiä & Koob, 1995), spinal cord (Ziskind-Conhaim, Gao, & Hinckley, 2003), and ventral tegmental area (VTA) (Theile, Morikawa, Gonzales, & Morrisett, 2008). It was hypothesized that one mechanism of the rewarding properties of ethanol could be a result of a disinhibitory mechanism involving opioid receptors that are expressed on VTA interneurons (Xiao, Zhang, Krnjević, & Ye, 2007).

AUDs are heterogeneous in etiology, pathophysiology, and response to treatment. In addition to environmental factors, AUDs are genetically complex, without an obvious pattern of Mendelian inheritance, and are moderately heritable as observed from family-based twin studies (~49%) (Kendler, Heath, Neale, Kessler, & Eaves, 1992; McGue, Pickens, & Svikis, 1992; Reed, Page, Viken, & Christian, 1996; Heath et al., 1997; Prescott & Kendler, 1999; True et al., 1999; Verhulst, Neale, & Kendler, 2015). It is likely that a combination of genetic variants and environment, each responsible for a small effect, are involved. Genome-wide association studies (GWAS) have identified several risk loci for AUDs (Dick et al., 2006; Edenberg, 2007; Hart & Kranzler, 2015). One variant associated with AUDs is a non-synonymous single nucleotide polymorphism (SNP) in OPRM1, which encodes the μ-opioid receptor (MOR), A118G (rs1799971; Asn40Asp or N40D), although this remains controversial (LaForge, Yuferov, & Kreek, 2000; Mague & Blendy, 2010; Sloan et al., 2018). The A118 OPRM1 major allele that codes for the MOR (N40) is found at the highest frequency in individuals of all ethnic backgrounds, and the minor allele G118 OPRM1 (D40 MOR) is found at a lower frequency overall. However, the minor allele SNP occurs in different frequencies in different ethnic groups, from ~3% in African, ~16% in European, and 39–42% in Asian ancestry (Zerbino et al., 2018). There have been several studies aimed at understanding the functional consequences of the MOR D40 variant on receptor activation in mouse models, primates, and humans (Bond et al., 1998; Befort et al., 2001; Beyer, Koch, Schröder, Schulz, & Höllt, 2004; Miller et al., 2004; Zhang, Wang, Johnson, Papp, & Sadée, 2005; Kroslak et al., 2007; Mague et al., 2009; Mague & Blendy, 2010; Mahmoud et al., 2011; Margas, Zubkoff, Schuler, Janicki, & Ruiz-Velasco, 2007). Both animal and human studies reveal that the N40D substitution destroys an N′-terminal glycosylation site and reduces the surface expression for MORs (Ray et al., 2011; Wang, Huang, Ung, Blendy, & Liu-Chen, 2012; Wang, Huang, Blendy, & Liu-Chen, 2014; Halikere et al., 2019). However, another study using a humanized mouse model of MOR N40D is not in complete agreement with this finding (Bilbao et al., 2015). Thus, to gain insight into mechanisms underlying drug abuse, it is imperative to understand how the D40 variant impacts MOR signaling and synaptic function in a human neuronal context.

Extensive work has explored the relationship between OPRM1 A118G and various substance abuse disorders, specifically alcohol abuse disorders (Bergen et al., 1997; Gelernter, Kranzler, & Cubells, 1999; Town et al., 1999; Schinka et al., 2002; Crowley et al., 2003; Luo, Kranzler, Zhao, & Gelernter, 2003; Kim et al., 2004; Loh, Fann, Chang, Chang, & Cheng, 2004; Nishizawa et al., 2006; Szeto, Tang, Lee, & Stadlin, 2001; Türkan et al., 2019). This has profound treatment implications, since naltrexone, an opioid receptor antagonist, decreases relapse in alcoholic individuals (Jonas et al., 2014) and is one of the few adjunctive therapies approved for the treatment of subjects with AUDs. Analysis of G-allele carriers diagnosed with an AUD responded more strongly to naltrexone, with lower rates of relapse and a longer time to return to heavy drinking (Oslin et al., 2003). However, a more recent study failed to observe the same effect (Oslin et al., 2015; Sloan et al., 2018), but reported that G-allele carriers consume more drinks per day, on average. This study's findings may differ from previous meta-analyses because the authors omitted studies that were not randomized clinical trials, which could explain the discrepancies between studies. This could be explained by multiple genetic components, for example, the selection of specific ethnic backgrounds included in the study, since the minor allele frequency of rs1799971 is variable among different ethnic groups, and potentially confounded by the presence or absence of other linked genetic variants. To control for this possibility, we have modeled contrasting rs1799971 genotypes in a CRISPR-edited, isogenic set of human-induced pluripotent stem (iPS) cells (Halikere et al., 2019).

Several studies have employed the use of patient-specific iPS cell technologies to model and elucidate the mechanisms underlying AUDs (reviewed by Prytkova, Goate, Hart, & Slesinger, 2018; Scarnati, Halikere, & Pang, 2019). Electrophysiological recordings conducted in several brain regions, from animal models, report a potentiation of GABA_A receptor response following exposure to alcohol (Aguayo, 1990; Jia, Chandra, Homanics, & Harrison, 2008; Nie et al., 2004; Nie, Madamba, & Siggins, 2000; Yeh & Kolb, 1997). However, recordings performed in iPS cell-derived human neuronal cells contradict these findings (Lieberman, Kranzler, Levine, & Covault, 2017). However, these recordings were performed in mixed excitatory/inhibitory co-cultures, and a puffing procedure was used to specifically measure the effect of ethanol on postsynaptic GABA_A receptors. There was no apparent strengthening of the GABA_A response following acute exposure to alcohol. This suggests species-specific mechanisms governing GABA_A receptor function, which supports the importance of human neuronal models in understanding the synaptic role of alcohol. Finally, the mechanisms of how GABAergic release and GABA_A receptor function are impacted by alcohol application in human neurons carrying functionally important and commonly found SNPs that have been linked to alcohol abuse are critical for understanding the initiation and progression of AUDs. This includes OPRM1 A118G, which has been linked to AUDs through GWAS (Hoehe et al., 2000; Berrettini, 2013).

The aim of this study is to elucidate ethanol's cellular and synaptic impact, both acutely and chronically, on human neurons of the GABAergic variety, carrying MOR N40D gene variants. Specifically, we were focused on the effect of ethanol on GABAergic transmission in iNs expressing MOR N40D, independent of opioidergic signaling. We found that acute application of ethanol caused an increase in sIPSC and mIPSC frequency for N40-harboring iNs, while only a modest increase was observed in D40 human iNs. In agreement with these data, we also observed a significant decrease in spontaneous action potential firing frequency for N40-containing iNs, due to an increased GABA release. Interestingly, we observed a significant increase in inhibitory synaptic release exclusively in iNs harboring D40 MOR allelic variants, following a 10-day chronic intermittent ethanol (CIE) exposure paradigm, intended to mimic a diurnal drinking pattern common in AUD subjects. Our findings suggest that ethanol treatment alone results in a differential sensitivity to acute and chronic treatment between genotypes. These results may provide a better mechanistic understanding of the interaction between alcohol and opioid signaling in humans.