Neurosteroids (allopregnanolone) and Alcohol Use disorder: From mechanisms to potential pharmacotherapy

Abstract

Alcohol Use Disorder (AUD) is a multifaceted relapsing disorder that is commonly comorbid with psychiatric disorders, including anxiety. Alcohol exposure produces a plethora of effects on neurobiology. Currently, therapeutic strategies are limited, and only a few treatments — disulfiram, acamprosate, and naltrexone — are available. Given the complexity of this disorder, there is a great need for the identification of novel targets to develop new pharmacotherapy. The GABAergic system, the primary inhibitory system in the brain, is one of the well-known targets for alcohol and is responsible for the anxiolytic effects of alcohol. Interestingly, GABAergic neurotransmission is fine-tuned by neuroactive steroids that exert a regulatory role on several endocrine systems involved in neuropsychiatric disorders including AUD. Mounting evidence indicates that alcohol alters the biosynthesis of neurosteroids, whereas acute alcohol increases and chronic alcohol decreases allopregnanolone levels. Our recent work highlighted that chronic alcohol-induced changes in neurosteroid levels are mediated by epigenetic modifications, e.g., DNA methylation, affecting key enzymes involved in neurosteroid biosynthesis. These changes were associated with changes in GABA_A receptor subunit expression, suggesting an imbalance between excitatory and inhibitory signaling in AUD. This review will recapitulate the role of neurosteroids in the regulation of the neuroendocrine system, highlight their role in the observed allostatic load in AUD, and develop a framework from mechanisms to potential pharmacotherapy.

1. Introduction

Alcohol Use Disorder (AUD) is a multifaceted relapsing disorder, one of the most prevalent psychiatric disorders (Grant et al., 2004; Rehm et al., 2015), and a considerable economic burden (Rehm and Shield, 2019). According to a recent survey, 14.5 million people over the age of 12 had an AUD in the United states in 2019 (National Survey on Drug Use and Health, SAMHSA), a number that has significantly worsened during the COVID-19 pandemic (Pollard et al., 2020; Yazdi et al., 2020). Individuals suffering from AUD have an increased vulnerability to develop other psychiatric disorders, including depression and anxiety (Birrell et al., 2015; Lai et al., 2015; Swendsen et al., 1998) and alterations of executive functions (Fernández-Serrano et al., 2010). Conversely, negative emotional states, including stress and anxiety, lead to higher consumption of alcohol (Koob, 2015; Peltier et al., 2019; Pandey et al., 2017). The complex interaction of environmental and genetic factors can predispose an individual to the development of AUD (Schuebel et al., 2016; Starkman et al., 2012). While early evidence indicated an increased likelihood of developing the disease if AUD cases exist in the family (Cotton, 1979; Devor & Cloninger, 1989), specific loci mediating the risk to develop an AUD have been difficult to identify. Genome-wide association studies conducted in the past decades pointed to risk loci in genes involved in alcohol metabolism, including the genes for alcohol dehydrogenase (ADH) and the mitochondrial form of aldehyde dehydrogenase (ALDH2) (Carvalho et al., 2019; Sanchez-Roige et al., 2019). In addition, a growing body of evidence has implicated epigenetic mechanisms, e.g., DNA methylation, histone modifications and noncoding RNAs, in the development and maintenance of alcohol addiction (Bohnsack & Pandey, 2021; Gatta et al., 2021; Kyzar et al., 2022). Recent epigenome-wide association studies of alcohol consumption identified significant differentially methylated CpGs in the cystine/glutamate transporter SLC7A11 gene (Lohoff et al., 2021) and in glucocorticoid signaling genes (Lohoff et al., 2020) in individuals with AUD when compared to controls, suggesting that alterations in excitatory pathways along with environmental stimuli are key players in the disease. The dysregulations of the neurocircuitry underpinning AUD have been characterized as an ‘allostatic load,’ a term coined by Dr. Bruce S. McEwen to define an inadequate and pathological response to chronic stress with excessive release of stress hormones and the emergence of a pro-inflammatory state (McEwen, 1998; McEwen & Stellar, 1993). This concept has been extended to AUD (Koob, 2003). Indeed, chronic alcohol use is known to affect both the reward and stress systems (Gatta et al., 2019; Koob, 2015) and impair numerous physiological functions including immune response (Crews & Vetreno, 2014). While there is a growing understanding of the neurobiological mechanisms of AUD, only a few therapeutic strategies are currently available for the treatment of AUD, i.e., disulfiram, acamprosate and naltrexone (O’Malley & O’Connor, 2011). Given the complex pathophysiology of AUD (Koob & Lemoal,1997; Pandey et al., 2017), there is a great need for the identification of new targets to develop improved pharmacotherapy for AUD. This review has discussed the role of neurosteroids, specifically allopregnanolone, in the regulation of the neuroendocrine system, in particular the stress system, and highlighted their role in the allostatic load observed in AUD. We also discussed how the neurosteroid system is epigenetically regulated by ethanol and whether this could serve as a potential target for pharmacotherapy development to prevent or treat AUD.

2. Stress systems in alcohol addiction: a spiraling interplay

Koob and Lemoal (1997) characterized addiction as a three stage cycle, i.e., preoccupation/anticipation, binge/intoxication, and withdrawal/negative affect. These stages contribute to the development of a pathological state by aggravating each other. The addiction cycle involves key brain regions that encode for reward, motivation as well as stress (Koob & Volkow, 2016), including, among others, the prefrontal cortex, the hypothalamus, the nucleus accumbens and the extended amygdala. It is important to note that all drugs of abuse stimulate the mesolimbic dopamine system that mediate their rewarding properties (Di Chiara & Imperato, 1988; Koob & Volkow, 2010). The transition from controlled to compulsive drinking is mediated by neuroadaptive changes of the dopaminergic mesolimbic and the GABAergic hypothalamic pathways resulting in an allostatic overload of both reward and neuroendocrine circuits (Sinha, 2013). The existence of a frequent comorbidity between stress-related disorders (Conway et al., 2006; Grant et al., 2015), including anxiety, depression and AUD, suggests common underlying mechanisms in the development of these psychiatric disorders.

Epidemiological studies have reported a long standing association between stress and AUD (Ayer et al., 2021; Becker, 2017; San José et al., 2000), with women being four times more likely to have an AUD diagnosis after a stressful life event when compared to men (Verplaetse et al., 2018). Environmental stress and adverse life events may be a trigger for excessive alcohol consumption (Enoch, 2011; Koob, 2008, 2013; Uhart & Wand, 2009), and some individuals drink alcohol in an attempt to self-medicate and attenuate stress-related symptoms (Becker, 2017; Bolton et al., 2009; Kushner et al., 2000). But, the early view of alcohol as an anxiolytic and tension-reducing agent (Cappell & Herman, 1972; Greeley & Oei, 1999) has been contrasted by evidence that higher doses of alcohol act as a stressor. Alcohol exposure and withdrawal from chronic alcohol activate the hypothalamic-pituitary-adrenal (HPA) axis (Blaine & Sinha, 2017; Richardson et al., 2008; Rose et al., 2010), leading to increased release of corticotropin-releasing factor (CRF), adrenocorticotropic hormone (ACTH) from the paraventricular nucleus (PVN) within the hypothalamus, as well as glucocorticoids released from the adrenals, which are key mediators of the neuroadaptations underlying addictive behaviors (Richardson et al., 2008; Rivier, 2014; Stephens & Wand, 2012). This increase is particularly evident as a subject goes through the drinking cycle with repeated episodes of alcohol intoxication and withdrawal (Adinoff, 1990; Adinoff et al., 2003). Moreover, individuals suffering from AUD develop a high tolerance to the rapid effects of alcohol, including sedation and motor impairments, and a higher vulnerability to withdrawal symptoms, e.g., anhedonia and anxiety (Becker, 2008; Chen et al., 2019; Hatzigiakoumis et al., 2011; Martinotti et al., 2008; Pandey et al., 2017). Hence, in recent decades, AUD has been defined as a stress surfeit disorder (Koob, 2008, 2013) with high comorbidities with other psychopathological conditions, such as major depression disorder (MDD), posttraumatic stress disorder (PTSD), and anxiety disorder (Grant et al., 2015).

The relationship between alcohol and stress has been shown to be bidirectional. Intracerebral infusion of glucocorticoids increases voluntary alcohol intake (Fahlke et al., 1996), while suppression of glucocorticoids release by adrenalectomy induces a reduction in alcohol intake in alcohol preferring rats (Fahlke & Eriksson, 2000). In alcohol-dependent rats, acute withdrawal was associated with a reduction of glucocorticoid receptor (GR, a key component of the stress system) expression in stress/reward brain regions, such as the prefrontal cortex and the amygdala (Roy et al., 2002; Vendruscolo et al., 2012). The use of a GR antagonist, i.e. mifepristone, prevented alcohol intake and compulsive behavior in both rodents and human subjects (Vendruscolo et al., 2012, 2015), strengthening the important overlap existing between stress and alcohol.

The development of mood and anxiety disorders appears to be a predictor of alcohol consumption (Moscato et al., 1997). Individuals suffering from stress-related disorders (e.g., MDD and PTSD) also show impairments in the reward system (Grant et al., 2004; Petrakis & Simpson, 2017; Russo & Nestler, 2013). The comorbidity of AUD with other psychiatric disorders leads to a worse prognosis than either disorder alone (Conway et al., 2006; Grant et al., 2004; Hasin et al., 2007). Moreover, there is a reciprocal interaction between these pathologies, with depression increasing the risk of AUD and vice versa (McHugh & Weiss, 2019). The relationship between alcohol and other psychiatric disorders is also relevant for PTSD. Individuals suffering from PTSD are 1.2 times more likely to also meet the criteria for AUD when compared to healthy controls (Simpson et al., 2019). This prevalence is significantly higher in veterans (Chen et al., 2018; Seal et al., 2011). It should be noted that alterations in the HPA axis are a common neurobiological underpinning for these disorders. Specifically, central and peripheral glucocorticoids levels have been key determinants in the development of a pathological state in both depression (Stetler & Miller, 2011; Young et al., 2001) and PTSD (Daskalakis et al., 2013). Preclinical models of PTSD show that female mice exposed to chronic stressors increase their alcohol self-administration when compared to males (Cozzoli et al., 2014). Similarly, exposure to a traumatic stress paradigm increased ethanol consumption in alcohol-dependent adult male mice (Piggott et al., 2020). These observations suggest that therapeutic strategies targeting the stress system could be promising for the treatment of co-occurring stress-related disorders.

3. Key role of altered GABAergic neurotransmission on the stress system

Neurosteroids and glucocorticoids are involved in number of crucial central and peripheral functions, including the regulation of metabolism, immune and inflammatory mechanisms as well as mood and anxiety disorders (Morrow et al., 2020). The biological mechanisms modulating the function of the stress system have been investigated for decades (Jones et al., 1976; Makara & Stark, 1974). Regulation of the HPA axis activity occurs via a bottom-up control exerted by glucocorticoids, which have both genomic and non-genomic actions (de Kloet et al., 2008). Specifically, the GR regulates stress response by acting as a negative feedback loop on the HPA axis, in addition to its metabolic and immune suppressant actions (de Kloet and Derijk, 2004; Silverman and Sternberg, 2012). Furthermore, GABAergic projections originating from the posterior bed nucleus of the stria terminalis (BNST) and the peri-PVN regions mediate the inhibitory control of the HPA axis (Herman, 2012). The limbic and forebrain regions, including the hippocampus and the prefrontal cortex, also regulate the HPA axis by projecting to GABAergic nuclei (Radley et al., 2006; Ulrich-Lai & Herman, 2009). GABA released by these projections primarily binds to synaptic and extra-synaptic GABA_A receptors (GABA_ARs), which are expressed throughout the stress neurocircuitry (Cullinan et al., 2008; Maguire, 2014). The pharmacological and biophysical properties of GABA_ARs are determined by the combination of specific subunits, 19 identified so far (α_1-6, β_1-3, γ_1-3, δ, ε, θ π and ρ_1-3), which form a pentameric structure (Olsen & Sieghart, 2008, 2009) belonging to the family of ionotropic receptors, ligand-gated chloride anion channels (Nayeem et al., 1994). The expression profile of these subunits varies depending on the brain region; most GABA_ARs isoforms include two α, two β and a single γ, δ or ε subunit (Sente et al., 2022).

Two main GABA_ARs isoforms have been characterized (Fig. 1): synaptic receptors, which mediate phasic GABAergic transmission and mostly contain α, β and γ subunits, and the extrasynaptic receptors, which contain α, β and δ subunits responsible for tonic (Farrant & Nusser, 2005) and “spill-over” inhibition (Herd et al., 2013, Figure 1). It has recently been demonstrated that the neuronal location of GABA_AR isoforms is the result of a dynamic trafficking between synaptic and extrasynaptic locations (Davenport et al., 2021; Wu et al., 2021). In addition, slow synaptic GABAergic inhibition is mediated by metabotropic GABA_B receptors (Chebib & Johnston, 1999; Li & Slesinger, 2022), which are expressed both pre-and post-synaptically throughout the CNS.

Figure 1. GABAergic neurotransmission and modulation.

GABA acts on both synaptic and extrasynaptic GABA_A receptors (GABA_ARs) as well as on metabotropic GABA_B receptors (GABA_BRs). Synaptic GABA_ARs are responsible for phasic current, which results from the releasing of a high, short-lasting concentration of GABA into the synaptic cleft. It acts on a relatively small number of GABA receptors on the postsynaptic membrane. Extrasynaptic GABA_ARs are, instead, responsible for tonic inhibition; a low concentration of GABA, persisting in the extracellular ambient despite the activity of GABA transaminases, is responsible for the tonic current. GABA tonically activates extrasynaptic high-affinity GABA_ARs, which are located far from the synaptic cleft. GABA binds between α and β subunits of both synaptic and extrasynaptic receptors. Neurosteroids bind at a site between α and β subunits of both synaptic and extrasynaptic receptors and show a greater affinity for the extrasynaptic receptor, where they act at nM concentrations. Several drugs known to be used in the treatment of alcohol use disorder (AUD) target GABAergic neurotransmission. Benzodiazepines (BDZ) act between α and γ subunits of synaptic receptors. Gabapentin binds the β subunit of the extrasynaptic receptor, while Topiramate only binds β 2/3 subunits. On the other hand, Sodium oxybate and Baclofen bind GABA_BR. Interestingly, ethanol binds both synaptic and extrasynaptic GABA_ARs and GABA_BRs.

Pharmacological studies have further highlighted the role of GABAergic neurotransmission in the inhibition of glucocorticoids release. Microinfusion of bicuculine, a GABA_ARs antagonist, into the PVN enhanced stress-induced glucocorticoid plasmatic levels, while the GABA_ARs agonist muscimol blunted the stress response in adult rats exposed to restraint stress (Cullinan, 1998). The importance of GABAergic neurotransmission in the regulation of the HPA axis is also supported by the evidence that γ₂-deficient mice have neuroendocrine abnormalities which have been linked to HPA axis hyperactivity (Shen et al., 2010). γ₂-containing receptors are the most abundant GABA_ARs in the brain, mediating fast phasic inhibition in response to synaptic GABA release (Belelli et al., 2021). Given the importance of HPA axis dysregulations in stress-related disorders, including depression and anxiety, modulators of the GABAergic transmission have been considered as putative therapeutic options. Considering that AUD has been labeled a “stress surfeit disorder” (Koob, 2013), this therapeutic strategy could also be promising for the treatment of alcohol dependence and withdrawal.

The balance between pro and anti-stress neuropeptides (e.g., CRF and Neuropeptide Y (NPY), respectively), which are highly expressed in the amygdala (Koob, 2015; Palmisano & Pandey, 2017), has also been shown to modulate inhibitory GABAergic currents. The central nucleus of the amygdala (CeA) is an anatomical structure functioning at the interface between the stress-system and addictive processes. Most neurons in the CeA co-release GABA and neuropeptides, which appear to have a key regulatory role in anxiety-like and alcohol-related behaviors (Walker, 2021). Both alcohol withdrawal and stress induce an increase in CRF production and release within the CeA, leading to stress-related behaviors via a robust increase in GABAergic transmission. Of note, CRF receptor 1 (CRF1) antagonists reverse this phenotype (Funk et al., 2007; Knapp et al., 2004; Overstreet et al., 2004). Conversely, NPY has a strong anxiolytic effect resulting from its action on both NPY presynaptic (Y₂R) and postsynaptic (Y₁R) receptors in both the CeA and the basolateral amygdala (BLA; Gilpin, 2012). Ablation of Y₂R results in increased depression and anxiety-like behaviors, a response that might be associated with a dysregulation of GABAergic neurotransmission (Tasan et al., 2010). Thus, an ethanol withdrawal-induced decrease in NPY levels in the CeA may play a significant role in the increase of GABAergic tone in alcohol-dependent rats (Gilpin et al., 2011). Despite their opposite behavioral effects, NPY and CRF have a remarkably similar anatomical pattern of distribution; this, along with electrophysiological data, suggests that NPY and CRF interact on the regulation of the GABAergic output in the CeA leading to the development of chronic alcohol intake and AUD (Gilpin, 2012).

4. Modulatory role of neurosteroids (allopregnanolone) on the stress response

Neurosteroids are endogenous GABA_AR positive allosteric neuromodulators and include allopregnanolone (3α,5α-tetrahydroprogesterone) and its potent stereoisomer pregnanolone (Belelli & Lambert, 2005; Figure 2). Unlike glucocorticoids, neurosteroids (progesterone metabolites 5α-pregnan-3α-tetrahydroprogesterone [allopregnanolone], 5β-pregnan-3α-tetrahydroprogesterone and the deoxycorticosterone [DOC] metabolite, 5α,3α-tetrahydrodeoxycorticosterone [5α3α-THDOC]) are produced in the periphery (predominantly by the adrenal cortex and the ovaries (Paul & Purdy, 1992)), in the peripheral (Giatti et al., 2015; Mensah-Nyagan et al., 2008; Patte-Mensah et al., 2006) as well as the central nervous system (Baulieu et al., 1981; Cheney et al., 1995; Do Rego et al., 2009) where they mediate both paracrine and autocrine actions (Agís-Balboa et al., 2006; Belelli & Lambert, 2005). In the CNS, neurosteroid biosynthesis begins with the transport of cholesterol within glial cells from the outer to the inner mitochondrial membrane by the 18kDa translocator protein (TSPO) (Costa & Guidotti, 1991; Rupprecht et al., 2009), and the steroidogenic acute regulatory (StAR) protein (Clark et al., 1994; Lacapere et al., 2020) (Fig. 2). While the origin of brain TSPO has been identified in a specific subtype of glial cells, e.g. microglia cells, recent evidence suggests that TSPO is also expressed in other brain cells, such as astrocytes and neurons (Nutma et al., 2021). Interestingly it has been demonstrated that numerous brain cells, including neurons, oligodendrocytes, pinealocytes and glial cells, possess all the enzymatic machinery necessary for the local biosynthesis of neurosteroids (Agís-Balboa et al., 2006; Baulieu & Robel, 1998; Kimoto et al., 2001; King et al., 2002; Lloyd-Evans & Waller-Evans, 2020; Melcangi et al., 1993; Mellon & Deschepper, 1993; Mellon & Vaudry, 2001). Although TSPO is expressed in glial cells, the synthesis of neurosteroids most likely occurs in neurons (Porcu et al., 2016). Additionally, diazepam binding inhibitor (DBI) binds to TSPO to stimulate the transport of cholesterol to the mitochondria, thus increasing the concentration of this rate limiting component for neurosteroid synthesis (Costa et al., 1994; Costa & Guidotti, 1991; Midzak et al., 2015). The mitochondrial cholesterol side-chain cleavage enzyme (P450scc) then converts cholesterol into pregnenolone, which is the precursor for all neurosteroids. Pregnenolone diffuses into the cytosol, where it is further metabolized into progesterone by the 3β-hydroxysteroid dehydrogenase (3β-HSD). Type-1 5α-reductase (5α-R1) and 3α-HSD then convert progesterone into allopregnanolone (Do Rego et al., 2009; Locci & Pinna, 2017). 5α-1 and 3α-HSD also convert peripherally derived glucocorticoid metabolite, DOC into 5α3α-THDOC (Karavolas & Hodges, 1990).

Figure 2. Alcohol-induced epigenetic modifications of neurosteroidogenesis at the GABAergic synapse.

GABA is released from GABAergic interneurons and acts on synaptic (phasic current) and extrasynaptic (tonic current) GABA_A receptors (GABA_ARs). GABA_ARs are modulated by the neurosteroids, including allopregnanolone. The synthesis of neurosteroids occurs both in the periphery (where they are produced from circulating progesterone) and the central nervous system. In the brain, neurosteroidogenesis starts in neurons or glial cells, where cholesterol is transported from the outer to the inner mitochondrial membrane by the translocator protein (TSPO) and is converted into pregnanolone. Pregnanolone is further transported into the cytosol and converted into Progesterone, 5α-Dihydroprogesterone and finally into allopregnanolone, which is a positive allosteric modulator of GABA_ARs. Both alcohol and stress activate the hypothalamic-pituitary-adrenal (HPA)-axis. Allopregnanolone acting on corticotropin releasing factor (CRF)-secreting neurons via GABAergic current-mediated mechanisms dampens the hyperactivation of the stress system. Chronic alcohol consumption alters GABAergic transmission and neurosteroidogenesis via epigenetic mechanisms, leading to the loss of inhibition of HPA-axis GABA-mediated control, physiologically modulated by allopregnanolone. PE=pregnanolone, Prog=progesterone, 5α-DHP=5α-dehydroprogesterone, TET=ten-eleven translocation methylcytosine dioxygenases, DNMT=DNA methyltransferase.

Neurosteroids exert their biological function by binding with high affinity to allosteric modulatory sites located on GABA_ARs. Unlike benzodiazepines, which exert their actions through different GABA_ARs subtypes (e.g. α₁ and α₂ exert an anxiolytic and sedative effect, while β₃ exerts an anesthetic action), in vitro experiments have demonstrated that neurosteroids act at physiologically nanomolar concentrations on a variety of GABA_ARs subtypes (Lambert et al., 2003). In addition, α subunit of GABA_ARs does not influence the positive allosteric modulatory action of allopregnanolone on GABA_ARs-mediated Cl-influx, when expressed with β and γ subunit, strengthening the hypothesis that the neurosteroid modulatory effect on GABA complex action may be independent of the GABA_ARs complex subunit composition. Therefore, the modulatory effects of neurosteroids on GABAergic neurotransmission are dependent on cell and neuronal types, brain regions, and distribution density of GABA_ARs, expressed throughout the CNS. The binding of allopregnanolone to the transmembrane domains of α₆/α₄ and δ subunits containing extrasynaptic GABA_ARs results in a highly efficacious positive allosteric modulatory GABAergic inhibitory neurotransmission (Concas et al., 1998; Hosie et al., 2006; Brickley & Mody, 2012; Callachan et al., 1987; Lambert et al., 1995; Shu et al., 2004). Indeed, the δ subunit of the GABA_ARs complex plays a pivotal role in the sensitivity of the receptor to the modulatory effect of neurosteroids on GABAergic neurotransmission. Most importantly, this subunit is highly expressed in thalamus, granule cells of the cerebellum, and hippocampal dentate gyrus, where it acts as a major component of the tonic inhibitory current (Follesa et al., 2015). Of note, in δ subunit knock-out mice, the modulatory effects of neurosteroids are significantly attenuated (Mihalek et al., 1999; Spigelman et al., 2003; Stell et al., 2003), and the replacement of γ subunit with δ subunit enhances the sensitivity of the receptor to neurosteroid modulatory actions (Belelli et al., 2002; Brown et al., 2016; Wohlfarth et al., 2002). A similar reduction in tonic inhibitory current was also reported in mice lacking the α₄ subunit, which is often co-assembled with the δ subunit (Chandra et al., 2006).

By modulating neuronal activity, neurosteroids are also key players in the regulation of the stress response as they participate in a feed-forward process of the HPA axis (Tonhajzerova & Mestanik, 2017). Exposure to stressful stimuli has been shown to increase allopregnanolone levels in the plasma and the central nervous system (Barbaccia et al., 1996, 1997; Purdy et al., 1991; Serra et al., 2002). In vitro and in vivo experiments have demonstrated that allopregnanolone dampens the endocrine response to stress by reducing CRF, ACTH and glucocorticoids release (Owens et al., 1992; Patchev et al., 1994, 1996), resulting in anxiolytic and anti-depressant effects (Bitran et al., 1991, 1999; Carboni et al., 1996; Crawley et al., 1986). In mice, systemic administration of trilostane, a 3β-HSD inhibitor, reduced progesterone levels in the brain and produced antidepressant-like effects (Espallergues et al., 2012). Conversely, the administration of finasteride, a 5α-R1 inhibitor that blocks neurosteroidogenesis, increases anxiety/depressive-like behaviors (Pallarès et al., 2015; Yoshizawa et al., 2017). In addition to these neuromodulatory roles, early evidence also highlighted the transcriptional effect of allopregnanolone on CRF gene expression in the hypothalamus (Patchev et al., 1994). The regulation of the stress system appears to be influenced by a sex-dependent mechanism; it has been showed that allopregnanolone administration decreased CRF synthesis and release in hippocampus and amygdala of male but not female rats. This evidence suggests a more complex mechanism in need of further investigation (Boero et al., 2021).

Thus, allopregnanolone has an important role in restoring the physiological response of the organism after stress exposure (Gunn et al., 2015) through regulating the HPA axis, both directly by impacting genomic mechanisms and indirectly by modulating GABAergic neurotransmission. Interestingly, Deo and colleagues (Deo et al., 2010) showed that allopregnanolone could also exert its anxiolytic-like effect by increasing the expression of NPY Y1 receptors in CeA in mice. The intra-CeA administration of either allopregnanolone, NPY or NPY Y1/5-receptor agonists resulted in anxiolytic effects in a dose-dependent manner, while an intra-CeA injection of NPY Y1 antagonists resulted in a significant increase in anxiety-like behavior. The anxiolytic effect of allopregnanolone was even more significant when administrated with effective doses of NPY, thus empowering the hypothesis that there is an interaction between neuropeptides and the neurosteroid system in selective nuclei of the amygdala.

As described above, impairment in HPA axis regulation is one of the underlying causes for the development of psychiatric disorders, including depression, PTSD and AUD. Mounting evidence indicate that allopregnanolone might serve as a biomarker for these diseases (for review, Aspesi and Pinna, 2018; Tomaselli and Vallée, 2019; Almeida et al., 2021). Individuals suffering from depression present with reductions in peripheral allopregnanolone levels in blood (Romeo et al., 1998; Ströhle et al., 1999) as well as in cerebrospinal fluid (CSF, Uzunova et al., 1998). Similarly, both men and women with PTSD have decreased allopregnanolone levels in the CSF (Rasmusson et al., 2006, 2019). These changes are associated with reduced expression and activity of GABA_ARs (Croarkin et al., 2011; Sanacora & Saricicek, 2007). Recent studies have shown that the intravenous infusion of brexanolone, a formulation of the endogenous allopregnanolone, reduces emotional disturbances in women with postpartum depression for more than a month (Dacarett-Galeano & Diao, 2019; Kanes et al., 2017; Meltzer-Brody et al., 2018). These findings have led to the FDA approval of this drug for the treatment of post-partum depression (Powell et al., 2020) and strengthen the hypothesis that targeting the GABAergic transmission is a potential therapeutic strategy for the treatment of stress-related psychiatric disorders.

5. GABAergic transmission impairments in AUD: current pharmacological approaches

The GABAergic system is a well-known target for alcohol (Liang & Olsen, 2014). Specifically, GABA_ARs have been identified as a central target for the action of ethanol (Becker et al., 1998; Boehm et al., 2004; Koob, 2004; Olsen & Spigelman, 2012), and single nucleotide polymorphisms of the α₂ and γ₁ subunit have been associated with alcohol dependence (Edenberg et al., 2004; Ittiwut et al., 2012; Soyka et al., 2008). Both low doses and intoxicating high doses of alcohol bind to synaptic and extrasynaptic GABA_ARs (Davies, 2003; Olsen, 2018), which mediate its pharmacological effects (Figure 2). To better understand the development of alcohol tolerance and dependence, an understanding of the distinctive roles of the synaptic and the extrasynaptic GABA_ARs in mediating the modulatory effects of alcohol on neuronal signaling appears to be crucial (Figure 2). By binding to these receptors, acute alcohol induces anxiolysis, sedation as well as impairments in motor coordination. Further, chronic alcohol consumption results in lethargy, confusion and loss of sensation which may eventually lead to death (Valenzuela, 1997).

A plethora of preclinical studies have investigated the effects of ethanol in the brain. Acute ethanol potentiates GABAergic neurotransmission (Harris & Allan, 1989; Suzdak et al., 1986), and in the basolateral amygdala, this has been related with the anxiolytic effects of this drug (Silberman et al., 2012). Interestingly, these changes have been associated with epigenetic mechanisms regulating the expression of neuropeptide Y in the amygdala (Berkel et al., 2019; Sakharkar et al., 2012). Some studies have demonstrated that a single alcohol injection could change the expression of GABA_AR subunits. Specifically, in the hippocampus, acute ethanol reduces the surface expression of extrasynaptic receptors containing α₄βδ within minutes. However, while this effect is also visible for synaptic receptors (α₁βγ), it requires a longer time (Gonzalez et al., 2012; Liang et al., 2007). One or two days following ethanol exposure, an upregulation of synaptic receptors expression is observed (Liang et al., 2007). These changes highlight that the regulation of GABA_AR subunits is complex and dynamic, thus representing an interesting target for understanding the development of ethanol dependence.

Several animal models are used to investigate the long-term consequences of chronic alcohol exposure and to investigate the association of GABAergic plasticity with the development of ethanol dependance. Rats exposed to chronic intermittent ethanol consumption have shown significant changes in the expression of synaptic GABA_aR subunits, with a reduction of α₁ subunit (Cagetti et al., 2003; Kumar et al., 2003) and an increase in the synaptic expression of α₄ (Liang et al., 2006; Papadeas et al., 2001). Of note, these subunits have been identified as being involved in the sedation, anticonvulsant activity (Rudolph & Knoflach, 2011), and in changes of mood and anxiety (Liang & Olsen, 2014). Our recent work highlighted a reduced α₂ (synaptic) and δ (extrasynaptic) mRNA expression in the cerebellum and prefrontal cortex of subjects suffering from AUD, suggesting a loss of tonic inhibition after chronic and excessive alcohol consumption. These changes were associated with altered DNA methylation at the promoter levels of these genes (Gatta et al., 2017, 2020). Mounting evidence has demonstrated the loss of GABAergic inhibition in postmortem brain of individuals with AUD, with alteration in both synaptic and extrasynaptic GABA_AR subunits expression (Behar et al., 1999; Jin et al., 2012; Lingford-Hughes et al., 2005; Mitsuyama et al., 1998; Volkow et al., 1993). GABA_ARs downregulation after abstinence from chronic alcohol use contributes to many of the symptoms of alcohol withdrawal syndrome ([AWS], Liang and Olsen, 2014), which include disordered sleep, delirium tremens, and status epilepticus. Indeed, the loss of GABAergic inhibition is thought to contribute to sleep loss and increased wakefulness later in night (Koob & Colrain, 2020). During alcohol withdrawal, the loss of the modulatory effect of alcohol on GABA_ARs is likely to lead to an additional dysregulation of GABAergic neurotransmission and the simultaneous hyperactivation of glutamatergic transmission and HPA axis (Adinoff et al., 2005). Similarly, the development of status epilepticus is characterized by alterations in GABA_AR subunit composition (e.g., internalization and desensitization of synaptic subunits, α and β), leading to a resistance to benzodiazepine (BDZ) treatment.

The use of modulators of the GABA receptors (Figure 1) to reverse the effects of alcohol dependence and withdrawal has been investigated (Burnette et al., 2022; Farokhnia et al., 2018, 2019; Liang & Olsen, 2014). GABA_ARs have a number of binding allosteric sites that allow their modulation by endogenous or non-endogenous ligands (Liang & Olsen, 2014). BDZ, one of the most well-known molecules for the modulation of GABAergic transmission, binds between the α and the γ subunit of GABA_AR (Griffin et al., 2013) and thus enhances phasic inhibition but not tonic inhibition (Stell et al., 2003). In the clinic, BDZs are traditionally used for decreasing the risk of seizures, the emergence of panic attacks, and are indicated for generalized anxiety disorder (GAD), insomnia, and catatonia. They are also known to reduce AWS symptoms. However, because BDZ possess abuse potential, addictive sedative effects on the CNS when combined with alcohol, and dependance and cross-tolerance with alcohol (Devaud et al., 1996), they are unlikely to be used as a chronic treatment for AUD and withdrawal, as guidelines recommend their use for no longer than a few weeks (Liang & Olsen, 2014).

Topiramate, a fructopyranose derivative, potentiates GABA_ARs and inhibits AMPA/kainate glutamatergic transmission. It is currently FDA approved for the treatment of epilepsy and migraine. Topiramate is also used off-label for psychiatric disorders such as bipolar disorder, bulimia nervosa, PTSD, binge-eating disorder, and obesity (Marcotte, 1998; Nickel et al., 2005; Vieta et al., 2002). Recent evidence indicates that topiramate could serve as a putative treatment for substance related disorders, including alcohol dependance and withdrawal, nicotine, cocaine, benzodiazepine dependance and ecstasy abuse (Shinn & Greenfield, 2010). Clinical trials comparing the efficacy of Topiramate to FDA approved drugs for the treatment of AUD (e.g., disulfiram and naltrexone) show that Topiramate decreases alcohol consumption and intake (Baltieri et al., 2008; De Sousa et al., 2008). Although promising in terms of efficacy, Topiramate administration was associated with serious adverse events (including paresthesia, memory or concentration impairment) as well as a greater likelihood of relapse when compared to disulfiram (De Sousa et al., 2008; Shinn & Greenfield, 2010).

Gabapentin is a GABA analog with anticonvulsant properties. While its effect on alcohol intake has raised some controversies in preclinical models (Ozburn et al., 2020; Roberto et al., 2008), clinical trials showed a decrease in the percentage of heavy-drinking and craving, with an increase in abstinent days and a longer time-to-relapse in patients with AUD when compared to controls (Furieri & Nakamura-Palacios, 2007; Mason et al., 2014). Contraindicating its use, however, the administration of gabapentin is associated with dizziness, ataxia, somnolence, and gait disorders. Additionally, there is great potential for misuse and abuse of this drug, especially in patients with substance use disorder (Smith et al., 2016).

The GABAergic transmission can also be modulated by targeting metabotropic GABA_BRs. Sodium oxybate, the sodium salt of γ-hydroxybutyrate (GHB), is approved in Italy and Austria for the maintenance of abstinence in subjects suffering from AUD (Addolorato et al., 2020; Guiraud et al., 2021). Sodium oxybate exerts an alcohol mimetic action on the GABAergic system, binding with low affinity GABA_BRs and with high affinity GHB-specific receptors. Clinical trials showed the superiority of sodium oxybate in maintaining a sustained abstinence when compared to naltrexone (Caputo et al., 2003). Despite these encouraging results, sodium oxybate shows adverse effects which could limit its therapeutic indications, including dizziness and vertigo, the potential of abuse and misuse (especially in patients with psychiatric comorbidities as well as those suffering from cocaine or heroin dependance), and the risk of CNS depression and dependence/withdrawal (van den Brink et al., 2018).

One of the selective agonists for the metabotropic GABA_BRs is Baclofen, a derivative of GABA. Baclofen is FDA approved for the treatment of muscle spasticity. In France, it is also approved for the treatment of AUD, while it is used off-label in other European countries and in Australia (Addolorato & Leggio, 2010; Naudet & Braillon, 2018). The therapeutic effect of Baclofen in reducing alcohol intake has been demonstrated in some human studies, depending on doses and timing of administration (Farokhnia et al., 2018; Garbutt et al., 2021), while other studies have not observed an improvement in abstinence rates when compared to placebo-treated individuals (Beraha et al., 2016; Reynaud et al., 2017). To date, Baclofen is the only compound capable of safely reducing alcohol intake in patients with liver alcohol-associate diseases (Morley et al., 2018), but many adverse side effects have been observed, particularly in women, including sedation, headache, vertigo, and confusion. Furthermore, a certain level of tolerance to baclofen can develop if chronically administrated at high doses (Santos & Olmedo, 2017).

Although promising, the existing pharmacotherapy for AUD needs to be adapted to account for individual variability, considerable adverse effects, and the risk of relapse. As described above, GABAergic neurotransmission can also be fine-tuned by neuroactive steroids, like allopregnanolone, that exert a regulatory role on several endocrine systems underpinning the development of neuropsychiatric disorders, including substance use disorder and alcohol use disorder (Morrow et al., 2006).

6. Allopregnanolone alterations in neuropsychiatric pathologies: focus on alcohol use disorder

Since the pioneering work of Purdy and colleagues in the 90’s, a number of studies have investigated the effect of adverse events on neurosteroid levels along with the anxiolytic and anti-depressant properties of neurosteroids (for review, Tomaselli and Vallée, 2019). Mounting evidence indicates that, like stress, alcohol alters the biosynthesis of neurosteroids (Peltier et al., 2019, 2021). These effects are complex, however, with acute alcohol increasing allopregnanolone levels in the brain of rodents (VanDoren et al., 2000) as well as plasma of male and female subjects (Torres & Ortega, 2003, 2004) and chronic alcohol reducing these levels (Cagetti et al., 2004; Maldonado-Devincci et al., 2014). In addition, a decrease in allopregnanolone concentration has been observed during alcohol withdrawal (Romeo et al., 1996). The infusion of a low dose of alcohol reduces plasmatic allopregnanolone levels in both women suffering from PMDD and healthy controls (Nyberg et al., 2007). Alcohol consumption also reduced allopregnanolone in both men and women, and low allopregnanolone levels were associated with alcohol craving in men (Pierucci-Lagha et al., 2006). However, this reduction was not observed for a moderate dose of alcohol in healthy subjects (Holdstock et al., 2005). Our recent work highlighted that chronic alcohol induced changes in neurosteroid biosynthesis and affected the cerebellar levels of allopregnanolone and pregnanolone. These changes are associated with epigenetic modifications, DNA methylation in particular, affecting neurosteroidogenic enzymes, i.e., TSPO and 3α-hydroxysteroid dehydrogenase (Gatta et al., 2020). In addition, we observed a reduced expression (both mRNA and protein) in GABA_AR α₂ and δ subunits in association with increased DNA methylation at the gene promoter region in the cerebellum (Gatta et al., 2017, 2020). Preclinical studies also demonstrated that ethanol-induced epigenetic modifications mediated changes in GABA_AR expression (Bohnsack et al., 2017, 2018). Altogether, these data strengthen the hypothesis of an imbalance between excitatory and inhibitory signaling in AUD.

Adding further complexity, neurosteroid levels are influenced by the phase of the menstrual cycle, as progesterone (the precursor of all neurosteroids) fluctuates throughout the cycle and significantly rises after ovulation (i.e., the luteal phase). High progesterone and increased allopregnanolone levels are associated with the emergence of negative moods (Bäckström et al., 1983; Rubinow & Schmidt, 2018; Sundström Poromaa & Gingnell, 2014), which are particularly evident during the luteal phase in premenstrual dysphoric disorder (PMDD) (Bäckström et al., 2015). This observation suggests that the neuromodulatory effect of neurosteroids on GABAergic transmission depends on their concentration. Likely, in these conditions, the binding capacity of the neurosteroids to the GABA_ARs is saturated, and this may prevent further activation of the GABA_ARs. Alternatively, the paradoxical symptoms induced by neurosteroids are also dependent on an individual’s vulnerability and their environment (Bäckström et al., 2015), with the existence of an inverted U-shaped relationship between the occurrence of negative symptoms and the levels of allopregnanolone (high and low concentrations being less determinant for the onset of dysphoria) (Andréen et al., 2006). This counterintuitive relationship exists for all GABAergic modulators, including benzodiazepines, barbiturates as well as alcohol (Gourley et al., 2005; Miczek et al., 2003).

The promise of neurosteroids for the treatment of substance use disorder is strengthened by the evidence that stimulation of neurosteroidogenesis reduces drug intake. Brain and plasma levels of allopregnanolone and 5α3α-THDOC are decreased in patients and in preclinical models of cocaine and alcohol-addiction (Milivojevic et al., 2019). Schmoutz and colleagues (2014) showed that an intraperitoneal injection of metyrapone decreased cocaine self-administration in rats. Metyrapone is an 11β-hydroxylase inhibitor that blocks the synthesis of pro-stress neurosteroids (i.e., glucocorticoids) while shifting the production toward anti-stress products (e.g., allopregnanolone and 5α3α-THDOC) (Rupprecht et al., 1998). Of note, both bicuculline (an antagonist of GABA_A receptor) and finasteride (that blocks 5α-R1) partially reverse the effect of metyrapone (Schmoutz et al., 2014), demonstrating the importance of allopregnanolone and 5α3α-THDOC in restoring GABAergic inhibition and in modulating cocaine-related behaviors. Interestingly, allopregnanolone’s effect on cocaine-primed reinstatement and cue-induced reinstatement has been found in women more often than in men, suggesting a sex specific effect of neurosteroids on reducing cocaine-induced effects (Milivojevic et al., 2016).

Recently, ganaxolone, a synthetic derivative of allopregnanolone, has been filed for New Drug Application at the FDA for the treatment of seizures associated with CDKL5 deficiency disorder (CDD), a rare, genetic epilepsy (FDA, 2022). Kaminski and colleagues (2003) demonstrated that ganaxolone inhibited the expression and development of cocaine-kindled seizure in male mice. In addition, ganaxolone reverses behavioral symptoms in rodent models of kindling and bicuculline-treated animals (Beekman et al., 1998) suggesting a promising therapeutic effect in AWS.

7. Conclusion

Mounting evidence highlights the central role of an imbalance of GABAergic neurotransmission in the development of AUD. Excessive and protracted alcohol consumption leads to changes in synaptic and extrasynaptic subunit composition of GABA_ARs, resulting in loss of GABAergic inhibition and disruption of GABAergic control on the stress system. This imbalance is likely to be connected to an altered production of neurosteroids (e.g. pregnanolone and allopregnanolone) both in the brain and the periphery, which is influenced by alcohol consumption through epigenetic mechanisms, among others (Fig.2). Indeed, active neurosteroids are endogenous GABA_ARs positive allosteric neuromodulators, and recent studies suggest that their administration can reduce alcohol intake and craving by restoring the physiological tone of GABAergic neurotransmission and its control on the stress system and the HPA-axis, specifically. Interestingly, an alteration in neurosteroidogenesis has also been reported in several neuropsychiatric conditions that often are comorbid with AUD. The FDA approval of Brexanolone, an endogenous formulation of allopregnanolone, for the treatment of women suffering from PPD is an encouraging result because, unlike BDZ, Brexanolone exerts a long-lasting effect without induction of tolerance or dependence liability. This evidence suggests that neurosteroids represent a neuroregulatory system that should be further investigated as a novel treatment for people suffering from AUD, anxiety, epilepsy and depression.

Abbreviations:

AUD

Alcohol Use Disorder

ADH

alcohol dehydrogenase

ALDH2

Aldehyde Dehydrogenase

HPA

Hypothalamic-Pituitary-Adrenal axis

CRF

Corticotropin-Releasing Factor

ACTH

Adrenocorticotropic Hormone

PVN

Paraventricular Nucleus

MDD

Major Depression Disorder

PTSD

Post-Traumatic Stress Disorder

GR

Glucocorticoid Receptor

BNST

Bed Nucleus of the Stria Terminalis

GABA_ARs

Extrasynaptic GABA_A Receptors

CNS

Central Nervous System

NPY

Neuropeptide Y

CeA

Central Nucleus of the Amygdala

CRFR1

CRF receptor 1

Y₂R

NPY receptor type 2

Y₁R

NPY receptor type1

BLA

Basolateral Amygdala

DOC

Deoxycorticosterone

α3α-THDOC

5α,3α-tetrahydrodeoxycorticosterone

TSPO

Translocator Protein

StAR

Steroidogenic Acute Regulatory Protein

DBI

Diazepam Binding Inhibitor

3β-HSD

3β-hydroxysteroid dehydrogenase

5α-R1

Type-1 5α-reductase

CSF

Cerebrospinal Fluid

AWS

Alcohol Withdrawal Syndrome

BDX

Benzodiazepine

GAD

Generalized Anxiety Disorder

GHB

γ-hydroxybutyrate

PMDD

Premenstrual Dysphoric Disorder

PPD

Post-Partum Depression