4.1 GPR6
GPR6 is a constitutively active Class C GPCR that couples to a stimulatory G-protein (GS) leading to increased cAMP levels at a similar amplitude of fully activated GPCRs (Tanaka et al., 2007; Uhlenbrock et al., 2002) and enhanced neurite outgrowth in vitro (Tanaka et al., 2007). GPR6 mRNA is predominantly expressed in neurons in the brain, particularly in the striatum (caudate, putamen, nucleus accumbens, and olfactory tubercle) and to a lesser extent the frontal cortex, retrosplenial cortex, hippocampus, amygdala, and hypothalamus (Heiber et al., 1995; Marchese et al., 1994; Song et al., 1994). Within the striatum GPR6 is localised on dopamine D2 receptor-expressing striatopallidal medium spiny neurons (MSNs; Heiman et al., 2008; Lobo et al., 2007). Controversy about the endogenous ligand for GPR6 still persist. Initial studies showed agonism of GPR6 by sphingosine-1-phosphate (S1P) but these results were not replicated (Ignatov et al., 2003; Yin et al., 2009), leaving GPR6 as an orphan receptor (Alexander et al., 2017).
GPR6 expression is most dense within the striatum, a brain region important for reward behaviours, decision making and motor control (Lobo et al., 2007). Two intermingled but distinct populations of MSNs, differing in dopamine receptor subtype expression control behavioural output from the striatum. Dopamine D1receptor-expressing MSNs in the dorsal striatum project into and inhibit the substantia nigra pars reticulata (SNr; direct, or striatonigral pathway), releasing inhibition of thalamic activity and therefore prompting behaviour. In contrast, D2 receptor-expressing MSNs project to and inhibit the external globus pallidus (indirect or striatopallidal pathway), disinhibiting the subthalamic nucleus (STN) and exciting the downstream SNr, which ultimately inhibits the thalamus and supresses behaviour (Kreitzer & Malenka, 2008). These pathways are postulated to antagonise each other to allow a balanced striatal output (Albin et al., 1989).
Dopamine is released in the dorsal striatum by neurons located in the substantia nigra pars compacta (SNpc) and acts upon D1dopamine receptors (which depolarise the cell in response to dopamine) and D2 dopamine receptors (which hyperpolarise the cell in response to dopamine). Drugs of abuse acutely increase dopamine release in the striatum, and thus have the dual effect of exciting the direct pathway while simultaneously inhibiting the indirect pathway (Kreitzer & Malenka, 2008). GPR6 is specifically expressed in striatopallidal, D2 receptor-positive neurons (Heiman et al., 2008; Lobo et al., 2007), and their deletion leads to adaptive changes in both striatopallidal-specific genes (Drd2 andAdora2a ) and a striatonigral-specific gene (Tac1; Lobo et al., 2007). Further, GPR6 deficient mice have increased in dopamine and metabolite levels in the striatum (Oeckl et al., 2014) and enhanced instrumental responding for a sucrose reward, without altered motor co-ordination (Lobo et al., 2007).
Genome-wide RNA sequencing has recently shown GPR6 is downregulated in the dorsal striatum of individuals with alcohol use disorder (Walker et al., 2020). In the prefrontal cortex (PFC) however, sequencing revealed Gpr6 was upregulated in both food and cocaine- “addicted” compared to “non-addicted” mice (Navandar et al., 2021). However, the exact role of GPR6 in driving alcohol and substance use is not known; whether dysregulation of GPR6 signalling is causal or a consequence of substance use is not established and whether targeting GPR6 may have potential to reduce alcohol and substance use requires elucidation.
Recently several small molecule inverse agonists have been identified and developed to interact with GPR6. Phylogenetically GPR6 is closely related to the cannabinoid receptors, and cannabidiol (CBD), several synthetic cannabinoids and endocannabinoid-like N-acylamides act as inverse agonists at GPR6 (Laun et al., 2019; Laun & Song, 2017; Shrader & Song, 2020). Preclinical studies have shown CBD may be effective to reduce opioid, psychostimulant, nicotine and alcohol use (Nona et al., 2019; Prud’homme et al., 2015). However, the mechanisms that CBD acts through are widespread, and whether any actions are mediated via GPR6 would require further examination. A novel compound CVN424 has recently been developed and shown to be a potent, orally active, and brain-penetrant selective inverse agonist for GPR6, which is effective in reducing Parkinson’s-like symptoms in a rodent model (Brice et al., 2021). This compound has successfully undergone Phase I safety trials and is currently in Phase II trials (Margolin et al., 2022), highlighting a potential future opportunity of repurposing for other indications. However, given the opposing regulation of GPR6 in the striatum and PFC in response to drugs of abuse, whether inverse agonism would further exacerbate symptoms is a possibility that may limit development in this regard.