Introduction
Regulators of G-protein signaling (RGS) are signal transduction proteins which couple to heterotrimeric G-protein coupled receptors (GPCRs). RGS proteins bind to the Gα-GTP complex and accelerate its intrinsic GTPase activity, promoting the formation of Gα-GDP and the reassembly of Gαβγ trimer. This causes the termination of GPCR-driven intracellular signaling (Berman et al. , 1996; Tesmer et al. , 1997). RGS proteins are grouped into five subfamilies (R4, R7, R12, RA and RZ) based on sequence homology. They interact with Gαi, Gαo, Gα12/13 but not Gαs, and show a variable degree of receptor and G-protein specificity (Kimple et al. , 2011; Sjogren, 2017). Different subtypes of RGS have been shown to modulate opioid receptors (Traynor, 2012), and have been proposed as a novel therapeutic targets for pain, depression and addiction (Gross et al. , 2019; Sakloth et al. , 2020; Senese et al. , 2020; Stratinaki et al. , 2013; Traynor et al. , 2005). In particular, RGS type 4 (RGS4) has been shown to regulate µ opioid (Xie et al. , 2005) and δ opioid (Dripps et al. , 2017) receptors, RGS type 9 isoform 2 (RGS9-2) regulates µ opioid receptors (Garzon et al. , 2001; Psifogeorgou et al. , 2007; Zachariou et al. , 2003) and RGS type 12 (RGS12) regulates κ opioid receptors (Gross et al. , 2019). Preliminary in vitro evidence suggests that RGS type 19 (RGS19) modulates the nociceptin/orphanin FQ (N/OFQ) opioid peptide (NOP) receptors (Xie et al. , 2005). The NOP receptor is a “non-opioid” member of the opioid receptor family and is endogenously activated by its natural heptadecapeptide ligand N/OFQ. N/OFQ regulates several central and peripheral functions (Toll et al. , 2016) and is involved in motor disorders, such as Parkinson’s disease (PD) and L-Dopa induced dyskinesia (LID) (Mercatelliet al. , 2020), a major disabling complication of L-Dopa pharmacotherapy of PD (Bastide et al. , 2015). Specifically, NOP receptor antagonists proved effective in attenuating motor symptoms and neuropathology associated with experimental parkinsonism whereas NOP receptor agonists proved effective as antidyskinetic agents in animal models of LID (Arcuri et al. , 2018; Marti et al. , 2012). However, small molecule NOP receptor agonists also produced strong hypolocomotion/sedation which partly masked their antidyskinetic effects (Arcuri et al. , 2018). Therefore, in view of the fact that RGS can modulate specific behavioral outcomes of δ opioid and µ opioid receptor stimulation (Jutkiewicz et al. , 2005), we hypothesized that targeting RGS4 would improve the antidyskinetic effects of NOP receptor agonists relative to their hypolocomotive/sedative effects which would widen their safety window. In the present study, we investigated the interaction of NOP with a specific subtype of RGS, namely RGS4, and its relevance for LID in vivo. RGS4 and NOP receptor are expressed in brain areas relevant to LID, such as cortex, striatum and substantia nigra (Ebert et al. , 2006; Gold et al. , 1997; Neal et al. , 1999). Moreover, both RGS4 and NOP are expressed by medium-sized GABAergic striatal neurons which are the main neurobiological substrate of LID (Ebert et al. , 2006; Neal et al. , 1999). RGS4 is involved in LID development and expression, and blockade of RGS4 with an antisense nucleotide improved dyskinesia in a rat model of LID (Ko et al. , 2014).
In this study, the interaction between RGS4 and NOP receptor was investigated first in a cellular model (HEK293T cells) artificially overexpressing both proteins, then in native tissues, i.e. rat primary striatal cultures and striatal slices of adult mouse. In native tissues, the RGS4 selective small molecule antagonist CCG-203920 (Turner et al. , 2012) was used to pharmacologically inhibit RGS4 and potentiate NOP receptor responses. The ability of CCG-203920 to potentiate the inhibitory effect of AT-403 (Arcuri et al. , 2018) on LID and its biochemical correlates (i.e. pERK and pGluR1 upregulation) was then tested in vivo. The levels of RGS4 protein in the dyskinetic striatum, both OFF and ON L-Dopa were also measured.