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.