Conclusions
Here, we provide strong evidence of a NOP-RGS4 receptor interaction in a cell line and in native tissues and its relevance for the therapy of LID. An RGS4 inhibitor potentiated the antidyskinetic effect of NOP agonists without worsening its primary sedative/hypolocomotor properties, possibly preventing the effects resulting from up-regulation of RGS4 induced by L-DOPA in striatum. RGS4 plays an important role in regulating striatal functions and plasticity under parkinsonian conditions (Lerner et al. , 2012; Shen et al. , 2015). The present study confirms the role of RGS4 in LID (Koet al. , 2014; Shen et al. , 2015) and lends support to the therapeutic potential of RGS4 inhibitors in the therapy of neuropsychiatric disorders (Ahlers-Dannen et al. , 2020).
Figure legends
Figure 1. RGS4 and RGS19 reduced the NOP receptor agonist ability to stimulate cAMP accumulation in cells. Concentration–response curve of N/OFQ (0.001 nM-1 µM; A) and AT-403 (0.001 nM- 1 µM; B) in HEK-293T cells transfected with NOP and RGS4 or RGS19. Data are mean ± SEM of n=7 experiments per group (one outlier in the NOP group of panel B).
Figure 2. RGS4 and RGS19 reduced the NOP receptor agonist ability to stimulate cAMP accumulation in striatal primary neurons. Neurons were treated with SKF-38393 (100 µM) and N/OFQ (0.01-1 nM) in the presence or the absence of CCG-203920 (100 nM). Data are mean ± SEM of n=8-10 experiments per group, namely N/OFQ 0.001 nM n=9, N/OFQ 0.003 n=8, N/OFQ 0.01 nM n=10, N/OFQ 0.03 nM n=10 (no CCG203920) and n=8 (+ CCG1203920), N/OFQ=0.1 nM (n=9).
Figure 3 . CCG-203920 potentiated the AT-403 driven inhibition of D1 receptor-stimulated ERK signalling in striatum. Number of ERK-positive cells in striatal slices of naïve mice following application of SKF-38393 (100 μM), AT-403 (30 nM) and CCG203920 (500 nM) alone and in combination. Data are mean ± SEM of n=7 mice per group. *p< 0.05, different from unstimulated vehicle; °p<0.05, different from SKF-38393 alone; # p<0.05 different from AT-403 alone. §p<0.05, different from AT-403 + SKF-38393. Three-way ANOVA followed by the Bonferroni post hoc test.
Figure 4 . CCG-203920 reversed raclopride-induced-akinesia in wild-type (WT) but not in RGS4-/- mice. Immobility time in the bar test (in s; A) and number of steps in the drag test (B) were monitored before (baseline, time 0) and after the administration of 1 mg kg-1 raclopride (i.p.), followed 30 min later by CCG-203920 10 mg kg-1 or saline (i.p.). Arrows indicate the time of drug administration. Data are mean ± SEM of n=10 determinations per group, obtained from 10 WT and 10 RGS4-/- mice treated under a crossover design. *p< 0.05, different from baseline; °p<0.05, different from raclopride in WT mice (2-way RM ANOVA followed by the Tukey post hoc test).
Figure 5 . CCG-203920 extended the antidyskinetic effect of AT-403 without worsening motor performance on the rotarod. (A) ALO AIMs were scored in 6-OHDA hemilesioned dyskinetic rats following challenge with L-Dopa (6 mg kg-1 plus benserazide 15 mg kg-1, s.c.) combined with vehicle, AT-403 (0.03 mg kg-1, s.c.) or CCG-203920 (10 mg kg-1, i.p) (A). Values are mean ± SEM of 13 rats per group. *p<0.05, **p<0.01, different from L-Dopa; ## p<0.01, different from L-Dopa + CCG-203920; °p<0.05, different from L-Dopa + AT-403. Two-way repeated measure ANOVA followed by the Tukey post hoc test. (B) Rotarod performance was evaluated as time on rod in seconds, before (OFF) and 60 min after (ON) L-Dopa (6 mg kg-1 plus benserazide 15 mg kg-1, s.c.) combined with vehicle, AT-403 (0.03 mg kg-1, s.c.) or CCG-203920 (10 mg kg-1, i.p.). Values are mean ± SEM of 11 rats per group. *p<0.05, different from OFF L-Dopa (Student t-test, two tailed for unpaired data).
Figure 6 . AT-403 in combination with CCG-203920 inhibited L-Dopa-induced ERK phosphorylation in the striatum of dyskinetic rats. Western blot representative images (upper panel) and quantification (lower panel) of pERK (A) and total ERK (B) in the lesioned and unlesioned striatum of 6-OHDA hemilesioned L-Dopa-naïve or dyskinetic rats. Dyskinetic rats were treated with AT-403 (0.03 mg kg-1, s.c.) or vehicle and, 10 min later, challenged with L-Dopa (6 mg kg-1, i.p.). CCG-203920 (10 mg kg-1, i.p.) or vehicle were administered 5 min before AT-403. Values are mean ± SEM of 6 rats per group. *p<0.05, different from unlesioned striatum (Student t-test, two-tailed for unpaired data).
Figure 7 . AT-403 inhibited the L-Dopa-stimulated pGluR1 phosphorylation in the striatum of dyskinetic rats. Western blot representative images (upper panel) and quantification (lower panel) of pGluR1 (A) and total GluR1 (B) in the striatum of 6-OHDA hemilesioned, L-Dopa-naïve or dyskinetic rats. Dyskinetic rats were treated with AT-403 (0.03 mg kg-1, s.c.) or vehicle and, 10 min later, challenged with L-Dopa (6 mg kg-1, i.p.). CCG-203920 (10 mg kg-1, i.p.) or vehicle were administered 5 min before AT-403. Values are mean ± SEM of 6 rats per group. *p<0.05, different from unlesioned striatum (Student t-test, two-tailed for unpaired data).
Figure 8 . RGS4 levels were modulated by 6-OHDA and L-DOPA treatment. Western blot representative images (upper panel) and quantification (lower panel) of RGS4 in the striatum of naïve, 6-OHDA hemilesioned L-Dopa naive, or dyskinetic rats. In dyskinetic rats, RGS4 analysis was carried out both OFF and ON L-Dopa. RGS4 values were normalized to α-tubulin as housekeeper and expressed as absolute values (A) or percentage of RGS4 in the lesioned relative to unlesioned striatum (B). Tyrosine hydroxylase levels normalized to α-tubulin are also shown (C). Values are mean ± SEM of 7 rats per group (n=6 in the ON L-Dopa group due to animal loss. In panel B, one outlier was removed from the 6-OHDA group). *p<0.05, different from naïve (A-B) or unlesioned striatum (C); #p<0.05 different from 6-OHDA (B). One-way ANOVA followed by the Newman-Keuls test (A), Kruskal-Wallis test followed by the Dunn test (B). Student t-test, two-tailed for unpaired data (C).