Posted on Authorea 26 Feb 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161437480.01864791/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. lrsaAn Pisan`o dyskinesia Anna Clarissa induced L-Dopa for implication signaling: opioid receptor FQ Nociceptin/Orphanin modulates negatively RGS4 Morella ihr Neubig D Richard podrcpos(ryo,21) n aebe rpsda oe hrpui agt o an depression pain, for targets therapeutic homology. novel a sequence as on (Gross proposed based been addiction have RZ) and and and 2012), (Traynor, RA receptors R12, opioid R7, (R4, (Kimple specificity subfamilies G five with (Berman into interact They signaling grouped are intracellular proteins GPCR-driven G RGS of of G- G termination formation heterotrimeric the the the to to couple bind promoting which proteins activity, RGS proteins GTPase transduction (GPCRs). signal receptors are coupled (RGS) protein signaling G-protein of Regulators the mitigating agonists, Introduction striatum. expression. receptor NOP dyskinesia lesioned NOP during enhances the of occurring inhibitor potential in levels RGS4 RGS4 antidyskinetic RGS4 an striatal the upregulated and of improved upregulation acutely signaling inhibitor of receptor Levodopa RGS4 effects NOP an inhibits levodopa. Furthermore, and bilateral and physiologically by neurons, caused RGS4 dyskinesia responses. reversed striatal depletion of was Implications: dopamine rat inhibition and which Unilateral primary mediated Conclusions levels AT-403 effects. in AT- RGS4 motor-improving enhanced potency and its of CCG-203920 NOFQ N/OFQ compromising reduction reduced increased without slices. expression CCG-203920 correlates, striatal RGS4 biochemical mouse results: its cells. in Key HEK293T response measured. in AT-403 were potentiated efficacy levels dyskinesia and RGS4 CCG- of striatal inhibitor, potency correlates AT-403 RGS4 biochemical with striatal and 403 striatal co-administered the levels) rat movements, was of pGluR1 Primary dyskinetic CCG-203920 presence and and vivo, the rats, In readout. (pERK in hemilesioned monitored. a AT-403 6-hydroxydopamine were to as or responses levodopa N/OFQ levels pERK and with induced cAMP or challenged cAMP D1-stimulated L-Dopa were D1-stimulated using slices and for with AT-403, 203920, striatal challenged relevance agonist mouse were has adult NOP/RGS19 NOP and or modulation molecule NOP/RGS4 neurons small NOP, this intrinsic with the accelerates whether transfected and that and cells N/OFQ protein HEK293T signaling transduction approach: receptor Experimental signal (NOP) a dyskinesia. is opioid (RGS4) FQ 4 nociceptin/orphanin G signal of G-protein activity of Regulator GTPase purpose: and Background Abstract 2021 26, February 5 4 3 2 1 ihgnSaeUniversity State Michigan Therapeutics Astraea University Cardiff Cork College University Ferrara of University 3 tfnaFasano Stefania , α tal. et / n G and i/o 5 α tal. et n ihl Morari Michele and , i G , 1 01 jge,21) ieetsbye fRShv ensont modulate to shown been have RGS of subtypes Different 2017). Sjogren, 2011; , ail Mercatelli Daniela , α o α G , 09 Sakloth 2019; , uuis upesn PRsgaig eew netgt hte G4modulates RGS4 whether investigate we Here signaling. GPCR suppressing subunits, q 3 uuanZaveri Nurulain , α 12/13 u o G not but tal. et 1 α- α 1 atn Mazzocchi Martina , s D n h esebyo G of reassembly the and GDP 4 n hwavral ereo eetradG-protein and receptor of degree variable a show and , icroBrambilla Riccardo , 00 Senese 2020; , 1 α- T ope n ceeaeisintrinsic its accelerate and complex GTP tal. et tal. et 2 00 Stratinaki 2020; , 96 Tesmer 1996; , let Brugnoli Alberto , 3 eadO’Keeffe Gerard , αβγ rmr hscauses This trimer. tal. et tal. et 1 Ilaria , 1997). , 2 2013; , , Posted on Authorea 26 Feb 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161437480.01864791/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. n icmot aswr arfie iha vroeo sflrn rwt sflrn netei olwdby followed anaesthesia isoflurane with pain were or animal Milanese, Animals isoflurane minimize of Settimo cycle). to overdose Mucedola, an taken dark/light with were hr diet; sacrificed measures (12 were standard Adequate Rats conditions (4RF21 enrichments. lighting discomfort. environmental food regular and (LARP) with under to facility groups kept standard access in and (SPF) housed free water, pathogen-free with and specific Ferrara Italy) a Milan, of in University housed were the Animals of RGS4 Ferrara. analysis. of University blot RGS4 Western the (Cifelli 6 Toronto) for other of and used (University CCG-203920, were with Heximer RGS4 controls studies 10 behavioral Specifically, C57BL/6J for for 6 S1). used 24 were and (Fig. controls specificity and C57BL/6J antibody 10 studies RGS4 and and WB mice CCG-203920 for of selectivity kept RGS4 of were analysis 35 treatment, RGS4 which L-Dopa (16) of of Sixteen [?]100), for end used score the were At AIM L-Dopa. 7 (ALO with analysis. below): rats underwent treatment behavioral (see 103 dyskinetic chronic whereas criteria underwent 59 7) 78 selection obtained (Fig. used and the we levels were 5-7) RGS4 passed Italy) (Figs. of rats Lecco; studies analysis 6-OHDA Calco, WB WB Lab, (85) for River Eighty-five kept Charles were lesioning. na¨ıve g; rats (7) 6-OHDA (150 Seven rats study. Sprague-Dawley this male in (110) ten hundred One measured. The also vivo. were in L-Dopa subjects ON tested Animal and then OFF was both methods upregulation) (Arcuri striatum, pGluR1 and AT-403 dyskinetic and Materials of the pERK effect in (i.e. protein inhibitory antagonist RGS4 correlates the molecule of biochemical potentiate small levels its to selective striatal and CCG-203920 RGS4 primary model LID of the on rat cellular ability tissues, a native The i.e. in In tissues, responses. first mouse. native investigated adult in was (Turner of then CCG-203920 receptor slices proteins, NOP striatal both and and overexpressing cultures RGS4 artificially between cells) improved interaction nucleotide (HEK293T the antisense study, an (Ko this with LID In RGS4 of areas of model brain blockade rat (Ebert and in a LID expression, in expressed of and dyskinesia are development substrate receptor LID neurobiological in NOP main involved and the (Ebert RGS4 nigra are substantia RGS, which of and vivo. subtype striatum in specific al. cortex, LID a as et their with such for NOP widen LID, relevance of would to interaction its which relevant the and effects investigated we hypolocomotive/sedative RGS4, study, their present namely of the to In relative outcomes window. agonists behavioral safety receptor specific NOP modulate of can effects RGS that (Arcuri fact (Jutkiewicz LID stimulation the (Arcuri of of effects models antidyskinetic view animal their in in masked parkinsonism partly agents which experimental antidyskinetic hypolocomotion/sedation with as effective associated al. proved neuropathology et agonists and receptor symptoms NOP motor whereas attenuating (Bastide in (Mercatelli PD effective (LID) central of dyskinesia several pharmacotherapy induced regulates L-Dopa N/OFQ L-Dopa N/OFQ. and receptors ligand (PD) (Toll (NOP) heptadecapeptide functions peptide natural peripheral opioid and its (N/OFQ) by FQ activated nociceptin/orphanin endogenously the modulates (RGS19) (Xie 19 type RGS regulates (RGS12) (Garzon receptors and 2005) , Traynor tal. et 99.Mroe,bt G4adNPaeepesdb eimszdGBegcsraa neurons striatal GABAergic medium-sized by expressed are NOP and RGS4 both Moreover, 1999). , 08 Marti 2018; , tal. et 05.TeNPrcpo sa“o-pod ebro h podrcpo aiyadis and family receptor opioid the of member “non-opioid” a is receptor NOP The 2005). , δ pod(Dripps opioid 05.I atclr G ye4(G4 a ensont regulate to shown been has (RGS4) 4 type RGS particular, In 2005). , -/- tal. et κ tal. et tal. et podrcpos(Gross receptors opioid n 6C7L6 idtp aemc 2-6g 01 ek l)wr sdfrthe for used were old) weeks 10-12 g, (24-26 mice male wild-type C57BL/6J 16 and tal. et 01 Psifogeorgou 2001; , 02 a sdt hraooial nii G4adptnit O receptor NOP potentiate and RGS4 inhibit pharmacologically to used was 2012) , 02.Hwvr ml oeueNPrcpo gnssas rdcdstrong produced also agonists receptor NOP molecule small However, 2012). , 05,w yohszdta agtn G4wudipoeteantidyskinetic the improve would RGS4 targeting that hypothesized we 2005), , tal. et tal. et 07 eetr,RStp sfr RS-)regulates (RGS9-2) 2 isoform 9 type RGS receptors, 2017) , 06 n sivle nmtrdsres uha akno’ disease Parkinson’s as such disorders, motor in involved is and 2016) , tal. et tal. et tal. et 2014). , 08,wr rvddb rR ebgadcln asdat raised colony and Neubig RR Dr by provided were 2008), , tal. et tal. et 05.Seicly O eetratgnssproved antagonists receptor NOP Specifically, 2015). , 2 09.Peiiayi ir vdnesget that suggests evidence vitro in Preliminary 2019). , 07 Zachariou 2007; , tal. et tal. et 00,amjrdsbigcmlcto of complication disabling major a 2020), , 06 Neal 2006; , -/- tal. et ie rgnlydvlpdb rS Dr by developed originally mice, tal. et 06 Gold 2006; , δ podand opioid 03 n G ye12 type RGS and 2003) , tal. et tal. et μ 08.Therefore, 2018). , tal. et 99.RS is RGS4 1999). , pod(Xie opioid μ podreceptor opioid tal. et 97 Neal 1997; , μ -/- 2018) , opioid tal. et mice -/- Posted on Authorea 26 Feb 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161437480.01864791/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. PA o 5mna omtmeaue lcswr isdtretmsi B n rortce n30% in cryoprotected and PBS in times paraformaldehyde three 4% rinsed in were fixation After slices temperature, vehicle. 4 10 or room at overnight NaHCO3, nM) at (100 solution mM (500 sucrose min SKF-38393 25 CCG203920 NaH2PO4, agonist 15 nM), mM receptor for (30 1.2 (PFA) D1 AT-403 MgSO4, The of mM Mis- 32 1.3 CaCl2). presence Inc., at KCl, the mM design mM h 2.4 5 System 1 D-glucose, NaCl, Scientific for mM mM recover chamber-BSC1, 124 to slice (ACSF: subsequently allowed CSF (Brain 10 and artificial and chamber NaHCO3, CO2) Canada) slice mM 200- 5% ON, 25 brain Microsystems); O2, sucrose, sissauga, a (95% VT1000S-Leica mM dissecting into carbogenated (Vibratome, 75 sucrose-based transferred acid), stage NaH2PO4, ice-cold kynurenic and mM vibratome with mM 1 the filled 2 MgCl2, on plate CaCl2, mM mounted glass 7 mM KCl, cool 0.5 mM a were D-glucose, 2.5 on slices mM NaCl, put brain (Arcuri mM and and described (87 removed dislocation, solution previously rapidly cervical protocol were and brains the anaesthesia The to after Biotek, according decapitated NEO; prepared were freshly (Synergy mice reader C57Bl6 microplate male Adult TR-FRET a vitro on in read measurement was Finally, ERK plate manual. The the to temperature. VT). according room Winooski, triplicate 5 at in nM; (2,000 h generated 40 cells was 1 final (5 Then curve agonist; tracer standard 1M. (Eu)-cAMP receptor Versene indicated cAMP europium (D1 using A as SKF-38393 dishes transfected temperature. and from were room N/OFQ dissociated cells of man- were concentrations HEK293T the cells ous assay, with experiment, 5 accordance the of in in day before cells/well performed the day were On the MA) Briefly, above. Waltham, Elmer; instructions. (Perkin ufacturer’s assays of cAMP success Ultra the LANCE establish cells to HEK293T in used measurements were cAMP levels of ligands cAMP lack specific in The change with DNA. receptors. of robust NOP treatment amount the after total and (Feng the cells For cells transfection adjust transfected transfected transfection. to (pcDNA3.1+) after D1/NOP used vector h was in control 24 (pcDNA3.1+) 6 run in vector at were stimulation Empty constant kept Experiments cAMP was used. FBS. DNA was % dishes. plate before 10 6-mm were h per in with 4-5 plated transfections DMEM were for cells All to proceed assays, back to cAMP allowed changed protocol. were was Transfections recommended media Opti-MEM. manufacturer’s the in the conditions with serum-free to supplemented under ml 5% (DMEM) according performed U medium with 2000 100 Eagle’s 37degC Lipofectamine (FBS), modified at using serum Dulbecco’s incubator in bovine humidified confluence fetal a 90-95% in 10% to maintained grown were and cells CO2 (HEK293T) kidney embryonic Human in transfection h and (12 slices culture light in of Cell (license studies conditions regular below ERK under experiments outlined for University, libitum. from vitro Cardiff ad used as laparotomy In water at were by striatum and facility removed mice rat standard food were a C57BL6 with the total) in cycle), male of light/dark housed in 12-week-old cultures were (106 Seven Mice primary embryos vitro. Unit, 14, generated I304). Service (E) with to AE19130/ Biological day cycle) used the number embryonic light/dark and in (12h On housed lights dams were of water. euthanized There conditions and rats regular of Sprague-Dawley striatum. food under Ministry the female to Italian Cork of pregnant the College cultures time-mated University and primary (10) Ferrara at generate Ten of to University 368/2018). used Experimental the were and of dislocation. Committee 714/2016-PR cervical Ethical (licenses by the followed Health by anaesthesia approved isoflurane were with protocols mice studies), (WB decapitation μ )wr rnfre oawie34wl irpae(eknEmr n nuae ihvari- with incubated and Elmer) (Perkin microplate 384-well white a to transferred were l) tal. et 07.Ti spsil eas E23 el ontntvl xrs 1and D1 express natively not do cells HEK293T because possible is This 2017). , ° .O h olwn a,sie eefrhrctit 18- into cut further were slices day, following the On C. μ )adUih-nicM (5 ULight-anti-cAMP and l) -1 eiilnad100 and penicillin 3 ° ,wt osatpruino carbogenated of perfusion constant a with C, μ μ )wr de oec eladicbtdfor incubated and well each to added were l) ml g -1 tetmcn el eetransfected were Cells streptomycin. tal. et μ μ n 6 and g )wsapidfr1 i in min 10 for applied was M) 08 Marti 2018; , μ μ -hc lcswr cut were slices m-thick fLipofectamine2000 of l μ μ -hc lcsuiga using slices m-thick /el o 0mnat min 30 for l/well) dlibitum ad tal. et 2012). , access Posted on Authorea 26 Feb 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161437480.01864791/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. 090o aie ordy ae,tetet eecosd h aepooo a dpe o 0wild-type 10 for adopted was protocol same The crossed. were treatments later, mice. days control Four saline. or RGS4 203920 10 of dragged cohort of and together. A number table) average the the and observers on measures blinded forepaws test separate design the drag two Experimental s (allowing by The of cm tail counted recorded together. blocks 20 was the was (about forepaw on pooled by paw each speed each lifted alternately and by by constant gently maximum) placed seconds) a when were s (in at animal forepaws blocks 60 backward the the left block, by on Each (or and per made spent posture. bar right time s steps static Total The the 20 imposed cm). reproducible. and externally time 6 an became table and (cutoff to 3, a performance (1.5, respond motor on heights to gently their increasing animal until placed the of tests was ability behavioral mouse the the measures on test week catalepsy) a kg for mg daily 10 by later, RGS4 (Marti min 5 tests in drag activity and motor bar (Blazer the in reported previously mice we in As CCG-203920 guidelines. of BJP selectivity and RGS4 ARRIVE of the Evaluation with accordance in performed software. were analysis Experiments J Image in using A11005) densitometry experiments or by vivo measured A32814 In was 1:500 cells (Invitrogen; individual of antibodies 3 intensity 4 secondary fluorescence to at 594-conjugated prior MAB2146; PBS in BSA (R&D mM subsequently incubated cAMP 1% 10 were were 1:500), Cultures in AF6259; temperature. cells BSA room (R&D Fol-T, 1% at DARPP32 in h paraformaldehyde. antibodies: 1 diluted 4% primary for 1:500), in PBS-T following mM min the 10 15 in (100 in for incubated (BSA) SKF-38393 fixed albumin with serum then min bovine were 30 5% Cultures for nM). treated were 3 (0.01-1 lowing cells N/OFQ indicated and/or where nM) culture, (100 of 8 7DIV. day for On culture neurons in striatal (Sig- maintained primary were plates in Cells 24-well L-glutamine measurements FBS. coated 1% cAMP 1% (Sigma), poly-D-lysine and penicillin/streptomycin in (Invitrogen) 1% plated B27 with After supplemented 2% were Committee. (Sigma) (Sigma), neurons Ethics media and DMEM:F12 Experimentation dissociated in Animal ma) was local Communities tissue European by the the approval with (from accordance dissection, and (Schmidt striatum) in (86/609/EEC) license described as a previously Directive to under as Council referred performed dissected were (hereafter were procedures eminence Cork) scientific All College ganglionic 2012). University rat Unit, 14 Service slice. day Biological each (E) the embryonic in counted of was cultures striatum Primary the in cultures cells neuron positive striatal pERK Primary of number 40 the room method at Images and at peroxidase-based solution. System), striatum h H2O2 standard Imaging the and 2 a (DAB) from for using 3,3’-diamino-benzidine acquired 1000) out a carried were by #BA incu- followed was cat. then Labs), antibodies Vector were Laboratories, Vectastain, bound Vector Sections (ABC-kit, the (1:200, 4 L). of IgG at #4370 Detection anti-rabbit overnight cat. temperature. goat Technology incubated biotinylated Signaling were with Cell slices bated (1:1000, solution, (Thr202/Tyr204) X-100 kinase (Papale Triton MAP Immunohistochem- described 0.1% Scientific). previously and (Thermo as slides serum Plus performed SuperFrost was onto mounted istry and CM1850) (Leica cryostat × i ahsi 0m B- 00%Tio -0 n1 MPS,clue eeicbtdin incubated were cultures PBS), mM 10 in X-100 Triton (0.02% PBS-T mM 10 in washes min 5 × -/- i ahs el eeiae sn nOypsI7 netdmcocp.The microscope. inverted IX71 Olympus an using imaged were Cells washes. min 5 -1 iewsalte ntogop n5ec)rciigrcord olwdb CCG- by followed raclopride receiving each) (n=5 groups two in allotted was mice -/- C-090o aie(...Pirt hraooia etn,mc eetrained were mice testing, pharmacological to Prior (i.p.). saline or CCG-203920 iead5wl-yecnrl rae ih1m kg mg 1 with treated controls wild-type 5 and mice tal. et tal. et × 04 Viaro 2004; , anfiainuigabihfil irsoe(ec Macro/Micro (Leica microscope brightfield a using magnification 05 htRS niiosrvrercord-nue akinesia raclopride-induced reverse inhibitors RGS4 that 2015) , -1 ° o xddsac 10c) h ubro tp made steps of number The cm). (100 distance fixed a for ) vrih.Floig3 Following overnight. C 4 tal. et tal. et 06:1hatrbokn n5 omlgoat normal 5% in blocking after h 1 2016): , 08,tesm et eeue omonitor to used were tests same the 2008), , × i ahsi 0m PBS- mM 10 in washes min 5 ° ihanti-phospho-p44/42 with C -1 ip)rcord n,30 and, raclopride (i.p.) μ ) CCG-203920 M), tal. et , Posted on Authorea 26 Feb 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161437480.01864791/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. ieetehlgcltss(h a n rgtss (Marti tests) drag and bar (the > tests (Paxinos ethological mm different -2.4 (AP) antero-posterior at (Esteve bar solution mm): tooth (Synulox (in antibiotics -7.8, surface with (DV) dural medial dorso-ventral (Arcuri the (right) described 1.2, and the previously (ML) bregma in from medio-lateral anesthesia coordinates isoflurane -4.4, Parkinson’s stereotaxic under of following injected model the unilaterally 12 rodent were with validated bundle g) best forebrain (150 and na¨ıve rats popular (103) most three (Cenci the LID rat, and lesioned disease 6-OHDA unilaterally The lesion 6-OHDA Unilateral oeet AM) orlt fLD(Cenci for LID treated of were test correlate drag and a kg bar mg (AIMs), the (6 movements in values L-Dopa threshold with the days passed 21 successfully that rats rating (78) Seventy-eight movement involuntary abnormal and treatment L-Dopa 2020). ratio paw contralateral/ipsilateral a (Brugnoli eaaechr f1 aswssbetdt h aetetet saoefrteaayi fmotor of analysis global the of L-Dopa). improvement for (ON an above with administration as associated L-Dopa was treatments after effect treatments. same and antidyskinetic between the L-Dopa) potential (Arcuri allowed (OFF activity to the washout motor whether before 3-day subjected evaluate a both was to with rotarod, This rats times, the 12 four on of tested performance was cohort were animal separate Experimenters Each A kg guidelines. saline. mg (6 BJP or L-Dopa to 203920, and randomized were ARRIVE kg rats mg dyskinetic the fully 15 (12) with Twelve accordance treatments. to in blinded performed 4 were to (Cenci Experiments observation 0 each from of frequency scale and design a amplitude Experimental on between product measured the was of AIMs by al. sum interrupted these et the (Cenci not as of scale obtained and amplitude validated were continuous value the previously 4, occasional; a addition, distraction; (1, forelimb 4 on external In to based (ii) an 0 by distraction). from lesion; scale interrupted external and frequency the but movements a an continuous masticatory to on rated 3, purposeless contralateral the was twitching, frequent; to subtype muscle side contralateral 2, AIM orofacial forelimb Each the is, the protrusion. toward of that twisted tongue grabbing AIM, contralateral body is, purposeless orolingual that upper and/or (iii) movements AIM, and and dystonic axial lesion; and neck (i) jerky the subtypes: is, that three movements of AIM, Dyskinetic into turning ceased. distribution movements or topographical dyskinetic their until posture or on injection based L-DOPA classified followed were that h 3 the during aeedons wnysvn(7 aswr lotdi rusadtetdwt -oaaoe( mg (6 alone L-Dopa with hu- treated reaching and for groups sacrificed kg 4 was mg in rat (0.03 allotted one AT-403 were but rats experiment, (27) this kg Twenty-seven for saved endpoints. were mane rats min dyskinetic 60-80 (35) peak Thirty-five a showed course time analysis AIMs blot was ALO Western performance the motor Rotarod since a (Arcuri performance. administration administration motor whether L-Dopa L-Dopa with whereas after along after min performance AIMs 60 reduce rotarod at would improve agent assessed sedative consequently a and or LID inhibiting alleviate would compound 0sca h otaaea a ntebrts,and test, bar the in paw contralateral the at sec 20 -1 esrzd 5m kg mg 15 benserazide + 07,adflydsiei assoe [?]100. scored rats dyskinetic fully and 2007), , -1 tal. et .. ncmiainwt T43(.3m kg mg (0.03 AT-403 with combination in s.c.) , 00 Marti 2020; , TM .Towesltr aswr cendb sesn h oo smer cr ntwo in score asymmetry motor the assessing by screened were rats later, weeks Two ). -1 =)o C-090+A-0 n7.I obnto tde,CG232 was CCG-203920 studies, combination In (n=7). AT-403 + CCG-203920 or n=7) , tal. et tal. et TM μ f6OD yrboie(isle n00%acraesln) codn to according ascorbate-saline), 0.02% in (dissolved hydrobromide 6-OHDA of g 50 , 08 Marti 2018; , tal. et -1 tal. et -1 08 Duty 2018; , .. =) rLDp obndwt C-090(0m kg mg (10 CCG-203920 with combined L-Dopa or n=6), s.c., , μ esrzd 5m kg mg 15 benserazide + /g ..,adtewudwsstrdadifitae ih2 lidocaine 2% with infiltrated and sutured was wound the and i.p.), l/Kg, 02 Paolone 2012; , 08 Marti 2018; , < tal. et 0)wr sdi h td (Brugnoli study the in used were 50%) tal. et 08 Marti 2018; , tal. et tal. et tal. et 02 Paolone 2012; , tal. et 01 Schwarting 2011; , tal. et 07.Ail ibadOoiga AO Istotal AIMs (ALO) Orolingual and Limb Axial, 2007). , 5 98 Cenci 1998; , 02 Pisan`o 2012; , < 05.Rt eeosre o i,eey2 min, 20 every min, 1 for observed were Rats 2015). , -1 tal. et tp ttecnrltrlpw(ralternatively (or paw contralateral the at steps 3 .. nediy oidc bomlinvoluntary abnormal induce to daily) once s.c., , -1 tal. et ,CG232 1 gkg mg (10 CCG-203920 ), 02 Paolone 2012; , tal. et 05.Rt hwn moiiytime immobility showing Rats 2005). , tal. et tal. et tal. et 05.I at rl antidyskinetic truly a fact, In 2015). , 00.Aiaswr pretreated were Animals 2020). , 07,a rvosydescribed previously as 2007), , 96 a sd One-hundred used. was 1996) , tal. et tal. et 2015). , 00 Pisan`o 2020; , -1 ,A-0 CCG- + AT-403 ), tal. et -1 benserazide + -1 .. n=7), i.p., , 96,as 1986), , tal. et , Posted on Authorea 26 Feb 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161437480.01864791/v1 — This a preprint and has not been peer reviewed. 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Tissues challenge and until group). last dissected OFF -80degC (receiving rapidly (L-Dopa was rats at striata analysis dyskinetic stored and blot additional decapitation and Western seven by nitrogen L-Dopa. sacrificed by liquid isoflurane, later in with min anaesthetized 10 were and (Arcuri AT-403 rats described by previously as later out min carried 5 followed first, administered 08 Paolone 2018; , 50 tal. et tal. et copne y9%cndneitra nprnhss I5 n E and pIC50 parenthesis. in interval confidence 95% by accompanied 08.Aldt eeaaye sn rpPdPim843(rpPd LaJolla, (GraphPad; 8.4.3 Prism GraphPad using analyzed were data All 2018). , 2015). , tal. et 08 Paolone 2018; , < .5wr osdrdsaitclysignificant. statistically considered were 0.05 6 tal. et 05.Tit iue fe L-Dopa, after minutes Thirty 2015). , α uui ( tubulin α u)rabbit Tub) max values Posted on Authorea 26 Feb 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161437480.01864791/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. eyn ntewl-salse O eetrihbtr ciiyo 1sgaig(Olianas signaling D1 on activity inhibitory receptor NOP well-established the slices on striatal Relying mouse 11.55 to in response pIC50 N/OFQ NOP of potentiated 2B). shift CCG-203920 leftward (Fig. a efficacy caused N/OFQ CCG-203920 changing 2B). stimulation without (Fig. the pIC (12.43-10.67), levels of inhibited an cAMP increase N/OFQ with of 83% 2A). manner inhibition an concentration-dependent (Fig. maximal caused a CCG-203920 SKF-38393 in by SKF-38393 nM). affected by (100 significantly induced CCG-203920 not inhibitor was with RGS4 that stimulated levels the neurons cAMP of DARPP32+ absence in (100 the measured cultures SKF-38393 was or primary accumulation pharma- agonist presence in cAMP of receptor neurons 2). impact DARPP32+ D1 the in (Fig. the investigated striatum tissues, was rat native responses E14 in the NOP interaction of on receptor RGS4 RGS4-NOP of a inhibition of cological neurons occurrence striatal the primary investigate rat To AT-403 in response the NOP right the the (pIC potentiated to potency CCG-203920 AT-403 shifted of t=3.24). t=4.10) also (pIC df=11, (df=11 RGS19 reduction potency (44%, of significant of efficacy a Co-transfection and reduction to leading significant t=3.79). 1B), a df=11 1B) (Fig. with curve (50%, 2), (Fig. efficacy AT-403 (Fig. and curve t=2.71) investigated. AT-403 next the higher of was slightly AT-403 shift showing manner, of concentration-dependent (pIC effect a (pIC potency in the potency production N/OFQ cAMP modulate of D1-stimulated to also E reduction the inhibited RGS19 N/OFQ reported significant of and was a reduction RGS4 and with of non-significant Whether RGS4 (Fig.1), presence a right to and the the t=3.093) similar in (df=12, to very N/OFQ 9.88-7.55) shifted structurally of 8.71, was is response curve (Xie RGS19 the N/OFQ receptor fact, proteins, the NOP In RGS the 1A). a other with with by interact (Fig. (Fig.1A) regulated assessed N/OFQ be was of might RGS19 curve receptor concentration-response (Fig.1), NOP the of cells the pIC shift NOP-transfected (E of rightward were In reduction a production AT-403, significant pIC caused cAMP RGS4. a agonist, receptor of showed with NOP NOP inhibition N/OFQ or the maximal molecule manner. alone concentration-dependent a small receptor a and the (Fig. in NOP (10.15-8.70) readout and production the biochemical cAMP N/OFQ, inhibition with a inhibited the ligand, as transfected N/OFQ measuring nM) endogenous cells by (40 receptor in cells SKF-38393 tested NOP HEK273 agonist receptor in The investigated D1 by first 1). stimulated was production RGS4 cAMP and of NOP between crosstalk HEK293T The in production cAMP D1-stimulated of inhibition cells receptor-driven NOP modulates negatively RGS4 experiments vitro In purchased water, (Bloomsburg, was center assay DMSO Resource Results cAMP cDNA 4% Ultra from purchased LANCE and were water. plasmids 1M USA). 0.02% All in PA, with MA). Texas dissolved CH3COOH saline (Waltham, were of in Elmer 2% SKF-38393 (University dissolved Perkin was Mumby from and in 6-OHDA N/OFQ S saline, dissolved Dr were in acid. was dissolved from of ascorbic benserazide were AT-403 gift purchased CCG-203920 and generous and were USA). benserazide a L-Dopa N/OFQ L-DOPA, TX, was and USA). antibody Dallas, hydrobromide MI, U1079 6-OHDA Southwestern, UK). Lansing, (Bristol, (East Italy). Bioscience University (Milan, Tocris (Arcuri Sigma-Aldrich State USA) from Michigan CA, purchased at View, (Mountain Core Medicinal Therapeutics Chemistry the Astraea by synthesized at was Zaveri (4-(2-(methoxyethyl-)-2-ethyl-1,2,4-thiadiazolidine-3,5-dione) CCG-203920 NT was Dr by synthesized (2-(1-(1-((1s,4s)-4-isopropylcyclohexyl)piperidin-4-yl)-2-oxoindolin-3-yl)Nmethylacetamide) AT-403 50 99,1.890)adecc 7% hnNOQ G4c-rnfcincue rightward a caused co-transfection RGS4 N/OFQ. than (75%) efficacy and 10.78-9.06) =9.92, 50 o86 96-.9 d=2 =.6 n E and t=3.56) (df=12, (9.62-7.69) 8.66 to tal. et μ )cmie ihicesn ocnrtoso /F,i the in N/OFQ, of concentrations increasing with combined M) 05.We O eetradRS9wr co-transfected, were RGS19 and receptor NOP When 2005). , 7 max f6% otaseto fRS with RGS4 of Co-transfection 61%. of ) max 50 f1.1(13-02)ada70% a and (11.36-10.26) 10.81 of o4% oivsiaewhether investigate To 45%. to max 50 92,97-.6 (df=11, 9.77-8.66) =9.22, o39%. to 50 89,9.92-7.93) =8.93, tal. et tal. et 50 2008), , 2018). , f9.43 of 50 Posted on Authorea 26 Feb 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161437480.01864791/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. O gns nue niykntceet sgicn ffc ftm F potentiates time F blockade of interaction RGS4 treatment effect that without x (significant suggesting appearance, time effects AIM 5A), and antidyskinetic in (Fig. delay AT-403 induced of severity min to agonist and 20 response duration NOP further overall overall the a the to affecting caused affecting model, without CCG-203920 significantly min, same of 40 by Co-administration the AIMs response. in of the kg onset Here, the mg delayed sedation. (0.03 kg alone AT-403 of AT-403 mg challenged 5A). absence we 0.03 dose, the kg AT-403 of in mg this dose (6 rat of effect the effect 6-OHDA antidyskinetic antidyskinetic at transient the Conversely, the improve and in effect. AT-403 mild antidyskinetic of a the exerted effect overlapped antidyskinetic that (Arcuri dose-dependent effects vivo sedative the in described LID of we study, model previous a AT-403 In of effect antidyskinetic the extended CCG-203920 RGS4 wild-type in in hypokinesia ineffective raclopride-induced was reversed but significantly animals administration CCG-203920 4). (Fig. mice F ietcneuneo h yeatvt fDRP3 steicesdpopoyaino R12 well- a ERK1/2, of phosphorylation increased (Pavon rodents the in is LID DARPP-32 (Bastide of in of DARPP-32 correlate alterations hyperactivity and accepted to the leads PKA of and as consequence LID such direct with kinases, A downstream associated several is MSNs of pathway activity direct striatum phosphorylating in in transmission signaling receptor D1 ERK Aberrant AT-403, of of inhibition effect AT-403 condition promoting the sedation. potentiated motor baseline AT-403-induced CCG-203920 the to potentiate worsen respect not not with did to did blockade the reduction administration due AT-403, RGS4 significant CCG-203920 baseline indicating with no 5B). to pretreated and (Fig. compared were improved observed reduced animals was L-Dopa dramatically When ON was t=4.075). performance administration df=10 rotarod L-Dopa (-76%; after appearance movement rod dyskinetic the administration on L-Dopa after spent and AT- time L-Dopa) of (Arcuri OFF effect rats (baseline, antidyskinetic before dyskinetic the monitored in with AT-403 was along performance by effects induced rotarod sedative the L-Dopa the 403, ON increased performance CCG-203920 rotarod whether of investigate To improvement the affect not did CCG-203920 the of increase marked and prolonged a F caused action F raclopride (time that revealed time ANOVA immobility Two-way each). (14.45 raclopride-induced n=20 test ly; reversed bar CCG-203769 (6.59 the test inhibitor drag in model RGS4 time akinesia/catalepsy the immobility neuroleptic-induced that greater (Blazer the reported mice vivo, previously in in we akinesia CCG-20920 since of used selectivity was RGS4 the confirm To vivo in RGS4 targets CCG-203920 experiments vivo In ANOVA, (three-way 56% stimulation. by response F receptor-mediated ANOVA, D1 (three-way reduced but levels SKF- pERK agonist F basal F receptor on ANOVA, D1 effect (three-way the no basal of had over Application cells SKF-38393-induced 3). immunoreactive the (Fig. pERK of striatum inhibition mouse mediated (100 of AT-403 slices 38393 the in affect number could cell RGS4 ERK-positive whether investigated next we 3.36 1,42 1.5 p =18.95, 1276.CG232,wihwsieetv ln,sgicnl oetae h ffcso AT-403 of effects the potentiated significantly alone, ineffective was which CCG-203920, =102.746). 9,108 -1 esrzd 5m kg mg 15 benserazide + μ 63,p =6.35, )t titlsie fn¨v iecue napoiaeyfu-odices ntenme of number the in increase four-fold approximately an caused na¨ıve mice of slices striatal to M) < ± .01 ieXtetetitrcinF interaction treatment X time 0.0001, .6ses =0 oprdt otos(5.7 controls to compared n=20) steps, 0.56 < .01 n euto fsepn ciiy(ieF (time activity stepping of reduction a and 0.0001) 1,42 tal. et 3,108 tal. et 922.I at hnc-ple ihCG232,A-0 ul niie D1 inhibited fully AT-403 CCG-203920, with co-applied when fact, In =9.222). 173 p =117.3, tal. et 05.RGS4 2015). , 08 Marti 2018; , -1 24,432 08.A h ihs oetse T43(. gkg mg (0.1 AT-403 tested dose highest the At 2018). , -/- .. ntepeec n nteasneo C-0901 gkg mg 10 CCG-203920 of absence the in and presence the in s.c.) < ie ugsigRS agtn tti dose. this at targeting RGS4 suggesting mice, =43.06). .01 ramn F treatment 0.0001; tal. et -/- tal. et iewr lgtyhpkntca aeie showing baseline, at hypokinetic slightly were mice ± 06 Santini 2006; , .0s =0 n 0 eue tpigatvt nthe in activity stepping reduced 40% and n=20) s, 1.40 02 Paolone 2012; , 8 9,108 40,p000)i ohwl-yeadRGS4 and wild-type both in p=0.0002) =4.09, 3,36 1,42 ± 1.4 p =11.34, tal. et .8sad10.08 and s 0.48 2399 i.3.A-0 ln 3 nM) (30 alone AT-403 3). Fig. =213.909; tal. et 07.I rvossuyw showed we study previous a In 2007). , 8,432 05 Fg B.A xetd the expected, As 5B). (Fig. 2015) , < 3,95 .01 ieXtetetinter- treatment X time 0.0001, 3.0 ramn F treatment =37.00, 6.0 p =67.50, ± .5ses respective- steps, 0.45 -1 < -1 .01 treatment 0.0001; .. ihL-Dopa with s.c.) xre strong exerted ) tal. et 3,432 -1 AT-403 , > 2015). , -1 =6.86, 2-fold (Fig. -/- Posted on Authorea 26 Feb 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161437480.01864791/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. nta td igecnetaino /F a etd hc a orymdltdb G4 In RGS4. by modulated poorly was which tested, was (Xie subtypes N/OFQ receptor of opioid concentration with the transfected single of cells a COS-7 each study in and evaluated that RGS4 originally containing In was plasmid receptor NOP dual-expression and a agonist. RGS4 DOP between but this interaction antihyperalgesia, of The and action antinociception pro-convulsant SNC80-induced the affect increased not CCG-203769, did with (Han RGS4 tolerance of or inhibition analgesia morphine-induced al. affect et not did but al. morphine agonist preferential fine-tunes RGS4 receptor These that properties. reports (Dripps al. previous sedative agonist behaviors to its et add and NOP worsening and signaling a interaction, without receptor receptor of vivo, RGS4-NOP opioid ability in functional Moreover, the correlates a vitro. to neurochemical potentiates point in its RGS4 data responses and of receptor LID inhibition NOP attenuate pharmacological inhibits to that RGS4 demonstrates that evidence study strong this provides study present The L-Dopa. by dyskinetic signaling of levels receptor striatum RGS4 D1 lesioned Discussion of the aberrant increase in of upregulated 44% stimulation rapidly a is acute causing RGS4 following that it, suggests animals reversed This group) striatum. (ON lesioned detected the L-Dopa was na¨ıvein counterparts acute (H reduction However, rats respective significant ratio. naive a with the to ratio, normalized compared compared lesioned-to-unlesioned rats as when hemilesioned expressed 6-OHDA were animals, of in levels 6-OHDA specificity RGS4 effect If of The strong 8A). a L-Dopa. striatum (Fig. revealed OFF ANOVA (-51%) 1). and Fig. unlesioned ON (Supp and mice both knockout measured RGS4 (F were in treatment confirmed levels of first RGS4 dyskinetic was and rats, used consequence hemilesioned antibody dyskinetic na¨ıve na¨ıve,the 6-OHDA a In L-Dopa of as 8). striatum occurring (Fig. the RGS4 rats (Geurts in analysis of administration Western adaptation L-Dopa by plastic measured and/or a depletion corrects DA treatment inhibition L-Dopa of RGS4 and whether depletion investigate DA To by modulated were levels or amounts RGS4 df=10) protein Striatal total t=2.239, affected (+52%; treatments increase over neither induced receptors Again, NOP 7B). L-Dopa levels. of (Fig. the pGluR1 influence able alter of inhibitory was normalization not well-known alone AT-403-driven the did AT-403 the with ERK, CCG-203920 line on signaling. effect in D1 the 7A), canonical to AT- (Fig. contrast levels of in pGluR1 However, ability normalize 7A). the to Fig. potentiates df=12; by t=2.272, CCG-203920 activated (+52%; PKA whether stratum by kg investigated phosphorylated mg therefore physiologically (0.03 We is 403 receptors. which (Santini receptor, D1 rodents AMPA via in glutamate LID the dopamine of of correlate subunit biochemical a another to is is levels due pGluR1 were of striatum increase observed in The changes phosphorylation the pGluR1 receptor-stimulated that expression. combined D1 suggesting protein inhibited was 6B), not Pharmacological AT-403 L-Dopa and 6A). (Fig. pathway (Fig. levels when striatum the However, protein lesioned of the total activation 6A). in the affect Fig. rise not not p=0.08; which did did df=10, levels df=12), significance treatments pERK t=3.584 t=1.947, (+117%; AT-403, (+47%; CCG-203920 and value CCG-203920 or striatum threshold with increase df=10) unlesioned significant the t=2.261, the a (+79%; above to with AT-403 associated relative just with was striatum pretreatment LID by lesioned expected, unaffected the As was 6). in (Fig. levels rats pERK dyskinetic of of striatum the in pERK kg mg (0.03 AT-403 (Arcuri striatum DA-depleted kg mg (0.1 AT-403 that 00.RS lodffrnilymdltdDPrcpo-eitdbhvoa ucmsi ie(Dripps mice in outcomes behavioral receptor-mediated DOP modulated differentially also RGS4 2010). , 07 Stratinaki 2017; , 05.I at G4ngtvl euae eadadpyia eedneidcdb h MOP the by induced dependence physical and reward regulated negatively RGS4 fact, In 2005). , 7,46 -1 omdlt Gu1lvl.A xetd -oaeeae Gu1lvl ntelesioned the in levels pGluR1 elevated L-Dopa expected, As levels. pGluR1 modulate to ) 79) akddces nRS rti eeswsfudi ohtelsoe (-64%) lesioned the both in found was levels protein RGS4 in decrease marked A =7.95). -1 ln ri obnto ihCG232 ol omlz -oaidcdices of increase L-Dopa-induced normalize could CCG-203920 with combination in or alone ) -1 tal. et a bet omlz h -oaidcdpR eesi h -HAlesioned, 6-OHDA the in levels pERK induced L-Dopa the normalize to able was ) tal. et 03.Ide,gntcdlto fRS swl sauepharmacological acute as well as RGS4 of deletion genetic Indeed, 2013). , 08.I h rsn td,w netgtdwehralwrds of dose lower a whether investigated we study, present the In 2018). , tal. et 9 07 Han 2017; , 3,20 tal. et 1.3 Fg8) hoi -oa(F group) (OFF L-Dopa Chronic (Fig.8B). =13.73) 03 Ko 2003; , tal. et 00 Stratinaki 2010; , tal. et 04,RS eeswere levels RGS4 2014), , tal. et tal. et 07.GluR1 2007). , tal. et 03 Xie 2013; , 2005). , et Posted on Authorea 26 Feb 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161437480.01864791/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. neetnl,peiu tde niae h novmn fRS nteptoeei fLD hwn that showing LID, of pathogenesis (Ko the effects in antidyskinetic RGS4 therapeutic of induced involvement blockade NOP the RGS4 potent indicated of studies effects previous pharmacodynamic Interestingly, for and as sedation agonist that between AT-403. window receptor suggests like therapeutic This NOP agonists the a pGluR1 effects. widen by sedative of perhaps its induced and rise (Dripps these amplifying effect the agonists Overall, without antidyskinetic receptor inhibited pathways, CCG-203920. the opioid signaling fully by improves underlying alone inhibition blockade the AT-403 further RGS4 and concerned, precluded that changes have are pathway indicate might MAPK levels confirming data which modulate pGluR1 CCG-203920, to dyskinesia, as agonist with inhibitor receptor far associated RGS4 NOP As a with of LID. combined ability underlying the when potentiates levels blockade RGS4 pERK that (Pavon (Arcuri normalized ERK LID and alone, blunted receptor AMPA ineffective and glutamate levels effectors of downstream pERK subunit several normalized GluR1 of the phosphorylation as al. the such et MSNs, to pathway leads direct pathways striatal D1 in pathways non-canonical molecular and (Santini cascades the canonical MAPK is the with and LID cAMP/PKA interference AT-403. the G by by as induced of such mediated phosphorylation enhancement truly ERK aberrant of is targeting. by inhibition RGS4 showcharacterized CCG-203920 the by we potentiated mediated of is CCG-203920 Since effect CCG-20920 LID, of underlying interaction properties. the effect selective antidyskinetic antidyskinetic that through AT-403 the its mice confirm also of of in that To effect akinesia confident potentiation positive are raclopride-induced true we reversed the RGS4, CCG-203920 a of with of worsening due dose the likely same by is the onset accompanied CCG-203920 that AIM it not in that performance, was Consistently, delay show effect rotarod alone. the this now on response extending Since we D1 significantly AT-403, the AT-403. model, affecting of by this without effect induced AT-403 antidyskinetic In of the dose activity. potentiated submaximal CCG-203920 antidyskinetic a (Arcuri of of slices effect striatal predictor the in biochemical potentiates mice. SKF-38393 a adult agonist neurons and in 2012), receptor striatal also active ERK-positive D1 , but constitutively of by rats increase is induced the in but inhibited MSNs) tissues AT-403 cells (likely developing and HEK273 N/OFQ in that only in demonstrated overexpression not previously D1- We occurs protein inhibiting interaction of This in artifact relevant. indicating potency an neurons physiologically N/OFQ not GABAergic, likely is increased DARRP32-positive, interaction in CCG-203920 neurons occurs this since interaction striatal This Primary responses levels. evoked RGS4. cAMP stimulated receptor of activity D1 (Buzas endogenous modulate NOP the to and and block neurons RGS4 to primary striatal the inhibitor both by RGS4 i.e. express systems, an receptor native using NOP in confirmed slices, the was striatal of of AT-403. interaction RGS4-NOP activation like than a and AT-403 ligand of interaction occurrence of nonpeptidic The of efficacy (Xie small-molecule modes the a vitro different efficiently versus the in N/OFQ more to signaling ligand modulate due endogenous to receptor be seem NOP could RGS19 This modulate and N/OFQ. and RGS4 negatively curves Thus, functionally to to AT-403 RGS19, Gi/o efficacy. reported protein, and N/OFQ to RGS was N/OFQ couples another negatively which of by RGS4 shared shift RGS4, al. that is rightward to suggests effect The RGS4 close this However, of very receptor. presence signaling. NOP the receptor the in NOP N/OFQ efficacy inhibit at AT-403 for agonist of reported full reduction those the potent with a consistent is are [ 403 values but the (Feng respectively).These in efficacy molecule 9.92, function AT-403 the small N/OFQ and intracellular and inhibited the affect AT-403 (9.43 protein-mediated of and not Gi/o potencies N/OFQ potency similar a did cells, and HEK293T production, RGS4 efficacy forskolin- NOP-transfected cAMP the cells, In of D1-stimulated AT-403. both HEK293T inhibition agonist reduced in selective N/OFQ it and study unaffected Moreover, potent potency. left present its but the reduced activity In significantly GTPase levels. N/OFQ cAMP 28% induced by increased RGS4 fact, 05.Itrsigy G1 eue h ffiayo T43 u hwdol rn o euto of reduction for trend a only showed but AT-403, of efficacy the reduced RGS19 Interestingly, 2005). , 06 Santini 2006; , 35 tal. et S]GTP 07 epeiul eotdta oeo T43a iha . gkg mg 0.1 as high as AT-403 of dose a that reported previously We . 2007) , γ n acu oiiainasy (Ferrari assays mobilization calcium and S tal. et tal. et 07,RS lcaemgtdffrnilyipc O behaviors, NOP impact differentially might blockade RGS4 2017), , α 98 Runne 1998; , n G and tal. et 10 βγ tal. et inln ahasdwsra fteD receptor, D1 the of downstream pathways signaling 08.W o eotta -odlwrdose, lower 3-fold a that report now We 2018). , 07.Seicly h nrae ciiyalong activity increased the Specifically, 2007). , tal. et tal. et 08 n h w ucinlyinteract functionally two the and 2008) , 04 Shen 2014; , tal. et 07 ofimn htAT- that confirming 2017) , tal. et tal. et 05.Specifically, 2015). , 08 Marti 2018; , tal. et 07 with 2017) , tal. et et -1 δ Posted on Authorea 26 Feb 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161437480.01864791/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. p SKF-38393 # of *p application group. following na¨ıve per mice of mice n=7 slices striatal in cells n=10 (100 ERK-positive nM of 0.03 Number N/OFQ striatum. in n=10, (n=9). nM nM 0.01 3 N/OFQ=0.1 N/OFQ mean Figure CCG1203920), n=8, are (+ 0.003 Data n=8 N/OFQ and nM). n=9, CCG203920) (100 nM (no (100 CCG-203920 0.001 SKF-38393 of N/OFQ with namely absence group, treated the were or Neurons presence the neurons. primary striatal B). mean panel in are of Data group 2. NOP RGS19. 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G4adRS9rdcdteNPrcpo gns blt osiuaecM accumulation cAMP stimulate to ability agonist receptor NOP the reduced RGS19 and RGS4 G4adRS9rdcdteNPrcpo gns blt osiuaecM accumulation cAMP stimulate to ability agonist receptor NOP the reduced RGS19 and RGS4 .5 ieetfo baseline; from different 0.05, tal. et -1 rsln ip) rosidct h ieo rgamnsrto.Dt r mean are Data administration. drug of time the indicate Arrows (i.p.). saline or 2020). , < .5 ieetfo ntmltdvehicle; unstimulated from different 0.05, tal. et tal. et 03 Ko 2003; , § ° p p < < .5 ieetfo T43+SF333 he-a ANOVA Three-way SKF-38393. + AT-403 from different 0.05, .5 ieetfo alpiei Tmc 2wyR ANOVA RM (2-way mice WT in raclopride from different 0.05, 04.Pamclgclbokd fRS sepce to expected is RGS4 of blockade Pharmacological 2014). , tal. et 11 04.Mr pcfial,te ieycomplement nicely they specifically, More 2014). , -1 alpie(..,floe 0mnltrb CCG- by later min 30 followed (i.p.), raclopride μ tal. et ;A n T43(.0 M 1 nM- (0.001 AT-403 and A) M; ° p tal. et < 04,weeRS xrsinwas expression RGS4 where 2014), , .5 ieetfo K-89 alone; SKF-38393 from different 0.05, ± -/- μ E fn81 xeiet per experiments n=8-10 of SEM 04 Shen 2014; , )adNOQ(.11n)in nM) (0.01-1 N/OFQ and M) ietetdudracrossover a under treated mice ± E fn7experiments n=7 of SEM tal. et tal. et 02 Shen 2012; , 05 and 2015) , ± μ ;B in B) M; -/- E of SEM ± mice. tal. et SEM Posted on Authorea 26 Feb 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161437480.01864791/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. nidsiei rfie ftonvlptn n eetv oietnopai Qrcpo agonists. receptor FQ nociceptin/orphanin selective and I potent Morella novel CA, Pisano two Pharmacol D, of Mercatelli Function profiles M, Motor Frassineti anti-dyskinetic of S, Regulators Novello Critical L, as Arcuri Disease. Proteins Parkinson’s RGS in (2020). Implications RA Their Fisher and Student MM, (B). Spicer KE, test Ahlers-Dannen Dunn the by followed *p test References group). (C). Kruskal-Wallis 6-OHDA data (A), the unpaired from test for removed two-tailed Newman-Keuls was t-test, the outlier by one levels #p B, followed (C); hydroxylase panel VA striatum Tyrosine In unlesioned (B). or na¨ıve loss. striatum (A-B) from animal and different unlesioned to OFF to due both relative group out lesioned L-Dopa carried the was in to hemile- RGS4 analysis na¨ıve, to normalized 6-OHDA of RGS4 of normalized percentage rats, striatum were or dyskinetic the values (A) In in RGS4 rats. RGS4 L-Dopa. dyskinetic of ON or panel) naive, (lower L-Dopa quantification sioned and panel) (upper images 8 Figure data). unpaired rats Dyskinetic mean rats. kg and dyskinetic L-Dopa-na¨ıve mg (A) or hemilesioned, pGluR1 (0.03 kg 6-OHDA AT-403 of of panel) with striatum (lower treated quantification the were and in panel) (B) GluR1 (upper images total representative blot Western rats. data). 7 unpaired Figure for mean two-tailed hemilesioned are t-test, 6-OHDA Values kg (Student kg of AT-403. striatum mg mg striatum before unlesioned (6 (0.03 unlesioned L-Dopa min AT-403 and with 5 with challenged lesioned administered treated later, the were (lower min in quantification 10 rats (B) and and, Dyskinetic ERK panel) rats. total (upper and dyskinetic images L-Dopa-na¨ıve (A) representative or blot pERK Western of rats. kg panel) dyskinetic mg of (6 striatum the L-Dopa (ON) 6 after Figure min data). 60 unpaired and for (OFF) tailed two mean before are seconds, Values performance in i.p.). Rotarod rod kg (B) mg on test. 15 hoc time benserazide post Tukey as the evaluated by followed was ANOVA measure repeated Two-way AT-403. p ## L-Dopa; with kg from challenge different mg following rats (10 dyskinetic CCG-203920 hemilesioned 6-OHDA kg or in mg scored were (6 AIMs L-Dopa ALO (A) rotarod. the on rgoiA iaoC,Mrr 22) titladnga ucrnctp n ye4rcposmodulate receptors 4 type and 1 type muscarinic nigral and Striatal (2020). M Morari CA, SM Pisano Wade A, M, Brugnoli inhibitors. Calcagno Gi RGS4 EM, the thiadiazolidinone Turner of for EM, potential proteins Jutkiewicz anti-Parkinson’s GTPase-activating AJ, are Storaska RGS4 LL, Blazer and subunits. GAIP alpha (1996). protein AG G of Gilman subfamily TM, Wilkie disease. DM, M Parkinson’s Feyder Berman in PO, complications Fernagut non-motor S, Fasano and B, motor Picconi L-dopa-induced WG, Meissner MF, Bastide -1 ...CG232 1 gkg mg (10 CCG-203920 i.p.). , ± E f6rt e ru.*p group. per rats 6 of SEM T43ihbtdteLDp-tmltdplR hshrlto ntesrau fdyskinetic of striatum the in phosphorylation pGluR1 L-Dopa-stimulated the inhibited AT-403 . G4lvl eemdltdb -HAadLDP ramn.Wsenbo representative blot Western treatment. L-DOPA and 6-OHDA by modulated were levels RGS4 . T43i obnto ihCG232 niie -oaidcdEKpopoyainin phosphorylation ERK L-Dopa-induced inhibited CCG-203920 with combination in AT-403 . 175 α- (5) uui r loson() ausaemean are Values (C). shown also are tubulin -1 : lsbneaie1 gkg mg 15 benserazide plus 782-796. -1 .. obndwt eil,A-0 00 gkg mg (0.03 AT-403 vehicle, with combined s.c.) , ± E f1 asprgop *p group. per rats 11 of SEM -1 .)() ausaemean are Values (A). i.p) , < .1 ieetfo -oa+CCG-203920; + L-Dopa from different 0.01, -1 < .. rvhcewr diitrd5mnbfr T43 ausare Values AT-403. before min 5 administered were vehicle or i.p.) , .5 ieetfo neindsrau Suettts,totie for two-tailed t-test, (Student striatum unlesioned from different 0.05, -1 Cell .. rvhcead 0mnltr hlegdwt -oa( mg (6 L-Dopa with challenged later, min 10 and, vehicle or s.c.) , 86 (3) -1 o Pharmacol Mol α- .. obndwt eil,A-0 00 gkg mg (0.03 AT-403 vehicle, with combined s.c.) , uui shueepradepesda bouevalues absolute as expressed and housekeeper as tubulin : 445-452. -1 12 ...CG232 1 gkg mg (10 CCG-203920 i.p.). , ± < E f6rt e ru.*p group. per rats 6 of SEM ± < .5 ieetfo F -oa(tdn t-test, (Student L-Dopa OFF from different 0.05, .5dffrn rm6OD B.OewyANO- One-way (B). 6-OHDA from different 0.05 E f1 asprgop *p group. per rats 13 of SEM ± 98 C hmclneuroscience chemical ACS E f7rt e ru n6i h ON the in (n=6 group per rats 7 of SEM (6) : tal. et , 730-738. -1 .. rCG232 1 gkg mg (10 CCG-203920 or s.c.) , tal. et , ° p rgNeurobiol Prog < 21) niPrisna and Anti-Parkinsonian (2018). tal. et , .5 ieetfo -oa+ L-Dopa from different 0.05, 21) ahpyilg of Pathophysiology (2015). -1 21) eetvt and Selectivity (2015). < .. rvhcewere vehicle or i.p.) , .5 ieetfrom different 0.05, -1 < .. rvehicle or s.c.) , .5 **p 0.05, 6 . (6) : 911-919. -1 -1 < < s.c.) , rJ Br 0.01, 0.05, plus -1 , Posted on Authorea 26 Feb 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161437480.01864791/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. cin frgltro rti inln poemrhn eadaddpnec u rmt analgesia. promote but dependence and reward morphine D oppose Howland 4 signaling K, Psychiatry protein Psifogeorgou Biol G Z, of Rahman regulator RH, of Ring actions K W, Renthal Wix MH, KM, Han Boyt behavior. ES, and signaling Cogan receptor-dependent 44 KT, opioid kappa Schmidt of regulation SW, region- differential proteins: Kaski (RGS) brain. signaling JD, rat G-protein Gross of in Regulators subtypes (1997). nine EJ of Nestler expression HG, (RGS) specific Dohlman signaling YG, depletion. G-protein Ni dopamine of SJ, undergoing regulators Gold rats of of expression striatum Altered the (2003). acute E in facilitate Hermans mRNAs JM, proteins Maloteaux RGS9 M, (2001). Geurts P Sanchez-Blazquez effects. A, mu-opioid Lopez-Fando to M, tolerance Rodriguez-Diaz J, Garzon AT-403. agonist nonpeptide R receptor-selective Guerrini perspectives NOP C, unbiased & Trapella novel research MF, a Bird of characterization BV, gical Journigan D, Malfacini F, Ferrari regions. dynamic Subcortical of II. analysis development. transgenic during chromosome 4 signaling artificial to G-protein Bacterial clues of (2006). and roscience regulator treatments P of novel Levitt patterns of DB, expression source Campbell a G PJ, disease: of Ebert Parkinson’s regulator of disease. of models the role of Animal The cause (2011). (2017). the P EM Jenner Jutkiewicz S, JR, behaviors. Duty Traynor receptor-mediated KC, delta-opioid Rice in reviewers. 4 RR, peer signaling Neubig and protein authors Q, for Wang guidance IJ, MA simplified Dripps Giembycz and CH, updated II: George Pharmacol reporting JR, J their Docherty Br and G, analysis Cirino and S, design node. sinoatrial Alexander PH the MJ, Backx in Curtis SS, control Bolz rate lesion J, heart Voigtlaender-Bolz 6-OHDA and H, unilateral signaling Zhang sympathetic the RA, in Rose mice. C, dyskinesia and Cifelli L-DOPA-induced rats striatal of in with disease Ratings Parkinson’s associated (2007). of is M model rat Lundblad the MA, in mRNA. Cenci decarboxylase dyskinesia acid L-DOPA-induced glutamic (1998). and A prodynorphin- Bjorklund of disease. nocicep- overexpression CS, Parkinson’s Lee in of MA, dyskinesia regulation Cenci l-dopa-induced of AMP-dependent models cyclic Animal (2018). and Disord AR cultures. Activity Crossman astrocyte MA, (1998). and neuronal Cenci BM primary in Cox expression gene J, FQ tin/orphanin Rosenberger B, Buzas rats. hemilesioned 6-hydroxydopamine in Dis activation pathway Neurobiol striato-nigral and dyskinesia levodopa-induced ukeizE,Rc C ryo R od H(05.Sprto ftecnusosadantidepressant- and convulsions rats. in the SNC80 of agonist Separation delta-opioid (2005). the JH by Woods produced JR, effects Traynor like KC, Rice EM, Jutkiewicz encepha- GNAO1 in disorder Movement (2017). RR Neubig A, mutations. Gezer gain-of-function V, Shaw with B, associated lopathy Karaj B, Sjogren H, Feng (10) : 33 1728-1741. 142 (6) : 144: (4) 889-899. 67 175 : 1163-1181. (8) 105044. (7) : 761-769. : 5 rJPharmacol J Br 987-993. (4). u Neurosci J Eur 164 (4) 13 urPoo Neurosci Protoc Curr : Neurosci J (4) 1357-1391. Neurology 13 : 801-811. 17 89 scohraooy(Berl) Psychopharmacology scohraooy(Berl) Psychopharmacology ice Pharmacol Biochem (20) (8) icRes Circ : tal. et , : hpe 9: Chapter 8024-8037. 762-770. tal. et , tal. et , tal. et , u Neurosci J Eur 21) oeo G1 nthe in RGS12 of Role (2019). Neurochem J 103 20) G4rgltspara- regulates RGS4 (2008). tal. et , 21) nvtopharmacolo- vitro In (2017). 21) ri einspecific region Brain (2010). Neuropsychopharmacology nt925. 9 Unit (5) 66 : 21) Experimental (2018). 527-535. (7) 10 182 : 71 234 1163-1170. (8) Pharmacology (2) (4) : (1) 2694-2706. : : 556-563. 588-596. : 29-39. Neu- Mov Posted on Authorea 26 Feb 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161437480.01864791/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. alt ,Plz ,BrhrtF ahro 22) euaoso rti inln naagsaand analgesia in signaling protein G of Regulators (2020). V Zachariou striatal F, the Bertherat addiction. on C, effects Polizu circuitry. polyglutamine-related F, brain that Sakloth on shows V dependent Perrin disease be O, Huntington’s not Gokce of may D, transcriptome models Zala neuron A, primary Kuhn in E, Regulier H, SJ Runne Gold D, function. Kardassis receptor RL, mu-opioid Neve of SJ, modulator disease. Russo Parkinson’s P, E Papakosta of K, Melloni model Psifogeorgou C, rat a Keywood in C, release Caccia glutamate S, vivo Novello A, Brugnoli Pisan`o CA, are expression FosB (1986). and Orlando. C phosphorylation Watson ERK G, mice. (2006). Paxinos hemiparkinsonian R in Moratalla dyskinesia L-DOPA-induced A, with Mendialdua associated F AB, Marchisella inhibitors. Martin Ras-ERK L, N, relevant Marrone Pavon clinically RE, by Bernardi levodopa-induced mice prevents MT, in behaviours Indrigo Eltoprazine cocaine-mediated IM, (2015). activity. Morella M pathway A, Morari direct Papale D, and glutamate Mercatelli striatal L, reducing Arcuri by A, dyskinesias Brugnoli G, Paolone ON moving disease: Parkinson’s Managing (2020). M Morari NT, Zaveri NOP. R, I with Eleopra E, Morella Bezard S, D, Fasano Mercatelli R, Guerrini dyskinesias. FQ Q, L-DOPA-induced Li nociceptin/orphanin attenuate D, agonists of glutamate Blockade Rodi nigral (2004). M, normalizes M Marti and Morari akinesia C, haloperidol-induced Bianchi reverses G, nigra release. Calo substantia R, rat in Guerrini transmission F, Mela M, disease. Parkinson’s Marti with associated J neurodegeneration Witta and JM, rosci Brown symptoms S, attenuates Zucchini transmission M, Fantin FQ susceptibility F, and Mela LTD M, striatal Marti of the control dopaminergic in deficits. for involved required motor is is parkinsonian RGS4 to RGS4 (2012). AC Parkinson’s (2014). Kreitzer of TN, P model Lerner rat Ravenscroft 6-OHDA-lesioned AR, unilateral the Crossman in E, movements disease. their involuntary Bezard abnormal and of ML, signaling generation targets. Martin-Negrier G-protein discovery WK, drug of as Ko Regulators use (2011). their DP in Siderovski challenges 728-749. and PM, promises Giguere substrates: DE, Galpha Bosch AJ, Kimple niistrsn yrxls hshrlto,dpmn ytei,addpmn ()rcpo inln in system signaling striatum. receptor receptor dorsal FQ-NOP D(1) and nociceptin/orphanin dopamine accumbens of and Activation nucleus synthesis, (2008). rat dopamine mRNA P phosphorylation, Onali receptor hydroxylase M, tyrosine ORL1 Boi inhibits of S, comparison Dedoni H MC, system: Olianas nervous Akil central O, binding. rat Civelli FQ HP, (125)I-[(14)Tyr]-orphanin the with Nothacker in expression R, distribution Reinscheid receptor A, (ORL1) like Mansour Jr., CR, Neal 25 Neurochem J erbo Dis Neurobiol (42) o Pharmacol Mol rJPharmacol J Br : 9591-9601. 91 70: (6) . 138-148. : 177 1501-1504. h a ri nseetxccoordinates stereotaxic in brain rat The Neuron (1) : 28-47. 73 Neurochem J (2) Neurochem J : 347-359. Neurosci J 14 opNeurol Comp J 103 Neurosci J tal. et , 107 tal. et , tal. et , (2) 32 Neuropharmacology (2) : (46) 617-625. 21) oietnopai Qreceptor FQ Nociceptin/orphanin (2012). : 20) yrglto fgn expression gene of Dysregulation (2008). 20) lcaeo nociceptin/orphanin of Blockade (2005). 544-556. 28 : tal. et , 16106-16119. n d.Aaei rs:Sde ; Sydney Press: Academic edn. 2nd . 412 (39) o Disord Mov ilPsychiatry Biol tal. et , : tal. et , (4) 9723-9731. tal. et , 22) anmd niisin inhibits Safinamide (2020). : 20) G92i negative a is RGS9-2 (2007). 563-605. eLife 167 19) podreceptor- Opioid (1999). 30 hrao Rev Pharmacol 21) maretof Impairment (2016). 5 . (13) 59 . (1) : 1728-1738. : 64-74. 63 Neu- J (3) : Posted on Authorea 26 Feb 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161437480.01864791/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. iue7 Figure 6 Figure 5 Figure 4 Figure 3 Figure 2 Figure O Berton R, 1 DiLeone Figure Z, Rahman action. N, opiate Sanchez in RGS9 D, Georgescu receptor V, ORL1 Zachariou regulating in protein KJ GAIP/RGS19 Kim full-length X, of selectivity Han signaling. lacks K, GAIP/RGS19 Maruyama FQ mouse E, the Nociceptin/orphanin of Ito (2008). G Y, M Yanagisawa of Morari GX, O, Xie Regulator Isacson of parkinsonism. C, MPTP-induced Trapella Inhibitors attenuates M, Molecule blockade Marti receptor Small R, Sanchez-Pernaute (2012). R, SM Viaro Husbands RR, Proteins. abuse. (RGS) of Neubig Signalling drugs LL, Protein & signaling Blazer sym- protein EM, a G Turner of from Regulators proteins: (2005). (RGS) RR signaling Neubig protein pharmacology. JR, Traynor mu-opioid G Structure, in of Receptor concepts regulators FQ new and on Nociceptin/Orphanin receptors posium (2016). mu-Opioid NT (2012). Systems. Zaveri J Opioid Traynor BM, with Interactions Cox G(i and G, AlF4–activated Functions, to Calo Ligands, bound Signaling, MR, RGS4 hydrolysis. Bruchas of GTP L, for Structure Toll state (1997). transition SR the Sprang of AG, stabilization Gilman alpha1): models. DM, pain Berman neuropathic and JJ, A depression Tesmer in S action U drug in Sci antidepressant years Acad of C 20 Natl modulator Dias Proc - crucial J, Magida a targets S, is drug Ghose [corrected] as V, 4 Mitsi proteins A, signalling Varidaki M, protein Stratinaki G 21. of Review regulators IUPHAR of making: evolution the The Q Dyskinesia. (2017). Li L-DOPA-Induced B Z, of Sjogren Xie Models WK, in Deficits Ko Plasticity V, Striatal Francardo Ameliorates JL, Plotkin W, Shen Management. Pain Protein for (RGS) Target Signaling Potential G-Protein a brain of as neuroscience Regulator Signaling behavioral (2020). Receptor JR Opioid in Traynor of KE, Modulation model Kochan R, lesion Kandasamy treatments. 6-hydroxydopamine NB, and Senese recovery unilateral deficits, The functional (1996). of striatal Analysis JP and research. dopaminergic Huston mouse RK, of Schwarting culture assays. and matrix Dissection collagen (2012). three-dimensional RJ in Pasterkamp explants F, Morello ER, JA Schmidt Girault A, dyskinesia. Borgkvist L-DOPA-induced in Neurosci M, signaling J Carta kinase protein A, signal-regulated extracellular Usiello and cAMP/DARPP-32 E, Valjent E, Santini o Biol Mol J 27 13: (26) 5. : 6995-7005. 353 rcNt cdSiUSA S U Sci Acad Natl Proc (5) 110 : 1081-1092. (20) rJPharmacol J Br C eiia hmsr letters chemistry medicinal ACS : 8254-8259. 100 174 15 rgAchlDepend Alcohol Drug ora fvsaie xeiet JoVE : experiments visualized of Journal (23) (6) tal. et , : : 427-437. 13656-13661. erbo Dis Neurobiol tal. et , tal. et , Cell 21) 4Msaii eetrSignaling Receptor Muscarinic M4 (2015). rgNeurobiol Prog 21) euao fGpoensignaling protein G of Regulator (2013). 3 tal. et , 20) -emnlytuctdvariant truncated N-terminally (2005). hrao Rev Pharmacol 89 (2) (2) : 30 146-150. tal. et , 121 : 20) rtclivleetof involvement Critical (2007). 251-261. (3) (3) : 430-438. 50 20) seta oefor role Essential (2003). : Neuron 173-180. o Interv Mol (2-3) 68 rnir nmolecular in Frontiers (2) : 88 275-331. : 419-457. (4) 5 (61). (1) : 762-773. : 30-41. Posted on Authorea 26 Feb 2021 — The copyright holder is the author/funder. All rights reserved. No reuse without permission. — https://doi.org/10.22541/au.161437480.01864791/v1 — This a preprint and has not been peer reviewed. Data may be preliminary. iue8 Figure 16