β-Arrestin2 Regulates Cannabinoid CB1 Receptor Signaling and Adaptation in a Central Nervous System Region–Dependent Manner

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<ul><li><p> abS aRPe , DaLa</p><p>Ba ofma epenpro ecepthe</p><p>Me ia, cwil ]GTPpa</p><p>Re tivityveh -medcat reducau to THwa ra ofTH</p><p>Co te ansug obseres effede ithred</p><p>Kepa</p><p>Cenproingpamodugioconun</p><p>cycpo(7)(do</p><p>curs by G-protein-coupled receptor (GPCR) kinase (GRK)-mediated</p><p>Fro</p><p>Ad</p><p>Rec</p><p>00doy Words: Beta-arrestin, cannabinoid receptors, GPCR, statisticalrametric mapping, tolerance</p><p>B1 receptors (CB1Rs) are widely distributed in the centralnervous system (CNS) (1) and mediate the central effects of</p><p>9-tetrahydrocannabinol (THC) and cannabinoids (2). Thedocannabinoid system is implicated in numerous physiologicalcesses and is a potential therapeutic target for disorders includ-neurodegenerative and neuropsychiatric diseases and chronic</p><p>in (3). However, therapeutic use is limited by psychoactive andtor side effects.Moreover, repeated cannabinoid treatment pro-ces tolerance to cannabinoid-mediated in vivo effects (4). Re-n-specific CB1R desensitization and downregulation occur injunction with tolerance (5), but themolecular mechanisms thatderlie CB1R adaptations and tolerance are not well defined.CB1Rs primarily activate Gi/o-proteins, which regulate adenylyllases, ion channels, and kinases (6). Persistent cannabinoid ex-sure induces CB1R uncoupling from G-proteins (desensitization), with subsequent receptor internalization (8) and degradationwnregulation) (9,10). Onemechanism for these adaptations oc-</p><p>phosphorylation of activated receptors and subsequent -arrestinbinding (11). -arrestin2 is one of two arrestin isoforms in the brain(12), and findings in cell models support a role for -arrestin2 inCB1R adaptations. Coexpression of GRK3 and -arrestin2 was re-quired for rapid desensitization of CB1R-mediated potassium cur-rents following exposure to WIN55,212-2 in Xenopus oocytes (8).Similarly, expression of dominant negative -arrestin2 attenuateddesensitization ofWIN55,212-2-mediated inhibition of glutamater-gic neurotransmission in hippocampal neurons (13). Immunohisto-chemical studies show that CB1Rs are codistributed with -arres-tin2 in certain CNS regions (12,14), suggesting that -arrestin2might regulate CB1R signaling in the CNS.</p><p>Because there are no pharmacological -arrestin inhibitors,-arrestin2 knockout (arr2-KO)mice (15) provide amodel to studyits role in regulatingGPCRs in vivo (16). Acute administrationof THCto arr2-KO mice revealed enhanced sensitivity to its antinocicep-tive and hypothermic effects (17). However, direct evidence for therole of -arrestin2 in CB1R adaptations and tolerance followingrepeated THC is lacking.</p><p>We adapted statistical parametric mapping (SPM) to analyze[35S]GTPS autoradiography (18). SPMhas the advantage of assess-ing changes in G-protein activation in an unbiased and anatomi-cally inclusive manner. We applied SPM to examine the role of-arrestin2 in CB1R regulation by comparing cannabinoid-stimu-lated [35S]GTPS binding in brains from vehicle and THC-treatedarr2-KO and wild-type (WT) littermates. Combining this approachwith behavioral assessment following THC administration allowedus to compare CB1R signaling with behavioral responses observedin thearr2-KOmice.Wedemonstrate region-specific regulation of</p><p>m the Department of Pharmacology and Toxicology and Institute forDrug and Alcohol Studies (PTN, DES, LJS-S), Virginia CommonwealthUniversity, Richmond, Virginia; and Departments of Molecular Thera-peutics and Neuroscience (CLS, KMR, LMB), The Scripps Research Insti-tute, Jupiter, Florida.dress correspondence to Laura J. Sim-Selley, Ph.D., Box 980524, 1112 E.Clay Street, Richmond, VA 23298; E-mail: ljsimsel@vcu.edueived Aug 4, 2011; revised Oct 25, 2011; accepted Nov 30, 2011.</p><p>BIOL PSYCHIATRY 2012;71:71472406-3223/$36.00i:10.1016/j.biopsych.2011.11.027 2012 Society of Biological Psychiatry-Arrestin2 Regulates Cannignaling and Adaptation inegionDependent Mannerter T. Nguyen, Cullen L. Schmid, Kirsten M. Raehalura J. Sim-Selley</p><p>ckground: Cannabinoid CB1 receptors (CB1Rs) mediate the effectsrijuana. Repeated THC administration produces tolerance and dduces motor and psychoactive side effects. -arrestin2 mediates rse CB1R effects and receptor regulation is unclear.</p><p>thods: CB1R signaling and behaviors (antinociception, hypothermd-type mice after THC administration. Cannabinoid-stimulated [35Srametric mapping and region-of-interest analysis.</p><p>sults: -arrestin2 deletion increased CB1R-mediated G-protein acicle-treated mice. arr2-KO mice exhibited enhanced acute THCalepsy. After repeated THC administration, arr2-KOmice showeddal periaqueductal gray, and spinal cord and attenuated tolerances found in hypothalamus, cortex, globus pallidus, and substantia nigC-induced catalepsy was observed in arr2-KO mice.</p><p>nclusions: -arrestin2 regulation of CB1R signaling following acugest that multiple, overlapping mechanisms regulate CB1Rs. Theponses toacuteTHCanddecreased tolerance to theantinociceptivevelopment of cannabinoid drugs thatminimize CB1R interactionswuced motor suppression.inoid CB1 ReceptorCentral Nervous System</p><p>na E. Selley, Laura M. Bohn, and</p><p>9-tetrahydrocannabinol (THC), the psychoactive component indence, which limit therapeutic development. Moreover, THCtor desensitization, internalization, and signaling, but its role in</p><p>atalepsy) were assessed in -arrestin2-knockout (arr2-KO) andS and [3H]ligand autoradiography were assessed by statistical</p><p>in subregions of the cortex but did not affect CB1R binding, iniated antinociception and hypothermia, with no difference inced CB1R desensitization and/or downregulation in cerebellum,C-mediated antinociception. In contrast, greater desensitizationarr2-KO comparedwithwild-typemice. Enhanced tolerance to</p><p>d repeated THC administration was region-specific, and resultsrvations that arr2-KO mice display enhanced antinociceptivectsof thedrug, yet enhanced tolerance to catalepsy, suggest that-arrestin2might produce improved cannabinoid analgesicswith</p><p>Original text: givenname</p><p>Original text: surname</p><p>Original text: givenname</p><p>Original text: surname</p><p>Original text: givenname</p><p>Original text: surname</p><p>Original text: givenname</p><p>Original text: surname</p><p>Original text: givenname</p><p>Original text: surname</p><p>Original text: givenname</p><p>Original text: givenname</p><p>Original text: surname</p><p>Original text: surname</p></li><li><p>CB1Rs by-arrestin2 that parallel changes in THC-mediated behav-ior</p><p>Me</p><p>Mi</p><p>injethtrechintaftHe</p><p>Be</p><p>imlininjimmebo(15spiFocenimsibanCa</p><p>[35</p><p>hocongeWIbaPa(21wamoNe</p><p>[35</p><p>duCPjacact(18[3H(1)[3Hmestmeagvarho</p><p>Results</p><p>TH</p><p>trebaswitlate.09prosubmaF (5Thibeofinteacchrcurcom</p><p>indeurep treimacubeF (1act53.F (5theptiTHshi</p><p>adecomF (1tiosigtyp32.tre.00grolateHocan[gesugTHdia</p><p>-A</p><p>whsugspi</p><p>P.T. Nguyen et al. BIOL PSYCHIATRY 2012;71:714724 715and tolerance.</p><p>thods andMaterials</p><p>Detailed Methods are provided in Supplement 1.</p><p>ceMale WT and arr2-KO mice (littermates, 4 months) (15) wereected intraperitoneally with THC (10 mg/kg) or vehicle (1:1:18anol:cremaphor: .9% saline) twice daily for 6.5 days (subchronicatment). Twenty-four hours after the final injection, mice wereallengedwith increasing doses of THC (3, 7, 20, 26, and 44mg/kg,raperitoneally) every 40 min, with responses assessed 30 miner each injection. Studies followed the National Institutes ofalthGuidelines for the Care and Use of Laboratory Animals.</p><p>haviorAntinociception was assessed using the warm-water (52C) tail-mersion assay (19). Duplicate measurements determined base-e responses, but mice were assessed only once following eachection to minimize tissue damage. A trained observer assessedmobility by determining the time mice spent motionless on atal ring-stand over 5 min (20). Mice were gently restrained, anddy temperaturewasmeasuredusinga rectal probe thermometer).Micewere sacrificedbydecapitation24hours after testing. Thenal cord and brain were extracted, frozen, and stored at80C.r antinociception and catalepsy, data are presented as the per-tageof themaximumpossible effect (%MPE)100% [(exper-ental response latency basal response latency)/(maximal pos-le response basal response latency)]. Nonlinear regressionalysis was calculated using GraphPad Prism software (La Jolla,lifornia).</p><p>S]GTPS and [3H]SR141716A BindingWhole spinal cord was collected (Supplement 1), tissue wasmogenized, and agonist-stimulated [35S]GTPS binding wasducted as published (10). Concentration-effect curves were</p><p>nerated using .013 mol/L CP55,940 or .0310 mol/LN55,212-2. Percent stimulation [(agonist-stimulated basal)/sal] 100%. Curveswere fit using nonlinear regression inGraph-d Prism. [3H]SR141716A binding was performed as published) using [3H]SR141716A (.12.5 nmol/L), and nonspecific bindingsmeasuredwith 5mol/L SR141716A. Datawere fit to a one-sitedel in GraphPad Prism. Statistical comparisons used Student-wman-Keuls post hoc test.</p><p>S]GTPS and [3H]CP55,940 AutoradiographyAgonist-stimulated [35S]GTPS autoradiography was con-cted on duplicate serial sections as published (7,18). Basal and55,940-stimulated [35S]GTPS binding were conducted in ad-ent sections. CP55,940 is a high-efficacy agonist for G-proteinivation but does not activate non-CB1 sites in brain sections). Net stimulation (nCi/g) (agonist-stimulated basal).]CP55,940autoradiographywasmodified fromHerkenham et al.and Moise et al. (22). Total binding was assessed with 3 nmol/L]CP55,940 and nonspecific binding was measured using 10ol/L CP55,940. Image reconstructions, SPM, and region-of-inter-(ROI) analysis were conducted as published (18,23). The ROIasurements were made on original unprocessed images, aver-ed across hemispheres, and analyzed by two-way analysis ofiance (significance p .05) and Student-Newman-Keuls postc comparisons.C-Induced Responses inarr2-KOMiceTHC-mediated antinociception was assessed in vehicle- or THC-ated WT and arr2-KO mice by cumulative dosing of THC. Theal latencies in WT and arr2-KO mice subchronically treatedh vehiclewere 1.650 .176 and 1.625 .251, respectively. Basalncies for WT and arr2-KOmice treated with THCwere 1.4382 and 1.788 .210, respectively. Cumulative dosing of THCduced a greater degree of antinociception in arr2-KO micechronically treated with vehicle, compared with their WT litter-tes [Figure 1A; for genotype: F (1,78) 8.95, p .0037; for dose:,78)92.00,p .0001; for interaction: F (5,78)5.11,p .0004].s difference was due to a difference in potency (ED50 value)tweenWT andarr2-KOmice (Table 1). To determine the degreeantinociceptive tolerance, the ED50 of THC and 95% confidenceervals were calculated using nonlinear regression analysis ofh curve. Comparison between genotypes revealed that sub-onic THC treatment shifted the antinociceptive dose-responseve to the right to a much greater extent in WT (8.45-fold shift)pared to arr2-KO mice (1.68-fold shift; Table 1).</p><p>Cannabinoids decrease rodent motility measured as time spenta cataleptic state. Cumulative dosing of THC induced a similargree of catalepsy in vehicle-treated WT and arr2-KO mice [Fig-1B; genotype: F (1,78) 2.53, p .1159; dose: F (5,78) 65.19,.0001; interaction: F (5,78) .71, p .6193]. Subchronic THC</p><p>atment reduced the time that bothWT andarr2-KOmice spentmobile compared with vehicle-treated mice given the samete dose of THC, indicating that both WT and arr2-KO micecome tolerant to THC-mediated catalepsy [WT, treatment:,84) 18.13, p .0001; dose: F (5,84) 84.00, p .0001; inter-ion: F (5,84) 2.49, p .0376; arr2-KO, treatment: F (1,72) 27, p .0001; dose: F (5,72) 54.97, p .0001; interaction:,72) 4.91, p .0006]. Comparison of the THC-induced shift inED50 for each genotype revealed that, in contrast to antinocice-on, arr2-KO mice displayed a greater degree of tolerance toC-induced catalepsy (4.76-fold shift) than WT mice [2.12-foldft; (F (1,70) 7.873; p .01 sum of least squares F test; Table 1].THC-induced hypothermia was also assessed in WT andrr2-KO mice. Vehicle-treated arr2-KO mice displayed greatercreases in body temperature across the THC dosing regimen,pared with vehicle-treated WT mice [Figure 1C; genotype:</p><p>,78) 8.15, p .0055; dose: F (5,78) 41.96, p .0001; interac-n: F (5,78) .16, p .9775]. Subchronic THC treatment inducednificant tolerance to THC-mediated hypothermia in both geno-es [WT, treatment: F (1,84) 51.15, p .0001; dose: F (5,84) 38, p .0001; interaction: F (5,84) 13.26, p .0001; arr2-KO,atment: F (1,72) 65.02, p .0001; dose: F (5,72) 14.76, p 01; interaction: F (5,72) 6.94, p .0001]. The data for THCups did not converge, therefore ED50 values could not be calcu-d, and comparison of the degree of tolerance was not possible.wever, statistical analysis of the two curves revealed no signifi-t difference between THC-pretreated WT and arr2-KO micenotype: F (1,78) .18, p .6765]. Collectively these studiesgest region-specific CB1R regulation by -arrestin2, becauseC-induced antinociception, catalepsy, and hypothermia are me-ted by different neuronal populations in the CNS (2426).</p><p>rrestin2 Regulates CB1R Desensitization in Spinal CordThe finding that THC-mediated antinociception is enhanced,ereas development of tolerance is reduced, in arr2-KO micegests that -arrestin2 might negatively regulate CB1Rs in thenal cord because CB1Rs in this region contribute to tail-flickwww.sobp.org/journal</p></li><li><p>antbinBatypag).levCPvehbewitbintorzatCPfro(Ta</p><p>valcanciedru[35</p><p>Figtre ollowtoT onofhoc ive thpos ent [11. or prehoc t hypring s dispinte ferroF(5 HCpreffe onferdis erminp theirdis nteraBon t: F(5pre indicpro</p><p>716 BIOL PSYCHIATRY 2012;71:714724 P.T. Nguyen et al.</p><p>wwinociception (27). Therefore, cannabinoid-stimulated [35S]GTPSding was assessed in spinal cords from WT and arr2-KO mice.sal [35S]GTPS binding did not significantly differ between geno-es or treatments (WT/vehicle 215 11, WT/THC 208 18,rr2-KO/vehicle 221 20, and arr2-KO/THC 198 15 nCi/Residual THC would stimulate [35S]GTPS binding above basalels in vehicle-treated mice, thus THC washout was sufficient.55,940-mediated G-protein activity was first compared betweenicle-treated WT and arr2-KO mice and showed no differences</p><p>tween genotypes (Figure 2A, Table 2). Subchronic treatmenth THC significantly reduced CP55,940-stimulated [35S]GTPSding in WTmice, with an approximately 38% decrease in recep--mediated activity (Figure 2A, Table 2, p .001). CB1R desensiti-ion was attenuated in arr2-KO mice, where the Emax value for55,940-stimulated [35S]GTPS binding did not significantly differm its vehicle control, but differed from THC-treated WT miceble 2, p .001). Half-maximal effective concentration (EC50)</p><p>ure 1. Cumulative9-tetrahydrocannabinol (THC) doseresponse curvesatedwith either vehicle or THC (10mg/kg twice daily, intraperitoneally). (A) FHCcomparedwithWTmice in the tail-flick antinociceptive test [for interactianalysis]. FollowingTHCpretreatment,arr2-KOmice remainmore responst hoc analysis]. Both genotypes display tolerance following THC pretreatm25, p .0001, ^p .05, ^^^p .001 Bonferroni post hoc analysis.arr2-KO: fanalysis]. (B) Following vehicle treatment, arr2-KOmice display equivalentest for catalepsy [for genotype: F(1,78) 2.53, p .1159]. Both genotyperaction of dose and pretreatment:...</p></li></ul>

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