Nuclear receptor Nurr1 agonists enhance its dualfunctions and improve behavioral deficits in ananimal model of Parkinson’s diseaseChun-Hyung Kima,b,1,2, Baek-Soo Hana,c,1, Jisook Moona,d,1, Deog-Joong Kima,1, Joon Shine, Sreekanth Rajane,Quoc Toan Nguyene, Mijin Sohnc, Won-Gon Kimc, Minjoon Hana, Inhye Jeonga, Kyoung-Shim Kimc, Eun-Hye Leef,Yupeng Tug, Jacqueline L. Naffin-Olivosg, Chang-Hwan Parkf, Dagmar Ringeg, Ho Sup Yoone,h, Gregory A. Petskog,i,2,and Kwang-Soo Kima,2

aMolecular Neurobiology Lab, McLean Hospital and Program in Neuroscience, Harvard Medical School, Belmont, MA 02478; bInstitute of Green Bio Scienceand Technology, Seoul National University, Kangwon-Do, Korea; cFunctional Genomics Research Center, Korea Research Institute of Bioscience andBiotechnology, Daejeon, Korea; dDepartment of Biotechnology, College of Life Science, CHA University, Seoul, Korea; eSchool of Biological Sciences,Nanyang Technological University, Singapore; fGraduate School of Biomedical Science and Engineering, Hanyang University, Seoul, Korea; gDepartments ofBiochemistry and Chemistry, Brandeis University, Waltham, MA 02453; hDepartment of Genetic Engineering, College of Life Sciences, Kyung Hee University,Seoul, Korea; and iHelen and Robert Appel Alzheimer’s Disease Research Institute, Weill Cornell Medical College, New York, NY 10065

Contributed by Gregory A. Petsko, May 28, 2015 (sent for review August 1, 2013)

Parkinson’s disease (PD), primarily caused by selective degenera-tion of midbrain dopamine (mDA) neurons, is the most prevalentmovement disorder, affecting 1–2% of the global population overthe age of 65. Currently available pharmacological treatments arelargely symptomatic and lose their efficacy over time with accom-panying severe side effects such as dyskinesia. Thus, there is anunmet clinical need to develop mechanism-based and/or disease-modifying treatments. Based on the unique dual role of the nu-clear orphan receptor Nurr1 for development and maintenance ofmDA neurons and their protection from inflammation-induceddeath, we hypothesize that Nurr1 can be a molecular target forneuroprotective therapeutic development for PD. Here we showsuccessful identification of Nurr1 agonists sharing an identicalchemical scaffold, 4-amino-7-chloroquinoline, suggesting a criticalstructure–activity relationship. In particular, we found that twoantimalarial drugs, amodiaquine and chloroquine stimulate thetranscriptional function of Nurr1 through physical interaction withits ligand binding domain (LBD). Remarkably, these compoundswere able to enhance the contrasting dual functions of Nurr1 byfurther increasing transcriptional activation of mDA-specific genesand further enhancing transrepression of neurotoxic proinflamma-tory gene expression in microglia. Importantly, these compoundssignificantly improved behavioral deficits in 6-hydroxydopaminelesioned rat model of PD without any detectable signs of dyskine-sia-like behavior. These findings offer proof of principle that smallmolecules targeting the Nurr1 LBD can be used as a mechanism-based and neuroprotective strategy for PD.

NR4A2 | Nurr1 | Parkinson’s disease | agonist | drug target

PD is primarily caused by selective degeneration of midbraindopamine (mDA) neurons and is the most prevalent movement

disorder, affecting 1–2% of the global population over the age of65 (1–3). Currently available pharmacological treatments [e.g.,L-3,4-dihydroxyphenylalanine (L-DOPA)] are largely symptomaticand lose their efficacy over time, with accompanying severe side ef-fects such as dyskinesia. Thus, there is an unmet clinical need to de-velop mechanism-based and/or disease-modifying treatments (2, 3).During the last two decades, many intrinsic signals and extrinsic

transcription factors have been identified to play critical roles formDA neuron development (4–6). In particular, development ofmDA neurons is dependent on two major signaling molecules,Sonic hedgehog (Shh) and wingless-type MMTV integration sitefamily, member 1 (Wnt1), and their downstream factors. These twocritical pathways (i.e., Shh-FoxA2 and Wnt1-Lmx1a) merge tocontrol the expression of the orphan nuclear receptor related 1protein (Nurr1) (7), suggesting that Nurr1 is a key regulator of

mDA neurons. Indeed, Nurr1 [also known as nuclear receptorsubfamily 4, group A, member 2 (NR4A2)] is essential not only fordevelopment (8–10) but also for maintenance of mDA neurons inadult brains (11). In addition, a recent study demonstrated thatNurr1 plays critical roles in both microglia and astrocytes to repressproinflammatory genes and protects mDA neurons from in-flammation-induced death (12).In line with the relevance of Nurr1 to PD, postmortem studies

showed that Nurr1 expression is diminished in both aged and PDpostmortem brains (13, 14). Furthermore, functional mutations/polymorphisms of Nurr1 have been identified in rare cases of fa-milial late-onset forms of PD (15) although their significance re-mains unclear (16, 17). In addition, Nurr1 heterozygous null micebehave like an animal model of PD, as they exhibit a significantdecrease in both rotarod performance and locomotor activitiesassociated with decreased levels of DA in the striatum and de-creased number of A9 DA neurons (18). Taken together, thesefindings strongly suggest that disrupted function/expression ofNurr1 is related to neurodegeneration of DA neurons and itsactivation may improve the pathogenesis of PD (19). To addressthis possibility, we established efficient high throughput screening

Significance

Parkinson’s disease (PD) is the most prevalent movement dis-order with no available treatments that can stop or slow downthe disease progress. Although the orphan nuclear receptorNurr1 is a promising target for PD, it is thought to be a ligand-independent transcription factor and, so far, no small moleculehas been identified that can bind to its ligand binding domain.Here, we established high throughput cell-based assays andsuccessfully identified three Nurr1 agonists among FDA-approveddrugs, all sharing an identical chemical scaffold. Remarkably,these compounds not only directly bind to Nurr1 but alsoameliorate behavioral defects in a rodent model of PD. Thus,our study shows that Nurr1 could serve as a valid drug targetfor neuroprotective therapeutics of PD.

Author contributions: C.-H.K., G.A.P., and Kwang-Soo Kim designed research; C.-H.K., B.-S.H.,J.M., D.-J.K., M.H., and E.-H.L. performed research; C.-H.K., J.S., S.R., Q.T.N., andM.S. contributednew reagents/analytic tools; C.-H.K., M.S., W.-G.K., I.J., Kyoung-Shim Kim, Y.T., J.L.N.-O., C.-H.P.,D.R., and H.S.Y. analyzed data; and C.-H.K., G.A.P., and Kwang-Soo Kim wrote the paper.

The authors declare no conflict of interest.1C.-H.K., B.-S.H., J.M., and D.-J.K. contributed equally to this work.2To whom correspondence may be addressed. Email: kskim@mclean.harvard.edu, chkim@mclean.harvard.edu, or gpetsko@med.cornell.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1509742112/-/DCSupplemental.

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assays for small molecules that can boost the transcriptional ac-tivity of Nurr1. We successfully screened a US Food and DrugAdministration (FDA)-approved drug library and identified threehit compounds exhibiting Nurr1-activating function. Strikingly, allthree hit compounds share an identical chemical scaffold, 4-amino-7-chloroquinoline, strongly supporting the structure–activity relation(SAR). Furthermore, these compounds activated Nurr1 functionthrough binding to its ligand binding domain (LBD) and significantlyimproved behavioral deficits in an animal model of PD, 6-OHDA-lesioned rats without any sign of dyskinesia-like side effects.

Results and DiscussionIdentification of Small Molecules That Can Activate the Nurr1 Function.To identify small molecules that can activate Nurr1, we establishedefficient high-throughput assay systems based on our previousstudy showing that Nurr1 prominently activates the tyrosine hy-droxylase (TH) gene promoter function (20). We used the reporterconstruct p4xNL3-Luc (containing four copies of the NGFI-B re-sponsive element (NBRE)-like motif (NL3), identified in the THupstream promoter positioned immediately proximal to the TATAbox and linked to the luciferase gene) along with pCMV-Nurr1construct (full-length Nurr1-expression vector) in human neuro-blastoma SK-N-BE(2)C cells, followed by treatment with eachcompound for 24 h followed by measurement of luciferase activity.Using these cell-based assay systems, we screened a chemical librarycomposed of 960 FDA-approved drugs (MicroSource DiscoverySystems) and successfully identified three hit compounds (i.e., twoantimalaria drugs amodiaquine (AQ) and chloroquine (CQ) and apain-relieving drug (glafenine). Remarkably, all three compoundsshare an identical chemical scaffold, 4-amino-7-chloroquinoline,suggesting a SAR associated with this motif (Fig. 1A).In our original assays using the reporter construct p4xNL3-Luc

containing four copies of the NBRE-like NL3 motif residing inthe TH promoter (20), AQ and CQ activated luciferase reporteractivity up to approximately 3-fold in a dose-dependent manner(Fig. 1B and SI Appendix, Fig. S1 A and B). In the present studywe focused on AQ and CQ because glafenine showed a weakeractivity (∼1.5-fold). To investigate whether AQ and CQ activateNurr1 function through its LBD or DNA binding domain(DBD), we generated additional reporter constructs in which theyeast transcription factor GAL4’s DBD is fused to either Nurr1LBD or DBD [pGAL-Nurr(LBD) and pGAL-Nurr(DBD)].Notably, AQ and CQ stimulated Nurr1-dependent transcrip-tional activity through its LBD, whereas no response was ob-served when GAL4 or GAL4-Nurr1 DBD was used (Fig. 1B,Right). AQ and CQ induced Nurr1 LBD-based reporter activityup to ∼15- and 10-fold with an EC50 of ∼20 and 50 μM, re-spectively (Fig. 1B and SI Appendix, Fig. S1 C and D).To test if reporter gene activation by AQ and CQ is through

Nurr1, we treated cells with Nurr1-specific siRNA and confirmeddecreased levels of Nurr1. Nurr1 siRNAs, but not scrambledRNAs, reduced Nurr1 protein expression as well as AQ/CQ-inducedluciferase activation by more than 80%, strongly suggesting thattranscriptional activation by AQ and CQ is indeed through modu-lation of Nurr1 function (Fig. 1C and SI Appendix, Figs. S2 and S3).The recruitment of transcriptional coregulators such as steroidreceptor coactivator (SRC) is a crucial mechanism of nuclearreceptors (NRs) (21). To test if AQ/CQ modulates Nurr1’s in-teraction with SRC, a coimmunoprecipitation assay was per-formed. We found that Nurr1 weakly interacts with SRC-1 andSRC-3 in SK-N-BE(2)C cells and that these interactions were sig-nificantly enhanced by AQ and CQ (SI Appendix, Fig. S4). We nexttested the effects of AQ and CQ on Nurr1 transcriptional functionfollowing SRC-1 or SRC-3 overexpression. Whereas SRC-1/SRC-3overexpression itself did not greatly influence Nurr1 function,AQ and CQ further enhanced Nurr1’s transcriptional functionin the presence of SRC-1/SRC-3 overexpression (Fig. 1D). Takentogether, our data suggest that AQ and CQ induce Nurr1’s

Fig. 1. Identification of AQ and CQ as Nurr1 activators. (A) Chemical structuresof three hit compounds that activate Nurr1’s transcriptional function. Notably, allcompounds contain an identical scaffold, 4-amino-7-chloroquinoline (high-lighted by red color). (B) AQ, CQ, and glafenine increase the transcriptionalactivity of Nurr1-based reporter constructs: full-length Nurr1-dependent(fNurr; Left) and Nurr1 LBD-dependent (Right) transcriptional activities.(C) Effect of Nurr1-specific siRNA on Nurr1 LBD’s transactivation activity. Aknockdown of Nurr1 expression by treatment with Nurr1-specific siRNA re-duced the reporter gene activity of the Nurr1 LBD construct. (D) Effect of SRCproteins on AQ- and CQ-induced Nurr1 transactivation. AQ and CQ enhancedNurr1’s transcriptional function in the presence of SRC-1/SRC-3 overexpression.The fold induction was derived by comparing each luciferase activity to basallevel obtained by non-SRC transfection. (E) Target selectivity of AQ/CQ forLBDs of various NRs. A total of 30 μM AQ and 100 μM CQ robustly activatesLBD function of Nurr1, but not other NRs tested here, indicating a highspecificity. The positive reactivity of these NR constructs was confirmed by theknown activators (2 nM dexamethasone, 20 nM retinoic acid, 2 μM GW3965,50 nM GW7647, and 5 nM GW1929 for glucocorticoid receptor (GR), retinoid Xreceptor-α (RXRα), liver X receptor-α (LXRα), peroxisome proliferator-activatedreceptor-α (PPARα), and PPARγ, respectively). The basal level of transcriptionalactivity was normalized to 1. Bars represent means ± SEM from three in-dependent experiments.

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transactivation function through its Nurr1 LBD by facilitatingthe recruitment of coactivators like SRC-1/SRC-3.We next tested if AQ and CQ are able to nonspecifically ac-

tivate the LBD function of other NRs. As shown in Fig. 1E, AQand CQ were unable to induce the transcriptional activity of anyof these constructs, showing high selectivity. We also tested ifvarious quinoline compounds have similar Nurr1-activatingfunction. Remarkably, none of these compounds showed anydetectable transactivation function in a wide range of concen-trations (SI Appendix, Fig. S5). Because only AQ and CQ containthe 4-amino-7-chloroquinoline entity, these results further sup-port its SAR.

AQ/CQ Physically Binds to the Nurr1. Our findings are surprisingbecause Nurr1 lacks a “classical” binding pocket due to thepresence of bulky hydrophobic side chain residues and is thoughtto be a ligand-independent and constitutively active NR (22, 23).Thus, we further tested whether AQ and CQ physically interactwith Nurr1’s LBD. Toward this goal, we purified the Nurr1 LBDpolypeptide (amino acids 328–598) and examined its bindingwith AQ. First, we analyzed their physical binding using theBiacore S51 SPR sensor, which has a higher sensitivity and im-proved fluidics, enabling the monitoring of small signal changesderived from binding of compounds to proteins. As shown in SIAppendix, Fig. S6 A and B, AQ specifically bound to Nurr1-LBD

in a dose-dependent manner but not to the retinoid X receptor(RXR)-LBD. Next, we performed fluorescence quenching anal-ysis. The Nurr1-LBD displayed maximal fluorescence at 336 nm,whereas AQ itself had no fluorescence at this wavelength. Whenthe Nurr1-LBD was incubated with increasing amounts of AQ,fluorescence intensity gradually decreased (SI Appendix, Fig. S6C).In contrast, RXR-LBD’s fluorescence emission was quenched by9-cis retinoic acid but not by AQ over a wide range of concen-trations (SI Appendix, Fig. S6 D and E). In addition, we performeda radioligand binding assay using [3H]-CQ. [3H]-CQ showed sat-urable binding to Nurr1-LBD with a dissociation constant (Kd) of0.27 μM and a maximal binding capacity (Bmax) of 13.9 μM (Fig.2A). Furthermore, competition-binding assay showed that un-labeled AQ/CQ can compete for binding with [3H]-CQ with Kivalues of 246 and 88 nM for AQ and CQ, respectively (Fig. 2B).In contrast, unlabeled primaquine, which could not enhanceNurr1’s transcriptional function (SI Appendix, Fig. S5), was un-able to compete. To further investigate the molecular interactionbetween AQ and the Nurr1 LBD, we examined the chemicalshift perturbations (CSPs) on 2D 1H-15N heteronuclear singlequantum correlation (HSQC) spectra of the 15N-labeled Nurr1-LBD in the presence of AQ. A number of LBD residues showednotable CSP upon addition of AQ (Fig. 2 C and D and SI Appendix,Fig. S7). The overlay of free and ligand-bound spectra of the Nurr1LBD showed that perturbed residues are mainly located in the

Fig. 2. Interaction of AQ/CQ with the Nurr1-LBD protein. (A) Nurr1-LBD protein was incubated with increasing concentrations (3.9, 7.8, 15.5, 31, 62.5, 125,250, 500, and 1,000 nM) of [3H]-CQ. The Inset indicates Scatchard analysis of the specific binding. (B) Competition of AQ, CQ, and primaquine (PQ) for bindingof [3H]-CQ to Nurr1-LBD. Increasing concentrations of unlabeled AQ, CQ, or PQ were incubated with 500,nM [3H]-CQ and Nurr1-LBD. (C) Molecular interactionof the Nurr1-LBD and AQ by NMR titration experiments using uniformly 15N-labeled Nurr1-LBD. The 2D 1H-15N TROSY-HSQC spectra of Nurr1-LBD wererecorded on a Bruker Avance 700 spectrometer at 298K in the absence (red) and presence of AQ at molar ratios of 1–1 (magenta), 1–2 (black), and 1–5 (blue).Expanded sections of overlaid 2D 1H-15N TROSY-HSQC spectra show concentration-dependent chemical shift perturbations upon AQ binding. Amino acidsshowing chemical shift perturbations with increasing concentration of AQ are indicated by arrows. Disappeared resonance of I403 by addition of AQ ismarked as rectangular box. (D) Mapping of the interaction sites between Nurr1-LBD and AQ. (Left) Surface mapping of AQ binding site and interactionresidues on the crystal structure of Nurr1-LBD based on 2D 1H-15N HSQC titration data. Perturbed amino acid residues were displayed according to theirchemical shift perturbation values: red (Δδ > 0.1), blue (0.08 < Δδ < 0.1), and green (disappeared), respectively. (Right) Expanded view of potential bindingpocket for amodiaquine on the Nurr1-LBD. Perturbed amino acid residues were displayed by the same manner on the Right. (E) Functional effects of mu-tations in the potential AQ binding residues on Nurr1’s transcriptional activity. Wild-type and mutant constructs were tested by transient transfection assaywith or without AQ. The mutations at I403, L409, Y575, or D580 significantly reduced both basal transcriptional activity and its activation by AQ.

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helix α2 region (H402, I403, Q404, Q405, D408, and L409) andsome are in α11 (V468, Y575, and D580) (SI Appendix, Fig.S7B). Thus, it appears that AQ binding at α2 interacts with resi-dues Q474 and D408 (within the vicinity of L409). However, as AQat this binding pose is unlikely to form any direct interactions withC-terminal helix 11/12 residues, CSPs observed in Y575 and D580might be attributable to either conformational changes or an allo-steric effect upon AQ binding. To assess the functional contribu-tion, we mutated the perturbed residues in H2 and H11 to alanineand tested their transcriptional activities. As shown in Fig. 2E,mutants I403A, L409A, Y575A, and D580A exhibited profoundreduction of transcriptional activity, whereas other mutationslargely retained the activity. This functional analysis supports thatI403, L409, Y575, and D580, identified from nuclear magneticresonance (NMR) experiments, are critical residues involved in thephysical interaction of AQ and activator recognition. Together,these data strongly suggest that AQ activates Nurr1 function viadirect and specific binding to the Nurr1 LBD.

AQ/CQ Promotes mDA Neurogenesis. We next sought to test whetherthese compounds exhibit biological effects on mDA neurons inmore physiological contexts. First, we tested if they can increase thegeneration of TH+ neurons and/or expression of the mDA-specificgenes during in vitro differentiation of neural stem cells. Indeed,we found that AQ enhanced Nurr1’s function both in terms of thenumber of TH+ neurons generated from neural stem cells (Fig. 3 Aand B) and mRNA expression levels of the TH, dopamine trans-porter (DAT), vesicular monoamine transporter (VMAT), andaromatic amino acid decarboxylase (AADC) genes (Fig. 3C). Next,we performed qRT-PCR in the absence or presence of siRNA toNurr1 to test whether induction of gene expression by AQ/CQ isabolished by siRNA. As shown in Fig. 2C, siRNA to Nurr1 abro-gated gene expression of TH, DAT, VMAT2, and AADC induced

by AQ/CQ (Fig. 3C). In vivo chromatin immunoprecipitation(ChIP) assay clearly showed that Nurr1 is recruited to both NL1and NL3 sites of the TH promoter in AQ- and CQ-treated cells(20), suggesting that Nurr1 is directly involved in AQ- and CQ-mediated induction of TH gene expression (Fig. 3D).

Neuroprotective and Inflammation-Modulating Effects of AQ/CQ.Next, we tested if AQ and CQ prevent neurotoxin (6-OHDA)-induced death in primary DA neurons and rat PC12 cells. AQand CQ significantly inhibit 6-OHDA–induced cell death inprimary DA cells as examined by the number of TH+ neurons(Fig. 4A) and DA uptake (Fig. 4B). The neuroprotective effect ofAQ and CQ was also observed in rat PC12 cells (Fig. 4C). Be-cause Nurr1 has opposite transrepression activity in microgliaand astrocytes (12), we also analyzed the effect of AQ on ex-pression of proinflammatory cytokine genes in primary microgliaderived from P1 rat brains. When these cells were treated withthe inflammation-inducing lipopolysaccharide (LPS; 10 ng/mL)for 8 h, expression of proinflammation genes tested [interleukin-1β(IL-1β), interleukin-6, tumor necrosis factor-α (TNF-α), and in-ducible nitric oxide synthase (iNOS)] was dramatically induced

Fig. 3. Functional effects of AQ and CQ. (A and B) AQ stimulated thegeneration and gene expression of DA neurons from neural progenitorsisolated from E14.5 rat cortex in a dose-dependent manner. Differentiationwas induced by withdrawal of bFGF and AQ was added for 2 h during thedifferentiation. Immunocytochemical analyses for TH (A) and yields of TH+

/DAPI cells (B) for each treatment group were obtained following in vitrodifferentiation for 3 d and 9 d. (C) Real-time PCR analysis shows that AQtreatment enhances expression of the mDA-specific genes TH, dopaminetransporter (DAT), vesicular monoamine transporter (VMAT), and aromaticamino acid decarboxylase (AADC) during in vitro differentiation of neuralstem cells at 9 d. (D) Rat PC12 cells were treated with 20 μM AQ or 70 μM CQ.ChIP assay shows AQ- or CQ-dependent Nurr1 recruitment to the TH pro-moter. *P < 0.05 and **P < 0.005 versus untreated. Results are expressed asthe average of three independent experiments. Error bars represent SDs.

Fig. 4. Neuroprotective effects of AQ and CQ. (A–C) Primary cultures of ratmesencephalic DA neurons were treated with 20 μM 6-OHDA for 24 h in thepresence or absence of 5 μM AQ and 20 μM CQ. (A) The number of TH+

neurons and (B) the rate of [3H]DA uptake were measured. (C) Cell survivalwas measured by the MTT reduction assay in PC12 cells treated with 6-OHDAalone or in combination with AQ. Values from each treatment expressed as apercentage of untreated control for the MTT assay. (D) AQ suppresses LPS-induced expression of proinflammatory cytokines. Primary microglia from P1rat brains were treated with 10 ng/mL LPS for 4 h in the presence or absenceof AQ (10, 15, and 20 μM). Levels of mRNA expression were analyzed byquantitative real-time PCR and normalized with GAPDH. This experiment hasbeen repeated three times in triplicate using independently prepared RNAs.Each bar represents means ± SEM of n = 4–5. *P < 0.05, **P < 0.01,***P <0.001, compared with the LPS-only–treated group.

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(Fig. 4D). Remarkably, AQ treatment prominently reduced theexpression of all these genes in a dose-dependent manner (>10-fold). AQ and CQ showed very similar effects for repressingthese cytokine genes in both primary microglia and BV-2microglial cell line (SI Appendix, Fig. S8 A and B). Taken to-gether, our data show that AQ and CQ are able to enhance thecontrasting dual roles of Nurr1: (i) they increase Nurr1’s trans-activation of mDA-specific genes in mDA neurons, and (ii) theyalso further enhance Nurr1’s transrepression of proinflammatorycytokine gene expression in microglia.

AQ Improves Behavioral Deficits in a 6-OHDA–Lesioned Rat Model ofPD. The above findings prompted us to test whether these com-pounds can ameliorate motor behavior deficits in a PD animalmodel. Toward this goal, we tested the effects of AQ adminis-tration in an animal model of PD, 6-OHDA–lesioned rats. Ratswith unilateral intrastriatal 6-OHDA lesion were administeredwith saline or AQ for two weeks, starting from 1 d beforeintrastriatal 6-OHDA lesion (Fig. 5A). As expected, amphet-amine administration produced rotation behavior toward thelesion side in control rats, when measured at 4 and 6 wk post–6-OHDA lesion, indicating unilateral damage to the right striatum(Fig. 5 A and B). Remarkably, AQ administration significantlyameliorated 6-OHDA–induced rotation behavior at 4 and 6 wkpost–6-OHDA lesion (P < 0.06 and P < 0.0003, respectively). Toaddress if AQ treatment triggers abnormal involuntary move-ments (AIMs), well-validated dyskinesia-like behaviors (24), wemeasured AIM scores such as axial, limb, locomotive, and oro-lingual dyskinesias. In L-DOPA–treated rats (8 mg/kg for 2 wkbefore scoring), as expected, the animals showed dramaticallyincreased AIM scores [F(2,71) = 33, P < 0.0001], classified as theaxial, limb, locomotive, and orolingual dyskinesias (Fig. 5C). Incontrast, both saline- and AQ-treated 6-OHDA rats did notshow any detectable AIM behavior.Immunohistochemistry of the substantia nigra (SN) and the

striatum (STR) of these animals showed that AQ-treated ratsretained a significant number of TH+ cells in the lesion side SN,whereas saline-treated control rats lost the great majority of TH+

cells (Fig. 6A). Similarly, abundant TH+ fibers were spared in theSTR of AQ-treated brains, whereas they were mostly lost in theSTR of control brains. To quantitatively analyze these data, TH+

Fig. 5. Effects of AQ treatments on a 6-OHDA–lesioned rat model of PD.(A) Schematic representation of the administration of AQ to 6-OHDA–lesionedrats. Unilateral striatal 6-OHDA–lesioned rats were treated with AQ or saline for2 wk, starting from 1 d before lesioning. The gray shade indicates L-DOPAtreatment for 2 wk as control of AIMs test. (B) Amphetamine-induced rotationaltest was performed at 4 and 6 wk post–6-OHDA lesion. (C) 6-OHDA–lesioned ratstreated with L-DOPA, but not with AQ, exhibited severe side effects, as measuredby AIMs scores. Four types of AIMs were monitored (axial, forelimb, orolingual,and locomotor) at 2 and 6 wk postlesioning. Each AIM behavior was monitoredand scored on a 0–4 scale and summed. Bars represent the mean + SEM (#P <0.06, *P < 0.01, ***P < 0.0001).

Fig. 6. Immunocytochemical analysis of 6-OHDA–lesioned rat administratedby AQ. (A) TH+ fibers and neurons were plentiful in the STR and SN ofnormal site, whereas their marked depletion was observed in the right STRand SN by 6-OHDA lesioning. In contrast, abundant TH-immunopositive cellswere spared not only in the striatum but also in the SN in the AQ-treatedgroup. [Scale bar, 200 μm (black), 100 μm (white), 20 μm (red).] (B–E) AQreduces microglial activation in 6-OHDA–injected rat brains. Brain sectionsincluding the SN (B and D) and STR (C and E) regions were immunostainedwith anti–Iba-1 antibody, and Iba-1+ cells were counted in both lesion andintact sides. Data represent the mean ± SEM (**P < 0.01, ***P < 0.001).(Scale bar, 200 μm.)

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cells in the SN were stereologically counted in a blind manner.As shown in SI Appendix, Fig. S9, the number of TH+ neurons inthe lesion side of AQ-treated rats was ∼60% of those from theintact side at 6 wk post–6-OHDA lesion (P < 0.0003), whereas itremained less than 20% in saline-treated animals. Importantly,these spared TH+ cells coexpressed other DA markers such asFoxA2 and AADC (SI Appendix, Fig. S10). We also examinedmicroglial activation by immunohistochemistry of the microglialmarker ionized calcium binding adaptor molecule-1 (Iba-1). Asshown in Fig. 6 B–E, microglial activation was prominently ob-served in the ipsilateral SN (Fig. 6B) and STR regions (Fig. 6C) of6-OHDA–lesioned rats. In contrast, following AQ treatment, thenumbers of Iba-1+ microglia in the ipsilateral SN (Fig. 6 B and D)and STR regions (Fig. 6 C and E) decreased to the levels of thecontralateral sides, indicating a robust suppression of neuro-inflammation by Nurr1 activation (12).In summary, using our high throughput cell-based assays, we

identified small molecules, AQ and CQ, which can activateNurr1 through its LBD and significantly improve motor im-pairments in PD animal model without dyskinesia-like side ef-fects. Our experimental evidence supports that these compoundsenhanced the contrasting dual functions of Nurr1, activation ofmDA neuron-specific function (e.g., TH expression) and re-pression of microglial activation and neurotoxic cytokine geneexpression, leading to significant neuroprotective and/or neuro-restorative effects in a PD animal model. Notably, based onselective binding to neuromelanin to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) metabolite (25), a previous studydemonstrated that administration of CQ to monkeys protectedthem from MPTP-induced parkinsonian motor abnormalities(26). Our findings corroborate CQ’s neuroprotective effect andfurther suggest the involvement of alternate mechanism related to

Nurr1 activation. Together with this interesting primate study,our results provide a preclinical “proof of concept” that Nurr1could serve as a valid target for further development of mech-anism-based, disease-modifying therapeutics of PD.

Materials and MethodsAnimal Experiments. Animal experiments were performed in accordance withMcLean’s Institutional Animal Care and Use Committee and followed theNational Institutes of Health guidelines.

Chemical Library. A total of 960 compounds from The Genesis Plus Collection(MicroSource Discovery Systems) were used to identify Nurr1 activators. Thecompounds in this library are primarily FDA-approved compounds. A total of720 compounds containing pure natural products and their derivatives(MicroSource Discovery Systems) were also tested.

Statistical Methods. Statistical analyses were conducted using the StatisticalAnalysis System (Version 9.1; SAS Institute). Performance measures of in vitrooutcomes, cell counting, and behavioral outcomes were analyzed using thePROC TTEST, ANOVA, MIXED, or GLIMMIX program, a generalized linearmixed models procedure for conducting repeated measures analyses fol-lowed by Fisher’s least significant difference (LSD) post hoc tests (LSD is adefault post hoc test provided in SAS). Means were calculated for each an-imal for each testing condition, defined by the following variables (whereappropriate): treatment (AQ, L-Dopa, or saline) and time (2, 4, or 6 wk after6-OHDA lesion).

ACKNOWLEDGMENTS. We thank Dr. Stacie Weninger for advice and encour-agement and the Fidelity Bioscience Research Initiative. This work wassupported by National Institutes of Health Grants NS084869 and NS070577; agrant from the Michael J. Fox Foundation; Medical Research Center GrantsNRF-20080062190, NRF-2012M3A9C7050101, and NRF-20110030028; andCooperative Research Program for Agriculture Science and TechnologyDevelopment (Project no. PJ008022032012) Rural Development Administra-tion, Republic of Korea.

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