Gadd45β ameliorates L-DOPA-induced dyskinesia in a Parkinson's disease mouse model

Neurobiology of Disease 89 (2016) 169–179

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Neurobiology of Disease journal homepage: www.elsevier.com/locate/ynbdi

Gadd45β ameliorates L-DOPA-induced dyskinesia in a Parkinson's disease mouse model Hye-Yeon Park a, Young-Kyoung Ryu a, Yong-Hoon Kim a, Tae-Shin Park a,c, Jun Go a, Jung Hwan Hwang a, Dong-Hee Choi a, Myungchull Rhee c, Chul-Ho Lee a,b,⁎, Kyoung-Shim Kim a,b,⁎ a b c

Laboratory Animal Resource Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 34141, Republic of Korea Department of Functional Genomics, University of Science and Technology, Daejeon 34113, Republic of Korea College of Biosciences & Biotechnology, Chung-Nam National University, Daejeon 34134, Republic of Korea

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Article history: Received 15 August 2015 Revised 20 January 2016 Accepted 9 February 2016 Available online 10 February 2016 Keywords: GADD45β L-DOPA Dyskinesia Parkinson's disease

a b s t r a c t The dopamine precursor 3,4-dihydroxyphenyl-L-alanine (L-DOPA) is currently the most efficacious pharmacotherapy for Parkinson's disease (PD). However, long-term L-DOPA treatment leads to the development of abnormal involuntary movements (AIMs) in patients and animal models of PD. Recently, involvement of growth arrest and DNA damage-inducible 45β (Gadd45β) was reported in neurological and neurobehavioral dysfunctions. However, little is known about the role of Gadd45β in the dopaminergic nigrostriatal pathway or L-DOPAinduced dyskinesia (LID). To address this issue, we prepared an animal model of PD using unilateral 6hydroxydopamine (6-OHDA) lesions in the substantia nigra of Gadd45β+/+ and Gadd45β−/− mice. Dyskinetic symptoms were triggered by repetitive administration of L-DOPA in these 6-OHDA-lesioned mice. Whereas dopamine denervation in the dorsal striatum decreased Gadd45β mRNA, chronic L-DOPA treatment significantly increased Gadd45β mRNA expression in the 6-OHDA-lesioned striatum of wild-type mice. Using unilaterally 6-OHDA-lesioned Gadd45β+/+ and Gadd45β−/− mice, we found that mice lacking Gadd45β exhibited longlasting increases in AIMs following repeated administration of L-DOPA. By contrast, adeno-associated virusmediated expression of Gadd45β in the striatum reduced AIMs in Gadd45β knockout mice. The deficiency of Gadd45β in LID increased expression of ΔFosB and c-Fos in the lesioned striatum 90 min after the last administration of L-DOPA following 11 days of daily L-DOPA treatments. These data suggest that the increased expression of Gadd45β induced by repeated administration of L-DOPA may be beneficial in patients with PD. © 2016 Elsevier Inc. All rights reserved.

1. Introduction Parkinson's disease (PD) is the second most common neurodegenerative disorder affecting dopamine-producing neurons in the substantia nigra pars compacta (SNc) of the brain (Kish et al., 1988). Treatment with the dopamine precursor levodopa (L-DOPA) is the primary noninvasive therapeutic choice for patients with PD. However, prolonged administration of L-DOPA leads to the development of abnormal involuntary movements (AIMs) known as dyskinesia (Nutt, 1990). L-DOPA exerts its main actions via stimulation of the medium-sized spiny neurons expressing dopamine D1 receptors (D1R) and D2 receptors (D2R) in the striatum (Gerfen et al., 1990). Repeated exposure to LDOPA triggers enhancement of D1R-mediated responses in the DAdenervated striatum (Aubert et al., 2005; Gerfen et al., 2002; Guigoni

⁎ Corresponding authors at: Laboratory Animal Resource Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Gwahak-ro 125, Yuseong-gu, Daejeon 34141, Republic of Korea. E-mail addresses: [email protected] (C.-H. Lee), [email protected] (K.-S. Kim). Available online on ScienceDirect (www.sciencedirect.com).

http://dx.doi.org/10.1016/j.nbd.2016.02.013 0969-9961/© 2016 Elsevier Inc. All rights reserved.

et al., 2007; Picconi et al., 2003). L-DOPA causes an abnormally large and prolonged activation of cAMP- and MAPK-dependent signaling pathways in striatal neurons (Cenci and Konradi, 2010). The dysregulation of D1R transmission, including altered cAMP production by the olfactory type G-protein (Golf)-mediated stimulation of adenylyl cyclase and enhanced phosphorylation of cAMP-dependent phosphoprotein of 32 kDa, extracellular signal-regulated protein kinases 1 and 2 (ERK1/ 2), mitogen- and stress-activated kinase-1, and histone H3, was shown to be involved in L-DOPA-induced dyskinesia (LID) (Zhuang et al., 2000; Picconi et al., 2003; Corvol et al., 2004; Santini et al., 2007; Rangel-Barajas et al., 2011; Alcacer et al., 2012). In addition, dramatically altered expression and regulation of transcription factors were reported in the dyskinetic brain (Cenci and Konradi, 2010; Heiman et al., 2014). The upregulation of FosB-related proteins and prodynorphin is related to LID, and the targeted decrease of FosB/ΔFosB mRNA attenuates the dyskinesia severity induced by chronic L-DOPA administration (Andersson et al., 1999). The growth arrest and DNA damage-inducible (Gadd)45 family of genes (α, β, and γ) is associated with numerous cellular mechanisms, including cell-cycle regulation, stressor response, and molecular

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epigenetics (Salvador et al., 2013; Sultan and Sweatt, 2013). In the central nervous system (CNS), roles for Gadd45β in the epigenetic control of gene expression, adult cognition, and CNS diseases have been investigated (Ma et al., 2009; Day and Sweatt, 2011). Knockout of Gadd45β impairs activity-driven proliferation of neural progenitors and dendritic development of newborn neurons (Ma et al., 2009; Sultan et al., 2012). Recently, an upregulation of Gadd45β expression was detected in dopamine-depleted striatal spiny neurons following L-DOPA treatment in a 6-OHDA-induced hemiparkinsonism animal model (Heiman et al., 2014). In particular, the mRNA expression of Gadd45β was significantly changed in direct-pathway spiny projection neurons (dSPNs) expressing dopamine type 1 receptors (Heiman et al., 2014). However, the role of Gadd45β on LID in PD has not been addressed. In the present study, we used the unilateral 6-OHDA lesion mouse model of PD (Ungerstedt, 1968) to examine a potential role for Gadd45β on LID in PD. We demonstrated that L-DOPA treatment increased the mRNA level of Gadd45β in the 6-OHDA-lesioned striatum. Further, we provided the first evidence that Gadd45β expression played a protective role in motor complications arising from prolonged administration of L-DOPA in PD. 2. Materials and methods 2.1. Animals Male wild-type, and growth arrest and DNA damage-inducible 45β (Gadd45β) knockout mice (Jackson Laboratory, Bar Harbor, ME, USA; Lu et al., 2004) were used in the present study. All mice were housed in standard polycarbonate plastic cages in a temperature (21–22 °C)and humidity (50–60%)-controlled environment with a 12 h light/ dark cycle (lights on at 7 a.m.), and maintained on an ad libitum diet of gamma-irradiated laboratory chow (Purina Inc., Hwasung-si, Korea) and water. Cages were filled to an approximate depth of 1.5 cm with bedding made of chopped wood particles (JRS, Rosenberg, Germany). All materials used were autoclaved. The animal room was maintained under specific-pathogen-free (SPF) conditions. The animals used in the experimental group were from 3 to 4 litters. Unless indicated otherwise, 9-week-old male mice were used. All animals were handled in accordance with The Guidelines of Animal Care recommended by the Korea Research Institute of Bioscience and Biotechnology (KRIBB). 2.2. Drugs The 6-hydroxydopamine (6-OHDA) was purchased from SigmaAldrich Co. LLC (St. Louis, MO, USA) and diluted with 0.02% ascorbic acid in saline. Desipramine (25 mg/kg), 3,4-dihydroxyphenyl-Lalanine (L-DOPA, 20 mg/kg), and the peripheral DOPA decarboxylase inhibitor benserazide hydrochloride (12 mg/kg) were purchased from Sigma-Aldrich Co. LLC and dissolved in saline immediately before use. D-Amphetamine (AMPH) was purchased from USP (Rockville, MD, USA) and dissolved in saline.

− 1.3 mm; and dorsoventral (DV), − 4.7 mm. Mice were kept on a warming plate until they awoke from the anesthesia. Mice that awoke from anesthesia were returned to their home cages until used in the experiment. To avoid dehydration, lesioned mice received 5% glucose in sterile saline (10 mL/kg, s.c.) for 3 days. In addition, during the first week post-surgery, food pellets soaked in water were placed in a shallow vessel on the floor of the cages in the evening. The lesions were assessed at the end of the experiments by determining the striatal levels of tyrosine hydroxylase (TH) with immunohistochemistry. Only those animals with TH depletions over 80% in the striatum and TH reductions over 80% in the lesioned SN pars compacta (SNc) area compared with the control side were included in the analyses. 2.4. Cylinder test Before surgery, all mice were subjected to the grip strength test and cylinder test (Pre-test). The behavioral effects of the 6-OHDA-induced lesions and subsequent treatments with L-DOPA on sensorimotor function were examined with the cylinder test. Two weeks after the 6-OHDA infusion (6-OHDA) and 30 min before the first injection of L-DOPA (6OHDA/L-DOPA), each mouse was placed in a transparent acrylic cylinder (diameter, 15 cm; height, 27 cm), and the number of contacts with the right or left forepaw on the wall was counted for 5 min by observers who were blind to mouse genotype and drug treatment. The use of the impaired (right) forelimb was expressed as a percentage of the total number of supporting wall contacts. 2.5. Grip strength test The grip strength test was conducted with a grip strength machine (10 cm × 16 cm test grid, Bioseb, CCE). The grip strength was measured in g, as shown on the screen of the machine. Each mouse was allowed to grasp the test grid with his forelimbs and was then pulled by the tail. Each mouse was tested ten times, with each test conducted twice before the 6-OHDA injections and 2 weeks after the 6-OHDA injections. 2.6. Abnormal involuntary movement test Four weeks after the 6-OHDA injections, lesioned mice were treated with L-DOPA (20 mg/kg) plus benserazide hydrochloride (12 mg/kg) for 10 days. On the day 5 and day 10 of the L-DOPA injections, abnormal involuntary movements (AIMs) were assessed by observers who were blind to mouse genotype. Mice were individually placed in a separate glass cylinder, and dyskinetic behaviors were assessed for 1 min (monitoring period) in every 20 min block for a total of 120 min. The AIM score comprises the sum of the individual scores for each AIM subtype. A composite score was obtained by the addition of the scores for axial, limb, and orofacial AIMs (ALO score) in consideration of the report that composite AIM scores more closely reflect human dyskinetic behavior compared with the locomotive (LOC) AIM score (Lundblad et al., 2002, 2005; Alcacer et al., 2012; Park et al., 2014). 2.7. D-amphetamine-induced rotation test

2.3. Procedure for 6-OHDA lesions Mice were anesthetized with a mixture of ketamine hydrochloride and xylazine hydrochloride as described previously (Park et al., 2014) and mounted in a stereotactic frame (Stoelting Europe, Dublin, Ireland) that was equipped with a mouse adaptor. The 6-OHDA lesions were performed as described previously (Park et al., 2014). Mice were pretreated with desipramine (25 mg/kg, intraperitoneal) 30 min before the surgery to prevent noradrenergic neuron damage. Mice received unilateral injections of 6-OHDA in 3 μL (5 μg/μL, at the injection speed of 1 μL/min) into the left side of the substantia nigra (SN) at the following coordinates derived from the mouse brain atlas by Paxinos and Franklin (2008): anteroposterior (AP), − 3.0 mm; lateral (L),

For mice injected with adeno-associated virus (AAV), Damphetamine (D-AMPH)-induced rotation was measured 2 weeks after the 6-OHDA injections. Turning behaviors that were induced after D-AMPH (5 mg/kg) administration were recorded for 60 min in an observation cylinder (diameter, 20 cm; height 13 cm). The number of ipsilateral rotations was analyzed by a SMART video-tracking program (Panlab S.I., Barcelona, Spain). 2.8. Immunohistochemistry Immunohistochemistry was conducted as described previously (Kim et al., 2014). Briefly, 30 min after acute or chronic L-DOPA

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injections, mice were transcardially perfused with phosphate-buffered saline (PBS), followed by 4% paraformaldehyde in PBS. Their brains were removed, postfixed overnight, and then cut into 40 μm coronal sections with a vibratome (Vibratome VT1000A, Leica Microsystems GmbH, Wetzlar, Germany). Free-floating sections were incubated in PBS containing 3% hydrogen peroxide (v/v), rinsed three times in PBS, and blocked with 5% horse serum (HS) or goat serum (GS) for 1 h at room temperature. Sections were incubated overnight at 4 °C with primary antibodies. The primary antibodies used were rabbit polyclonal antibodies against TH (5% HS, Pel-Freez, catalog no. P40101-0), phospho-PKA substrate (5% HS, Cell Signaling Technology, catalog no. 9621S), and phospho (Ser10) acetyl (Lys14) histone H3 (pACH3, 5% GS, EMD Millipore Corporation, catalog no. 05-1315). After being washed, the sections were incubated with biotinylated secondary anti-rabbit IgG (Vector Laboratories, Inc., Burlingame, CA, USA), which was followed by incubation with an avidin-biotinylated peroxidase complex (ABC kit, Vector Laboratories, Inc.) and 3,3 -diaminobenzidine (Sigma-Aldrich Co. LLC). Sections containing the dorsal striatum (0.70 mm × 0.52 mm) at AP + 1.0 to + 0.5 mm and the SNc at AP − 3.0 to − 3.6 mm from bregma were selected. The number of pPKA substrate-, pAcH3-, and TH-positive cells from the lesioned and unlesioned striatum and SNc were counted under a microscope (Olympus Corporation). The TH-stained area in the dorsal striatum (+1.0 mm to +0.5 mm from the bregma) was measured in the 6-OHDA-lesioned side, and pPKA substrate-, pAcH3-, and TH-stained cells in the left and right striatum or SNc were counted for two–three sections for each assay per animal following the procedure described previously (Granado et al., 2008). To avoid double counting of neurons with unusual shapes, TH-stained cells were counted only when their nuclei were visualized in the focal plane. Assessments of the TH-stained area in the dorsal striatum were performed using the MetaMorph image analyzer (Molecular Devices Inc., Downingtown, PA, USA). Qualitative evaluations of immunoreactive cells were performed by investigators blind to genotype and treatment following the procedure introduced by Kim and Han (2009). 2.9. Western blot analysis Western blot analysis was described in a previous study (Kim et al., 2012). Thirty minutes after the last L-DOPA injection, mice were sacrificed, and the brain tissue was quickly removed and homogenized in a homogenization buffer (50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 0.1% SDS, and 0.1% sodium deoxycholate) containing a cocktail of protease inhibitors (Roche, Mannheim, Germany). Protein samples were resolved using SDS-PAGE and then were transferred onto PVDF membranes (Bio-Rad Laboratories, Inc., CA, USA). Blots were incubated with primary antibodies, followed by secondary antibodies, and specific signals were visualized using an enhanced chemiluminescence (ECL) kit (Intron Biotechnology, Gyeonggi-do, Korea). Western blot images were quantified using Quantity One 1-D analysis software version 4.6.1 (Bio-Rad Laboratories, Inc.). Phospho-ERK1/2 (Thr202/Tyr204, Cell Signaling Technology, catalog no. 9101S), ERK1/2 (Cell Signaling Technology, catalog no. 9102S), phospho-GluA1 (5% BSA, Millipore, catalog no. 04-1073), GluA1 (a gift from Dr. JR Lee, KRIBB, Korea), ΔFosB (D3S8R, Cell Signaling Technology, catalog no. 14,695), c-Fos (5% Skim Milk, Santa-Cruz, catalog no. sc-52), and actin (Millipore, catalog no. MAB1501) were used in western blotting. 2.10. Intrastriatal injections of AAV-Gadd45β The AAV-Gadd45β and AAV-green fluorescent protein (GFP) control were injected into the dorsal striatum 3 weeks after 6-OHDA lesions. For AAV2 production, the pAAV-MCS plasmid (Stratagene, La Jolla, CA, USA) was used. Production and concentration of recombinant AAV-GFPcontrol and AAV-Gadd45β were performed in the KIST virus facility

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(Seoul, Korea; Paydar et al., 2014). The AAV viral system contained the CMV promoter to drive the expression of Gadd45β and GFP. For recombinant virus generation, AAV-293 cells were cotransfected with pAAVRC (Stratagene) encoding the AAV genes rep and cap and the helper plasmid (Stratagene) encoding E24, E4, and VA. The produced viruses were harvested 72 h after transfection. The virus was concentrated using CsCl gradient purification. For high purity, the virus was concentrated using two rounds of ultracentrifugation. Fluorescent expression of the transduced cells was observed and photographed using a fluorescence microscope. The AAV viral titer was determined using a QuickTiter AAV Quantitation Kit (Cell Biolabs, San Diego, CA, USA), and a titer of 1010–1011 TU was obtained. Intrastriatal injections of AAV-Gadd45β and AAV-GFP control were performed as described previously (Kim et al., 2008; Park et al., 2014). In brief, unilaterally 6-OHDA-lesioned Gadd45β+/+ and Gadd45β−/− mice were anesthetized with intraperitoneal injections of a mixture of ketamine hydrochloride and xylazine hydrochloride, and placed in a stereotaxic apparatus (Stoelting Europe). The mice were intrastriatally injected unilaterally on the lesioned side (stereotaxic coordinates in mm with reference to bregma: AP, + 1; ML, − 1.8; and DV, − 3.6) with a total of 4 μL of AAV-Gadd45β (1 × 1010 TU in total) or AAV-GFP with the same titer (1 × 1010 TU) at the speed of 1 μL/min with a 28gauge needle. After 5 min, the needle was removed with three intermediate stops for 3 min to minimize backflow. The injected mice were kept on a warming pad until they awoke. Surgically manipulated mice that woke from anesthesia were returned to their home cages. The mice received L-DOPA (20 mg/kg) starting 7 days after the viral injection and continuing for 10 days. The behavioral, histological, and molecular analyses were conducted 17 days after the viral injection.

2.11. Real-time RT-PCR analysis The RNA preparation and real-time qPCR were performed as described previously (Kim et al., 2012). Total RNA was purified from the dorsal striatum using TRI reagent (Sigma, MO, USA). Reverse transcription was conducted using a Promega RT-PCR kit (Promega, Madison, WI, USA). The PCR was prepared with a mixture of 5 μL of 2× SYBR Green mix (Applied Biosystems, USA), 1 μL each of 5 pmol/mL forward and reverse primers, and 2 μL of cDNA (1/50 dilution of the cDNA synthesized from 1 μg of total RNA and eluted in 15 μL) in a volume of 10 μL using the StepOne Real-Time PCR system as follows: 10 min at 95 °C, followed by 41 cycles of 20 s at 95 °C, 30 s at 60 °C, and 20 s at 72 °C. The difference in amplification fold was calculated based on real-time qPCR amplification of the target gene vs. GAPDH as a reference using Gene Expression Analysis for StepOne software v2.1 (Applied Biosystems). The following primer sets were used: Gadd45α (5′-CTG CCA AGC TGC TCA ACG TA3′ and 5′-ACG GAT GAG GGT GAA ATG GA-3′), Gadd45β (5′-GTT CTG CTG CGA CAA TGA CA-3′ and 5′-TTG GCT TTT CCA GGA ATC TG-3′), Gadd45γ (5′-GCA TTG CAT CCT CAT TTC GA-3′ and 5′-CCT CGC AGA ACA AAC TGA GCT T-3′), FosB/ΔFosB (5′-AAC GGT CAC CGC AAT CAC3′ and 5′-GGC ATG TCA TAA GGG TCA AC-3′), and GAPDH (5′-AGG TCG GTG TGA ACG GAT TTG-3′ and 5′-TGT AGA CCA TGT AGT TGA GGT CA-3′).

2.12. Statistical analysis GraphPad PRISM software (GraphPad Software, Inc., La Jolla, CA, USA) was used to perform statistical analyses. Two-sample comparisons were conducted with Student's t tests, whereas multiple comparisons were made with a two-way analysis of variance (ANOVA), followed by a Bonferroni post hoc test or one-way ANOVA, followed by a Tukey– Kramer's post hoc test. All of the results are presented as means ± standard error of the mean. Any difference with a p value less than 0.05 was considered statistically significant.

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3. Results 3.1. Regulation of Gadd45β mRNA by DA denervation and L-DOPA treatment in the striatum Recent study showed increased expression of Gadd45β induced by chronic L-DOPA following dopamine depletion and a correlation between Gadd45β expression levels and levodopa dose (Heiman et al., 2014). However, the alteration in Gadd45β mRNA transcription profiles observed using GeneChip microarray was not validated with another method. In the present study, we examined changes in Gadd45β expression following DA denervation induced by 6-OHDA and following the administration of L-DOPA to mice with the DA-denervated striatum (Fig. 1A and B). The neurotoxin was unilaterally injected into the SNc of wild-type mice. Two weeks after the lesion, the efficacy of the 6-OHDA lesions was verified using western blotting to detect the depletion of tyrosine hydroxylase (TH) levels in the striatum. Mice showing TH depletion levels N 80% were included in the final analysis. The TH levels were reduced by 95 ± 1.21% in the DA-denervated striatum compared with the intact striatum. Two weeks after the 6-OHDA injection, the Gadd45β mRNA level was significantly decreased in the lesioned striatum compared with that on the intact side (Fig. 1A). However, 30 min after a single administration of L-DOPA 4 weeks after the 6-OHDA lesion, the Gadd45β mRNA level was significantly increased in the 6OHDA-lesioned striatum compared with that in the intact striatum (Fig. 1B). Thirty minutes after the last administration of L-DOPA in mice subjected to the 11 day L-DOPA treatment schedule, the Gadd45β expression was further enhanced by repeated administration of L-DOPA (20 mg/kg/day, i.p.) in mice with the 6-OHDA-lesioned striatum (Fig. 1B).

3.2. Gadd45β deficiency does not affect the development of LID but distinctly exacerbates AIM scores in long-lasting manner Because the Gadd45β mRNA level was high in mouse striatum after chronic L-DOPA treatment, we examined whether Gadd45β was associated with the development of LID. To address this question, we generated a 6-OHDA-lesion-based model of PD and LID in Gadd45β+/+ and Gadd45β−/− mice. The Gadd45β+/+ and Gadd45β−/− mice showed a significant decrease in forelimb grip strength after the 6-OHDA lesion (Fig. 2A). Injection of 6-OHDA into SNc reduced TH immunoreactivity in the SNc and in most areas of the dorsal striatum in these Gadd45β+/+ and Gadd45β−/− mice (Fig. 2D). TH levels in the lesioned

Fig. 1. Gadd45β expression is altered by 6-OHDA lesion and L-DOPA treatment in dorsal striatum. A. Expression levels of Gadd45β mRNA in 6-OHDA-lesioned and unlesioned striatum (n = 8; Student's t test, **p b 0.01). B. Expression levels of Gadd45β mRNA in striatum in response to acute (30 min on the first administration of L-DOPA) and chronic (30 min on the last L-DOPA administration for 11 days) L-DOPA treatment in unilaterally 6-OHDA-lesioned wild-type mice (n = 7–15; Student's t test, *p b 0.05 and **p b 0.01).

SNc and striatum were depleted by 90.73 ± 3.57% and 94.74 ± 1.33% in Gadd45β+/+ and 91.38 ± 1.86% and 94.14 ± 1.94% in Gadd45β−/− mice (Fig. 2B and C). Meanwhile, to examine the pharmacotherapeutic effects of L-DOPA in 6-OHDA-lesioned Gadd45β+/+ and Gadd45β−/− mice, we assessed the animals for 30 min as they performed the cylinder test following the first L-DOPA treatment. The 6-OHDA-lesioned Gadd45β+/+ and Gadd45β−/− mice showed a significant reduction in the use of the forelimb on the side contralateral to the lesion; however, a marked recovery in forelimb usage was observed for mice with both genotypes following L-DOPA administration (Fig. 3A). To investigate the role of Gadd45β in LID, we treated 6-OHDA-lesioned mice with daily L-DOPA (20 mg/kg with 12 mg/kg benserazide) and determined their AIM scores 5 and 10 days later. A significant difference was observed between mice in scores for axial, limb, and orofacial (ALO) AIMs and locomotive (LOC) AIMs in response to the 10 day L-DOPA treatment (Fig. 3B and C). The total AIM scores on day 10 were also significantly increased (Fig. 3D). At 20 and 40 min after the L-DOPA injection, AIM scores are similar in the two groups. Interestingly, an increase of AIM scores in Gadd45β−/− mice was caused by long-lasting AIMs during one measurement period (Fig. 3E). Gadd45β deficiency also significantly affected the time course for the L-DOPA effects on LOC scores (Fig. 3F) and ALO scores (Fig. 3G). No correlation was observed between the reduction of TH levels and the severity of AIMs in either genotype (Fig. 3H and I).

3.3. Gadd45β deficiency does not alter hyperactivation of the D1R/protein kinase A/ERK1/2 response 30 min after L-DOPA administration Repeated exposure to L-DOPA triggers enhancement of D1Rmediated responses in the DA-denervated striatum (Aubert et al., 2005; Guigoni et al., 2007; Picconi et al., 2003). L-DOPA causes an abnormally large and prolonged activation of cAMP- and MAPK-dependent signaling pathways in striatal neurons (Cenci and Konradi, 2010). There is an increase in the phosphorylation of ERK1/2 and histone H3, a downstream target of ERK involved in transcriptional regulation, associated with LID (Santini et al., 2007). We therefore examined whether the enhanced D1R-mediated responses produced by repeated administration of L-DOPA in Gadd45β−/− mice were upregulated. Immunohistochemical and immunoblotting methodologies were used to determine the effects of L-DOPA on the D1R signaling in 6-OHDAlesioned mice. Thirty minutes after the last administration of L-DOPA in mice subjected to the 11 day L-DOPA treatment schedule, the number of phospho-protein kinase A (PKA) substrate-positive cells, the phosphorylated form of the PKA substrate consensus sequence (Sindreu et al., 2007), was increased in the 6-OHDA-lesioned striatum of mice with both genotypes (Fig. 4A and B). Dopamine D1 receptor-mediated phosphorylation of GluR1 at Ser845 was also increased in the lesioned striatum of mice with both genotypes (Fig. 4E). Western blot analysis showed L-DOPA-induced activation of ERK1/2 in the DA-denervated striatum of Gadd45β+/+ and Gadd45β−/− mice (Fig.4F and G). However, there was no significant difference in the phospho-ERK1/2 level in the 6-OHDA-lesioned striatum between the two genotypes. Similarly, the number of neurons that were positive for pAcH3 in the lesioned striatum was increased in both Gadd45β+/+ and Gadd45β−/− mice (Fig. 4A and C). The expression of FosB/ΔFosB, a marker for the functional changes that induce dyskinesia and a target of the activated ERK1/2 (Andersson et al., 1999; Pavón et al., 2006; Fasano et al., 2010; Lindgren et al., 2011), was examined using the immunoblotting method in wild-type and Gadd45β−/− mice with LID. Thirty minutes after the last administration of L-DOPA, the expression of ΔFosB detected using immunoblotting analysis were significantly increased in the DA-denervated striatum in mice of both genotypes (Fig. 4H), with no significant difference detected between the two genotypes.

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Fig. 2. 6-OHDA-lesion-based model of PD in Gadd45β+/+ and Gadd45β−/− mice. A. Forelimb grip strength in Gadd45β+/+ and Gadd45β−/− mice before (Pre-test) and after 6-OHDA lesions (6-OHDA) (n = 21–25). **p b 0.01. Two-way ANOVA: effect of genotype, F(1,44) = 0.71, p = 0.40; effect of surgery, F(1,44) = 174.9, p b 0.0001; and interaction, F(1,44) = 0.41, p = 0.52, followed by the Bonferroni test. B and C. Percentage loss of substantia nigra pas compacta (SNc) tyrosine hydroxylase (TH)-positive cells (B) and percentage loss of striatum TH-fiber (C) on the lesioned side compared with that on the unlesioned side in Gadd45β+/+ and Gadd45β−/− mice. D. TH immunoreactivity in the 6-OHDA-lesioned and unlesioned hemispheres in the striatum and substantia nigra of Gadd45β+/+ and Gadd45β−/− mice. Scale bar, 500 μm.

Fig. 3. L-DOPA-induced dyskinesia is long-lasting in GADD45β−/− mice. A. Right forelimb use was measured in the cylinder test in Gadd45β+/+ and Gadd45β−/− mice before (Pre-test) and after 6-OHDA lesions (6-OHDA), and following the first administration of L-DOPA (6-OHDA/L-DOPA) (n = 15–19). **p b 0.01, one-way ANOVA, followed by Tukey's post hoc test. B-D. Sum of axial, limb, and orofacial (ALO) abnormal involuntary movements (AIMs) (B), locomotive (LOC) AIMs (C), and total AIMs (D) that were scored for 120 min after L-DOPA administration for 5 and 10 days in Gadd45β+/+ and Gadd45β−/− mice (n = 27–28; Student's t test, **p b 0.01). E-G. Time course of total, LOC, and ALO AIMs that were scored every 20 min for 120 min after the last L-DOPA administration (n = 27–28). Total AIMs (E); two-way ANOVA: effect of genotype, F(1,53) = 18.79, p b 0.0001; effect of time, F(5,265) = 27.65, p b 0.0001; and interaction, F(5,265) = 12.00, p b 0.0001, followed by Bonferroni test. LOC AIMs (F); two-way ANOVA: effect of genotype, F(1,53) = 3.48, p = 0.0677; effect of time, F (5,265) = 19.20, p b 0.0001; and interaction, F(5,265) = 6.02, p b 0.0001, followed by Bonferroni test. ALO AIMs (G); two-way ANOVA: effect of genotype, F(1,53) = 14.80, p = 0.0003; effect of time, F(5,265) = 5.28, p b 0.0001; and interaction, F(5,265) = 3.30, p = 0.0066, followed by Bonferroni test. H and I. Simple linear regression analyses showing the absence of a correlation between the depletion of TH and AIM scores in 6-OHDA-lesioned Gadd45β+/+ (H: r = −0.0058, p = 0.9722) and Gadd45β−/− (I: r = −0.1526, p = 0.4381) mice.

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Fig. 4. Thirty minutes after last L-DOPA administration, L-DOPA-induced D1R signaling changes are not affected by Gadd45β deficiency. Thirty minutes after the last injection of L-DOPA combined with benserazide, immunohistochemical (A-C) and immunoblotting (D-H) analyses were performed in 6-OHDA-lesioned and unlesioned striatum of Gadd45β+/+ and Gadd45β−/− mice. A–C. Phospho-PKA substrate, and phospho-Ser10-acetyl-Lys14 histone H3 (pAcH3) immunoreactivity in the 6-OHDA-lesioned dorsal striata of Gadd45β+/+ and Gadd45β−/− mice (A). Quantification of phospho-PKA substrate- (B), and pAcH3- (C) positive cells (n = 9–11) in the dorsal striatum (positive cells/0.70 mm × 0.52 mm). Two-way ANOVA results reveal a significant increase (lesion effect) in the number of phospho-PKA substrate-, and pAcH3-positive cells in the lesioned striatum of mice with both genotypes. However, no interaction is detected for genotype and lesion. Scale bar, 100 μm. D–H. Western blot analysis showing the levels of pGluR1 (E), pERK1/2 (F and G), and ΔFosB (H) (n = 6–10). Two-way ANOVA results reveal a significant increase (lesion effect) in immunoreactivity for pGluR1, pERK1/2, and ΔFosB in the lesioned striatum of mice with both genotypes. However, there is no interaction between genotype and lesion. **p b 0.01.

3.4. Gadd45β deficiency enhances ΔFosB and c-Fos levels at 90 min of LID Despite high AIM scores in the Gadd45β−/− mice, D1R hyperactivation was not observed 30 min after L-DOPA administration. Thus, we investigated whether the L-DOPA-mediated upregulation of D1R signaling was long-lasting in the lesioned striatum of Gadd45β−/− mice, similar to their AIM scores. Ninety minutes after the last administration of L-DOPA in mice subjected to the 11 day L-DOPA treatment schedule, the enhancement of D1R-mediated responses in the DAdenervated striatum was investigated using immunohistochemical and immunoblotting analyses. The number of phospho-PKA substrateand pAcH3-positive cells was increased in the lesioned striatum of Gadd45β+/+ and Gadd45β−/− mice (Fig. 5A-C), but the level was not significantly different between the two genotypes. Phosphorylation of ERK1, but not ERK2, was also slightly increased in the lesioned striatum of Gadd45β−/− mice compared with that of Gadd45β+/+ mice (Fig. 5F and G). In the immunoblotting assay, D1R-mediated phosphorylation of GluR1 and ERK1/2 in the DA-denervated striatum in Gadd45β−/− mice was similar to those levels in the lesioned striatum in wild-type mice (Fig. 5D-G). At this time point, the level of ΔFosB expression in the DA-denervated striatum was significantly increased in Gadd45β−/− mice compared with that in Gadd45β+/+ mice (Fig. 5H). In addition to FosB, many activator protein-1 (AP-1) factors are markedly upregulated by high-dose L-DOPA treatment (Heiman et al., 2014), and activation of ERK1/2 leads to AP-1 dependent transcription factor changes (Cenci and Konradi, 2010). Therefore, we also examined whether AP-1 was upregulated in the DA-denervated striatum of Gadd45β−/− mice compared with that in Gadd45β+/+ mice 90 min after the last administration of L-DOPA to mice subjected to the

11 day L-DOPA treatment schedule. The expression of c-Fos was upregulated in the lesioned striatum of Gadd45β+/+ and Gadd45β−/− mice compared with that in the unlesioned striatum (Fig. 5I). Moreover, in the lesioned striatum, expression levels of c-Fos were significantly increased in Gadd45β−/− mice compared with that in Gadd45β+/+ mice. 3.5. Gadd45β deficiency does not alter acute L-DOPA-induced activation of D1R/PKA signaling Dopamine-denervation-induced supersensitivity of DA receptors in the striatum is detected following a single administration of L-DOPA in mice (Park et al., 2014). The effect of a single L-DOPA treatment in DA-denervated mice was shown to upregulate cAMP and ERK signaling pathways (Santini et al., 2007; Alcacer et al., 2012). To examine whether the acute response to L-DOPA was altered in Gadd45β deficient mice, we administered L-DOPA to 6-OHDA-lesioned Gadd45β+/+ and Gadd45β−/− mice and, 30 min later, counted the number of cells positive for the phosphorylation forms of the PKA substrates and AcH3 in both the 6-OHDA-lesioned and unlesioned striatum (Fig. 6A and B). The number of these positive cells was increased in the 6-OHDAlesioned striatum of Gadd45β+/+ and Gadd45β−/− mice, but no significant difference was detected between the two genotypes. The results of immunoblotting assays showed that the D1R-mediated phosphorylation of ERK1/2 in the DA-denervated striatum was markedly increased in the lesioned striatum of both wild-type and mutant mice (Fig. 6C and D). Levels of ΔFosB and cFos expression in the DA-denervated striatum were significantly increased in both Gadd45β+/+ and Gadd45β−/− mice (Fig. 6E and F), but the protein level of ΔFosB and cFos on the lesioned side was not different in the two genotypes.

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Fig. 5. Ninety minutes after last L-DOPA administration, L-DOPA-induced ΔFosB and c-Fos levels are increased in Gadd45β−/− mice. Ninety minutes after the administration of L-DOPA on the 11 day L-DOPA treatment schedule, immunohistochemical (A–C) and immunoblotting (D–I) analyses were performed in the 6-OHDA-lesioned and unlesioned striatum of Gadd45β+/ + and Gadd45β−/− mice. A–C. Phospho-PKA substrate, and pAcH3 immunoreactivity in the 6-OHDA-lesioned dorsal striata of Gadd45β+/+ and Gadd45β−/− mice (A). Quantification of phospho-PKA substrate- (B), and pAcH3- (C) positive cells (n = 11–13) in the dorsal striatum (positive cells/0.70 mm × 0.52 mm). Two-way ANOVA results reveal a significant increase (lesion effect) in the number of phospho-PKA substrate-, and pAcH3-positive cells in the lesioned striatum of mice with both genotypes. However, no interaction is observed between genotype and lesion. Scale bar, 100 μm. D–I. Western blot analysis showing the levels of pGluR1 (E), pERK1/2 (F and G), ΔFosB (H), and c-Fos (I) (n = 7–12). Two-way ANOVA results show no significant increase (lesion effect) in immunoreactivity for pGluR1 and pERK1/2 in the lesioned striatum of mice with either genotype. ΔFosB and c-Fos levels are significantly increased in the 6-OHDA-lesioned striatum of mice with both genotypes, and an interaction occurs between the two genotypes (ΔFosB: effect of genotype, F(1,14) = 12.72, p = 0.0031; effect of lesion, F(1,14) = 109.40, p b 0.0001; and interaction, F(1,14) = 12.72, p = 0.0031, and c-Fos: effect of genotype, F(1,18) = 4.90, p = 0.040; effect of lesion, F(1,18) = 55.32, p b 0.0001; and interaction, F(1,18) = 4.90, p = 0.040). *p b 0.05; **p b 0.01.

3.6. Expression of the Gadd45 gene family induced by chronic L-DOPA administration in Gadd45β+/+ and Gadd45β−/− mice with PD

expression levels in the striatum of L-DOPA-treated mice in an animal model of PD.

We investigated the expression levels of Gadd45α, Gadd45β, and Gadd45γ in 6-OHDA-lesioned and unlesioned striatum using RT-PCR 30 min after the last administration of L-DOPA in Gadd45β+/+ and Gadd45β−/− mice subjected to the 11 day L-DOPA treatment schedule (Fig. 7A). Chronic L-DOPA increased Gadd45β expression levels in Gadd45β+/+ mice without affecting Gadd45α expression levels in either genotype. However, Gadd45γ expression was significantly increased in the DA-denervated striatum compared with that in the unlesioned striatum following chronic treatment of L-DOPA in mice of both genotypes, with the expression level in the lesioned striatum slightly but not significantly lower in Gadd45β−/− than that in Gadd45β+/+ mice. To determine whether the L-DOPA-mediated upregulation of the Gadd45β and Gadd45γ genes was responsible for D1R signaling, we pretreated mice with the D1R antagonist SCH23390 (0.2 mg/kg) to block D1Rs on the last day of the 11 day L-DOPA treatment and measured Gadd45α, Gadd45β, and Gadd45γ gene expression levels in the 6-OHDA-lesioned striatum of wild-type mice (Fig. 7B). Thirty minutes after the administration of L-DOPA, L-DOPA-induced Gadd45β and Gadd45γ gene expression on the lesioned side was significantly reduced by the SCH23390 pretreatment (Fig. 7B). By contrast, SCH23390 did not alter Gadd45α gene expression. These results suggest that the D1R signaling pathway regulates Gadd45β and Gadd45γ gene

3.7. AAV-Gadd45β injection into dorsal striatum of Gadd45β−/− mice significantly decreases AIM scores To investigate whether striatal Gadd45β gene expression ameliorates the severity of LID, we injected adeno-associated virus (AAV)Gadd45β into the dorsal striatum of Gadd45β−/− and Gadd45β+/+ mice. Thus, 2 weeks after 6-OHDA-induced striatal lesions, we screened for mice showing high levels of asymmetric rotations in response to administration of D-amphetamine (D-AMPH). The selected mice were separated into two groups for an injection of either AAV-GFP or AAVGadd45β (Park et al., 2014). Seven days after the AAV injection, we assessed the behavior of the mice in the cylinder test. A behavioral improvement in this test was observed after the first administration of LDOPA in all groups (Fig. 8A). The 6-OHDA-lesioned mice were treated with L-DOPA (20 mg/kg with 12 mg/kg benserazide) daily for 10 days, and the AIM scores were then assessed. The total AIM score displayed for the AAV-Gadd45β group was significantly lower than that for the AAV-GFP group in the Gadd45β−/− mice, but not in Gadd45β+/+ mice (Fig. 8B). The time course for the L-DOPA effects was significantly affected by the local expression of Gadd45β gene in the dorsal striatum of Gadd45β−/− mice (Fig. 8E). At 20 min post L-DOPA injection, there is a statistical difference between AAV-GFP and AAV-Gadd45β in Gadd45β+/+ mice (Fig. 8E), but there was no significant difference

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Fig. 7. Expression levels of Gadd45 gene family mRNA (Gadd45α, β, and γ) in unilaterally 6-OHDA-lesioned mice treated with L-DOPA and L-DOPA in combination with the D1R antagonist SCH23390. A. Gadd45α, Gadd45β, and Gadd45γ expression levels in dorsal striatum in response to chronic L-DOPA treatment (11 days, 20 mg/kg/day) in unilaterally 6-OHDA-lesioned GADD45β+/+ and GADD45β−/− mice (n = 8–11). Twoway ANOVA, followed by Bonferroni test (**p b 0.01). B. Expression levels of Gadd45α, Gadd45β, and Gadd45γ genes were determined in unilaterally 6-OHDA-lesioned wildtype mice with L-DOPA in combination with saline or SCH23390 (0.2 mg/kg, i.p.). n = 11–15; Student's t test, **p b 0.01.

Fig. 6. Effects of short-term L-DOPA treatment on cAMP/PKA and ERK signaling in Gadd45β+/+ and Gadd45β−/− mice. Thirty minutes after a single administration of LDOPA, immunohistochemical (A and B) and immunoblotting (C–F) analyses were performed in 6-OHDA-lesioned and unlesioned striatum of Gadd45β+/+ and Gadd45β−/− mice. A–B. Quantification of phospho-PKA substrate- (A), and pAcH3(B) positive cells (positive cells/0.70 mm × 0.52 mm) in the unlesioned and 6-OHDAlesioned dorsal striatum of Gadd45β−/− mice in comparison with Gadd45β+/+ animals (n = 7–8). Two-way ANOVA results reveal a significant increase (lesion effect) in the number of phospho-PKA substrate-, and pAcH3-positive cells in lesioned striatum of mice with both genotypes. However, no interaction is observed between genotype and lesion. C–F. Western blots showing the levels of pERK1/2 (C and D), ΔFosB (E), and cFos (F) in unlesioned and 6-OHDA-lesioned striatum of Gadd45β+/+ and Gadd45β−/− mice (n = 7–8). Two-way ANOVA results reveal a significant increase (lesion effect) in immunoreactivity for pERK1/2, ΔFosB, and c-Fos in the lesioned striatum of mice with both genotypes. However, there is no interaction between genotype and lesion. **p b 0.01.

after 40 min the administration of L-DOPA. In the Gadd45β−/− mice, a significant difference was observed between AAV-GFP control and AAV-Gadd45β-injected mice in the scores for ALO AIMs (Fig. 8C) and LOC AIMs (Fig. 8D) in response to long-term L-DOPA treatment. However, the total ALO and LOC AIM scores were not decreased in the AAV-Gadd45β group compared with AAV-GFP group in Gadd45β+/+ mice (Fig. 8C and D). These results indicate that AAV-mediated overexpression of Gadd45β in wild type mice has little effect on LID. The day after the AIM test, the mice were killed and their brains were

removed. A histological examination determined that the TH level in the lesioned SNc was reduced by N 80% compared with that in the intact SNc (Fig. 8F). To examine the presence of Gadd45β in the striata of Gadd45β−/− mice injected with AAV-Gadd45β and the link between Gadd45β and ΔFosB in the LID model, 90 min after the last administration of LDOPA in Gadd45β−/− mice subjected to the 11 day L-DOPA treatment schedule, real-time PCR was performed on day 17 after the viral injection in 6-OHDA-lesioned Gadd45β−/− mice. AAV-Gadd45β-induced expression of Gadd45β in the dorsal striatum was confirmed (Fig. 8H). In the lesioned striatum of Gadd45β−/− mice, the expression of Gadd45β gene mediated by AAV significantly decreased the expression level of FosB/ΔFosB mRNA (Fig. 8I).

4. Discussion LID remains a major therapeutic problem in patients with PD. One of the best examples of a signaling pathway involved in the LID symptoms is associated with D1R supersensitivity (Santini et al., 2007; Cenci and Konradi, 2010). However, because the therapeutic action of L-DOPA is mediated through D1Rs, direct pharmacological antagonism of this receptor is not a viable option for preventing LID. Recent studies reported successful relief of LID symptoms by partially inhibiting the D1Rhyperactive signaling pathway without reducing the therapeutic effects of L-DOPA (Santini et al., 2007; Fasano et al., 2010; Lebel et al., 2010; Park et al., 2014). In the present study, we show that Gadd45β gene expression is induced by exposure to L-DOPA through D1R-mediated responses in the DA-denervated striatum. A Gadd45β deficiency leads to long-lasting LID and enhanced ΔFosB and c-Fos expression in an animal model of PD, and functional recovery of Gadd45β in the dorsal striatum of the DA-denervated area relieves the LID symptoms in Gadd45β−/− mice. These findings suggest that Gadd45β may have a novel protective

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Fig. 8. Gadd45β expression in dorsal striatum attenuates LID in GADD45β−/− mice. The AAV-GFP control and AAV-Gadd45β were injected into the dorsal striatum of Gadd45β+/+ and Gadd45β−/− mice 3 weeks after 6-OHDA lesions. Right forelimb use was measured in the cylinder test in Gadd45β+/+ and Gadd45β−/− mice before (6-OHDA) and after the first administration of L-DOPA (6-OHDA/L-DOPA). AIM scores were determined in the Gadd45β+/+ and Gadd45β−/− mice injected with AAV-GFP and AAV-Gadd45β. A. Right forelimb use was measured in the cylinder test in AAV-GFP- or AAV-Gadd45β-injected DA-denervated mice before (6-OHDA) and after (6-OHDA/L-DOPA) the first treatment of L-DOPA in Gadd45β−/− (n = 7–8) and Gadd45β+/+ mice (n = 14–17) (Student's t test, *p b 0.05 and **p b 0.01). B–D. Sum of total abnormal involuntary movements (AIMs) (B), axial, limb, and orofacial (ALO) AIMs (C), and locomotive (LOC) AIMs (D) that were scored for 120 min after 10 days of L-DOPA administration combined with benserazide in AAV-GFP- or AAVGadd45β-injected Gadd45β−/− (n = 7–8) and Gadd45β+/+ (n = 14–17) mice (one-way ANOVA, followed by a Tukey–Kramer's post hoc test, *p b 0.05 and **p b 0.01). E. Time course of total AIMs that were scored every 20 min over a period of 120 min after the last L-DOPA administration. Two-way repeated-measures ANOVA and post hoc Bonferroni test; interaction, F(15,210) = 2.29, p = 0.0051; time, F(5,210) = 12.78, p b 0.0001; and group, F(3,42) = 21.60, p b 0.0001. (AAV-GFP-injected Gadd45β−/− mice versus AAV- Gadd45β-injected Gadd45β−/− mice; ##p b 0.01, AAV-GFP-injected Gadd45β+/+ mice versus AAV-GFP-injected Gadd45β−/− mice; *p b 0.05; **p b 0.01, AAV-GFP-injected Gadd45β+/+ mice versus AAV-Gadd45β-injected Gadd45β+/+ mice, &&p b 0.01). F. Percent loss of tyrosine hydroxylase (TH)-positive cells in substantia nigra pas compacta of AAV-GFP- or AAV-Gadd45βinjected mice (n = 7–10). G. Photomicrograph showing GFP fluorescence in the dorsal striatum injected with AAV-GFP. cc, corpus callosum. The rectangle indicates the region examined in H and I Scale bar, 500 μm. H. Real-time PCR data showing the expression level of Gadd45β in the dorsal striatum of Gadd45β−/− mice injected with AAV-GFP control and AAV-Gadd45β and wild type mice (n = 7–8). I. Real-time PCR data showing the expression level of FosB/ΔFosB in the dorsal striatum of Gadd45β−/− mice injected with AAV-GFP control and AAV-Gadd45β (n = 8).

role for LID in PD and a negative regulatory role on transcriptional activity of specific genes altered by D1R hyperactivation. We observed that the level of Gadd45β expression in the striatum of 6-OHDA-lesioned wild-type mice was markedly increased in response to L-DOPA treatment (Fig. 1B). This response was enhanced by repeated administration of L-DOPA. The increased expression of Gadd45β mRNA on the 6-OHDA-lesioned side by repeated L-DOPA was effectively blocked by the D1R antagonist SCH23390 (Fig. 7B). Although little is known about the regulatory mechanism of Gadd45 gene expression in the brain, it was reported that increased expression of cAMP response element-binding protein (CREB) led to elevated expression levels of Gadd45β and Gadd45γ in neurons (Tan et al., 2012) and that Gadd45β was necessary for active demethylation of candidate sites, including CREB-binding protein (Sultan and Sweatt, 2013). Gadd45γ was also increased by repeated L-DOPA treatment in 6-OHDA-lesioned striatum of wild-type mice, but Gadd45β−/− mice did not show

compensatory regulation of other Gadd45 isoforms and normal induction of Gadd45γ by L-DOPA treatment (Fig. 7A). Gadd45α did not respond to L-DOPA treatment. Further studies are needed to investigate the potential contribution of Gadd45γ to LID. In the CNS, Gadd45β was implicated in the epigenetic control of the gene expression in adult cognitive function and CNS diseases (Ma et al., 2009; Gavin et al., 2012; Sultan and Sweatt, 2013). The regulator of active DNA demethylation enhances long-term memory and synaptic plasticity (Sultan et al., 2012). A recent study suggested that Gadd45β can promote epigenetic DNA demethylation of brain-derived neurotrophic factor (BDNF) and fibroblast growth factor (FGF)-1B in neurons (Ma et al., 2009). These genes have been implicated as potential neuroprotective genes and therapeutic targets in PD (Connor and Dragunow, 1998; Unsicker, 1994). In our LID model, gene expression of BDNF and FGF was not regulated by chronic L-DOPA treatment in 6-OHDAlesioned striatum in mice with either genotype (data not shown).

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Nevertheless, based on the regulation and function of Gadd45β in neuroepigenetic dynamics during activity-associated states such as seizures and memory formation (Ma et al., 2009; Gavin et al., 2012; Sultan et al., 2012), the increased expression of Gadd45β in LID may modulate the target gene transcription. In the present study, deficiency of the Gadd45β gene induced the upregulation of ΔFosB and c-Fos gene expression in the 6-OHDAlesioned striatum 90 min, but not 30 min (Figs. 4 and 5), after the administration of L-DOPA on the 11 day L-DOPA treatment schedule. Previously, no correlation between Gadd45β and other immediate early genes, such as FOS and JUN, was found in psychotic disorders (Gavin et al., 2012), and DNA methylation might regulate gene expression and have a gene-silencing effect (Moore et al., 2013). It was recently reported that Gadd45 was involved in RNA splicing and processing (Sytnikova et al., 2011) in post-transcriptional RNA regulation. Although Gadd45β was not thought of as an on–off switch within the FosB and c-Fos promoter regions, expression levels of these genes were affected by Gadd45β deficiency and the reinstatement of Gadd45β expression in Gadd45β−/− mice decreased the FosB mRNA level. Thus, increased expression of ΔFosB and c-Fos may affect the long-lasting LID symptoms in Gadd45β−/− mice (Cao et al., 2010). Gadd45 proteins have been associated with age-related pathologies and aging-associated conditions such as oxidative stress and chronic inflammation (Liebermann and Hoffman, 2008; Chung et al., 2009; Moskalev et al., 2012; Schmitz, 2013). The upregulation of Gadd45 was observed in neuroblastoma cells of dopamine-induced neurotoxicity and in neurons of patients with Alzheimer's disease, suggesting that the increase might be in response to stress to allow cells to repair damage (Torp et al., 1998; Stokes et al., 2002). An increased expression of Gadd45 in age-related neurodegeneration is likely a protective mechanism aimed at coping with the neurotoxic stress (Moskalev et al., 2012). In addition, a positive association between Gadd45β expression and stroke recovery has been reported (DeSmaele et al., 2001; Liu et al., 2012). A Gadd45β deficiency in mice impaired activity-driven proliferation of neural progenitors and dendritic development of newborn neurons (Ma et al., 2009; Sultan et al., 2012). Based on these reports, it would be interesting to test a strategy that upregulated Gadd45β in such neurodegenerative and neurological diseases and determine whether this upregulation resulted in beneficial outcomes. In conclusion, this study provided the first evidence for a critical role of Gadd45β expression in dyskinesia. Deficiency of Gadd45β led to severe LID in an animal model of PD. Unlike the reported targets for partial inhibitors of the D1R hyperactive signaling pathway (Santini et al., 2007; Fasano et al., 2010; Lebel et al., 2010; Park et al., 2014) used to alleviate LID symptoms, Gadd45β gene expression mediated by activated D1R signaling might relieve LID symptoms. Therefore, strategies for the specific regulation of Gadd45β gene expression warrant development as a therapeutic target of LID.

Acknowledgments This work was supported by a grant from KRIBB Research Initiative Program.

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