Pharmacologic antagonism of dopamine receptor D3 attenuates neurodegeneration and motor impairment in a mouse model of Parkinson's disease

Neuropharmacology 113 (2017) 110e123

Contents lists available at ScienceDirect

Neuropharmacology journal homepage: www.elsevier.com/locate/neuropharm

Pharmacologic antagonism of dopamine receptor D3 attenuates neurodegeneration and motor impairment in a mouse model of Parkinson's disease Daniela Elgueta a, b, María S. Aymerich c, d, e, Francisco Contreras a, Andro Montoya a,
lez a, Marta Celorrio c, Estefanía Rojo-Bustamante c, d, Eduardo Riquelme a, Hugo Gonza f g , h a , b , *
nica Va
squez , Rafael Franco , Rodrigo Pacheco Mo ~ n
n Ciencia & Vida, Nu ~ oa, Santiago 7780272, Chile Fundacio
gicas, Facultad de Ciencias Biolo
gicas, Universidad Andres Bello, Santiago 8370146, Chile Departamento de Ciencias Biolo c Division of Neurosciences, Center for Applied Medical Research (CIMA), University of Navarra, Pamplona 31008, Spain d Department of Biochemistry and Genetics, School of Science, University of Navarra, Pamplona 31008, Spain e IdiSNA, Navarra Institute for Health Research, Pamplona, 31008, Spain f Department of Molecular Genetics and Microbiology, Faculty of Biological Sciences, Pontificia Universidad Catolica de Chile, Av. Libertador Bernardo O'Higgins 340, Santiago, Chile g Department of Biochemistry and Molecular Biomedicine, University of Barcelona, Barcelona 08028, Spain h
n en Red. Enfermedades Neurodegenerativas, Instituto de Salud Carlos III, 28049, Madrid, Spain CIBERNED. Centro de Investigacio a

b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 12 February 2016 Received in revised form 25 August 2016 Accepted 27 September 2016 Available online 28 September 2016

Neuroinflammation involves the activation of glial cells, which is associated to the progression of neurodegeneration in Parkinson's disease. Recently, we and other researchers demonstrated that dopamine receptor D3 (D3R)-deficient mice are completely refractory to neuroinflammation and consequent neurodegeneration associated to the acute intoxication with 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine (MPTP). In this study we examined the therapeutic potential and underlying mechanism of a D3R-selective antagonist, PG01037, in mice intoxicated with a chronic regime of administration of MPTP and probenecid (MPTPp). Biodistribution analysis indicated that intraperitoneally administered PG01037 crosses the blood-brain barrier and reaches the highest concentration in the brain 40 min after the injection. Furthermore, the drug was preferentially distributed to the brain in comparison to the plasma. Treatment of MPTPp-intoxicated mice with PG01037 (30 mg/kg, administrated twice a week for five weeks) attenuated the loss of dopaminergic neurons in the substantia nigra pars compacta, as evaluated by stereological analysis, and the loss of striatal dopaminergic terminals, as determined by densitometric analyses of tyrosine hydroxylase and dopamine transporter immunoreactivities. Accordingly, the treatment resulted́ in significant improvement of motor performance of injured animals. Interestingly, the therapeutic dose of PG01037 exacerbated astrogliosis and resulted in increased ramification density of microglial cells in the striatum of MPTPp-intoxicated mice. Further analyses suggested that D3R expressed in astrocytes favours a beneficial astrogliosis with antiinflammatory consequences on microglia. Our findings indicate that D3R-antagonism exerts a

Keywords: Parkinson's disease Neuroinflammation Dopamine receptors Astrocytes Microglia

Abbreviations: AUC, area under curve; BDNF, brain-derived neurotrophic factor; DARs, dopamine receptors; DAT, dopamine transporter; DnR, dopamine receptor n; DnRKO, DnR knockout; FBS, fetal bovine serum; GAPDH, Glyceraldehyde 3phosphate dehydrogenase; GDNF, Glial cell-derived neurotrophic factor; GFAP, Glial Fibrillary Acidic Protein; HBSS, Hank's Balanced Salt Solution; IGF-1, Insulinlike growth factor 1; iNOS, inducible Nitric Oxide Synthase; MPTP, 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine; MPTPp, MPTP and probenecid; SNpc, substantia nigra pars compacta; TH, tyrosine hydroxylase; TLRs, Toll like receptors; TLRn, Toll like receptor n; RNS, reactive nitrogen species; ROS, reactive oxygen species. ~ n ~ artu 1482, Nu ~ oa, Santiago 7780272, Chile. * Corresponding author. Av. Zan E-mail addresses: [email protected], [email protected] (R. Pacheco). http://dx.doi.org/10.1016/j.neuropharm.2016.09.028 0028-3908/© 2016 Elsevier Ltd. All rights reserved.

D. Elgueta et al. / Neuropharmacology 113 (2017) 110e123

111

therapeutic effect in parkinsonian animals by reducing the loss of dopaminergic neurons in the nigrostriatal pathway, alleviating motor impairments and modifying the pro-inflammatory phenotype of glial cells. © 2016 Elsevier Ltd. All rights reserved.

1. Introduction Neuroinflammation has been recognised as a hallmark of many different neurological disorders (Glass et al., 2010) and evidence strongly suggests that neuroinflammation constitutes a fundamental process in the cascade of events leading to neuronal loss and progression of Parkinson's disease (Gonzalez et al., 2015; Lucin and Wyss-Coray, 2009; Sanchez-Guajardo et al., 2015). Since microglial cells may stimulate both innate and adaptive immunity in the central nervous system (CNS), these cells play a central role in neuroinflammation (Gonzalez et al., 2014; Harms et al., 2013; Noelker et al., 2013; Reynolds et al., 2008, 2009; SanchezGuajardo et al., 2015). Consequently, the functional phenotype acquired by microglia determines whether surrounding neurons survive or die. In this regard, depending on the integration of regulatory signals, microglial cells may undergo two different activation modes, acquiring either a neurotoxic or a neuroprotective phenotype that are, respectively, known as M1-like and M2-like phenotype by analogy with phenotypes in peripheral macrophages (Kettenmann et al., 2011). Whereas M1-like microglia generate a detrimental microenvironment for neurons by producing glutamate, TNF-a, and reactive oxygen (ROS) and nitrogen (RNS) species (Bedi et al., 2013; Burguillos et al., 2011; Ransohoff and Perry, 2009), M2-like microglia secrete anti-inflammatory mediators and neurotrophic factors, such as insulin-like growth factor-1 (IGF-1), brain-derived neurotrophic factor (BDNF) and glial-derived neurotrophic factor (GDNF), thus inducing a supportive microenvironment for neurons (Appel, 2009; Gonzalez and Pacheco, 2014; Lu et al., 2014; Reynolds et al., 2008, 2009). It is currently known that acquisition of different functional phenotypes of microglia can be strongly influenced by the action of cytokines, neurotransmitters and some key cells, such as astrocytes (Gonzalez et al., 2014). According to this notion, reactive astrogliosis constitutes a neuropathological feature in the substantia nigra pars compacta (SNpc) of Parkinson's disease patients (Hirsch and Hunot, 2009). Thereby, these findings support the notion that astrocytes may be active players in neurodegeneration associated to Parkinson's disease. In this regard, analyses of necropsies of Parkinson's disease patients have shown that neuronal death correlates inversely with the number of activated astrocytes (Damier et al., 1993), thus supporting the idea that astroglial activation in Parkinson's disease could play a beneficial role on attenuating neuroinflammation and consequent neurodegeneration. Dopamine is typically recognised for controlling complex processes such as locomotion, cognition, hormone secretion, renal function and intestinal motility; however, recent evidence points dopamine as a key regulator of inflammation (Gonzalez et al., 2013; Pacheco et al., 2014; Prado et al., 2013; Shao et al., 2013; TorresRosas et al., 2014; Yan et al., 2015). Dopamine operates through engagement of five different dopamine receptors (DARs), termed D1R-D5R (Sibley et al., 1993; Strange, 1993). All of these receptors are hepta-spanning membrane proteins that belong to the superfamily of G protein-coupled receptors. Based on their sequence homology, signal transduction machinery and pharmacological properties, DARs have been classified into two subgroups. D1R and D5R belong to type I DARs which often are coupled with

stimulatory Ga subunits (Gas), whilst D2R, D3R, and D4R constitute type II DARs, which generally couple to inhibitory Ga subunits (Gai) (Sibley et al., 1993). On the other hand, due to the fact that different DARs present different affinity for dopamine, differential stimulation of DARs is induced depending on dopamine levels. In this regard, D3R displays the highest affinity for dopamine (Ki z 27 nM), followed by D5R (Ki z 228 nM) and then D4R, D2R and D1R (Ki z 450, 1705 and 2340 nM, respectively) (Malmberg et al., 1993; Strange, 2001; Sunahara et al., 1991; Wu et al., 2005). Previous studies have addressed the expression of DARs in glial cells. These studies have shown that human microglia express D1R-D4R (Mastroeni et al., 2009), while D1R, D2R, D4R and D5R were found in rat microglia (Farber et al., 2005). On the other hand, all five DARs were detected in rat astrocytes obtained from basal ganglia (Miyazaki et al., 2004). In the case of mouse, the analysis of DARs in microglia has been controversial. Whereas an early study described the selective expression of only D1R and D5R in mouse microglia (Farber et al., 2005), a recent study showed that all five DARs are expressed in these cells (Huck et al., 2015). The latter study also found all five DARs expressed in mouse astrocytes (Huck et al., 2015). Interestingly, decreased dopamine levels are associated with inflammatory processes, such as those in the SNpc of Parkinson's disease patients or the gut mucosa of inflammatory bowel disease patients (Pacheco et al., 2014). This evidence strongly suggests that signaling triggered by high-affinity DARs becomes relevant in these inflammatory processes. According to this notion, we and other authors have recently shown that genetic deficiency of the highest affinity DAR, the D3R, attenuates neuroinflammation and subsequent neurodegeneration on a murine model of Parkinson's disease induced by acute intoxication with 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine (MPTP) (Chen et al., 2013; Gonzalez et al., 2013). In this study we evaluated the therapeutic potential and the associated mechanism of the pharmacologic antagonism of D3R in a mouse model of Parkinson's disease not only at the level of neuroinflammation and neurodegeneration, but also at the level of locomotor impairment. Since the acute regime of intoxication with MPTP is not strong enough to manifest locomotor alterations, in the present study the chronic regime of intoxication with MPTP and probenecid was used to induce a measurable locomotor impairment (Fernandez-Suarez et al., 2014). 2. Materials and methods 2.1. Animals Wild-type (WT) C57BL/6 adult male mice (25e30 g) of 3 months of age were obtained from Charles River (St Germain sur l’Arbresle, France) for MPTP-intoxication experiments and from Jackson Laboratories (Bar Harbor, ME, USA) for experiments analysing biodistribution of PG01073, glial phenotypes and 6hydroxydopamine-induced lesion of nigrostriatal pathway. Adult male D3R-knockout (D3RKO) mice (25e30 g) of 3 months of age in the C57BL/6 background, kindly donated by Dr. Marc Caron (Joseph et al., 2002), were used for experiments analysing glial phenotypes. For experiments using glial cell cultures, 1e3 days postnatal WT

112

D. Elgueta et al. / Neuropharmacology 113 (2017) 110e123

C57BL/6 mice (Jackson Laboratories) were used. Five mice per cage were housed at 21
C in a humidity-controlled environment, on a 12/12 h light/dark cycle with lights on at 8 a.m., with ad libitum access to food (Tecklad Global Diets, Harlan, Castellar, Spain) and water. All procedures involving animals were in accordance with the EU Directive 2010/63/EU for the care and use of laboratory animals. The experimental design was approved by the Ethical Committee for Animal Testing of the University of Navarra and
n Ciencia & Vida. Fundacio

2.2. Determination of brain and plasma distribution of PG01037 PG01037 (or (E)-N-(4-(4-(2,3-dichlorophenyl) piperazin-1-yl) but-2-enyl)-4-(pyridin-2-yl) benzamide) (Tocris Biosciences, Bristol, UK) was dissolved in 0.9% NaCl by incubating at 50
C for 10 min and a dose of 15 mg/kg was administered by intraperitoneal (i.p.) injection at a volume of 5 mL/kg. Cohorts of three C57BL/6 mice were sacrificed by carbon dioxide asphyxiation at predose and 5, 10, 30, 60, 120, 240 and 360 min postdose. Blood was collected by heart puncture using heparinized syringes and centrifuged for 10 min at 9500 g, the resulting plasma fraction was stored at 80
C until analysis. Brain tissue was dissected, blotted dry, weighed and stored at 80
C until analysis. Brain samples were homogenized in water for 12 min at 10 rpm, using a Bullet Blender (Next Advance, NY, USA). To quantify PG01037 content on these samples, a validated HPLC method with UV detector was used (Mason et al., 2010). All calibration standards, quality controls and samples were prepared by incubating 60 ml of each sample with 180 ml of internal standard solution (10 mg/mL PG01030; (E)-N-(4-(4-(2,3dichlorophenyl)piperazin-1-yl) but-2-enyl) benzo[b] thiophene-2carboxamide) in acetonitrile on ice. The samples were vortexed for 15 s and then were centrifuged at 4
C at 6000 g for 30 min. Supernatants were mixed with 10 mM ammonium acetate (pH 4.7) to yield a 46:54 (v/v) solution, to inject 50 ml aliquots on the HPLC system. The HPLC system consisted of a PU2089 quaternary pump (JASCO, MD, USA) and a manual injector with a 20 ml sample loop (Rheodyne, CA, USA), and the separation was performed on a Kromasil C-18 analytical column (4.6  150 mm, 5 mm) attached to a C-18 guard column (4.6  30 mm, 5 mm), both from Akzo Nobel (The Netherlands). The mobile phase was a 46:54 (v/v) isocratic condition of acetonitrile and 10 mM ammonium acetate (pH 4.7), at a flow rate of 1.0 mL/min. Detection was performed with a MD2010 diode array detector (JASCO) at 283 nm. The lower limit of quantification of all compounds was 0.05 mg/mL, with a linearity range of 0.05e10 mg/mL. The data obtained from the distribution studies were analysed by the naive averaging method. The plasma concentrations from three animals at each time point were averaged. Compartmental modeling was used to estimate various pharmacokinetic parameters by using the PKSolver add-in program for Microsoft Excel (Zhang et al., 2010). Several compartmental models were evaluated to determine the best fit model. A variety of weighting schemes were also analysed including equal weight, 1/y, 1/y^, 1/y2, and 1/y^2, where y is the observed drug concentration, and y^ is the model-predicted drug concentration. Goodness of fit was based on visual inspection, weighted residual sum of squares, random distribution of residuals, precision of parameter estimates, Akaike's information criteria, and Schwarz criteria. Brain uptake of compounds was represented as a brain-to-plasma (B/P) concentration ratio in accordance with the equation of B/P ¼ Cbrain/Cplasma, where Cbrain and Cplasma are the concentration in brain and plasma at a specific time point, respectively. The S.E. associated with the secondary pharmacokinetic parameter estimates is a measure of the accuracy of the model predictions.

2.3. MPTP intoxication and treatments with PG01037 Seven groups of animals were treated as outlined in Fig. 2A. Five groups received 10 intraperitoneal (i.p.) injections of MPTP hydrochloride (20 mg/kg in saline; Sigma-Aldrich-Aldrich, St. Louis, MO, USA) plus probenecid (250 mg/kg in saline; Life Technologies, Oregon, USA), administered twice a week throughout 5 weeks. Probenecid and MPTP were administered in two consecutive injections. Three of these five groups (MPTPp þ chronic PG) also received 9 i.p. injections of PG01037 (5, 15 or 30 mg/kg) dissolved in saline (Braun, Barcelona, Spain) starting with the first injection the day after the second administration of MPTPp; PG01037 was administered twice a week along the 5 weeks of the neurotoxic treatment. Another group of MPTPp animals received a single i.p. injection of PG01037 30 mg/kg administered the day after the second injection with MPTPp (MPTPp þ acute PG). A control MPTPp group was treated only with the vehicle of PG01037 using the same administration regime of chronic PG01037. Two additional control groups were used: a group of mice receiving PG01037 15 mg/kg, probenecid (250 mg/kg in saline) and the MPTP vehicle (saline) and another group receiving probenecid (250 mg/kg in saline) and the vehicles for PG01037 and for MPTP (saline). In all the groups receiving MPTP (or its vehicle) plus probenecid the two compounds were administered together but in two consecutive injections during the early morning. In the groups receiving PG01037 or its vehicle, administration took place during early morning. 2.4. Nigrostriatal lesions induced by 6-Hydroxydopamine (6OHDA) and treatments with PG01037 Adult male C57BL/J6 mice (27e30 g) were anaesthetised using ketamine/xylazine (ketamine 100 mg/kg, xylazine 10 mg/kg) and placed into a stereotaxic frame with nose and ear bars (Stoelting). Animals received unilateral injections of 2 mL of 5 mg/mL of 6-OHDA hydrobromide (Sigma-Aldrich) dissolved in physiological saline containing 0.02% ascorbic acid (Sigma Aldrich). Injections were placed in the right striatum using a 10 mL Hamilton syringe at the following coordinates: anteroposterior þ0.1 mm respect to Bregma; mediolateral 0.18 mm respect to the medial line; dorsoventral 0.35 mm respect to dura (Paxinos and Franklin, 2001). The injection was conducted at a rate of 0.5 mL/min and the needle was left in place for 3 min for diffusion before being removed. Two groups of animal were treated with PG01037 i.p. (30 mg/kg) dissolved in saline (Braun, Brasil) beginning with the first injection 24 h before the 6-OHDA lesion. Then, the animals received PG01037 i.p. (30 mg/kg) 24, 72 and 120 h after lesion. Mice were transcardially perfused at day 7 after the surgical procedure and brain removed for histological analysis. 2.5. Coat-hanger test To determine the motor performance, we used the coat-hanger test, which has been validated for detection of motor dysfunctions (Brooks and Dunnett, 2009; Cutando et al., 2013). Briefly, we used a steel coat hanger (diameter: 2 mm, length: 40 cm) and suspended at a height of 35 cm from a cushioned surface. The mice were placed in the middle of the hanger and the time taken to move from the middle of the hanger to an extreme was recorded (extreme latency). 2.6. Tissue processing Animals were sacrificed by transcardial perfusion 48 h after the last MPTPp injection. For histological techniques, mice were anesthetized with an overdose of 5% isoflurane (Sigma-Aldrich) and

D. Elgueta et al. / Neuropharmacology 113 (2017) 110e123

transcardially perfused with 4% paraformaldehyde (Merck, Darmstadt, Germany) in 0.125 M phosphate buffered saline (PBS, pH 7.4). Brains were removed and cryoprotected for 48 h in 20% glycerin and 2% DMSO in PBS. For flow cytometry analysis, mice were transcardially perfused with PBS instead paraformaldehyde, brains were rapidly removed, dissected, and immediately processed for flow cytometry as indicated below. 2.7. Histological techniques Immunohistochemistry was performed on free floating sections (40 mm thick) and for a given experiment all sections were processed at the same time with each primary antibody. Sections were washed with PBS and endogenous peroxidase activity was inactivated by 30 min incubation with 0.03% H2O2 in methanol (SigmaAldrich). After washing 3 times with PBS, the tissue was incubated for 40 min with blocking solution [4% normal goat serum, 0.05% Triton X-100 (Sigma-Aldrich) and 4% BSA (Merck, Darmstadt, Germany) in PBS], and exposed overnight to the primary antibodies diluted in blocking solution at room temperature. The primary antibodies used were: rabbit anti-tyrosine hydroxylase (TH, 1:1000; Millipore, Temecula, CA, USA), rat anti-dopamine transporter (anti-DAT, 1:500; Millipore), rabbit anti-GFAP (1:1000; Dako, Glostrup, Denmark) and rabbit anti-Iba1 (1:500; Wako, Osaka, Japan). For colorimetric immunohistochemistry, antibody binding was detected by incubating sections with biotinylated goat antirabbit (1:500; Jackson ImmunoResearch Laboratories, West Gore, PA, USA) in blocking solution for 2 h at room temperature. The biotinylated antibodies were detected with peroxidase-conjugated avidin (1:5000; Sigma-Aldrich) for 90 min at room temperature followed by incubation with 0.05% diaminobenzidine (SigmaAldrich) in 0.03% H2O2/Trizma-HCl buffer (pH 7.6). For fluorescent immunohistochemistry, antibody binding was detected by incubating sections with Alexa-633 donkey anti-goat (1:1000; Invitrogen, Madrid, Spain) and Alexa-488 donkey anti-rabbit secondary antibodies (1:1000; Invitrogen) for 2 h. Sections were mounted on glass slides in a 0.2% solution of gelatin in 0.05 M Tris (pH 7.6) (Sigma-Aldrich), dried and dehydrated in toluene (Panreac, Barcelona, Spain) for 12 min before coverslipping with DPX (BDH Chemicals, Poole, UK). 2.8. Stereological analysis 2.8.1. TH immunopositive neurons in the SNpc The number of TH immunopositive neurons present in the SNpc was determined by unbiased design-based stereology, as described in our previous work (Fernandez-Suarez et al., 2014). Stereological counting was performed in 6 coronal SNpc sections (40 mm thick) taken at uniform intervals (120 mm) that covered the entire rostrocaudal extent of the nucleus between 2.92 mm and 3.64 mm relative to Bregma (Paxinos and Franklin, 2001). The reference volume of the SNpc was calculated from images obtained with the 2 objective using a point count array according to Cavalieri principles (Gundersen and Jensen, 1987). The cross-sectional area of the nucleus was measured and the reference volume (Vr) for the entire SNpc was estimated using the following equation:

Vr ¼ T

aX Pi p

where T is the section thickness, a/p is the area of each point and Pi is the number of points falling within the SNpc. All stereological countings were performed using a Bx61 microscope (Olimpus) equipped with a camera DP71 (Olympus) and a stage connected to an xyz stepper (H101BX, PRIOR) and newCAST Visiopharm

113

software (Hørsholm). The SNpc was outlined at low magnification (4) to estimate the area. The number of labeled neurons was calculated at 100 magnification under oil immersion, using randomized meander sampling and optical dissector methods. The optical dissector height was set at 11 mm to count 100e150 cells per animal using a sampling frame of 5294 mm2 and sampling steps of 145 mm  145 mm (dx, dy). Unbiased counting was performed blindly and the total number of TH-positive neurons (N) was calculated using the following formula:

N¼

X

Q

t 1 1 h asf ssf

P where Q is the total number of particles counted, t is the mean section thickness, h is the height of the optical dissector, asf is the area sampling fraction, and ssf is the section sampling fraction. Neuronal density (D) was determined using the following formula: D ¼ N/Vr. 2.8.2. Analysis of Iba1 immunopositive cells The following parameters were estimated by unbiased stereology in striatal Iba1 immunopositive cells: cell density, soma area and the length of microglial processes. This stereological analysis was performed in 5 coronal striatal sections between þ1.34 mm and 0.26 mm relative to Bregma (Paxinos and Franklin, 2001). Approximately 100e150 cells per animal were counted using a sampling frame of 3781.6 mm2 and sampling steps of 434.84 mm  434.84 mm (dx, dy). The soma area was estimated with a 2D nucleator (Visiopharm). The total length of neurites was estimated by global spatial sampling and by counting intersections of fibers with isotropically oriented virtual planes within a virtual box in a thick section with arbitrary orientation. The total fibers length (L) was calculated according to Larsen et al. (Larsen et al., 1998).

L¼

X 1 1 1 dx2x Q hsf asf ssf

P  where d is the plane separation distance and Q is the total number of fiber-plane intersections counted (hsf, asf and ssf). Counting was performed at a height of 11 mm using a frame area of 453.80 mm2 and sampling steps of 550.3 mm  550.03 mm (dx, dy) and a plane separation distance (d) of 20 mm (Drojdahl et al., 2010). 2.9. Glial cultures Mixed glial cultures were generates from 1 to 3 days postnatal mouse. Brains were removed from skull, meninges were taken away and the striatum and mid-brain regions were dissected. The tissue was dissociated using trypsin 0.01% in Hank's Balanced Salt Solution (HBSS) during 10 min. Afterwards, cells were washed, resuspended in DME/F12 (HyClone, Utah, USA) media supplemented with 10% fetal bovine serum (FBS; Gibco, New York, USA) and tissue was completely dissociated by mechanical disaggregation using a glass Pasteur pipettes. Cells obtained from four brains were seeded onto T-75 flask in 10 mL of DMEM/F12 supplemented with 2.5 mM L-glutamine and 10% FBS. After incubation for 24 h, the media and tissue was removed and replaced by fresh media. Afterwards, half of the volume of media was renovated every 72 h until cells reached 90% confluence. At this point, cells were replated in a new T-75 flask. The mixed glial culture was maintained at 37
C in humidified atmosphere of 5% CO2 and used after 14e28 days in vitro. Microglia were obtained from 14 to 28 days mixed glial culture by mechanical extraction using a horizontal rotating shaker at

114

D. Elgueta et al. / Neuropharmacology 113 (2017) 110e123

200 rpm for 2 h. Microglial cells contained in the supernatant were centrifuged at 500 g, resuspended in DME/F12 media (supplemented with 2.5 mM L-glutamine and 10% FBS) and plated in T-25 flask until use. The purity of the microglial cells obtained was evaluated by CD11b immunostaining (98% CD11bþ cells). Purification of astrocytes was carried out from the mixed glial culture by depletion of microglial cells. For this purpose, after 7 days of incubation, mixed glial cultures were shaked at 180 rpm overnight twice per week along 2 weeks and the supernatant containing microglial cells was replaced with fresh media every time. Afterwards, astrocytes layer was detached by incubation with trypsin 0.01% in HBSS for 30 min, and seeded onto T-75 flask with DME/F12 media supplemented with 10% FBS. The purity of astrocytes culture was evaluated by GFAP immunostaining (96% GFAPþ cells). 2.10. RT-PCR analysis Total RNA was extracted from mixed glial, microglia or astrocytes cultured using the E.Z.N.A. total RNA kit I (Omega, Georgia, USA), according to the manufacturer's instructions. Reverse transcription was conducted using M-MLV Reverse Transcriptase (Invitrogen, California, USA) and oligo(dT) primers. Real-time PCR was performed using the Brilliant II Ultra Fast QPCR Master Mix (Agilent Technologies, California, USA), followed by detection using the Stratagene Mx 3000P QPCR System (Stratagene, California, USA). The Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA levels were examined for use as housekeeping control. The nucleotide sequences of the primers were: D3R-forward: 50 -GAA CTC CTT AAG CCC CAC CAT-30 , D3R-reverse: 50 -GAA GGC CCC GAG CAC AAT-30 , GAPDH-forward: 50 -TCC GTG TTC CTA CCC CCA ATG-30 , GAPDH-reverse: 50 -GAG TGG GAG TTG CTG TTG AAG-30 . PCR reactions were carried out including an initial heating for 5 min at 95
C, followed by 40 cycles of 30 s at 95
C, 30 s at 60
C, and 60 s at 72
C. Results were analysed with the MxPro e Mx 3000P Software (Stratagene). Amplimer sizes were verified by electrophoresis on a 1.5% agarose gel following ethidium bromide staining. 2.11. Flow cytometry and immunofluorescence analysis Brain sections that contain mid-brain and striatum from adult mice were minced and then disaggregated using Colagenase Type IV 1 mg/ml (Gibco, New York, USA) and DNase I 0.25 mg/mL (Roche, Mannheim, Germany). After enzymatic disaggregation, cells were passage through 70 mm-pore cell strainer to obtain a single-cell suspension. For intracellular detection of phenotypic markers in astrocytes, cells were previously fixed and permeabilised. Then, cells were immunostained using the following fluorochromecoupled antibodies: PE-coupled anti-mouse GFAP (BD, California, USA), FITC-coupled anti-mouse iNOS (BD), and APC-coupled antimouse arginase-1 (R&D Systems Inc., Minneapolis, USA). Flow cytometry analysis was performed with identical instrument settings on a FACS Canto II (BD). Data were analysed using the FlowJo software (Tree Star). For immunofluorescence analysis, cells extracted from mixed glial, microglia or astrocytes cultures were mounted onto cover glasses and fixed with 4% paraformaldehyde, permeabilised using 0.1% Triton X-100 in PBS and then saturated with blocking solution containing 5% BSA and 0.1% Triton X-100 in PBS. Afterward, cells were incubated over night at 4
C in incubating solution (1% BSA and 0.1% Triton X-100 in PBS) containing the following primary antibodies: rat anti-mouse CD11b (1:500; Serotec, Raleigh, USA), rabbit anti-mouse GFAP (1:1000; Synaptic System, Goettingen, Germany) and rabbit anti-mouse D3R (1:100; AbCam, Boston, USA). After three washes with incubating solution, the cells were

incubated 1 h at room temperature with secondary antibodies goat anti-rabbit Alexa-fluor 488 (for D3R immunostaining) or goat antirabbit Alexafluor 546 (for GFAP immunostaining) (1:200; Invitrogen, Oregon, USA) and goat anti-rat DyLigth 488 (for CD11b immunostaining) (1:200; Jackson ImmunoResearch Laboratories), and after repeated washes, the cells were mounted using DAKO Fluorescent Mounting Medium (Dako, California, USA). The nuclei of the cells were marked with DAPI. Images were acquired with an inverted fluorescence microscope Olympus IX71 (Olympus, Tokyo, Japan) coupled to a power supply unit (Olympus U-RFL-T). 2.12. Statistical analysis All values are expressed as the mean ± SEM. Normality was confirmed with the KolmogoroveSmirnov test. Statistical analysis were performed with two-tailed unpaired Student's t-test, when comparing only two groups and with one-way ANOVA followed by Bonferroni's or Tukey's pos-hoc test (as indicated in each figure legend), when comparing more than two groups (GraphPad Software). P values < 0.05 were considered significant. 3. Results 3.1. Pharmacologic analysis of PG01037 distribution in mouse plasma and brain Since the main aim of this study is to assess the therapeutic potential of the systemic administration of PG01037 in a mouse model of Parkinson's disease, we first performed a pharmacokinetic analysis of this drug in mice (Fig. 1). Importantly, when PG01037 was intraperitoneally administered, the drug was able to reach the brain. Pharmacokinetic behaviour of PG01037 in the brain was best described with a non-compartmental model. Intraperitoneal administration of 15 mg/kg PG01037 resulted in a peak of the drug concentration in the brain at around 40 min after injection (Fig. 1A and E; Brain Cmax ¼ 12.1 ± 0.60 mg/g, Brain tmax ¼ 0.67 ± 0.17 h), and remained relatively high (z2 mg/g; approximately 3 mM) after 6 h. Pharmacokinetics of PG01037 in plasma were best described with a one-compartment model. While the drug reached a maximum concentration with similar magnitude and kinetics in both compartments, brain levels of PG01037 showed a significantly slower decay when compared to plasma concentrations (Fig. 1AeC and E; Plasma t½ vs Brain t½ p < 0.01). In line with this, brain exposure (determined as area under curve, AUCbrain) to PG01037 was significantly higher than plasma exposure (AUCplasma), with an overall brain-to-plasma exposure ratio (AUCbrain/AUCplasma) of 2.05 (Fig. 1AeC and E; AUCplasma vs AUCbrain p < 0.0001). Brain uptake of PG01037 was estimated by brain-to-plasma concentration ratios at discrete sampling times after i.p. administration. As shown on Fig. 1D, brain uptake of PG01037 ranged from 0.48 to 2.23, with a significant increase at 6 h compared to the initial 10 min after injection (Fig. 1D; p < 0.05). Considering the high affinity of PG01037 for D3R (Ki ¼ 0.7 nM) and the half-life of the drug in blood and brain, it is possible to predict that, after i.p. administration of 15 mg/ kg PG01037, the drug concentration is maintained biologically relevant for several hours. This is an important point, as brain cells would be exposed to relevant concentrations of the D3R antagonist PG01037 for several hours after i.p. administration of 15 mg/kg. 3.2. PG01037 attenuates the motor impairment in MPTPpintoxicated mice After confirming that PG01037 penetrates into the CNS, we addressed the question of whether i.p. administration of this D3R antagonist is able to attenuate the neurodegenerative process and

D. Elgueta et al. / Neuropharmacology 113 (2017) 110e123

115

Fig. 1. Biodistribution of PG01037 in mouse plasma and brain. Male C57BL/6 mice received 15 mg/kg PG01037 via the intraperitoneal route and then plasma and brain samples were taken at indicated times points. Samples were subsequently quantified by HPLC coupled to UV detector. (A) Brain concentration versus time profiles. (B) Plasma concentration versus time profiles. (C) Brain and plasma concentration versus time profiles. (D) Brain to plasma ratio of concentrations determined at each sampling time. One-way ANOVA followed by Tukey's post hoc test. *, p < 0.05; **, p < 0.01. (E) Pharmacokinetic parameters of PG01037 in both plasma and brain after intraperitoneal administration in mice. Student's t-test (brain versus plasma), **, p < 0.01; ****, p < 0.0001 (mean ± SD, n ¼ 3/time point). AUC, area under curve.

Fig. 2. Systemic treatment with PG01037 improves the motor performance of mice intoxicated with MPTP. (A) Illustration of the experimental design. Control animals (without MPTP treatment) were treated with saline, probenecid, and PG01037 or the vehicle used to prepare PG01037. MPTPp animals were injected with MPTP (20 mg/kg), probenecid (250 mg/kg) and PG 01037 at 5, 15 or 30 mg/kg. All compounds were intraperitoneally administered. PG01037 administration started after the second MPTPp administration. All animals groups received PG01037 twice a week, except for one group that was acutely injected with a single 30 mg/kg dose (bold arrow). (B) Parkinsonian motor symptoms were evaluated 16 h after the last MPTPp and PG 01037 administration in the coat-hanger test. Mice were placed hanging from the front legs in the middle of the horizontal bar of a steel coat-hanger and the time to get to the end of the bar was measured. Data represent the mean with the SEM. Student's t-test was used to compare control and MPTPp animals and one-way ANOVA followed by Bonferroni's multiple comparison post-hoc test were used to determine statistical differences between MPTPp animals treated or untreated with PG01037 (F4,37 ¼ 0.04). n ¼ 7e8 animals per group. *, p < 0.05.

116

D. Elgueta et al. / Neuropharmacology 113 (2017) 110e123

the motor impairment in a mouse model of Parkinson's disease induced by chronic administration of MPTP and probenecid. For this purpose, mice received 10 i.p. injections of MPTP and probenecid (MPTPp) along five weeks. As indicated in the scheme of experimental design (Fig. 2A), we tested the therapeutic potential of PG01037 at three different doses (5, 15 and 30 mg/kg), which were i.p. administered twice a week starting after the second injection of MPTPp. In addition, we tested the therapeutic potential of an acute single i.p injection of 30 mg/kg PG01037 administered after the second injection of MPTPp (Fig. 2A, bold arrow). The motor performance of experimental mice was determined using the coathanger test. Previous to the administration of MPTPp, mice were pre-trained in a coat-hanger and the different experimental groups were homogeneously formed depending on their motor coordination performance. For the motor test, mice were placed in the middle of the hanger and the time they spent to reach to the end of the coat-hanger was measured. Healthy animals needed a significant shorter time than MPTPp animals to perform the task (Fig. 2B). Chronic administration of PG01037 (15 and 30 mg/kg) to MPTPp animals induced a statistically significant reduction in the time that mice needed to reach the end of the hanger (Fig. 2B). No significant differences in motor behaviour were detected in mice receiving the acute treatment with PG01037 (30 mg/kg) (Fig. 2B). These results show a marked improvement in motor coordination in parkinsonian mice treated chronically with PG01037. 3.3. PG01037 reduces the extent of nigrostriatal dopaminergic denervation in MPTPp-intoxicated mice To further explore the mechanism of motor improvement achieved by PG01037 treatment, the status of the nigrostriatal pathway was analysed by histological techniques. For this purpose, 36 h after the last MPTPp and PG01037 injections, mice were sacrificed, the brains were removed, and coronal sections comprising the striatum and the SNpc were used for immunohistochemistry analysis. We first determined the density of dopaminergic terminals in the striatum by tyrosine hydroxylase (TH) and dopamine transporter (DAT) immunostaining (Fig. 3AeC). The density of immunopositive dopaminergic terminals was analysed by optical density. MPTPp administration induced a marked loss of dopaminergic terminals that resulted in a significant decrease of both DAT and TH immunostaining (Fig. 3B and C). MPTPp-intoxicated mice treated with the highest dose of PG01037 (30 mg/kg) chronically or acute, showed a significant increase in the density of DATþ and THþ terminals. Furthermore, the density of THþ neurons present in the SNpc was quantified by unbiased design-based stereology. The injection of MPTPp induced a significant loss of nigral THþ neurons compared to control mice (Fig. 3DeE). Strikingly, the treatment of MPTPp-intoxicated mice with PG01037 30 mg/kg, irrespective of the administration regime (acute or chronic), resulted in an increase in the number of dopaminergic neurons in the SNpc (Fig. 3DeE). Thus, these results show that PG01037 exerts a neuroprotective effect when administered to MPTPp mice irrespective of the administration regime. Interestingly, the chronic administration of PG01037 correlates with an improved motor behaviour suggesting that the administration pattern of the drug is important to obtain a behavioural effect. 3.4. Analysis of glial phenotypes involved in the neuroprotective effect exerted by PG01037 In addition to the analysis of the therapeutic potential of PG01037 in a Parkinson's disease model, another aim of this study was to analyse the mechanism by which D3R blockade impacts on the physiopathology of this disorder. Therefore, to gain insight into

the mechanism by which PG01037 promotes a neuroprotective effect in the MPTPp-induced Parkinson's disease model, we also analysed neuroinflammation. For this purpose we determined the extent of microgliosis and astrogliosis in the striatum by immunohistochemical analyses of Iba-1 and GFAP respectively. Densitometric analysis of Iba1þ cells gave no clear differences in the intensity of Iba1-associated immunoreactivity (data not shown). For this reason, we performed stereological analysis of the microglial morphology (Fig. 4). In this regard, we observed no differences in the body area and cell density of Iba1-stained microglia among the different experimental groups (Fig. 4AeC). Conversely, the MPTP-induced decrease in the ramification density of Iba1þ cells was inhibited by the chronic administration of PG01037 (Fig. 4D). To better understand the therapeutic effect of the systemic administration of PG01037 in MPTPp-intoxicated mice, we also analysed astrocyte activation, which corresponds to another relevant process underlying neuroinflammation in Parkinson's disease. For this purpose, we performed immunohistochemical analysis of GFAPþ cells in coronal sections of the striatum. Interestingly, the results show that chronic systemic administration of PG01037 (at 30 mg/kg) exacerbates the astrogliosis induced by MPTPp, as determined by densitometric quantification of GFAP-labeling (Fig. 5AeB). Taken together these results indicate that chronic systemic administration of PG01037 (at 30 mg/kg) attenuates MPTPp-induced motor impairment and neurodegeneration, a therapeutic effect that is associated with an attenuated acquisition of activated phenotype by microglia and exacerbated astrogliosis in the striatum. In this regard, neuronal death has been inversely correlated with the number of activated astrocytes in necropsies of Parkinson's disease patients (Damier et al., 1993). 3.5. D3R is selectively expressed in astrocytes, where its signaling triggers an inhibitory effect on astrocyte activation Since the therapeutic effect exerted by the D3R-selective antagonist PG01037 at the level of neurodegeneration of the nigrostriatal pathway and motor coordination is associated with altered microglial activation and astrogliosis, we wanted to further explore the relationship between D3R-signaling and glial activation. In this regard we first addressed the expression of D3R in glial cells. For this purpose, we performed single and mixed cell cultures of astrocytes and microglia obtained from mid-brain and striatum (Fig. 6A). Quantitative RT-PCR analysis showed that astrocytes and mixed cultures but not microglia express the transcript for D3R (Fig. 6B). The expression of D3R was subsequently analysed at the protein level by immunostaining of D3R in cultures of astrocytes. Analysis of labeled astrocytes by fluorescence microscopy showed D3R immunoreactivity in these cells, which was not observed when immunostaining was performed with an isotype-matched irrelevant antibody (Fig. 6C). To investigate how D3R-signaling affects astrocyte phenotype, we determined in striatal astrocytes from WT or D3RKO mice the expression of inducible oxide nitric synthase (iNOS), a M1-like marker and arginase-1, a M2-like marker. The results showed that D3R-deficiency leads to an evident higher degree of astrogliosis, as astrocytes from D3RKO mice displayed an increased density of GFAP staining (Fig. 6D). Remarkably, D3Rdeficient astrocytes presented higher frequency of the M2-like marker Arginase-1 in the GFAPþ population (Fig. 6D). Unexpectedly, however, the results show that D3R-deficiency favours an enhanced intensity in the expression of iNOS by GFAPþ astrocytes (Fig. 6D), indicating an exacerbated M1-like astrocyte phenotype under steady-state conditions. Taken together, these results suggest that D3R-signaling in astrocytes would induce the attenuation of the acquisition of a phenotype with M1 and M2 features by astrocytes, which seems to exert a negative effect in microglia activation,

D. Elgueta et al. / Neuropharmacology 113 (2017) 110e123

117

Fig. 3. Systemic administration of PG01037 attenuates the nigrostriatal degeneration of MPTP mice. Analysis of the nigrostriatal pathway in MPTPp animals treated with PG01037. (A) The density of dopaminergic terminals in the striatum was analysed by DAT and TH immunohistochemistry. Representative photomicrographs showing DAT- and THimmunoreactivity in the striatum of control and MPTPp animals treated with PG01037 are shown. (B) Densitometric quantification of DAT-immunostained fibers in the striatum. (C) Densitometric quantification of TH-immunostained fibers in the striatum. (D) Representative photomicrographs showing THþ dopaminergic neurons in the SNpc are shown. (E) Quantification of TH-immunopositive neurons in the rostrocaudal extension of the SNpc by unbiased stereology. Student's t-test was used to compare control and MPTPp animals and one-way ANOVA followed by Bonferroni's multiple comparison post-hoc test were used to determine statistical differences between MPTPp animals treated or untreated with PG01037: (B) F4,37 < 0.001; (C) F4,37 < 0.001; (E) F4,37 < 0.001. n ¼ 7e8 animals per group. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. Scale bar: 0.5 mm.

118

D. Elgueta et al. / Neuropharmacology 113 (2017) 110e123

Fig. 4. Morphological analysis of microglia in MPTPp animals treated with PG01037. (A) Representative photomicrographs of Iba1-immunostained microglia in the striatum. Quantification by stereology of microglial cell density (B), body area (C) and length of ramifications (D). Student's t-test was used to compare control and MPTPp animals and oneway ANOVA followed by Bonferroni's multiple comparison post-hoc test were used to determine statistical differences between MPTPp animals treated or untreated with PG01037 (F4,37 ¼ 0.006). n ¼ 5 animals per group. *, p < 0.05; **, p < 0.01. Scale bar: 50 mm.

Fig. 5. Analysis of astrogliosis in MPTPp animals treated with PG01037. (A) Representative photomicrographs of GFAP-immunostained cells in the striatum. (B) Densitometric quantification of GFAP immunostaining. Student's t-test was used to compare control and MPTPp animals and one-way ANOVA followed by Bonferroni's multiple comparison posthoc test were used to determine statistical differences between MPTPp animals treated or untreated with PG01037 (F4,37 ¼ 0.003). n ¼ 7e8 animals per group. *p < 0.05; ***p < 0.001. Scale bar: 100 mm.

reducing neuroinflammation and consequent neurodegeneration.

4. Discussion The present study demonstrates that the intraperitoneal administration of a D3R-selective antagonist, PG01037, exerts a

significant improvement in the locomotor performance, a clear reduction in the loss of dopaminergic neurons in the nigrostriatal pathway, an exacerbated astrogliosis and an increased ramification density in microglial cells in a mouse model of Parkinson's disease induced by the chronic intoxication with MPTPp. Subsequent analyses suggested that lack of D3R-signaling in astrocytes promotes a

D. Elgueta et al. / Neuropharmacology 113 (2017) 110e123

119

Fig. 6. D3R is selectively expressed in astrocytes and its deficiency results in increased intensity of iNOS expression. (A) Mixed glial cultures (left panel), and microglia (middle panel) and astrocyte (right panel) primary cultures were prepared as described in Materials and Methods (section 2.9). Representative photomicrographs of cells with GFAP (red) or CD11b (green) immunoreactivity. Nuclei were stained with DAPI. (B) Total RNA was extracted from cultures of microglia, astrocytes or mixed glial cultures, and expression of the mRNA codifying for D3R was analysed by RT-PCR. RNA obtained from striatum was used as a positive control and the mRNA codifying for GAPDH was analysed as a house-keeping molecule. (C) Astrocyte cultures were immunostained with anti-D3R antibody or a isotype-matched control primary antibody followed by a Alexa488-coupled secondary antibody and the immunofluorescence associated was analysed by fluorescence microscopy. Nuclei were stained with DAPI. Representative photomicrographs are shown. (D) Brain sections containing midbrain and striatum were extracted from WT and D3RKO mice and a cell suspension was prepared as described in Materials and Methods (section 2.11.). Cells were permeabilised and immunostained for iNOS, arginase-1 and GFAP. Percentages and mean-fluorescence intensity (MFI) of GFAP, iNOS and Arginase-1 were analysed by flow cytometry in the GFAPþ population. Representative dot-plots are shown in the left panel, and quantification in the right panel. n ¼ 5e6 animals per group. Student's t-test was used to compare WT versus D3RKO. *, p < 0.05; ***, p < 0.001. Scale bar: 20 mm.

beneficial astrogliosis with anti-inflammatory consequences on microglial cells. Thus, this work constitutes the first study demonstrating a substantial therapeutic effect of D3R-antagonism in a model of Parkinson's disease. Our findings here confirm that MPTP treatment promotes a striatal microglia with less arborisation than homeostatic microglia (Fig. 4D), which has been previously associated to proinflammatory microglia (Gonzalez et al., 2013; Sanchez-Guajardo et al., 2010). Thereby, our results suggest that chronic systemic administration of 30 mg/kg PG01037 attenuates activation of M1like pro-inflammatory microglia. As for macrophages and microglia, two different alternativelyactivated phenotypes have been described for astrocytes, one of them with inflammatory features and detrimental consequences (M1-like, by analogy with macrophages and microglia) and another

one with anti-inflammatory and neuro-supportive effects (M2like). Pro-inflammatory astrocytes display decreased buffering capability for clearing extracellular glutamate, express high levels of iNOS and inflammatory cytokines (Schwartz et al., 2003; Shao et al., 2013; Zou and Crews, 2005), thus contributing with the production of neurotoxic and inflammatory mediators. Furthermore, activated astrocytes may express Major Histocompatibility Complex class II and costimulatory molecules on their surface. Consequently, the inflammatory potential of astrocytes in the CNS is not limited to the stimulation of innate immunity, but also to the activation of adaptive immunity (Nikcevich et al., 1997). On the other hand, anti-inflammatory astrocytes are characterized by the expression of arginase-1 and IL-4, present strong capability for clearing extracellular glutamate and produce neurotrophic factors supporting neuronal function and survival (Derecki et al., 2010;

120

D. Elgueta et al. / Neuropharmacology 113 (2017) 110e123

Pacheco et al., 2007; Schwartz et al., 2003). Thereby, despite microglia are the main mediators of the inflammatory damage of the CNS during neurodegeneration, astrocytes may display similar features and both glial cell types actively collaborate in promoting neuroinflammation or neuroprotection. Interestingly, our data associates the therapeutic effect of the D3R-selective antagonist PG01037 with exacerbated astrogliosis and attenuated acquisition of morphological features of M1-like microglial phenotype. Further analyses in glial cells showed a selective expression of D3R in astrocytes and genetic evidence indicates that inhibition of D3R-signaling in vivo results in an exacerbated intensity of iNOS expression and increased frequency of arginase-1 in midbrain/striatal astrocytes. Together these findings suggest that by inducing an intermediate M1/M2-like phenotype in astrocytes, D3R-inhibition favours an antiinflammatory behaviour in microglia, attenuating the neurodegenerative process in a mouse model of Parkinson's disease induced by chronic administration of MPTP. In agreement with our data, neuronal death has been found to correlate inversely with the number of activated astrocytes in necropsies of Parkinson's disease patients (Damier et al., 1993). Accordingly, it has been shown that astrocytes play an important neuroprotective role performing the clearance of ROS in the surrounding environment. Thus, neuronal regions displaying lower density of activated astrocytes are more susceptible to oxidative damage (Damier et al., 1993). Whereas some studies support a beneficial role for activated astrocytes in the neuroinflammation that is associated with neurodegenerative diseases, there are reports suggesting that activated astrocytes display a neurotoxic behaviour similar to that of activated microglia. In this regard, astrocytes express several toll-like receptors (TLRs), including TLRs16 and TLR9, which imply that these cells could respond to infections and to protein aggregates (Carpentier et al., 2005; Farina et al., 2007). Indeed, it has been reported that, in response to aggregated a-synuclein, astrocytes secrete inflammatory cytokines, such as IL-1b, lL-6 and IL-18 (Lee et al., 2010). A conciliatory hypothesis proposes a dual role of activated astrocytes depending on the levels of TNF-a. It has been shown that high extracellular levels of TNF-a favours the inflammation and neurodegeneration mediated by astrocytes and microglia, whilst low levels of TNF-a secreted mainly by astrocytes stimulate, in an autocrine manner, the secretion of neurotrophic factors, supporting neuronal survival (Kuno et al., 2006). In contrast with our findings, pramipexole, a drug characterized as a D3R agonist (Mierau et al., 1995), has been shown to exert a neuroprotective effect in dopaminergic neurons of MPTPintoxicated mice (Joyce et al., 2004). Nevertheless, pramipexole promoted a down-regulation of DAT (Joyce et al., 2004), suggesting a relationship between D3R-induced signaling and DAT expression. Since MPTP-mediated intoxication involves specific uptake of 1methyl-4-phenylpyridinium (MPPþ) by DAT, it is likely that pramipexole-induced neuroprotective effect was due to a reduced uptake of the toxic cation MPPþ by dopaminergic terminals, making them less susceptible to MPTP-induced neurodegeneration. The attenuated neurodegeneration of nigrostriatal pathway here reported cannot be attributed to an effect of PG01037 on DAT expression, as mice treated with PG01037 in the absence of MPTPpintoxication displayed similar levels of striatal DAT (Fig. 3A and B). Supporting the same idea, we have observed no difference on striatal DAT expression between WT and D3RKO mice (Fig. S1), thus ruling out the possibility that the therapeutic effect observed by the inhibition of D3R-signaling in MPTP-intoxicated mice is due to an altered MPPþ uptake by dopaminergic terminals. Furthermore, we (Gonzalez et al., 2013) and others (Chen et al., 2013) have demonstrated that the genetic deficiency of D3R (in the

absence of any treatment with dopaminergic drugs) results in attenuated neuroinflammation and neurodegeneration in a mouse model of Parkinson's disease induced by MPTP-intoxication. Thus, these studies associate a reduction in MPTP-induced neurodegeneration with the lack of D3R-signaling in the absence of additional drugs and therefore in the absence of any pharmacological interactions that could interfere in the mechanism of action of MPTP. To gain further evidence of the therapeutic effect of PG01037 in attenuating neurodegeneration in the context of Parkinson's disease, we analysed the therapeutic potential of this drug in the mouse model of dopaminergic neurodegeneration induced by unilateral injection of 6-hydroxydopamine (6-OHDA) in the striatum (Riquelme et al., 2012). In agreement with the therapeutic effect of D3R-antagonism in the MPTPp model (Fig. 3), the systemic administration of PG01037 in 6-OHDA-intoxicated mice ameliorated the extent of dopaminergic neurodegeneration in the striatum and SNpc (Fig. S2). Thus, these results indicate that the neuroprotective effect of PG01037 is not confined to the MPTPp model and it can be extended to another model of Parkinson's disease. Moreover, these results rule out again the possibility that the attenuation of neurodegeneration exerted by D3R-antagonism was due to a direct effect in the mechanism of action of MPTP rather than to an actual effect in the neurodegenerative process associated to Parkinson's disease. Regarding the analysis of DARs expressed on mouse glial cells, we detected transcripts for D3R only in astrocytes but not in microglial cells (Fig. 6B). In agreement with these results, a previous study indicates that mouse microglia expresses only type I DARs (Farber et al., 2005). In contrast to our results, a recent study found all five DARs expressed in mouse microglia and astrocytes (Huck et al., 2015). This discrepancy could be due to that the study of Huck et al. used glial culture prepared from the cerebral cortex, whilst in the present study glial cells were obtained from midbrain. Furthermore, the differences observed for DARs expression in cortical microglia could be explained by differences in mouse strains used; whereas Huck et al. used the inbred C57BL/6 mice strain, Farber et al. used the outbreed NMRI strain. On the other hand, despite D3R expression has been previously described in mouse astrocytes (Huck et al., 2015), some studies addressing the role of dopaminergic regulation of these cells have been focused in the effects exerted by D2R-mediated signaling, without analysing the role of D3R-mediated effects in these cells (Duffy et al., 2011; Shao et al., 2013). Addressing the role of dopaminergic regulation in astrocytes and its impact on neuroinflammation, Shao et al. found that D2Rstimulation in astrocytes triggers an up-regulation of aB-crystallin, attenuating neuroinflammation and consequent neurodegeneration in MPTP-intoxicated mice (Shao et al., 2013). In this regard, these authors demonstrated that specific ablation of D2R in astrocytes showed a remarkable inflammatory response in several areas of the CNS and display an increased vulnerability of nigrostriatal dopaminergic neurons in response to MPTP-intoxication. Moreover, the treatment of WT mice with a D2R-selective agonist, quinpirole, increased resistance of dopaminergic neurons to MPTP-intoxication by suppressing neuroinflammation. Thus, the study performed by Shao et al. (Shao et al., 2013) suggests that D2Rsignaling in astrocytes promotes a M2-like neuro-supportive phenotype. Taken together the data reported by Shao et al. (Shao et al., 2013) and those here presented it is tempting to propose that low dopamine levels (stimulating selectively the highestaffinity DAR, D3R with a Ki z 27 nM) favours the M1-like inflammatory phenotype in astrocytes, whereas high dopamine levels (stimulating the low-affinity D2R with a Ki z 1705 nM) trigger the acquisition of M2-like features in these cells. According to this idea,

D. Elgueta et al. / Neuropharmacology 113 (2017) 110e123

striatal dopamine levels are z50 times lower in MPTP-treated mice than those observed in the striatum of untreated healthy mice (Brochard et al., 2009), which is in agreement with the inflammatory process observed in this brain structure in MPTP-treated mice. Interestingly, a similar dopaminergic regulation of inflammation has been proposed in the gut. In this regard, healthy individuals display high dopamine levels in the gut mucosa, whereas patients with Crohn's disease or ulcerative colitis present inflamed gut mucosa, which is associated with low dopamine levels (Pacheco et al., 2014). Similarly, in the case of CD4þ T-cells, D3Rstimulation is associated with the production of inflammatory cytokines, including IFN-g and TNF-a (Franz et al., 2015; Gonzalez et al., 2013; Ilani et al., 2004), whilst D2R-stimulation has been related with production of anti-inflammatory mediators, including IL-10 (Besser et al., 2005; Ilani et al., 2004). In line with this idea, an allelic variant of the D2R gene that results in a reduced expression of this receptor has been reported as a risk factor to develop refractory Crohn's disease (Magro et al., 2006). Furthermore, a polymorphism of D3R in Hispanic white subjects has been linked to reduced risk of Parkinson's disease (McGuire et al., 2011). Together, these evidences strongly suggest a dual role of dopamine in the regulation of inflammation, in which the final outcome depends on dopamine levels. In this study we described the therapeutic effect of PG01037 systemically administrated in the attenuation of neurodegeneration in a mouse model of Parkinson's disease. Since the drug was administered systemically, it is reasonable to speculate that all cells expressing D3R in the CNS could contribute to the final outcome of the therapeutic treatment. In this regard, D3R is expressed in several brain structures, such as the shell of the nucleus accumbens, olfactory tubercle (Sokoloff et al., 1990; Xing et al., 2013), ventral striatum, SNpc, ventral tegmental area and cerebellum (Diaz et al., 1994, 1995). Importantly, D3R is not only expressed by astrocytes (Fig. 6B), but also in neurons. This receptor is located at both postsynaptic and presynaptic sites, being able not only to mediate dopaminergic transmission, but also to modulate dopamine synthesis and release (Joseph et al., 2002). For these reasons, the contribution of the altered functioning of neural dopaminergic circuits expressing D3R in the therapeutic effect exerted by the pharmacologic inhibition of D3R cannot be ruled out. Importantly, our data show that the D3R-selective antagonist PG01037 efficiently crosses the blood brain barrier when administered intraperitoneally. Brain penetrance represents a fundamental requirement of drugs addressed to combat CNS associated disorders. In this regard, our results are in agreement with what was previously reported in rats by Mason et al. (Mason et al., 2010). Even though the magnitude of the analysed parameters differed in rat and mouse, a key parameter such as the brain-to-plasma exposure ratio was similar in the two studies (Fig. 1D). Interestingly, some preclinical studies showing a neuroprotective effect in MPTP mouse models of Parkinson's disease have shown translational potential. A successful example is constituted by exendin-4 and its synthetic version, exenatide, which are analogues of the glucagon-like peptide-1 (GLP-1) that selectively bind to the GLP-1 receptor. In this regard, these drugs tested in MPTPintoxicated mice prevented the loss of nigrostriatal neurons, decreased neuroinflammation and improved motor impairment (Kim et al., 2009; Li et al., 2009). Few years later, the drugs were tested in a small cohort (45 patients) and the results encourage additional investment in the expensive double-blind placebocontrolled phase II/III studies required for approval as medicaments (Aviles-Olmos et al., 2013; Foltynie and Aviles-Olmos, 2014). Thus, therapeutic effects detected in mice models of Parkinson's disease induced by intoxication with MPTP, represent a solid basis for

121

promising treatments for patients. In conclusion, our findings demonstrated a significant therapeutic effect of D3R-antagonism in a mouse model of Parkinson's disease. This therapeutic effect was proved by improvement of motor coordination and by preventing neuronal death in the nigrostriatal pathway. Furthermore, our results suggest that this therapeutic effect could be mediated by the induction of an intermediate M1/M2-like phenotype in astrocytes, which could transmit an anti-inflammatory signal to microglial cells. These findings not only contribute to a better knowledge of the physiopathology of Parkinson's disease, but they also provide the clues for new therapeutic approaches for the treatment of this neurodegenerative disorder. Acknowledgements We thank Dr. Marc Caron for providing D3RKO mice, Dr. Amy Newman for providing PG01030 and PG01037 for HPLC analyses and Dr. María Rosa Bono for supporting A.M. thesis as advisor. We
n Valenzuela for his valuable veterinary also thank Dr. Sebastia assistance in our animal facility. This work was supported by grants 10332 from Michael J. Fox Foundation (Target Validation program), 1130271 from FONDECYT and PFB-16 from CONICYT. DE holds graduated fellowship from Universidad Andres Bello. AM holds a graduate fellowship number 21110148 from CONICT. ERB holds graduated fellowship from Colfuturo. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.neuropharm.2016.09.028. References Appel, S.H., 2009. CD4þ T cells mediate cytotoxicity in neurodegenerative diseases. J. Clin. Invest 119, 13e15. Aviles-Olmos, I., Dickson, J., Kefalopoulou, Z., Djamshidian, A., Ell, P., Soderlund, T., Whitton, P., Wyse, R., Isaacs, T., Lees, A., Limousin, P., Foltynie, T., 2013. Exenatide and the treatment of patients with Parkinson's disease. J. Clin. Invest 123, 2730e2736. Bedi, S.S., Smith, P., Hetz, R.A., Xue, H., Cox, C.S., 2013. Immunomagnetic enrichment and flow cytometric characterization of mouse microglia. J. Neurosci. Methods 219, 176e182. Besser, M.J., Ganor, Y., Levite, M., 2005. Dopamine by itself activates either D2, D3 or D1/D5 dopaminergic receptors in normal human T-cells and triggers the selective secretion of either IL-10, TNFalpha or both. J. Neuroimmunol. 169, 161e171. Brochard, V., Combadiere, B., Prigent, A., Laouar, Y., Perrin, A., Beray-Berthat, V., Bonduelle, O., Alvarez-Fischer, D., Callebert, J., Launay, J.M., Duyckaerts, C., Flavell, R.A., Hirsch, E.C., Hunot, S., 2009. Infiltration of CD4þ lymphocytes into the brain contributes to neurodegeneration in a mouse model of Parkinson disease. J. Clin. Invest 119, 182e192. Brooks, S.P., Dunnett, S.B., 2009. Tests to assess motor phenotype in mice: a user's guide. Nat. Rev. Neurosci. 10, 519e529. Burguillos, M.A., Deierborg, T., Kavanagh, E., Persson, A., Hajji, N., GarciaQuintanilla, A., Cano, J., Brundin, P., Englund, E., Venero, J.L., Joseph, B., 2011. Caspase signalling controls microglia activation and neurotoxicity. Nature 472, 319e324. Carpentier, P.A., Begolka, W.S., Olson, J.K., Elhofy, A., Karpus, W.J., Miller, S.D., 2005. Differential activation of astrocytes by innate and adaptive immune stimuli. Glia 49, 360e374. Chen, Y., Ni, Y.Y., Liu, J., Lu, J.W., Wang, F., Wu, X.L., Gu, M.M., Lu, Z.Y., Wang, Z.G., Ren, Z.H., 2013. Dopamine receptor 3 might be an essential molecule in 1methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced neurotoxicity. BMC Neurosci. 14, 76. Cutando, L., Busquets-Garcia, A., Puighermanal, E., Gomis-Gonzalez, M., DelgadoGarcia, J.M., Gruart, A., Maldonado, R., Ozaita, A., 2013. Microglial activation underlies cerebellar deficits produced by repeated cannabis exposure. J. Clin. Invest 123, 2816e2831. Damier, P., Hirsch, E.C., Zhang, P., Agid, Y., Javoy-Agid, F., 1993. Glutathione peroxidase, glial cells and Parkinson's disease. Neuroscience 52, 1e6. Derecki, N.C., Cardani, A.N., Yang, C.H., Quinnies, K.M., Crihfield, A., Lynch, K.R., Kipnis, J., 2010. Regulation of learning and memory by meningeal immunity: a key role for IL-4. J. Exp. Med. 207, 1067e1080.

122

D. Elgueta et al. / Neuropharmacology 113 (2017) 110e123

Diaz, J., Levesque, D., Griffon, N., Lammers, C.H., Martres, M.P., Sokoloff, P., Schwartz, J.C., 1994. Opposing roles for dopamine D2 and D3 receptors on neurotensin mRNA expression in nucleus accumbens. Eur. J. Neurosci. 6, 1384e1387. Diaz, J., Levesque, D., Lammers, C.H., Griffon, N., Martres, M.P., Schwartz, J.C., Sokoloff, P., 1995. Phenotypical characterization of neurons expressing the dopamine D3 receptor in the rat brain. Neuroscience 65, 731e745. Drojdahl, N., Nielsen, H.H., Gardi, J.E., Wree, A., Peterson, A.C., Nyengaard, J.R., Eyer, J., Finsen, B., 2010. Axonal plasticity elicits long-term changes in oligodendroglia and myelinated fibers. Glia 58, 29e42. Duffy, A.M., Fitzgerald, M.L., Chan, J., Robinson, D.C., Milner, T.A., Mackie, K., Pickel, V.M., 2011. Acetylcholine alpha7 nicotinic and dopamine D2 receptors are targeted to many of the same postsynaptic dendrites and astrocytes in the rodent prefrontal cortex. Synapse 65, 1350e1367. Farber, K., Pannasch, U., Kettenmann, H., 2005. Dopamine and noradrenaline control distinct functions in rodent microglial cells. Mol. Cell Neurosci. 29, 128e138. Farina, C., Aloisi, F., Meinl, E., 2007. Astrocytes are active players in cerebral innate immunity. Trends Immunol. 28, 138e145. Fernandez-Suarez, D., Celorrio, M., Riezu-Boj, J.I., Ugarte, A., Pacheco, R., Gonzalez, H., Oyarzabal, J., Hillard, C.J., Franco, R., Aymerich, M.S., 2014. The monoacylglycerol lipase inhibitor JZL184 is neuroprotective and alters glial cell phenotype in the chronic MPTP mouse model. Neurobiol. Aging 35, 2603e2616. Foltynie, T., Aviles-Olmos, I., 2014. Exenatide as a potential treatment for patients with Parkinson's disease: first steps into the clinic. Alzheimers Dement. 10, S38eS46. Franz, D., Contreras, F., Gonzalez, H., Prado, C., Elgueta, D., Figueroa, C., Pacheco, R., 2015. Dopamine receptors D3 and D5 regulate CD4(þ)T-cell activation and differentiation by modulating ERK activation and cAMP production. J. Neuroimmunol. 284, 18e29. Glass, C.K., Saijo, K., Winner, B., Marchetto, M.C., Gage, F.H., 2010. Mechanisms underlying inflammation in neurodegeneration. Cell 140, 918e934. Gonzalez, H., Contreras, F., Pacheco, R., 2015. Regulation of the neurodegenerative process associated to Parkinson's disease by CD4þ T-cells. J. Neuroimmune Pharmacol. 10, 561e575. Gonzalez, H., Contreras, F., Prado, C., Elgueta, D., Franz, D., Bernales, S., Pacheco, R., 2013. Dopamine receptor D3 expressed on CD4þ T cells favors neurodegeneration of dopaminergic neurons during Parkinson's disease. J. Immunol. 190, 5048e5056. Gonzalez, H., Elgueta, D., Montoya, A., Pacheco, R., 2014. Neuroimmune regulation of microglial activity involved in neuroinflammation and neurodegenerative diseases. J. Neuroimmunol. 274, 1e13. Gonzalez, H., Pacheco, R., 2014. T-cell-mediated regulation of neuroinflammation involved in neurodegenerative diseases. J. Neuroinflammation 11, 201. Gundersen, H.J., Jensen, E.B., 1987. The efficiency of systematic sampling in stereology and its prediction. J. Microsc. 147, 229e263. Harms, A.S., Cao, S., Rowse, A.L., Thome, A.D., Li, X., Mangieri, L.R., Cron, R.Q., Shacka, J.J., Raman, C., Standaert, D.G., 2013. MHCII is required for alphasynuclein-induced activation of microglia, CD4 T cell proliferation, and dopaminergic neurodegeneration. J. Neurosci. 33, 9592e9600. Hirsch, E.C., Hunot, S., 2009. Neuroinflammation in Parkinson's disease: a target for neuroprotection? Lancet Neurol. 8, 382e397. Huck, J.H., Freyer, D., Bottcher, C., Mladinov, M., Muselmann-Genschow, C., Thielke, M., Gladow, N., Bloomquist, D., Mergenthaler, P., Priller, J., 2015. De novo expression of dopamine D2 receptors on microglia after stroke. J. Cereb. Blood Flow. Metab. 35, 1804e1811. Ilani, T., Strous, R.D., Fuchs, S., 2004. Dopaminergic regulation of immune cells via D3 dopamine receptor: a pathway mediated by activated T cells. Faseb J. 18, 1600e1602. Joseph, J.D., Wang, Y.M., Miles, P.R., Budygin, E.A., Picetti, R., Gainetdinov, R.R., Caron, M.G., Wightman, R.M., 2002. Dopamine autoreceptor regulation of release and uptake in mouse brain slices in the absence of D(3) receptors. Neuroscience 112, 39e49. Joyce, J.N., Woolsey, C., Ryoo, H., Borwege, S., Hagner, D., 2004. Low dose pramipexole is neuroprotective in the MPTP mouse model of Parkinson's disease, and downregulates the dopamine transporter via the D3 receptor. BMC Biol. 2, 22. Kettenmann, H., Hanisch, U.K., Noda, M., Verkhratsky, A., 2011. Physiology of microglia. Physiol. Rev. 91, 461e553. Kim, S., Moon, M., Park, S., 2009. Exendin-4 protects dopaminergic neurons by inhibition of microglial activation and matrix metalloproteinase-3 expression in an animal model of Parkinson's disease. J. Endocrinol. 202, 431e439. Kuno, R., Yoshida, Y., Nitta, A., Nabeshima, T., Wang, J., Sonobe, Y., Kawanokuchi, J., Takeuchi, H., Mizuno, T., Suzumura, A., 2006. The role of TNF-alpha and its receptors in the production of NGF and GDNF by astrocytes. Brain Res. 1116, 12e18. Larsen, J.O., Gundersen, H.J., Nielsen, J., 1998. Global spatial sampling with isotropic virtual planes: estimators of length density and total length in thick, arbitrarily orientated sections. J. Microsc. 191, 238e248. Lee, H.J., Suk, J.E., Patrick, C., Bae, E.J., Cho, J.H., Rho, S., Hwang, D., Masliah, E., Lee, S.J., 2010. Direct transfer of alpha-synuclein from neuron to astroglia causes inflammatory responses in synucleinopathies. J. Biol. Chem. 285, 9262e9272. Li, Y., Perry, T., Kindy, M.S., Harvey, B.K., Tweedie, D., Holloway, H.W., Powers, K., Shen, H., Egan, J.M., Sambamurti, K., Brossi, A., Lahiri, D.K., Mattson, M.P., Hoffer, B.J., Wang, Y., Greig, N.H., 2009. GLP-1 receptor stimulation preserves primary cortical and dopaminergic neurons in cellular and rodent models of stroke and Parkinsonism. Proc. Natl. Acad. Sci. U. S. A. 106, 1285e1290.

Lu, L., Lan, Q., Li, Z., Zhou, X., Gu, J., Li, Q., Wang, J., Chen, M., Liu, Y., Shen, Y., Brand, D.D., Ryffel, B., Horwitz, D.A., Quismorio, F.P., Liu, Z., Li, B., Olsen, N.J., Zheng, S.G., 2014. Critical role of all-trans retinoic acid in stabilizing human natural regulatory T cells under inflammatory conditions. Proc. Natl. Acad. Sci. U. S. A. 111, E3432eE3440. Lucin, K.M., Wyss-Coray, T., 2009. Immune activation in brain aging and neurodegeneration: too much or too little? Neuron 64, 110e122. Magro, F., Cunha, E., Araujo, F., Meireles, E., Pereira, P., Dinis-Ribeiro, M., Veloso, F.T., Medeiros, R., Soares-da-Silva, P., 2006. Dopamine D2 receptor polymorphisms in inflammatory bowel disease and the refractory response to treatment. Dig. Dis. Sci. 51, 2039e2044. Malmberg, A., Jackson, D.M., Eriksson, A., Mohell, N., 1993. Unique binding characteristics of antipsychotic agents interacting with human dopamine D2A, D2B, and D3 receptors. Mol. Pharmacol. 43, 749e754. Mason, C.W., Hassan, H.E., Kim, K.P., Cao, J., Eddington, N.D., Newman, A.H., Voulalas, P.J., 2010. Characterization of the transport, metabolism, and pharmacokinetics of the dopamine D3 receptor-selective fluorenyl- and 2-pyridylphenyl amides developed for treatment of psychostimulant abuse. J. Pharmacol. Exp. Ther. 333, 854e864. Mastroeni, D., Grover, A., Leonard, B., Joyce, J.N., Coleman, P.D., Kozik, B., Bellinger, D.L., Rogers, J., 2009. Microglial responses to dopamine in a cell culture model of Parkinson's disease. Neurobiol. Aging 30, 1805e1817. McGuire, V., Van Den Eeden, S.K., Tanner, C.M., Kamel, F., Umbach, D.M., Marder, K., Mayeux, R., Ritz, B., Ross, G.W., Petrovitch, H., Topol, B., Popat, R.A., Costello, S., Manthripragada, A.D., Southwick, A., Myers, R.M., Nelson, L.M., 2011. Association of DRD2 and DRD3 polymorphisms with Parkinson's disease in a multiethnic consortium. J. Neurol. Sci. 307, 22e29. Mierau, J., Schneider, F.J., Ensinger, H.A., Chio, C.L., Lajiness, M.E., Huff, R.M., 1995. Pramipexole binding and activation of cloned and expressed dopamine D2, D3 and D4 receptors. Eur. J. Pharmacol. 290, 29e36. Miyazaki, I., Asanuma, M., Diaz-Corrales, F.J., Miyoshi, K., Ogawa, N., 2004. Direct evidence for expression of dopamine receptors in astrocytes from basal ganglia. Brain Res. 1029, 120e123. Nikcevich, K.M., Gordon, K.B., Tan, L., Hurst, S.D., Kroepfl, J.F., Gardinier, M., Barrett, T.A., Miller, S.D., 1997. IFN-gamma-activated primary murine astrocytes express B7 costimulatory molecules and prime naive antigen-specific T cells. J. Immunol. 158, 614e621. Noelker, C., Morel, L., Lescot, T., Osterloh, A., Alvarez-Fischer, D., Breloer, M., Henze, C., Depboylu, C., Skrzydelski, D., Michel, P.P., Dodel, R.C., Lu, L., Hirsch, E.C., Hunot, S., Hartmann, A., 2013. Toll like receptor 4 mediates cell death in a mouse MPTP model of Parkinson disease. Sci. Rep. 3, 1393. Pacheco, R., Contreras, F., Zouali, M., 2014. The dopaminergic system in autoimmune diseases. Front. Immunol. 5, 117. Pacheco, R., Gallart, T., Lluis, C., Franco, R., 2007. Role of glutamate on T-cell mediated immunity. J. Neuroimmunol. 185, 9e19. Paxinos, G., Franklin, K.B.J., 2001. The Mouse Brain in Stereotaxic Coordinates. Academic press. Prado, C., Bernales, S., Pacheco, R., 2013. Modulation of T-cell mediated immunity by dopamine receptor d5. Endocr. Metab. Immune Disord. Drug Targets 13, 184e194. Ransohoff, R.M., Perry, V.H., 2009. Microglial physiology: unique stimuli, specialized responses. Annu. Rev. Immunol. 27, 119e145. Reynolds, A.D., Glanzer, J.G., Kadiu, I., Ricardo-Dukelow, M., Chaudhuri, A., Ciborowski, P., Cerny, R., Gelman, B., Thomas, M.P., Mosley, R.L., Gendelman, H.E., 2008. Nitrated alpha-synuclein-activated microglial profiling for Parkinson's disease. J. Neurochem. 104, 1504e1525. Reynolds, A.D., Stone, D.K., Mosley, R.L., Gendelman, H.E., 2009. Nitrated {alpha}synuclein-induced alterations in microglial immunity are regulated by CD4þ T cell subsets. J. Immunol. 182, 4137e4149. Riquelme, E., Abarca, J., Campusano, J.M., Bustos, G., 2012. An NR2B-dependent decrease in the expression of trkB receptors precedes the disappearance of dopaminergic cells in substantia Nigra in a rat model of presymptomatic Parkinson's disease. Park. Dis. 2012, 129605. Sanchez-Guajardo, V., Febbraro, F., Kirik, D., Romero-Ramos, M., 2010. Microglia acquire distinct activation profiles depending on the degree of alpha-synuclein neuropathology in a rAAV based model of Parkinson's disease. PLoS One 5, e8784. Sanchez-Guajardo, V., Tentillier, N., Romero-Ramos, M., 2015. The relation between alpha-synuclein and microglia in Parkinson's disease: recent developments. Neuroscience 302, 47e58. Schwartz, M., Shaked, I., Fisher, J., Mizrahi, T., Schori, H., 2003. Protective autoimmunity against the enemy within: fighting glutamate toxicity. Trends Neurosci. 26, 297e302. Shao, W., Zhang, S.Z., Tang, M., Zhang, X.H., Zhou, Z., Yin, Y.Q., Zhou, Q.B., Huang, Y.Y., Liu, Y.J., Wawrousek, E., Chen, T., Li, S.B., Xu, M., Zhou, J.N., Hu, G., Zhou, J.W., 2013. Suppression of neuroinflammation by astrocytic dopamine D2 receptors via alphaB-crystallin. Nature 494, 90e94. Sibley, D.R., Monsma Jr., F.J., Shen, Y., 1993. Molecular neurobiology of dopaminergic receptors. Int. Rev. Neurobiol. 35, 391e415. Sokoloff, P., Giros, B., Martres, M.P., Bouthenet, M.L., Schwartz, J.C., 1990. Molecular cloning and characterization of a novel dopamine receptor (D3) as a target for neuroleptics. Nature 347, 146e151. Strange, P.G., 1993. New insights into dopamine receptors in the central nervous system. Neurochem. Int. 22, 223e236. Strange, P.G., 2001. Antipsychotic drugs: importance of dopamine receptors for

D. Elgueta et al. / Neuropharmacology 113 (2017) 110e123 mechanisms of therapeutic actions and side effects. Pharmacol. Rev. 53, 119e133. Sunahara, R.K., Guan, H.C., O'Dowd, B.F., Seeman, P., Laurier, L.G., Ng, G., George, S.R., Torchia, J., Van Tol, H.H., Niznik, H.B., 1991. Cloning of the gene for a human dopamine D5 receptor with higher affinity for dopamine than D1. Nature 350, 614e619. Torres-Rosas, R., Yehia, G., Pena, G., Mishra, P., del Rocio Thompson-Bonilla, M., Moreno-Eutimio, M.A., Arriaga-Pizano, L.A., Isibasi, A., Ulloa, L., 2014. Dopamine mediates vagal modulation of the immune system by electroacupuncture. Nat. Med. 20, 291e295. Wu, W.L., Burnett, D.A., Spring, R., Greenlee, W.J., Smith, M., Favreau, L., Fawzi, A., Zhang, H., Lachowicz, J.E., 2005. Dopamine D1/D5 receptor antagonists with improved pharmacokinetics: design, synthesis, and biological evaluation of phenol bioisosteric analogues of benzazepine D1/D5 antagonists. J. Med. Chem.

123

48, 680e693. Xing, B., Liu, P., Jiang, W.H., Liu, F., Zhang, H., Cao, G.F., Chen, T., Dang, Y.H., 2013. Effects of immobilization stress on emotional behaviors in dopamine D3 receptor knockout mice. Behav. Brain Res. 243, 261e266. Yan, Y., Jiang, W., Liu, L., Wang, X., Ding, C., Tian, Z., Zhou, R., 2015. Dopamine controls systemic inflammation through inhibition of NLRP3 inflammasome. Cell 160, 62e73. Zhang, Y., Huo, M., Zhou, J., Xie, S., 2010. PKSolver: an add-in program for pharmacokinetic and pharmacodynamic data analysis in Microsoft Excel. Comput. Methods Programs Biomed. 99, 306e314. Zou, J.Y., Crews, F.T., 2005. TNF alpha potentiates glutamate neurotoxicity by inhibiting glutamate uptake in organotypic brain slice cultures: neuroprotection by NF kappa B inhibition. Brain Res. 1034, 11e24.