Accepted Manuscript Title: Dopamine D2 and angiotensin II type 1 receptors form functional heteromers in rat striatum Author: E. Mart´ınez-Pinilla A.I. Rodr´ıguez-P´erez G. Navarro D. Aguinaga E. Moreno J.L. Lanciego J.L. Labandeira-Garc´ıa R. Franco PII: DOI: Reference:

S0006-2952(15)00252-X http://dx.doi.org/doi:10.1016/j.bcp.2015.05.006 BCP 12246

To appear in:

BCP

Received date: Accepted date:

10-3-2015 7-5-2015

Please cite this article as: Mart´inez-Pinilla E, Rodr´iguez-P´erez AI, Navarro G, Aguinaga D, Moreno E, Lanciego JL, Labandeira-Garc´ia JL, Franco R, Dopamine D2 and angiotensin II type 1 receptors form functional heteromers in rat striatum, Biochemical Pharmacology (2015), http://dx.doi.org/10.1016/j.bcp.2015.05.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Dopamine D2 and angiotensin II type 1 receptors form functional heteromers in rat striatum Martínez-Pinilla E1&, Rodríguez-Pérez AI2&, Navarro G3,4&, Aguinaga D3,4 , Moreno E3,4,

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Lanciego JL1,4, Labandeira-García JL2,4* and Franco R3,4*

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1. Neuroscience Department, Center for Applied Medical Research (CIMA). University of Navarra. Pamplona. Spain.

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2. Laboratory of Neuroanatomy and Experimental Neurology. Department of Morphological Sciences, Center for Research in Molecular Medicine and Chronic Diseases (CIMUS).

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University of Santiago de Compostela. Santiago de Compostela. Spain.

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3. Laboratory of Molecular Neurobiology. Department of Biochemistry and Molecular Biology. Faculty of Biology. University of Barcelona. Barcelona. Spain.

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4. Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas

These authors contributed equally

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&

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(CIBERNED). Spain.

* Senior authors of the manuscript Corresponding author: Eva Martínez-Pinilla

Laboratory of Cell and Molecular Neuropharmacology Center for Applied Medical Research (CIMA)-University of Navarra Pio XII 55, 31008, Pamplona, Spain Email: [email protected]

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Abstract Identification of G protein-coupled receptors and their specific function in a given neuron becomes essential to better understand the variety of signal transduction mechanisms associated

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with neurotransmission. We hypothesized that angiotensin II type 1 (AT1) and dopamine D2 receptors form heteromers in the central nervous system, specifically in striatum. Using

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bioluminescence resonance energy transfer a direct interaction was demonstrated in cells transfected with the cDNA for the human version of the receptors. Heteromerization did not

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affect cAMP signaling via D2 receptors but attenuated the coupling of AT1 receptors to Gq. A common feature of heteromers, namely cross-antagonism, i.e. the blockade of the signaling of

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one receptor by the blockade of the partner receptors, was tested in co-transfected cells. Candesartan, the selective AT1 receptor antagonist, was able to block D2-receptor mediated

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effects on cAMP levels, MAP kinase activation and β-arrestin recruitment. This effect of candesartan, which constitutes a property for the dopamine-angiotensin receptor heteromer, was

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similarly occurring in primary cultures of neurons and rat striatal slices. The expression of

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heteromers in striatum was confirmed by robust labeling using in situ proximity ligation assays. The results indicate that AT1 receptors are expressed in striatum and form heteromers with

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dopamine D2 receptors that enable drugs selective for the AT1 receptor to alter the functional response of D2 receptors.

Keywords

GPCR heteromerization; basal ganglia; BRET; cAMP; calcium release.

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Abbreviations ATR, Angiotensin receptors; AT1R, Angiotensin II receptors (subtype 1); BRET, bioluminescence resonance energy transfer; CLSM, confocal laser-scanning microscope; CNS,

diethylpyrocarbonate;

DMEM,

Dulbecco's

modified

eagle

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central nervous system; D1R, dopamine D1 receptors; D2R, dopamine D2 receptors; DEPC, medium;

DMSO,

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dimethylsulphoxide; ERK1/2, extracellular-signal regulated kinase; FBS, fetal bovine serum;

GABABR, receptor for GABAB; GPCR, G protein-coupled receptor; HBSS, Hanks' balanced solution;

HEK-293T,

human

embryonic

kidney

293T;

L-DOPA,

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salt

L-3,4-

dihydroxyphenylalanine; MAPK, mitogen-activated protein kinase; mBU, milli BRET units;

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PB, phosphate buffer; PBS, phosphate buffered saline; PD, Parkinson’s disease; PEI, polyethylenimine; PLA: in situ proximity ligation assay; RAS, renin-angiotensin system; RLU,

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relative light units; TBS, Tris buffered saline.

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1. Introduction Dopamine is involved in almost every central action: motor control, reward, cognition, etc.

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Dopamine actions are mediated by five G protein-coupled receptors (GPCRs) that are classified as D1-like and D2-like depending on the coupling to, respectively Gs or Gi proteins. The

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presence of dopamine receptors in the central nervous system (CNS), mainly in the basal

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ganglia, reflects unequivocally their important role in motor function and dysfunction. In the current model of basal ganglia function, GABAergic dynorphinergic neurons constitute the direct pathway and mainly contain dopamine D1 receptors (D1R), and enkephalin-rich

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GABAergic striatal neurons constitute the indirect pathway and mainly contain dopamine D2 receptors (D2R). Motor control is achieved by a balance between dopamine acting on D1R of the

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direct pathway and on D2R in neurons of the indirect pathway [1-3]. The loss of this equilibrium in the basal ganglia circuits is associated with the development of motor complications in

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neurodegenerative disorders such as Parkinson’s disease (PD).

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Kidney is probably the main target of the renin-angiotensin system (RAS) that ultimate

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produces angiotensin II whose key role is the control of blood pressure and water homeostasis [4]. Over the last three decades, all components of the RAS have been also identified in the nervous system [5-7]. Thus, Sirett et al. (1981) quantitated angiotensin II in brain by radioimmunoassay showing the highest expression in hippocampus, the lowest in cortex and intermediate in striatum. The precursor protein, angiotensinogen is produced by astrocytes [8,9] and to a minor extent by neurons [10,11]. Moreover, brain levels of angiotensin II are higher than circulating levels [12]. This is due to the enzymatic action of renin and of complexes of pro-renin and renin receptors expressed in both neurons and glial cells [13]. Pro-renin to renin ratio is 5-10 times higher in brain than in the periphery [14] and hence, brain pro-renin appears as key to control angiotensin II production. Consistent with these findings, angiotensin II type 1 receptors (AT1R) have been observed both in neurons and glial cells from rodents and primates,

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including humans [15,16]. Radioligand binding to rat brain membranes showed heterogeneous CNS expression of angiotensin II type 1 and type 2 receptors (see [17] and references therein). Autoradiography demonstrated the expression of the two receptors in brain with predominance of type 1 in regions enriched in angiotensin II [18]. Subsequent to cDNA cloning, molecular

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biology tools and binding ligation assays confirmed expression of the two receptors in brain

[19-23]. Multiple observations suggest the existence of functional interactions between the RAS

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and the dopaminergic system. For example imbalance of the dopaminergic system has been

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shown to induce compensatory regulation of the RAS in the substantia nigra and the striatum [24]. A potential role for the angiotensin system in dopaminergic neurodegeneration [25] has

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been suggested by the neuroprotection afforded by captopril, an inhibitor of the angiotensinconverting enzyme [26]. In fact, in a chronic regime the compound protects dopaminergic

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neuronal cell bodies from death induced by intracerebral administration of 1-methyl-4phenylpyridinium in rats. Captopril also protects the striatum in the acute model of parkinsonism in mice, which consists of systemic administration of 1-methyl-4-phenyl-1,2,3,6-

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tetrahydropyrine [26]. The recently described beneficial effect of selective AT1R antagonists in

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animal models of PD [27,28] suggests that this receptor is important in the function of neurons

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of the basal ganglia circuits. A functional interaction between dopamine and angiotensin II receptors was first demonstrated in the periphery [29]. Interestingly, a functional interaction exists between dopamine and angiotensin systems, which counterbalance each other in renal cells but also in the striatum and substantia nigra [24,30,31]. A more specific interaction between D1R and AT1R is deduced from work in transfected human embryonic kidney 293T (HEK-293T) cells in which binding of the angiotensin antagonist, losartan, to AT1R leads to an increase in dopamine-receptor activation [32]. Moreover a direct interaction with the dopamine D1, D3 and D5 receptor subtypes has been demonstrated in renal proximal tubule cells [33-35]. The aim of the present study was to determine whether AT1 and D2 receptors form heteromers, whether this physical interaction has functional consequences and whether functional heteromers are present in the mammalian striatum.

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2. Methods 2.1. Drugs and Animals

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(-)-Quinpirole hydrochloride and candesartan were from Tocris Bioscience (Bristol, UK). Dopamine, raclopride, angiotensin II and forskolin were from Sigma (Sigma-Aldrich, St. Louis,

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MO, USA). 10 mM stock solutions were prepared in DMSO and aliquots were kept frozen at -

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20°C until use.

Sprague-Dawley female rats, 7-9 weeks old and weighing 200-250 g and pregnant rats

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(gestational period 18-20 days), were provided by the animal Service of the University of Navarra (Pamplona, Spain) and the University of Barcelona respectively. The animals were maintained in positive pressure-ventilated racks at 25 ± 1°C with a 12 h light/dark cycle, fed ad

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libitum with a standard rodent pellet diet (Global Diet 2014; Harlan), and allowed free access to

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filtered and UV-irradiated water. Animal procedures were conducted according to the

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Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research (2003) and approved by the Ethical Committees of the Institutions and of the regional Governments. 2.2. Primary cultures of neurons

Striatal neurons were prepared from Sprague Dawley embryos. Neurons were isolated as described in [36] and plated at 40,000 cells/0.32 cm2 confluence. Striatal cells were grown for 12 days in Neurobasal medium (NB) supplemented with 2 mM L-glutamine, 100 U/ml penicillin/streptomycin, and 2% (v/v) B27 supplement (Gibco, Life Technologies, Madrid, Spain) in a 6-well plate. 2.3. Rat perfusion and tissue processing

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Anesthetized animals were transcardially perfused with a fixative solution containing 4% paraformaldehyde in 0.125 M phosphate buffer (PB), pH 7.4. Perfusion was continued with a cryoprotectant solution containing 10% glycerin and 2% dimethylsulphoxide (DMSO) in 0.125 M PB, pH 7.4. Once perfusion was completed, the skull was opened, the brain was removed and

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stored for 48 hours in a cryoprotectant solution containing 20% of glycerin and 2% DMSO in

0.125 M PB, pH 7.4. All solutions used for perfusion and cryoprotection were treated with 0.1%

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diethylpyrocarbonate (DEPC) and autoclaved prior to use. Finally, frozen serial coronal sections

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(40 μm thick) were obtained on a sliding microtome and collected in 0.125 M PB, pH 7.4, as 10 series of adjacent sections.

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2.4. Rat brain slice preparation

Rats were anesthetized with 4% isoflurane (2-chloro-2-(difluoromethoxy)-1,1,1-trifluoro-

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ethane) and beheaded with a guillotine; brain was rapidly removed and placed in ice-cold oxygenated (O2/CO2: 95/5%) Krebs-HCO3- buffer (124 mM NaCl; 4 mM KCl; 1.25 mM

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NaH2PO4; 1.5 mM MgCl2; 1.5 mM CaCl2; 10 mM glucose; and 26 mM NaHCO3 pH 7.4).

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Brains were sliced at 4°C in a brain matrix (Zivic Instruments, Pittsburgh, PA) into 0.5 mm coronal slices and the striatum area was dissected. Slices were kept at 4°C in Krebs-HCO3-

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buffer during the dissection of the striatum. Each slice was transferred into an incubation tube containing 1 ml of ice-cold Krebs-HCO3- buffer. The temperature was raised to 23°C and after 30 min the medium was replaced by 2 ml Krebs-HCO3- buffer (23°C). Slices were incubated under constant oxygenation (O2/CO2: 95/5%) at 30°C for 4-5 hours in an Eppendorf thermomixer (5 Prime, Inc., Boulder, CO). The media was replaced by 200 µl of fresh KrebsHCO3- buffer and incubated for 30 min before the addition of ligands. Extracellular-signal regulated kinase (ERK1/2) phosphorylation was determined as described below. 2.5. Fusion proteins and expression vectors The human cDNAs for the long isoform of D2, AT1 or GABAB receptors (GABABR) cloned in pcDNA3.1 were amplified without their stop codons using sense and antisense primers

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harbouring either unique EcoRI and BamH1 (D2R, AT1R) or BamHI and HindIII (GABABR) sites. The fragments were then sub-cloned into EcoRI and BamH1 (AT1R) or BamHI and HindIII (GABABR) restriction sites of an Rluc-expressing vector (pRluc-N1, PerkinElmer, Wellesley, USA) and of an YFP-expressing vector (pYFP-N1, PerkinElmer, Wellesley, USA),

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or into the BamHI and EcoRI (D2R) restriction sites of an YFP-expressing vector to give the plasmids that express AT1R, GABABR or D2R fused to Rluc or YFP on the C-terminal end of

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the receptor (AT1R-Rluc, AT1R-YFP, GABABR-Rluc or D2R-YFP). Expression of receptors

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was tested by either fluorescent confocal microscopy or Rluc expression, and receptor function was tested by performing ERK1/2 activation assays. Human β-arrestin 2-Rluc, cloned in the

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pcDNA3.1 RLuc vector (pRLuc-N1 PerkinElmer, Wellesley, USA), was generously given by Dr. Marian Castro from University of Santiago de Compostela, Spain.

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2.6. Cell line cultures and transfection

HEK-293T cells were grown in Dulbeco’s modified Eagle’s medium (DMEM) supplemented

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with 2 mM L-glutamine, 1 mM sodium pyruvate, 100 units/ml penicillin/streptomycin, and 5%

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(v/v) heat-inactivated fetal bovine serum (FBS) (all supplements were from Invitrogen, Paisley, Scotland, UK). Cells were maintained at 37°C in a humidified atmosphere of 5% CO2 and were

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passaged when they reached 80-90% confluent, i.e. approximately twice a week. HEK-293T cells growing in 35-mm-diameter six-well plates were transiently transfected with the corresponding fusion proteins cDNAs (see figure legend) using ramified PEI (PolyEthylenImine, Sigma-Aldrich, St. Louis, MO, USA). Cells were incubated with the corresponding cDNA together with PEI (5 ml of 10 mM PEI for each mg of cDNA) and 150 mM NaCl in a serum-starved medium. After 4 hours, the medium was changed to a fresh complete culture medium. 48 hours after transfection, bioluminescence resonance energy transfer (BRET) was determined. Cells were washed twice in quick succession in Hanks' balanced salt solution (HBSS; 137 mM NaCl; 5 mM KCl; 0.34 mM Na2HPO4x12H2O; 0.44 mM KH2PO4; 1.26 mM CaCl2x2H2O; 0.4 mM MgSO4x7H2O; 0.5 mM MgCl2; and 10 mM HEPES

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pH 7.4) supplemented with 0.1% glucose (w/v), detached by gently pipetting and resuspended in the same buffer. To adjust the amount of protein of the different samples to 0.2 mg/mL, protein concentration was determined using a Bradford assay kit (Bio-Rad, Munich, Germany) with bovine serum albumin (Sigma-Aldrich, St. Louis, MO, USA) dilutions as standards. HEK-

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293T cell suspensions (20 µg of protein) were distributed into 96-well microplates; black plates with a transparent bottom (Porvair, Leatherhead, UK) were used for fluorescence whereas white

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2.7. Bioluminescence Resonance Energy Transfer (BRET) assay

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opaque plates (Porvair, Leatherhead, UK) were used for BRET experiments.

HEK-293T cells were transiently co-transfected with the indicated amounts of plasmid cDNAs

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corresponding to the indicated fusion proteins (see corresponding figure legends). To quantify receptor-fluorescence expression, cells (20 µg protein) were distributed in 96-well microplates

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(black plates with a transparent bottom; Porvair, Leatherhead, UK) and fluorescence was read using a Mithras LB 940 (Berthold, Bad Wildbad, Germany) equipped with a high-energy xenon

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flash lamp, using an excitation filter of 485 nm. Receptor-fluorescence expression was

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determined as fluorescence of the sample minus the fluorescence of cells expressing proteinRluc alone. For BRET measurements, the equivalent of 20 µg of cell suspension was distributed

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in 96-well microplates (white opaque plates; Porvair, Leatherhead, UK) and 5 µM Coelenterazine h (Molecular Probes, Life Technologies, Madrid, Spain) was added. 1 min after addition of compounds, readings were collected using a Mithras LB 940 (Berthold, Bad Wildbad, Germany) that allows the integration of the signals detected in the short-wavelength filter at 485 nm (440-500 nm) and the long-wavelength filter at 530 nm (510-590 nm). To quantify receptor-Rluc luminescence, readings were performed after 10 min of adding 5 µM Coelenterazine h. The net BRET is defined as [(long-wavelength emission)/(short-wavelength emission)]-Cf where Cf corresponds to [(long-wavelength emission)/(short-wavelength emission)] for the Rluc construct expressed alone in the same experiment. BRET curves were fitted by using a non-linear regression equation, assuming a single phase with GraphPad Prism

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software (San Diego, CA, USA). BRET is expressed as milli BRET units (mBU= 1,000

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netBRET).

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2.8. Immunocytochemistry

Immunocytochemistry assays were performed as previously described [37] using the primary

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mouse anti-D2R antibody (1/200; Santa Cruz, Texas, USA) and a secondary Cy3-conjugated anti-mouse antibody (1/200; Jackson ImmunoResearch, Baltimore, PA, USA). AT1R fused to

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YFP protein was detected by its fluorescence properties. Samples were observed in a Leica SP2

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confocal microscope (Leica Microsystems, Wetzlar, Germany). 2.9. cAMP measurements

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cAMP concentration was determined by an homogenous setup using the LANCE Ultra cAMP

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kit (PerkinElmer, Wellesley, USA) and a Pherastar FS Microplate Reader (BMG Labtech, Ortenberg, Germany), following the instructions of the supplier. Transfection was performed in

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a 384-well plate containing 5,000 cell/well for HEK-293T cells or 7,500 cell/well for primary cultures of striatal neurons. HEK-293T and primary culture cells in stimulation buffer (serumstarved DMEM medium supplemented with 50µM zardeverine, 5 mM HEPES and 0,1% BSA Stabilizer), containing or not 0.5 μM forskolin, were treated with the compounds at the concentrations indicated in Figure legends. 2.10. β-Arrestin recruitment assays Arrestin recruitment was determined by BRET assays in HEK-293T cells, 48 hours after transfection with the indicated amounts of cDNA corresponding to AT1R-YFP and β-arrestin-2Rluc. Cells (20 μg protein/well from a cell suspension in 96-well microplates) were treated for 10 min with 100 nM candesartan and 5 μM Coelenterazine H was added before stimulation with

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200 nM quinpirole for 10 min. The BRET signal between β-arrestin 2-Rluc and AT1R-YFP was measured as indicated above. 2.11. Calcium release assays

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Calcium release assays in HEK-293T cells were developed by using the ultrasensitive protein

calcium indicator GCaMP6s (Addgene, Cambridge, MA, USA) [38]. Around 500,000 HEK-

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293T cells plated in 6 well plates were transiently transfected with 1 µg cDNA encoding for

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AT1R, 0.75 µg cDNA encoding for D2R or both, plus 2 µg cDNA encoding for GCamP6s. 48 hours post transfection, cells were treated for 10 min with 10 nM candesartan or 1 µM raclopride before the addition of 250 nM angiotensin II. The fluorescence signal was determined

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using an EnSpire® Multimode Plate Reader (PerkinElmer, Waltham, MA, USA).

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2.12. ERK1/2 phosphorylation assays

ERK1/2 phosphorylation in striatal neurons was determined using AlphaScreen®SureFire® kit

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(Perkin Elmer, Wellesley, USA) following the instructions of the supplier and using an

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EnSpire® Multimode Plate Reader (PerkinElmer, Waltham, Wellesley, USA). Around 40,000 cell/well for primary cultures of striatal neurons were placed in 384-well plates with serum-

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starved DMEM medium. Striatal cells were activated for 10 min with 100 nM candesartan before the addition of 200 nM quinpirole. For HEK-293T-transfected cells and rat striatal slices, ERK1/2 phosphorylation levels were determined by immunoblotting. For that, both cells and striatal slices were treated or not with the indicated ligands (200 nM quinpirole or 10 or 100 nM candesartan for, respectively cells and slices) for the indicated time (see corresponding figure legends), rinsed with 100 µl ice-cold PBS and treated with 150 µl ice-cold lysis buffer (50 mM Tris-HCl pH 7.4; 50 mM NaF; 150 mM NaCl; 45 mM β-glycerophosphate; 1% Triton X-100; 0.4 mM NaVO4; and protease inhibitor mixture). The suspension was centrifuged at 13,000xg for 5 min at 4°C, and protein content was quantified by the bicinchoninic acid method using bovine serum albumin dilutions as standard. To determine the level of ERK1/2 phosphorylation, equivalent amounts of protein

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(15 µg) were mixed with 6X Laemli sample buffer, separated by electrophoresis on a denaturing 10% SDS-polyacrylamide gel and transferred onto nitrocellulose membranes (BioTrace™ NT Nitrocellulose Transfer Membrane, 66485, PALL). After that, Odyssey blocking buffer (LICOR Biosciences, Lincoln, Nebraska, USA) was added, and the membranes were rocked for 60

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min. The membranes were then probed with a mixture of a mouse anti-phospho-ERK1/2 antibody (1:1,000 dilution; M8159 Sigma-Aldrich, St. Louis, MO, USA) and rabbit anti-

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ERK1/2 antibody that recognizes both phosphorylated and non-phosphorylated ERK1/2

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(1:20,000; M5670 Sigma-Aldrich, St. Louis, MO, USA) in blocking buffer for 2-3 hours at room temperature. After washing three times in TBS/Tween20, membranes were incubated with

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a mixture of IRDye®CW 800 (anti-mouse) antibody (1:15,000; 926-32210 LI-COR Biosciences, Lincoln, Nebraska, USA) and IRDye® 680RD (anti-rabbit) antibody (1:15,000; 926-68071 LICOR Biosciences, Lincoln, Nebraska, USA) for 1 hour at room temperature. Bands were

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visualized by the Odyssey® Fc Imaging System (LI-COR Biosciences, Lincoln, Nebraska, USA) and their densities quantified using the Image Studio software 1.1 (LI-COR Biosciences,

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Lincoln, Nebraska, USA). The level of phosphorylated ERK1/2 isoforms was normalized for

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differences in loading using the total ERK1/2 protein band intensities.

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2.13. In situ proximity ligation assay (PLA) The PLA technique was carried out in rat paraformaldehyde-fixed brain sections containing the striatum. The presence/absence of receptor-receptor molecular interactions in the samples was detected using the Duolink II in situ PLA detection kit (developed by Olink Bioscience, Uppsala, Sweden; and now distributed by Sigma-Aldrich as Duolink® using PLA® Technology). To create our PLA probes we conjugated a rabbit anti-AT1R antibody (sc-579; Santa Cruz Biotechnology, Dallas, Texas, USA) with a PLUS oligonucleotide (Duolink®In Situ Probemaker PLUS DUO92009, Sigma-Aldrich, St. Louis, MO, USA) and a rabbit antidopamine D2R antibody (AB5084P; Millipore, Merck KGaA, Darmstadt, Alemania) with a MINUS oligonucleotide (Duolink®In Situ Probemaker MINUS DUO92010, Sigma-Aldrich, St. Louis, MO, USA) following the manufacturer´s instructions. Sections were washed in 50 mM

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Tris-HCl, 0.9% NaCl pH 7.4 buffer (TBS), permeabilized with TBS containing 0.1% Triton X100 for 12 min and finally washed with TBS. After permeabilization, sections were washed at room temperature, incubated in a preheated humidity chamber for 1 hour at 37°C with the blocking solution and then incubated overnight with the PLA probe-linked antibodies (1:50 and

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1:200 for anti-AT1R and anti-D2R antibodies, respectively) at 4°C. After washing with buffer A (Wash buffer A, DUO82047, Sigma-Aldrich, St. Louis, MO, USA) at room temperature,

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sections were incubated with the ligation solution for 1 hour at 37°C in a humidity chamber.

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Following washes with buffer A, sections were incubated with the amplification solution for 100 min at 37°C in a humidity chamber and then washed in buffer B (Wash buffer B,

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DUO82048, Sigma-Aldrich, St. Louis, MO, USA), followed by another wash with buffer B x0.01. Finally, samples were mounted using a mounting medium containing the nuclear marker

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DAPI (Duolink® In Situ Mounting Medium with DAPI DUO82040, Sigma-Aldrich, St. Louis, MO, USA). Appropriate negative control assays were carried out to ensure lack of nonspecific

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labeling and amplification.

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The specificity of the anti-AT1R antibody sc-579 was confirmed in previous studies using transgenic mouse models with AT1R targeted selectively to neurons [39], pre-adsorption with

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the corresponding synthetic peptide antigen [40] and by comparing regions that are known to express AT1R (positive controls) and regions that lack AT1R (negative controls; [40, 41]). In addition, we confirmed the PLA results with a second antibody (sc-31181; Santa Cruz Biotechnology, Dallas, Texas, USA) whose specificity was tested in our laboratory by western blot analysis of lysates from HEK-293T cells transfected with AT1R tagged to fusion tail (DDK, Origene Technologies, Rockville, USA). The results confirmed the specificity of the antibodies used as we observed a predominant immunoreactive band in positive-transfected lysate compared to negative control, which consisted in an empty vector transfected lysate. The specificity of the anti-dopamine D2R antibody was confirmed in several previous studies using transgenic mice overexpressing D2R and D2R KO mice [42], as well as pre-adsorption with the corresponding synthetic peptide antigen [43,44].

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Stained samples were inspected under a Zeiss 510 Meta confocal laser-scanning microscope (CLSM). To ensure appropriate visualization of the labeled elements and to avoid false positive results, the emission following excitation with the helium laser at 543 nm was filtered through a band pass filter of 560-615 nm and color coded in light red. Finally, a long pass filter of 650 nm

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was used to visualize the emission from the helium laser at 633nm and color coded in blue.

For

each

field

of

view,

a

stack

of

two

channels

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Images were acquired using the Zeiss software (Aim4). (one

per

staining)

and

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13 to 22 Z stacks with a step size of 0.7 µm were acquired. A quantification of cells containing one or more red spots versus total cells (blue nucleus) was determined considering a total 2200-

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2500 cells from 18 different fields within striatum from three different animals per group using the Fiji package (http://pacific. mpi-cbg.de/). Nuclei and red spots were counted on the

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maximum projections of each image stack. After getting the projection each channel was processed individually. The nuclei were segmented by filtering with a median filter, subtracting

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the background, enhancing the contrast and finally applying a threshold to obtain the binary

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image and the regions of interest (ROI) around each nucleus. Red spots images were also filtered and thresholded to obtain the binary images. Red spots were counted in each of the

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ROIs obtained in the nuclei images. 2.14. Statistical analysis

Data result from at least 4 independent experiments. The data in graphs are the mean ± SD. Statistical analysis was performed with SPSS 18.0 software. The test of Kolmogorov-Smirnov with the correction of Lilliefors was used to evaluate normal distribution and the test of Levene to evaluate the homogeneity of variance. Significance was analyzed by one-way ANOVA followed by multiple comparisons Tukey’s test, or by Student’s t-test. Significant differences were considered when p 80% of the maximal effect and the effect of 500 nM was not significantly different than that achieved by 200 nM of the compound. When candesartan, the AT1R antagonist was tested, we did not

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observe any effect in cells expressing D1, AT1 or the two receptors. Interestingly, the dopamine-

induced reduction in the forskolin-induced intracellular cAMP accumulation (Fig. 4A) was

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blocked in a dose-dependent fashion by candesartan in cells co-transfected with D2R and AT1R.

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The reversion induced by candesartan was partial at 10 nM and total at 100 nM (Fig. 4B). A similar experiment using 200 nM quinpirole as agonist led to a similar result, i.e. reversion by

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10 nM candesartan (Fig. 5Ai-Aiii). Another readout, D2R agonist-induced recruitment of βarrestins to D2R, was also assayed and the results were similar to those encountered in cAMP

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assays, i.e. candesartan blocking the recruitment induced by quinpirole in co-transfected cells but not in cells only expressing the D2R (Fig. 5Bi-Biii). Candesartan was neither able to recruit β-arrestins in cells only expressing AT1R (Fig. 5Biii). Finally, ERK1/2 phosphorylation assays

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showed that candesartan was able to blunt, in co-transfected and not in D2R-expressing cells, the

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MAP kinase phosphorylation induced by dopamine receptor activation (Fig. 6). These results

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point to a functional heterodimer unit in which the cross-antagonism, one of the common heteromer features, is identifiable.

Next, we investigated how D2R antagonists would affect signaling mediated by AT1R, which couple to Gq. Using a well-established fluorescene assay [38] that detects the increase of intracellular calcium levels, angiotensin II led to a transient peak in cells expressing AT1R but not in cells expressing D2R. The effect of angiotensin II in cells expressing AT1R was totally inhibited by candesartan but not by the D2R antagonist, raclopride. Interestingly, the angiotensin II-mediated signal in D2R transfected cells was qualitatively different in co-transfected cells and, also, raclopride affected it. The less robust signal in cells expressing heteromers indicates a less efficient coupling to Gq and the D2R antagonist led to a Gq coupling similar to that obtained in cells expressing only the AT1R. These data constitute an example of one of the

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common attributes of receptor heteromers, which is the ability that one receptor has to modify the downstream signaling of the partner receptor [45-47]. On the one hand, our data indicate that the calcium mobilization (Fig. 7) but not the cAMP signaling (Fig. 3) was qualitatively affected by AT1-D2 heteromerization. On the other hand, signaling via Gq-coupled AT1R or via

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Gi-coupled D2R was reciprocally affected by the antagonist of the partner receptor. Crossantagonism, which consists of the blockade of one receptor’s signaling by the antagonist of the

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3.3. AT1R and D2R heteromers are expressed in rat striatum

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partner receptor [48-50], is another frequently identified heteromer property.

To look for AT1R-D2R functional heteromers in the CNS, cross-antagonism was investigated in

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striatal neuron primary cultures. Neurons were treated with quinpirole and/or candesartan and forskolin-induced cAMP (Fig. 8A) and ERK1/2 phosphorylation levels (Fig. 8B) were

M

determined. The results are qualitatively similar to those encountered in co-transfected HEK293T cells, i.e. candesartan blocking the effect of quinpirole. We also investigated, in rat striatal

d

slices, whether the signaling pathway originated from D2R is regulated by co-expression of

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AT1R. We observed that the specific D2R agonist, quinpirole, led to an increase in the phosphorylation of ERK1/2 after 10 min of activation (Fig. 9). The significant ERK1/2

Ac ce p

phosphorylation obtained in slices treated with the specific D2R agonist was blocked by the AT1R antagonist, candesartan, which was not able to significantly affect the basal pERK1/2 levels (Fig. 9). This clear cross-antagonism indicates that D2R and AT1R are expressed and form functional heteromers in rat striatum. Using the in situ proximity ligation assay (PLA), a punctuate fluorescent signal reflecting an AT1R-D2R heteromer can be detected by confocal microscopy in assays performed in rat striatal sections. The PLA signal is only detectable if the two receptors are close enough to allow the two DNA-tagged probes to form double-stranded segments (Fig. 10C) [42, 51]. Pairs of D2R and AT1R are visualized as red spots surrounding DAPI-counterstained neuronal nuclei (Fig. 10A,D). A marked and significant reduction in the percentage of positive cells was observed in

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control sections when incubated with only one primary antibody, as shown (Fig. 10B,D). Taken together, data gathered from cross-antagonism experiments as well as from the PLA technique strongly supported the presence of AT1R-D2R heteromers in the rat CNS.

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4. Discussion Angiotensin receptors are expressed in cells of the basal ganglia thus suggesting that

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angiotensin II behaves there as a neuromodulator. Furthermore, it has been suggested that the

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density of AT1R is higher in human striatum and substantia nigra than in equivalent regions of other mammals [52,53]. In a series of studies [13,54], we demonstrated, by immunofluorescence and laser confocal microscopy, the presence of AT1R and AT2R receptors in nigral

an

dopaminergic neurons and glial cells (i.e. astrocytes and microglia) in rodents and primates, including human [16]. We have also observed AT1R and AT2R receptors in both types of striatal

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projection neurons constituting the direct and indirect pathways and in striatal glial cells (Labandeira-Garcia et al. unpublished observations). In the brain, an interaction between

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angiotensin II and dopamine was initially suggested from microdialysis studies showing that

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acute angiotensin II perfusion induces dopamine release, which may be blocked by AT1R antagonists [55,56]. The mechanism responsible for the angiotensin II-induced dopamine

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release has not been clarified, although the possible involvement of D2 autoreceptors was suggested [55]. More recently, we have shown functional interactions and counterbalancing mechanisms mediated by angiotensin and dopamine receptors in the striatum and substantia nigra of rodents [57,58]. Here, we report the presence of AT1R in striatum in cells expressing dopamine D2R. Moreover, we also tested the hypothesis of D2R and AT1R heteromerization in this specific CNS region.

The data obtained using different methods of receptor heteromer identification, shows that AT1R and D2R may form heteromers in both co-transfected cells and in rat striatum. The selective AT1R antagonist, candesartan, was able to blunt the effects of D2R activation in cotransfected cells, in brain slices and in primary cultures of neurons. Heteromer-specific

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signaling and cross-antagonism are often identified as fingerprints of the heteromer [45-50]. It should be noted that the two interacting receptors are coupled to different G proteins, Gi and Gq and that AT1R-mediated calcium mobilization was attenuated in cells expressing the two receptors, possibly due to allosteric interactions within the receptor heteromer-Gi-Gq complex.

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This hypothesis is supported by the finding that occupation of the D2R by the antagonist,

raclopride, resulted in better Gq coupling. It is likely that conformational changes induced by

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the antagonist leads to D2R conformational changes that result -by allosteric interactions- in a

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better coupling of AT1R and Gq, i.e. a coupling that becomes similar to that obtained in the absence of D2R. Conformational changes upon receptor heteromerization and allosteric within

the

heteromer-G

protein

complex

predict

heteromer-selective

an

interactions

pharmacological profiles. In fact heteromer-selective compounds have been described (e.g.

M

SKF83959 for dopamine D1-D2 receptor heteromers [59] and different affinities for a given antagonist upon binding to a given receptor (adenosine), in different heteromeric contexts, have been demonstrated [60]. These cross-modulations may be useful to match in vitro with in vivo

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dopaminergic transmission.

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pharmacological profiles to give a definite proof of heteromer-mediated regulation of

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From the blocking of D2R signaling by the antagonist candesartan, the natural AT1R agonist, angiotensin II, would lead to enhanced D2R-mediated dopaminergic transmission thus impacting on the basal ganglia system of motor control. In summary, angiotensin II levels would fine tune striatal dopaminergic neurotransmission and/or would serve to immediately regulate motor function in conditions leading to increased angiotensin II release into the synaptic extracellular milieu. Recently, positive candesartan effects on haloperidol-induced dyskinesia have been described in rats [61]. It should be noted that haloperidol-induced dyskinesia in rats may be a model of the repetitive and involuntary orofacial movements described in tardive dyskinesia, which is associated to chronic or high dosage anti-psychotic treatment [62,63]. These beneficial effects of candesartan would indicate that enhanced angiotensinergic tone causes abnormal motor control and dyskinesia, which also is a side effect

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appearing in parkinsonian patients subjected to dopamine-replacement therapy. In line with these recent findings we have previously reported an up-regulation of RAS in CNS under dopamine depletion, leading to increased dopaminergic vulnerability and progression of PD [30]. Up-regulation likely arises from compensation mechanism but a chronic high

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angiotensinergic tone may become detrimental for patients. Further studies are needed to

investigate whether the use of antagonists for AT1R may be useful in PD and/or in dyskinesia

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due to pharmacological treatment of patients. The demonstration of AT1R-D2R heteromerization

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in striatum, points to a cross-talk between the dopaminergic and angiotensinergic systems in the indirect basal ganglia pathway. Consequently, the use of AT1R antagonists, able to cross the

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blood brain, would be useful to better understand angiotensin-dopamine interactions in both health and disease.

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Acknowledgements

Authors would like to thank Ainhoa Oñatibia-Astibia for her technical support. Supported by

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grants from Eranet-Neuron (Heteropark), Ministerio de Economía y Competitividad (BFU2012-

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), XUGA and CiberNed.

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37907, BFU2012-37087, SAF2008-03118-E, SAF39875-C02-01 –including FEDER funding-

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Figure captions

Fig. 1. Co-localization of AT1 and D2 receptors. Immunocytochemistry assays performed in HEK-293T cells expressing AT1R-YFP (top left), D2R (top right) or co-expressing AT1R-YFP and D2R (bottom images). Confocal microscopy images are shown. Receptors were identified by immunocytochemistry (red) and proteins fused to YFP were identified by its own fluorescence (green). Co-localization is shown in yellow in the merge image (bottom right). Scale bar: 10 µm.

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Fig. 2. Direct interaction between AT1 and D2 receptors. Schematic representation of the BRET technology approach used (A) and BRET saturation curves in HEK-293T cells expressing AT1R-Rluc and D2R-YFP (B). BRET saturation experiments showing AT1R-D2R heteromerization were performed using cells transfected with 0.5 µg of cDNA corresponding to

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AT1R-Rluc and increasing amounts of cDNA (0 to 2 µg cDNA) corresponding to D2R-YFP (control, black) in the presence of 200 nM quinpirole (purple) or 50 nM candesartan (orange).

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For negative control, cells were co-transfected with cDNA corresponding to GABAB-Rluc (0.5

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μg) and to D2R-YFP (0 to 4 μg cDNA) and analyzed when they express the two receptors (grey). Both fluorescence and luminescence for each sample were measured before every

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experiment to confirm similar donor expressions (approximately 120,000 bioluminescence units) while monitoring the increase in acceptor expression (1,000 to 50,000 net fluorescence

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units). The relative amount of BRET (Y-axis) is given as the ratio between the fluorescence of the acceptor minus the fluorescence detected in cells expressing only the donor and the luciferase activity of the donor. YFP/Rluc ratio (X-axis) is given in percentage of the value for

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the 1:1 cDNA ratio (calculated for each data point as fluorescence of the sample minus the

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fluorescence of cells expressing protein-Rluc alone). Both YFP/Rluc ratios and BRET data are

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shown as mean ± SD (4-8 different experiments grouped as a function of the amount of BRET acceptor). mBU: milliBRET units. Fig. 3.

Dose-response curve of quinpirole for inhibiting forskolin-stimulated cAMP

accumulation in HEK-293T cells. Cells transfected with 0.4 µg of cDNA for D2R (A), 0.5 µg of cDNA for AT1R and 0.4 µg of cDNA for D2R (B) or 0.5 µg of cDNA for AT1R (C), were stimulated with increasing concentrations of the D2R agonist, quinpirole, for 7 min and cAMP levels were determined. Data (mean ± SD) are given in percentage of the 500 nM forskolininduced cAMP concentration. Significant differences were analyzed from 4 to 6 different experiments by a one-way ANOVA followed by post-hoc Tukey’s test. *p < 0.05, **p < 0.01 compared with control

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Fig. 4. Dose-response curve of candesartan for activating forskolin-stimulated cAMP accumulation in HEK-293T cells. Cells transfected with 0.4 µg of cDNA for D2R (A), 0.5 µg of cDNA for AT1R and 0.4 µg of cDNA for D2R (B) or 0.5 µg of cDNA for AT1R (C), were treated with increasing concentrations of the AT1R antagonist, candesartan, for 15 min in the

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presence or in the absence of 1 µM of the natural D2R agonist, dopamine, and the cAMP levels were determined. Data (mean ± SD) are given in percentage of the 500 nM forskolin-induced

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cAMP concentration. Significant differences were analyzed from 4 to 6 different experiments

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by a one-way ANOVA followed by post-hoc Tukey’s test.

#p < 0.05 compared with dopamine treatment.

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***p < 0.001 compared with control

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Fig. 5. cAMP levels and β-arrestin recruitment in HEK-293T cells. Cells transfected with 0.4 µg of cDNA for D2R (Panels Ai and Bi), 0.5 µg of cDNA for AT1R and 0.4 µg of cDNA for

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D2R (Panels Aii and Bii) or 0.5 µg of cDNA for AT1R (Panels Aiii and Biii), were stimulated

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with 200 nM of the D2R agonist, quinpirole, in the presence or in the absence of 10 nM of the AT1R antagonist, candesartan. cAMP levels (Panels Ai-Aiii) and β-arrestin recruitment (Panels

Ac ce p

Bi-Biii) were determined. cAMP levels (mean ± SD) are given in percentage of the 500 nM forskolin-induced cAMP concentration. BRET data are shown as mean ± SD. Significant differences were analyzed from 4 to 6 different experiments by a one-way ANOVA followed by post-hoc Tukey’s test.

**p < 0.01, ***p < 0.001 compared with control ##p < 0.01, ###p < 0.001 compared with quinpirole. Fig. 6. ERK1/2 phosphorylation in transfected HEK-293T cells. Cells transfected with 0.4 µg of cDNA for D2R (A), 0.5 µg of cDNA for AT1R and 0.4 µg of cDNA for D2R (B) or 0.5 µg of cDNA for AT1R (C), were stimulated with 200 nM of the D2R agonist, quinpirole, in the presence or in the absence of 10 nM candesartan and ERK 1/2 phosphorylation levels were

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determined by immunoblotting. ERK 1/2 phosphorylation basal levels are given as 100% and values (mean ± SD) are expressed as percentage over basal. A representative immunoblot is shown in each panel (bottom). Significant differences were analyzed from 4 to 6 different

ip t

experiments by a one-way ANOVA followed by post-hoc Tukey’s test. **p < 0.01, ***p < 0.001 compared with control

cr

##p < 0.01 compared with quinpirole.

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Fig. 7. Calcium release determination in transfected HEK-293T cells. Cells transfected with 0.75 µg of D2R (A), 1 µg of AT1R and 0.75 µg of D2R (B), or 1 µg of AT1R with 2 µg of

an

GCamP6s, were activated for 1 min with 10 nM candesartan or raclopride 1 µM before the addition of angiotensin 250 nM. The fluorescence signal was measured as described under

M

“Materials and Methods”. Calcium release values are expressed as percentage of fluorescence levels over basal levels found in untreated neurons.

d

Fig. 8. cAMP level determination and ERK1/2 phosphorylation assays in rat striatal

te

primary cultures of neurons. Rat striatal neurons were pretreated for 10 min with the AT1R antagonist candesartan (100 nM) prior to the addition of the D2R agonist, quinpirole (200 nM)

Ac ce p

for 10 min. cAMP levels (A) are given in percentage of the 500 nM forskolin-induced cAMP concentration. ERK1/2 phosphorylation was determined using an homogeneous assay as described under “Materials and Methods”. Data (mean ± SD) are given in percentage of the ratio pERK1/2 versus ERK1/2 relative to the basal levels found in untreated neurons (100%). Significant differences were analyzed by a one-way ANOVA followed by post-hoc Tukey’s test.

***p < 0.001 compared with control ###p < 0.001 compared with quinpirole. Fig. 9. ERK1/2 phosphorylation in rat striatal slices. Rat striatal slices were treated with the D2R agonist, quinpirole (200 nM) for 10 min or pretreated for 10 min with AT1R antagonist

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candesartan (100 nM) prior to agonist treatment. ERK1/2 phosphorylation was determined by immunoblotting as described under “Materials and Methods”. The immunoreactive bands from two different experiments performed in triplicate were quantified, and data (mean ± SD) are given in percentage of the ratio pERK1/2 versus ERK1/2 relative to the basal levels found in

cr

by post-hoc Tukey’s test. A representative immunoblot is shown (bottom).

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untreated slices (100%). Significant differences were analyzed by a one-way ANOVA followed

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***p < 0.001 compared with control ###p < 0.001 compared with quinpirole.

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Fig. 10. PLA assays in slices from rat striatum. A) Striatal sections were processed for PLA stain according to the guidelines issued by the manufacturer, and D2R and AT1R in close

M

proximity are detectable by confocal microscopy as a punctate red fluorescent signal. B) Sections incubated with anti-D2R antibodies alone/only led to a negligible amount of red dots

d

(negative control). Cell nuclei were stained with DAPI (blue) and the boxed areas are magnified (asterisks). C) Schematic representation of the PLA technology. D) The number of cells

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containing one or more red spots is expressed as the percentage of the total number of cells

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(blue nucleus). Data (% of positive cells) are the mean ± SD of counts in 18 different fields. From data taken from 18 fields Student’s t-test analysis was performed. *** p