Singular Location and Signaling Profile of Adenosine A2A-Cannabinoid CB1 Receptor Heteromers in the Dorsal Striatum

The dorsal striatum is a key node for many neurobiological processes such as motor activity, cognitive functions, and affective processes. The proper functioning of striatal neurons relies critically on metabotropic receptors. Specifically, the main adenosine and endocannabinoid receptors present in the striatum, ie, adenosine A2A receptor (A2AR) and cannabinoid CB1 receptor (CB1R), are of pivotal importance in the control of neuronal excitability. Facilitatory and inhibitory functional interactions between striatal A2AR and CB1R have been reported, and evidence supports that this cross-talk may rely, at least in part, on the formation of A2AR-CB1R heteromeric complexes. However, the specific location and properties of these heteromers have remained largely unknown. Here, by using techniques that allowed a precise visualization of the heteromers in situ in combination with sophisticated genetically modified animal models, together with biochemical and pharmacological approaches, we provide a high-resolution expression map and a detailed functional characterization of A2AR-CB1R heteromers in the dorsal striatum. Specifically, our data unveil that the A2AR-CB1R heteromer (i) is essentially absent from corticostriatal projections and striatonigral neurons, and, instead, is largely present in striatopallidal neurons, (ii) displays a striking G protein-coupled signaling profile, where co-stimulation of both receptors leads to strongly reduced downstream signaling, and (iii) undergoes an unprecedented dysfunction in Huntington’s disease, an archetypal disease that affects striatal neurons. Altogether, our findings may open a new conceptual framework to understand the role of coordinated adenosine-endocannabinoid signaling in the indirect striatal pathway, which may be relevant in motor function and neurodegenerative diseases.

individual) were from 10 patients with HD and 5 control subjects with no neurological disease. The characteristics of these samples are given in Supplementary Table S1.

Viral vectors
HA-tagged Cre recombinase, or EGFP as control, was subcloned in a recombinant adenoassociated virus (rAAV) expression vector with a minimal CaMKIIα promoter (kindly provided by Dr. Karl Deisseroth, Stanford University, Stanford, CA, USA) by using standard molecular cloning techniques. All vectors used were of an AAV1/AAV2 mixed serotype, and were generated by calcium phosphate transfection of HEK-293T cells and subsequent purification as described (Monory et al, 2006). Eight week-old CB 1 R floxed/floxed mice were injected stereotactically with CaMKIIα-Cre-rAAV or CaMKIIα-EGFP-rAAV (in 1.5 µl PBS) either into the dorsal striatum or into the motor cortex projecting onto the dorsal striatum (Chiarlone et al, 2014). In the case of the striatum, each animal received one bilateral injection at coordinates (to bregma): antero-posterior +0.6, lateral ±2.0, dorso-ventral -3.0. In the case of the cortex, each animal received 2 bilateral injections at coordinates (to bregma): antero-posterior +1.5, lateral ±1.2, dorso-ventral -1.7; and antero-posterior -0.5, lateral ±1.2, dorso-ventral -1.2. The placement of the rAAV vectors within the dorsal striatum has been previously described ( Figure S5A in Chiarlone et al, 2014;Figure 4b in Blazquez et al, 2015).
Six-eight weeks later mice were sacrificed by intracardial perfusion and their brains were excised for PLA analysis.

HIV TAT peptides
Peptides with the sequence of transmembrane domains (TM) of CB 1 R fused to HIV transactivator of transcription (TAT) peptide were used as heteromer-disrupting molecules. A cell-penetrating HIV TAT peptide (YGRKKRRQRRR) allows intracellular delivery of fused peptides (Schwarze et al, 1999). This HIV TAT peptide can be inserted effectively into the plasma membrane as a result of both the penetration capacity of the TAT peptide and the hydrophobic property of the TM domain (He et al, 2011). To obtain the right orientation of the inserted peptide, the HIV-TAT peptide was fused to the C-terminus of TM5 and TM7 or to the N-terminus of TM6 of CB 1 R (Viñals et al, 2015). The sequence of the fusion peptides was the following: TM5, ETYLMFWIGVTSVLLLFIVYAYMYILW GRKKRRQRRR TM6, GRKKRRQRRR KTLVLILVVLIICWGPLLAIMVYDVF TM7, LIKTVFAFCSMLCLLNSTVNPIIYALR GRKKRRQRRR

Cell culture and transfection
Conditionally immortalized striatal neuroblasts obtained from wild-type mice (STHdh Q7/Q7 cells) or knock-in mice expressing two copies of a mutant huntingtin allele (STHdh Q111/Q111 cells), thus expressing endogenous levels of full-length huntingtin with only 7 glutamines or with 111 glutamines in the protein N-terminal domain, respectively, were used (Trettel et al, 2000). Cells had been infected with a defective retrovirus transducing the temperaturesensitive A58/U19 large T antigen, and were grown at the permissive temperature of 33ºC in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 1 mM sodium pyruvate, 2 mM L-glutamine, 1% streptomycin/penicillin and 400 µg/ml geneticin. HEK-293T cells were grown in DMEM supplemented with 2 mM L-glutamine, 100 μg/ml sodium pyruvate, 100 U/ml penicillin/streptomycin, essential medium nonessential amino acids solution (1/100) and 5% (v/v) heat inactivated fetal bovine serum (all from Invitrogen, Paisley, UK) and were maintained at 37ºC in an atmosphere with 5% CO 2 .
Cells growing in 6-well dishes were transiently transfected with the corresponding protein cDNA by the polyethylenimine method (Sigma, Steinheim, Germany). Cells were incubated with the corresponding cDNA together with polyethylenimine (5.47 mM in nitrogen residues) and 150 mM NaCl in a serum-starved medium. After 4 h, the medium was changed to a fresh complete culture medium. Forty-eight h after transfection, cells were washed twice in quick succession in HBSS with 10 mM glucose, detached, and resuspended in the same buffer. To control cell number, sample protein concentration was determined using a Bradford assay kit (Bio-Rad, Munich, Germany).

In Situ Proximity Ligation Assays (PLA)
STHdh Q7/Q7 and STHdh Q111/Q111 cells were grown on glass coverslips and fixed in 4% paraformaldehyde for 15 min, washed with phosphate-buffered saline (PBS) containing 20 mM glycine, permeabilized with the same buffer containing 0.05% Triton X-100, and successively washed with PBS. Mice were deeply anesthetized and immediately perfused transcardially with PBS followed by 4% paraformaldehyde/phosphate buffer. Brains were removed and post-fixed overnight in the same solution, cryoprotected by immersion in 10, 20, 30% gradient sucrose (24 h for each sucrose gradient) at 4ºC, and then frozen in dry icecooled methylbutane. Serial coronal cryostat sections (30 μm-thick) through the whole brain were collected in cryoprotective solution and stored at -20ºC until PLA experiments were performed. Immediately before the assay, mouse brain sections were mounted on glass slides, washed in PBS, permeabilized with PBS containing 0.01% Triton X-100 for 10 min, and successively washed with PBS. Human brain slices were deparaffinized and antigen retrieval was performed with citrate buffer at pH 6.0. In all cases, heteromers were detected using the Duolink II in situ PLA detection Kit (OLink; Bioscience, Uppsala, Sweden) following supplier's instructions. A mixture of the primary antibodies [mouse anti-A 2A R antibody (1:100; Millipore, Darmstadt, Germany; cat #05-717) and rabbit anti-CB 1 R antibody (1:100; Thermo Scientific, Fremont, CA, USA; cat #PA1-745)] was routinely used to detect A 2A R-CB 1 R heteromers together with PLA probes detecting mouse or rabbit antibodies . The specificity of these antibodies for PLA assays has been previously reported by both us (Bonaventura et al, 2015;Viñals et al, 2015) and others (Trifilieff et al, 2011;Sierra et al, 2015). In addition, PLA experiments conducted with a different anti-CB 1 R primary antibody (1:100; Frontier Institute, Japan; cat #CB1-Rb-Af380) provided a similar A 2A R-CB 1 R heteromer detection in mouse dorsal-striatum preparations (Supplementary Figure S1d). Moreover, the specificity of the three aforementioned primary antibodies was also demonstrated by immunocytofluorescence studies, conducted under comparable conditions as the PLA assays, in HEK-293T cells transfected or not with cDNAs encoding human A 2A R or human CB 1 R (Supplementary Figure S1e). To detect CB 1 R-D 2 R heteromers, the anti-D 2 R antibody (1:100; Millipore; cat #AB5084P) was directly coupled to a plus DNA chain and the anti-CB 1 antibody was directly coupled to a minus DNA chain following supplier's instructions. Then, samples were processed for ligation and amplification with a Detection Reagent Red and were mounted using a DAPI-containing mounting medium.
Samples were analyzed in a Leica SP2 confocal microscope (Leica Microsystems, Mannheim, Germany) equipped with an apochromatic 63X oil-immersion objective (1.4 numerical aperture), and a 405 nm and a 561 nm laser lines. Three serial coronal sections (30-μm thick) per animal spaced 0.24 mm apart containing the dorsal striatum were used. For each field of view a stack of two channels (one per staining) and 9 to 15 Z-stacks with a step size of 1 µm were acquired. Images were opened and processed with Image J software (National Institutes of Health, Bethesda, MD). Quantification of cells containing one or more red dots versus total cells (blue nuclei) was determined by using the Fiji package (http://pacific.mpi-cbg.de). In some experiments, the total number of red dots found in stained cells versus total cells (blue nuclei) was determined. Nuclei and red dots were counted on the maximum projections of each image stack. After getting the projection, each channel was processed individually. The blue nuclei and red dots were segmented by filtering with a median filter, subtracting the background, enhancing the contrast with the Contrast Limited Adaptive Histogram Equalization (CLAHE) plug-in, and finally applying a threshold to obtain the binary image and the regions of interest (ROIs). When indicated, after image processing, the red channel was depicted in green color to facilitate detection on blue stained nuclei, always maintaining the color intensity settings constant for all images. Sample analysis and data acquisition were conducted in a blinded manner.

Fluorescence complementation assays
To obtain plasmids for fusion proteins expression, sequences encoding amino acid residues 1-155 and 156-238 of YFP Venus protein were subcloned in the pcDNA3.1 vector to obtain the YFP Venus hemi-truncated proteins (nVenus and cVenus). The cDNAs for human A 2A R and D 1 R were amplified without their stop codons using sense and antisense primers harboring unique EcoRI and BamHI sites. The cDNA for human CB 1 R was amplified without their stop codons using sense and antisense primers harboring unique EcoRI and KpnI. The amplified fragments were subcloned to be in-frame with restriction sites of pcDNA3.1-cVenus or pcDNA3.1-nVenus vectors to give the plasmids that express proteins fused to hemi-YFP Venus on the C-terminal end (A 2A R-cYFP, D 1 R-cYFP or CB 1 R-nYFP). For fluorescence complementation assays, HEK-293T cells were transiently co-transfected with the cDNA encoding receptor fused to nYFP and receptor fused to cYFP. After 48 h, cells were treated or not with the indicated TM-TAT peptides (4 μM) for 4 h at 37 °C. To quantify proteinreconstituted YFP Venus expression, cells (20 μg protein) were distributed in 96-well microplates (black plates with a transparent bottom, Porvair, King's Lynn, UK), and emission fluorescence at 530 nm was monitored in a FLUOstar Optima Fluorimeter (BMG Lab Technologies, Offenburg, Germany) equipped with a high-energy xenon flash lamp, using a 10 nm bandwidth excitation filter at 400 nm reading. Protein fluorescence expression was determined as the fluorescence of the sample minus the fluorescence of cells not expressing the fusion proteins (basal). Cells expressing A 2A R-cVenus and nVenus or CB 1 R-nVenus and cVenus showed similar fluorescence levels than non-transfected cells.

Dynamic Mass Redistribution (DMR) assays
The cell signaling signature was determined using an EnSpire® Multimode Plate Reader (PerkinElmer, Waltham, MA, USA) by a label-free technology. Refractive waveguide grating optical biosensors, integrated in 384-well microplates, allow extremely sensitive measurements of changes in local optical density in a detecting zone up to 150 nm above the surface of the sensor. Cellular mass movements induced upon receptor activation were detected by illuminating the underside of the biosensor with polychromatic light and measured as changes in wavelength of the reflected monochromatic light that is a sensitive function of the index of refraction. The magnitude of this wavelength shift (in picometers) is directly proportional to the amount of DMR. Briefly, 24 h before the assay, cells were seeded at a density of 10,000-12,000 cells per well in 384-well sensor microplates with 30 μl growth medium and cultured for 24 h (37°C, 5% CO 2 ) to obtain 70-80% confluent monolayers.
Previous to the assay, cells were washed twice with assay buffer (HBSS with 20 mM Hepes, pH 7.15) and incubated for 2 h in 30 μl per well of assay-buffer with 0.1% DMSO in the reader at 24°C. Hereafter, the sensor plate was scanned and a baseline optical signature was recorded before adding 10 μl of test compound dissolved in assay buffer containing 0.1% DMSO. Then, DMR responses were monitored for at least 5,000 s. Kinetic results were analyzed using EnSpire Workstation Software v 4.10.

Determination of cAMP concentration
Homogeneous time-resolved fluorescence energy transfer (HTRF) assays were performed using the Lance Ultra cAMP kit (PerkinElmer), based on competitive displacement of a europium chelate-labelled cAMP tracer bound to a specific antibody conjugated to acceptor beads. We first established the optimal cell density for an appropriate fluorescent signal. This was done by measuring the TR-FRET signal determined as a function of forskolin concentration using different cell densities. The forskolin dose-response curves were related to the cAMP standard curve in order to establish which cell density provides a response that covers most of the dynamic range of the cAMP standard curve. Cells (500-1000 per well) growing in medium containing 50 µM zardeverine were pre-treated with toxins or the corresponding vehicle in white ProxiPlate 384-well microplates (PerkinElmer) at 25°C for the indicated time, and stimulated with agonists for 15 min before adding 0.5 μM forskolin or vehicle, and incubating for an additional 15 min period. Fluorescence at 665 nm was analyzed on a PHERAstar Flagship microplate reader equipped with an HTRF optical module (BMG Lab technologies, Offenburg, Germany).

Determination of Ca 2+ concentration
To determine cytosolic Ca 2+ free concentration, striatal cells were transfected with 4 μg of GCaMP6 calcium sensor (Chen et al, 2013) using Lipofectamine 3000. After 48 h, cells were incubated (0.2 mg of protein/ml in 96-well black, clear bottom microtiter plates) with Mg 2+free Locke's buffer (in mM: 154 NaCl, 5.6 KCl, 3.6 NaHCO 3 , 2.3 CaCl 2 , 5.6 glucose and 5 HEPES, pH 7.4) supplemented with 10 μM glycine. After the addition of receptor ligands at the indicated concentrations, fluorescence emission intensity of GCaMP6 was recorded at 515 nm upon excitation at 488 nm on an EnSpire® Multimode Plate Reader (PerkinElmer) for
The cellular debris was removed by centrifugation at 13,000 g for 5 min at 4°C, and the protein was quantified by the bicinchoninic acid method using bovine serum albumin dilutions as standard. Equivalent amounts of protein (10 μg) were separated by electrophoresis on a denaturing 10% SDS-polyacrylamide gel and transferred onto PVDFfluorescence membranes. Odyssey blocking buffer (LI-COR Biosciences) was then added, and the membrane was rocked for 90 min. Membranes were routinely probed for 2-3 h with a mixture of a mouse anti-phospho-ERK1/2 antibody (1:2500; Sigma; cat #M8159), a rabbit anti-phospho-Ser473-Akt antibody (1:2500; SAB Signalway Antibody, Pearland, USA; cat #11054), and a rabbit anti-ERK1/2 antibody that recognizes both phosphorylated and nonphosphorylated ERK1/2 (1:40,000; Sigma; cat #M5670). Alternatively, when indicated, Akt activation was determined by probing membranes with a mixture of the aforementioned rabbit anti-phospho-Akt and anti-total-ERK antibodies, plus a mouse anti-total-Akt antibody (1:2500; Cell Signaling, Danvers, MA, USA; cat #2920). Bands were visualized by the addition of a mixture of IRDye 800 (anti-mouse) antibody (1:10,000; Sigma) and IRDye 680 (anti-rabbit) antibody (1:10,000; Sigma) for 1 h and scanned by the Odyssey infrared scanner (LI-COR Biosciences). Band densities were quantified at exposure times within the dynamic range (not saturated, not overexposed) using the scanner software exported to Excel (Microsoft). Due to the high sensitivity of the anti-total-ERK antibody, the levels of phosphorylated ERK1/2 or phosphorylated Akt were routinely normalized for differences in loading using the total ERK protein band optical density. The anti-total-ERK antibody works better in our hands than the anti-total-Akt antibody. In any event, we have not detected any significant variability with using anti-total-ERK, anti-total-Akt or anti-actin loading controls with respect to the pERK or pAkt signals in these short-term cell signaling experiments.

Determination of A 2A R and CB 1 R immunoreactivity
Wild-type Hdh Q7/Q7 and heterozygous mutant Hdh Q7/Q111 mice at 6 months of age were perfused transcardially with PBS followed by 4% paraformaldehyde/phosphate buffer. Brains were removed and postfixed overnight, cryoprotected, and frozen in dry ice-cooled methylbutane. Serial coronal cryostat sections (30 μm-thick) through the whole brain were collected in PBS as free-floating sections and stored at -20ºC. Immunofluorescence staining was performed as previously described (Puigdellivol et al, 2015). immunofluorescence. Brain slices were incubated with sodium citrate buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0) for 30 min at 90⁰C in a pre-heated water bath and then removed to room temperature for 20 min. Before permeabilization and blocking, the slices were washed 3 times in PBS. Images were acquired with Zeiss LSM510 META confocal microscope with argon and HeNe lasers. Images were taken using a 63X numerical aperture objective with 1X digital zoom and standard (one Airy disc) pinhole. Three serial coronal sections (30-μm thick) per animal spaced 0.24 mm apart containing the dorsal striatum were used. For each slice, we obtained 3 fields per dorsal striatum region. In each field, an entire Zstack was obtained, and optical sections (between 3 and 5 per field) of 0.5 μm were collected separately (4 μm) in order to avoid biased counting. CB 1 R and A 2A R immunoreactivity were quantified using ImageJ software. None of the secondary antibodies produced any signal in preparations incubated in the absence of the corresponding primary antibodies. Sample analysis and data acquisition were conducted in a blinded manner.

Statistics
Unless otherwise indicated, data are presented as mean ± SEM. Statistical analyses were conducted with unpaired Student's t test or with one-way ANOVA, followed by Bonferroni or Dunnett post hoc test, as appropriate. Details of the statistical analyses conducted for each set of data are given in Supplementary Table S2. A p value of less than 0.05 was considered significant.

References for Supplementary Materials and Methods
Bellocchio L, Lafenetre P, Cannich A, Cota D, Puente N, Grandes P et al ( HD, Huntington's disease; M, male; F, female; C, caudate; P, putamen; PMI, post-mortem interval; n.a., non-available information; PLA, proximity ligation assay (values are mean ± SEM of the number of cells containing one or more A 2A R-CB 1 R-positive dots expressed as the percentage of the total number of cell nuclei).  Further details of statistical analyses are given in Supplementary Table S2.
Further details of statistical analyses are given in Supplementary Table S2.
Supplementary Figure S8. A 2A R-CB 1 R heteromer expression in the caudate and the putamen of a representative control subject and a representative high-grade HD patient.
PLA assays performed in caudate-putamen sections of post-mortem samples from a representative control subject (a; Subject #5 in Supplementary Table S1) and a representative grade 3 HD patient (b; Subject #11 in Supplementary Table S1)