Adenosine A2A receptor inhibition reduces synaptic and cognitive hippocampal alterations in Fmr1 KO mice

In fragile X syndrome (FXS) the lack of the fragile X mental retardation protein (FMRP) leads to exacerbated signaling through the metabotropic glutamate receptors 5 (mGlu5Rs). The adenosine A2A receptors (A2ARs), modulators of neuronal damage, could play a role in FXS. A synaptic colocalization and a strong permissive interaction between A2A and mGlu5 receptors in the hippocampus have been previously reported, suggesting that blocking A2ARs might normalize the mGlu5R-mediated effects of FXS. To study the cross-talk between A2A and mGlu5 receptors in the absence of FMRP, we performed extracellular electrophysiology experiments in hippocampal slices of Fmr1 KO mouse. The depression of field excitatory postsynaptic potential (fEPSPs) slope induced by the mGlu5R agonist CHPG was completely blocked by the A2AR antagonist ZM241385 and strongly potentiated by the A2AR agonist CGS21680, suggesting that the functional synergistic coupling between the two receptors could be increased in FXS. To verify if chronic A2AR blockade could reverse the FXS phenotypes, we treated the Fmr1 KO mice with istradefylline, an A2AR antagonist. We found that hippocampal DHPG-induced long-term depression (LTD), which is abnormally increased in FXS mice, was restored to the WT level. Furthermore, istradefylline corrected aberrant dendritic spine density, specific behavioral alterations, and overactive mTOR, TrkB, and STEP signaling in Fmr1 KO mice. Finally, we identified A2AR mRNA as a target of FMRP. Our results show that the pharmacological blockade of A2ARs partially restores some of the phenotypes of Fmr1 KO mice, both by reducing mGlu5R functioning and by acting on other A2AR-related downstream targets.


Drugs and treatment
CHPG (mGlu5R agonist, Cat# 1049), ZM241385 (A2AR antagonist, Cat# 1036), DHPG (mGluR agonist, Cat# 0342), CGS21680 (A2AR antagonist, Cat# 1063), and istradefylline (A2AR antagonist, Cat# 5147) were purchased from Tocris Biosciences (Bristol, UK). CHPG, ZM241385, CGS21680, and DHPG, were dissolved in dimethyl sulfoxide (DMSO) or in distilled water to obtain stock solutions. Stock solutions were made to obtain concentrations of DMSO lower than 0.001% in the superfusing ACSF and cell cultures. This DMSO concentration did not affect basal synaptic transmission in hippocampal slices (data not shown). Istradefylline (hereinafter referred to as KW6002), a selective adenosine A2AR antagonist (permeable to the blood-brain barrier), was orally administered solubilized in the vehicle (the drug stock was prepared in 40% DMSO, 30% Cremophor EL and 30% mineral oil, and further diluted in 2% sucrose in water to prepare the final treatment solutions, which contained 0.2% DMSO, 0.15% Cremophor EL, and 0.15% mineral oil), in light protected bottles, and was continuously available. Istradefylline levels in the plasma and brain of mice exposed to similar doses and treatments have been accurately evaluated in 9 . The weight of the animals and the volume intake were assessed three times a week and the concentration of the solution adjusted so that the drug intake was maintained at 4 mg/kg per day. The control groups of mice were treated only with the vehicle.

Slice preparation and recordings
Hippocampal slices were prepared as follows. After the sacrifice of animals by cervical dislocation, the hippocampus was removed and 450-μm slices were cut with a McIlwain tissue slicer (Cat # TC752, The Mickle Lab, Guildford, United Kingdom). Slices were maintained at RT (22-24°C) in ACSF containing (in mM): 126 NaCl, 3.5 KCl, 1.2 NaH2PO4, 1.2 MgCl2, 2 CaCl2, 25 NaHCO3, 11 glucose (pH 7.3) saturated with 95% O2 and 5% CO2. After incubation in ACSF for at least 1 h, a single slice was transferred to a submerged recording chamber and continuously superfused at 32-33°C with ACSF at a rate of 2.6 ml/min. The drugs were added to this superfusion medium. The perfusion apparatus was made of chemically inert materials (silicone tubing). Extracellular fEPSPs were recorded in stratum radiatum of the CA1 hippocampus with a glass microelectrode filled with 2 M NaCl solution (pipette resistance 2-5 MΩ) upon stimulation of Schaffer collaterals with an insulated bipolar twisted NiCr electrode (50 μm OD). Each pulse was delivered every 20s (square pulses of 100 μs duration at a frequency of 0.05 Hz), and three consecutive responses were averaged.
The stimulation intensity used in the fEPSP recordings was always adjusted to obtain a submaximal fEPSP slope (~60% of maximum) with minimum population spike contamination. Signals were acquired with a DAM-80 AC differential amplifier (WPI) and analyzed with the LTP program. At least 10 min of stable baseline recording preceded drug application. Data were expressed as mean ± SEM of n experiments (one slice tested per experiment. Slices were obtained from at least two animals for each set of the experiment). To allow for comparisons between different experiments, slope values were normalized, taking the average of the baseline values to be 100%. The drug effect was expressed as the mean percentage variation of the slope from baseline over the last 5 min of drug perfusion. The washout period lasted at least 30 min.

Spine number and morphological analyses
The following protocol was used for the analysis of the spine number and morphology 1 . In brief, coronal brain sections were stained through consecutive steps in water (1 min), ammonium hydroxide

Behavioral evaluation.
Locomotor activity. The open field test was used to assess both locomotor activity and anxiety-related response. It was performed in a Plexiglass box (43x43x20cm) equipped with an overhead camera associated with the Anymaze video tracking system (Cat# 60000, UGO BASILE); the arena was arranged into a 16 square grid in the camera window with 4 squares in the center and 12 squares around the perimeter. Mice performed the test at PND 76, 97, and 118 for 20 min, and distance traveled in the center 4 squares (a measure of anxiety) and total distance (a measure of locomotion) 4 were compared between genotypes. Also, the center distance/total distance ratio was used as an index of anxiety.
Learning impairment. The Novel object recognition test (NORT) was performed as follows. The day after the open field test mice underwent the NORT: they were placed in the box facing the wall at the opposite end from the objects and were allowed to explore two identical objects located equidistant from the walls and the center of the open field for 10 min. One hour later, the trial was repeated with one of the objects being replaced with a novel object of similar size. The time spent exploring the familiar and novel objects was quantified. A mouse was considered to be exploring the object if it was sniffing, touching, or facing it within 2 cm or less, and measurements were recorded in seconds. Rotarod test. To evaluate if chronic treatment with KW6002 could affect motor coordination and skill learning of animals the accelerating rotarod test was performed. To this aim, animals were trained on a rotarod apparatus (UGO BASILE) four times a day on two consecutive days with a constant speed of 12 rounds per min for 120 s. During the test, the rotation was increased from 4 to 40 rounds in 30 s steps within 5 min, and latency to fall was automatically recorded (in seconds). The test was performed once weekly starting from PND68.

Protein extraction and Western blot analysis.
Hippocampal and cortical mouse tissue samples were homogenized in RIPA Buffer containing 1% 1X PBS, with freshly added protease and phosphatase inhibitor (Thermo Scientific, Milan, Italy), and kept on ice for 30 min. After the incubation they were centrifuged at 12.000 × g for 20 min at +4 °C; the pellet was discarded and the supernatant transferred into new tubes and stored at -80 °C. Protein content was determined using the BCA protein Assay (Thermo Scientific, Bremen, Germany).

STEP activity/expression analysis
The crude synaptosomal fraction was prepared as follows according to the published protocol 2 .
Briefly, the dissected hippocampus samples were homogenized in ice-cold buffer containing (in mM):

FMRP Immunoprecipitation
Brain extracts were prepared from cortex and hippocampus or striatum of WT and Fmr1 KO male mice 3-4 weeks old, using RIPA buffer plus Protease inhibitor cocktail (Roche), Phosphatase inhibitor cocktails II and III (Sigma), 40 U/ml RNaseOUT (Invitrogen). Protein extracts were used for RNA-Immunoprecipitation (RIP) using a specific anti-FMRP antibody. Dynabeads previously saturated with 1% BSA in 1X PBS were incubated with anti-FMRP antibody or normal rabbit IgG (Santa Cruz Biotechnology) for 1 hour at room temperature (RT). The beads were then washed in a buffer (250 mM NaCl, 20 mM Tris-HCl pH 7.4, 10 mM MgCl2 and 0.1% Triton X-100), the protein extract added to the Dynabeads and incubated for 1-2 hours at 4°C. RNAs were eluted in TRIzol (Invitrogen).
Upon RIP, the RNAs co-immunoprecipitated were extracted and analyzed by RT-qPCR, using the 7 StepOne Plus 7500 instrument (Life technologies) and specific oligonucleotides designed to amplify the RNAs of interest (Table S2).

Saturation binding experiments
Striatum, cortex, and hippocampus from WT and Fmr1 KO mice

Statistics
Results from electrophysiological experiments were expressed as mean ± SEM (independent number of slices). The number of animals from which the slices have been obtained is reported for each data set in the Results. Data from the behavioral, dendritic spine, and testis weight evaluations were presented as mean ± SEM (independent number of mice). Results from in vitro biochemistry experiments were expressed as a percentage of control, which was considered as 100%, and as mean ± SEM values of at least 3 independently performed experiments (each independent experiment corresponds to an independent tissue lysate preparation from each mouse, thus n indicates the number of mice). Statistical analysis of the electrophysiology, behavioral, dendritic spine, biochemistry, and testis weight data was performed by using Mann-Whitney test. For STEP activity analysis results are expressed as mean ± SEM values of at least 4 independently performed experiments (each independent experiment corresponds to an independent tissue lysate preparation). Results from immunoprecipitation and RT-qPCR were expressed as mean ± SEM, and p-values were calculated 8 by Student's t-test. Statistical analysis for behavioral evaluation was performed separately on male and female Fmr1 KO animals, and then results combined in case no differences were observed.
Dissociation equilibrium constants for saturation binding, affinity, or KD values, as well as the maximum densities of specific binding sites (Bmax), were calculated for a system of one-or twobinding site populations using a non-linear curve fitting. All binding data are reported as mean ± SEM of at least 3 independent experiments. Differences between the groups were analyzed with Student's t-test. A p-value <0.05 was considered to indicate a significant difference. Variances between groups were similar. Statistical tests were two-sided. Statistical test for the evaluation of KW6002 on macroorchidism was one-sided since no increase of testis weight has been reported in the preclinical and clinical characterization of the drug as a Parkinson's disease medication. All statistical analyses and curve fittings were obtained using GraphPad Prism software (RRID: SCR_002798). We did not apply any inclusion or exclusion criteria to samples or animals. index of lower anxiety, contrary to the FXS clinical phenotype 3 . As for the inability of KW6002 to prevent such an effect, it should be considered that A2AR antagonists do not affect the anxiety parameters in the elevated plus-maze test 4,5 . Interestingly, furthermore, in spontaneously hypertensive rats (SHR), a widely used animal model useful to study the neurodevelopmental disorder ADHD, chronic blockade of A2AR was able to improve NOR performance 6 , while being ineffective on hyperlocomotion and low anxiety behavior 7 . c Stereotyped behavior: the effect of treatment with KW6002 on stereotyped behavior was evaluated in marble-burying tests, but, at least in our experimental conditions, we did not observe any significant difference between KO/VEH and WT/VEH mice in terms of buried marbles and then it has not been possible to deduce the effect of KW6002. It should be taken into account that the reliability of the marble-burying test in mimicking obsessive-compulsive disorder (OCD) or anxiety is strongly debated; it has been observed that some anxiolytic compounds with no human anti-compulsive effects consistently pass this assay while some known anti-compulsive drugs consistently fail 8 . d Rotarod test: chronic treatment with KW6002 did not affect motor coordination and skill learning of animals assessed in the accelerating rotarod test.

Supplemental Figures and Tables
Motor coordination was almost comparable in Fmr1 KO and WT mice and, anyway, chronic drug treatment did not affect the rotarod performance. This is in line with the lack of effect on locomotor function already reported for KW6002, up to 10 mg/kg per day 9 .  (a, b) the histograms indicate the % of phosphoproteins normalized to respective total protein levels. The ERK signaling pathway is found hyper-responsive in Fmr1 KO mice 10 and increased in the frontal cortex of FXS patients 11 . Our biochemistry data confirm that ERK phosphorylation level is significantly higher specifically in the cortex of vehicle-treated Fmr1 KO animals compared to WT mice, but the chronic blockade of A2AR did not affect this hyper-phosphorylation.
12 Table S1 List of specific oligonucleotides designed to amplify the RNAs of interest in the FMRP-Immunoprecipitation experiment.
Data are expressed as the mean ± SEM of 3 independent experiments performed in duplicate.
Affinity and density of A2ARs are comparable in the brain areas of Fmr1 KO and WT mice. Saturation binding experiments performed with the antagonist radioligand [ 3 H]-ZM241385 revealed that no differences in A2AR affinity and density were observed in striatum, cortex, and hippocampus from WT and Fmr1 KO mice in basal conditions.