Upregulation of adenosine A2A receptor by astrocytes is sufficient to trigger hippocampal multicellular dysfunctions and memory deficits.

Upregulation of adenosine A2A receptor by astrocytes is sufficient to trigger hippocampal multicellular dysfunctions and memory deficits. | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Upregulation of adenosine A 2A receptor by astrocytes is sufficient to trigger hippocampal multicellular dysfunctions and memory deficits. David Blum, Agathe Launay, Kévin Carvalho, Thibaut Gauvrit, Victoria Gomez Murcia, and 13 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4791082/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 23 Jul, 2025 Read the published version in Molecular Psychiatry → Version 1 posted 18 You are reading this latest preprint version Abstract Adenosine is an ubiquitous neuromodulator that ensures cerebral homeostasis. It exerts numerous functions through the activation of G-protein-coupled adenosine receptors (ARs), in particular A 1 (A 1 R) and A 2A (A 2A R) receptors. Interestingly, A 2A R levels are upregulated in cortical and hippocampal regions in several pathological conditions such as Alzheimer’s disease, tauopathies or epilepsia. Such abnormal upregulations have been particularly reported in astrocytes, glial cells that play a key role in regulating synaptic plasticity. However, the overall impact and the underlying mechanisms associated with increased A 2A R in astrocytes remain poorly understood. In the present study, we induced the upregulation of A 2A R in hippocampal astrocytes using dedicated AAVs and comprehensively evaluated the functional consequences in 4 months-old C57Bl6/J mice. Our results show that A 2A R upregulation promotes cell-autonomous alterations of astrocyte reactivity, morphology and transcriptome, with a link to aging-like phenotype as well as non-cell autonomous impairments of neuronal excitability and microglial phenotype. These changes driven by a restricted A 2A R upregulation in hippocampal astrocytes were sufficient to induce impairments of short-term spatial memory (Y-Maze task) and spatial learning (Barnes Maze task). This study highlights the impact of astrocytic A 2A R upregulation, as seen in various neurological conditions, on the development of a detrimental multicellular response associated with memory alterations and provides an additional proof-of-concept for the value of targeting this receptor in different neurodegenerative conditions. Biological sciences/Neuroscience Health sciences/Diseases Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION Adenosine is a widespread modulator of the central nervous system (CNS) involved in a large number of key processes controlling cerebral homeostasis 1 . The effects of adenosine in the brain are mediated by the activation of G-protein-coupled adenosine receptors, in particular A 1 (A 1 R) and A 2A (A 2A R) receptors. Together, they fine tune synaptic plasticity through neuronal and glial-based mechanisms, by regulating both the pre-synaptic release and neurotransmitter uptake (e.g. glutamate and GABA) as well as post-synaptic activity, notably by affecting the function of other receptors, for instance D2R, mGluR5 or NMDAR 1 – 9 . These functions likely explain the modulatory impact of caffeine, a non-selective adenosine receptor antagonist, towards brain network activity, related molecular processes and cognitive functions in various species 1 , 10 – 14 . The parenchymal levels of adenosine receptors, especially the A 2A R subtype, are upregulated in several neurological conditions 15 , and particularly in the brain of patients with cognitive pathologies such has Alzheimer’s disease, other tauopathies, depression or seizures as well as related experimental models 16 – 23 . Accrediting a detrimental role of such A 2A R dysregulation, receptor overactivation using a selective agonist 24 or its selective upregulation in neurons 16 – 18 , 25 – 27 have been shown to favor the development of cognitive impairments. In line, normalizing A 2A R function was found to alleviate behavioral and plasticity impairments in various neurodegenerative models 17 , 19 , 21 , 28 – 31 . Besides neurons, pathological upregulation of A 2A R also occurs in astrocytes 18 , 22 , 32 – 34 , glial cells highly intertwined with synaptic processes 35 . Few in vitro studies demonstrated that A 2A R upregulation in astrocytes functionally alters several of their known functions such as the recycling of glutamate, the activity of Cx43 gap junction hemichannels and their pro-inflammatory profile 6 , 7 , 36 – 38 . In vivo , cell-autonomous and non-cell autonomous effects of such A 2A R astrocytic dysregulation remain largely ill-defined. To fill this gap, the present study aimed at investigating the functional outcomes of A 2A R astrocytic upregulation on hippocampal astrocyte biology as well as its consequences on neuronal and microglial biology and, ultimately, memory. MATERIALS AND METHODS Animals. Two-months male C57Bl6/J mice (from Charles Rivers) were maintained in groups of 5–6 in ventilated cages in a SOPF facility (12h/12h light/dark cycle, 22°C), with ad libitum access to food and water. The animals were maintained in compliance with European standards for the care and use of laboratory animals and experimental protocols were approved by the CEEA75 ethical committee (12787-2015101320441671v9). Astrocytic A 2A R hippocampal overexpression. Upregulation the murine A 2A R (mAdora2a), or the enhanced form of the green fluorescence protein (eGFP) as control, were obtained thanks to an adeno-associated virus (AAV) of 2/9 serotype with gene expressions under the control of a GfaABC1D astrocyte-selective promoter (AAV2/9-GfaABC1D-A2A or AAV-A2A; AAV2/9- GfaABC1D-GFP or AAV-GFP, respectively) (Fig. 1 ; Supplementary Fig. 1 ). For the surgical procedure, mice were deeply anesthetised with ketamine (150 mg/kg) and xylazine (10 mg/kg). Buprenorphine (0.05 mg/Kg) was injected subcutaneously 30 min before the beginning of surgery followed by lidocaine (5 mg/kg) applied on the scalp. The AAVs were bilaterally injected in the CA1 hippocampal area of 2 months old C57Bl6/J mice at the following stereotaxic coordinates: -2.5 mm rostro-caudal; +/-1.5 mm medio-lateral axis; -1.7 mm dorso-ventral) with a final concentration of 1.10 9 vg/µl and a total volume of 2 µl per injection site, at a rate of 0.25 µl/min. Before the AAVs injection, the needle was left in place 1 min and 5 min at the end, before being slowly removed. The skin was sutured and mice were allowed to recover. Except for behavioral studies where animals were investigated at the age of 3 to 4 months (i.e. 1 to 2m post-injection), all other evaluations were performed at 4 months of age (i.e. 2m post-injection). Behavioral evaluations. Behavioral experiments as detailed in Supplementary methods were conducted on animals randomly assigned by experimenters blinded to the injection group. Euthanasia. Mice were sacrificed 2 months post-AAVs injection. They were deeply anesthetized with Dolethal (200 mg/kg, intraperitoneal injection, i.p) and lidocaine was applied on the skin and ribs before transcardially perfusion with cold NaCl (0.9%). Brains were removed and post-fixed in 4% paraformaldehyde (PFA) (in PBS; pH 7.4) for 24h at 4°C and transferred into sucrose 30% overnight for saturation before being frozen. Coronal brains sections (35 µm) were cut using a Leica cryostat and stored in a PBS-azide (0,2%) at 4°C until immunohistochemical analysis (A 2A R, GFP, Iba1, GFAP, STAT-3, YAP). Immunohistological and quantification procedures related to these samples are described below. Transcriptomic analysis of hippocampal astrocytes. Tissue preparation. The cell sorting method was performed using the Neural Tissue Dissociation Kit (#130-092-628; Miltenyi Biotec; Supplementary Fig. 2A ). To do so, left and right hippocampi were dissected out following cervical dislocation and directly collected in HBSS buffer (1X; #55021C; sigma) and kept at 4°C. Hippocampi were then cut using dissecting scissors and incubated for 15 min at 37°C to facilitate enzymatic digestion. Additional cell dissociation was performed using polished Pasteur pipettes with three different diameters (from the largest 5–6 mm, to the medium 3–4 mm, to the smallest 1–2 mm). Homogenates were slowly aspirated 10 times in each pipette and then were incubated at 37°C for 10 min under slow and continuous rotation. Samples were then centrifuged (1249 rpm; 10min at room temperature, RT) in an HBSS solution (1X; #55037C; Sigma) and the supernatants discarded. Myelin removal with MS column. The pellet was resuspended in an enzymatic solution from the kit before being incubated with the magnetic myelin removal beads (#130-096-731; Miltenyi Biotec) for 15 min at 4° C. Cells were then washed by using Buffer myelin removal (BMR) solution and then centrifuged (1249 rpm; 10 min at RT). The pellet was re-suspended in BMR solution and loaded onto the MS column, placed in the magnetic field separator. The eluted solution (unlabeled cells) was collected, containing cells without the myelin, while the magnetically labeled cells (cells with myelin) were retained on the MS column. The eluted solution was centrifuged in order to eliminate dead cells (1249 rpm; 5 min at RT). Anti-ACSA-2 Microbeads labeling with MS column. The pellet was re-suspended in BMR solution with FcR blocking solution. Samples were incubated 10 min at 4°C before addition of the anti-ACSA-2 (Astrocyte Cell Surface Antigen 2; 39 Microbeads (#130-097-679; Miltenyi Biotec) and the solution was re-incubated for 15 min at 4°C then centrifugated (1249rpm; 10 min at 4°C). The pellet was re-suspended in BMR solution and loaded onto the MS column, placed in the magnetic field of the MACS separator. The magnetically labeled ACSA-2 cells were retained within the column, while the unlabeled cells ran through and were eluted. To increase purity, the positively selective fraction was separated over a second MS column. In order to perform the sequencing analysis, RNA was extracted from the ACSA-2 positive cell suspension corresponding of astrocytic cells and RNAseq performed as indicated in the Supplementary methods . 3D astrocytic morphology. At the time of AAV-A2A and AAV-GFP stereotaxic injections, a dedicated group of mice also received a retro-orbital (RO) venous sinus delivery of an AAV-PHP.eB at 5.10 10 vg in 50 µl total volume using a U100 insulin syringe (BD micro-fine 0.3 mL, 30-gauge needle). This AAV with a PHP.eB capsid (AAV-PHP.eB-GfaABC1D-Tomato or AAV-PHP.eB), known to cross the blood-brain barrier after intravenous injection 48 , was used to express the fluorescent Tomato protein in sparse astrocytes to allow 3D imaging afterwards (Fig. 2 E-G). For these mice, brains were removed and post-fixed in 4% PFA (in PBS; pH 7.4) for 24 h at 4°C and stored into PBS-azide until process. They were cut using a Leica vibratome (parameters: 0.12 speed and amplitude of 2 µm) at a thickness of 100 µm according to the coronal axis. The astrocytic 3D-reconstructions were performed thanks to the confocal microscope and the imaging software Imaris. Astrocytic 3D-reconstruction and analysis. The 100 µm mouse floating vibratome sections were directly mounted on slides and coverslips (High performance thickness 1 1/2 and diameter 0.170 ± 0.005mm; Zeiss). Tomato-positive astrocytes of the CA1 area were imaged on a Zeiss LSM-710 confocal microscope taking-up to 50 stacks at 0.43µm steps with a x63 oil objective (optimal frame size of 1348). The complex arborization of the CA1 astrocytes was 3D reconstructed using the “filament tracer” rendering function in Imaris (Bitplane) and 3D analyzed using the Imaris plug-in “X-tension” (4–6 astrocytes/mice). Then the 3D cell and soma volumes as well as the number, the length of the processes and the Sholl intersections were analyzed. Sholl intersections are the number of times a cell extension in a 2D representation intersects the concentric circles, or radius, positioned at a 1 µm interval and starting from the cell soma. 3D Microglia 3D-reconstruction and analysis. After performing Iba1 immunofluorescence on the 35 µm mouse floating sections, Iba1 + cells of the CA1 area were imaged using a Zeiss Spinning Disk high-resolution microscope taking-up to 15 z-stacks at 1.5 µm steps with a 40x oil objective. The complex arborization of the CA1 microglia was 3D reconstructed using the “filament tracer” rendering function in Imaris and 3D analyzed using the Imaris plug-in “X-tension” (10–15 microglia/mice). Then the Sholl intersections were analyzed. Determination of Neuronal excitability using DREADD. In order to evaluate the neuronal excitability in animals with astrocytic A 2A R upregulation, a serotype 5 AAV carrying hM3Dq DREADD receptor (Designer Receptor Exclusively Activated by Designer Drugs) and mCherry genes, under the control of the neuronal CaMKII promoter, was used (AAV5-CAMKII-hM3Dq-mCherry or AAV-hM3Dq). The AAV-hM3Dq was bilaterally co-injected with the AAV-A2A or AAV-GFP in the CA1 hippocampus area at the concentration of 1.10 9 vg/µl with a total volume of 2 µl per injection site at a rate of 0.25 µl/min (N = 5/group). Two months following the AAV stereotaxic injections, mice were i.p. administrated with clozapine-N-oxide (CNO; 5 mg/kg), an hM3Dq exogeneous and synthetic ligand, or saline (NaCl; 0,9%) as a control, 90 min before sacrifice (as described above) to acutely activate the hM3Dq-DREADD receptor and evaluate the immediate early gene (IEG) expression by mRNA and immunohistochemical analysis (Fig. 4 B). To validate the neuronal expression of hM3Dq-DREADD receptor, an RFP immunostaining was performed to amplify and visualize the mCherry signal. RNA extractions and RT-qPCR from whole hippocampi were performed as described previously 16 . Sequences of primers used in this study are given in Supplementary Table 1 . Cyclophilin A was used as a reference housekeeping gene for normalization. Immunohistological and analysis procedures regarding these samples are described in the Supplementary methods . Preparation of synaptosomes. One hippocampus per mouse was homogenized in 200µl of Tris buffer (pH 7.4) containing 10% sucrose using a Potter-Elvehjem on ice. Protein concentrations were quantified using the BCA assay (Pierce) and diluted in Tris buffer (pH 7.4) containing 10% sucrose to obtain a final concentration of 4ug/µl. 200µl of Syn-PER™ Synaptic Protein Extraction Reagent (#87793; Thermofisher, France) was then added to 100µl of the protein homogenates. The samples were homogenized again using a Potter-Elvehjem and then centrifuged (1200g; 10 min at 4°C). Cell debris were discarded, supernatants centrifuged (15000g; 20min at 4°C) and the resulting pellets resuspended in 100µl of Syn-PER™ solution. These samples were stored at -20°C until use. Related Western blot analysis is described in the Supplementary methods . Statistical analysis. The results were expressed as the mean ± SEM. Differences between groups were determined using either Student's t-test, One-Way ANOVA followed by a Tukey’s post-hoc test or by two-way ANOVA. All statistical analyzes were performed with GraphPad Prism version 8 software. A p-value (P) of less than 0.05 was considered significant. RESULTS Astrocytic upregulation of A 2A R in the mouse CA1. To determine the impact of astrocytic A 2A R upregulation in the mouse hippocampus, we bilaterally injected dedicated adeno-associated viral (AAV) vectors allowing the selective expression of A 2A R (AAV-A2A) or GFP (AAV-GFP), taken as a control, in the CA1 astrocytes of 2 months-old C57Bl6/J mice and animals studied 2 months post-injection (Fig. 1 A). Immunohistochemistry using antibodies against A 2A R or GFP allowed to visualize their hippocampal levels in the different CA1 sublayers i.e. stratum oriens (SO), pyramidal layer (PL) and stratum radiatum (SR) (Fig. 1 B; Supplementary Fig. 1 ). Co-immunostainings using antibodies directed against astrocytic (GFAP, Sox2, S100β), neuronal (NeuN) and microglial (Iba1) markers showed an exclusive expression of A 2A R by astrocytes, as expected (Fig. 1 C). Cell-autonomous impact of astrocytic A 2A R upregulation on reactivity and morphological complexity. Astrocytes can adopt a reactive phenotype which is notably characterized by an increased expression of GFAP, an intermediate filament protein. We found a significant increase of the GFAP + staining in the CA1 of AAV-A2A mice as compared to the AAV-GFP control (+ 100.2 ± 38.3%; P = 0.0034; Student’s t-test; Figs. 2 A-B). Recent studies have also identified the JAK2/STAT3 signaling pathway as a central player in the induction of the astrocytic reactive state in pathological conditions such as Alzheimer's disease and Huntington's disease 49 . Accordingly, we performed a GFAP/STAT3 co-immunostaining and observed a significant increase of STAT3 + staining within the GFAP + astrocytes of the CA1 area in the AAV-A2A condition as compared to the AAV-GFP control (+ 68.3 ± 35.9%; P = 0.0024; Student’s t-test; Figs. 2 C-D). Moreover, astrocytes continually adapt to their environment and exhibit morphological changes eventually associated with their reactive state 50 . In order to assess more finely the morphological changes of astrocytes upon A 2A R upregulation, a 3D-reconstruction of the latter was performed, following a retro-orbital injection of an AAV-PHP.eB allowing the selective expression of tdTomato in sparse CA1 astrocytes (cytosol and arborization), of both AAV-A2A and AAV-GFP-injected mice (Figs. 2 E-F). We thus imaged isolated tdTomato + astrocytes in the CA1 stratum radiatum sublayer by high-resolution imaging and performed their 3D-reconstruction using Imaris software (Fig. 2 G). Our results indicated a significant higher number of intersections, as shown by Sholl analysis, consistent with a higher number of the astrocytic processes in AAV-A2A vs. AAV-GFP mice (Figs. 2 H-I). A decreased astrocytic processes length was also observed (Fig. 2 J) without change of the overall astrocyte or soma volumes (Fig. 2 K-L). Together, these results support that astrocytic A 2A R upregulation promotes astrocyte reactivity and complexity. Aging-like molecular signature induced by astrocytic A 2A R upregulation. To gain molecular insights on the cell-autonomous impact of astrocytic A 2A R upregulation, we performed a RNA sequencing analysis from hippocampal astrocytes of AAV-A2A and AAV-GFP-injected mice. To do so, we used magnetic cell sorting thanks to ACSA-2 (Astrocyte Cell Surface Antigen 2 39 ; Supplementary Fig. 2A ) magnetic microbeads in order to isolate astrocytes from freshly dissected hippocampi. Following RNA sequencing, and using an available database 51 , we first validated our enrichment procedure by comparison of the expression of known genes specifically expressed by astrocytes with genes expressed by other cell types ( Supplementary Fig. 2B ). The first Principal Composant (PC1), which explained 40.24% of the variance, nicely separated our experimental conditions (Fig. 3 A). We found 1127 differentially expressed genes (DEG) in the AAV-A2A condition as compared to the AAV-GFP control, 916 being downregulated and 211 upregulated (|Log2 fold-change (FC)|>0.32; Adjusted P-value (Padj) < 0.05; Fig. 3 B; list of DEG available on Supplementary Table 3 ). The top ten of downregulated genes is given on Fig. 3 Ci . Functional enrichment analysis provided by DAVID and GSEA analysis indicated that downregulated astrocytic genes in AAV-A2A mice are associated with several metabolic processes such as insulin signaling as well as glucose and glutamate metabolisms. We also observed a significant impact on transcriptional processes (Fig. 3 D; Supplementary Fig. 3 ). Regarding upregulated genes, as expected, we found A 2A R ( Adora2a ). We also observed the upregulation of Timp1 (Log2 FC = 3.19; Padj = 1.59 E-07 ), a gene encoding a metalloproteinase inhibitor, being involved in astrocyte reactivity as well as in neurodegenerative conditions 52 , 53 (Fig. 3 Cii ). GSEA analysis supported that upregulated genes were associated with inflammatory processes (NFκB, TNFɑ and Il1) as well as oxidative stress and DNA repair pathways (Fig. 3 E; Supplementary Fig. 3 ), all previously linked to cellular processes associated with aging 54 , 55 . One of the hallmarks of aging, cellular senescence, has been particularly linked with astrocytes in AD 56 , 57 . Interestingly, it has been recently demonstrated that astrocytic senescence associates with a reduction of cdk6 through an impairment of the activity of YAP (Yes1 Associated Transcriptional Regulation) 58 . Accordingly, our data highlighted significant reductions in the expressions of cdk6 (Log2 FC=-3.03; Padj = 8.85 E-31 ; Fig. 3 Ci ) and yap1 , coding YAP (Log2 FC=-1.12; Padj = 6.93 E-05 ). In order to validate impairment of YAP, instrumental to astrocyte senescence 58 , we performed co-immunostainings against YAP and GFAP (Fig. 3 F). Our data showed a significant reduction in the percentage of YAP + cells among GFAP + cells in AAV-A2A vs. AAV-GFP mice (-25.0 ± 11.2%; P = 0.002; Student’s t-test) as well as a reduction of the nuclear YAP staining of GFAP + astrocytes (-45.0 ± 11.5%; P = 0.0183; Student’s t-test) in the CA1 of AAV-A2A vs. AAV-GFP mice (Fig. 3 G). Further, we also found that the volume of astrocyte nuclei was significantly enlarged in YAP + GFAP + of A 2A R expressing astrocytes as compared to GFP controls (+ 22.8 ± 8.0%; P = 0.0344; Student’s t-test; 6–8 nuclei per animals; N = 4–5/group; not shown), another hallmark of senescence 59 , 60 . Together, these data suggested that the upregulation of A 2A R in hippocampal astrocytes is sufficient to elicit an aged and senescent-like phenotype. In this sense, we compared our transcriptomic data with the signature of astrocytes isolated from aged mice 61 but also to a list of “astrocytic senescence-related genes” extracted from the literature (Fig. 3 H; Supplementary Table 4 ; Supplementary Table 5 ). We found an overlap with 33 genes (out of 358) varying similarly in the AAV-A2A animals when compared the “aged” signature. Same applied regarding 13 genes (out of 62) belonging the “astrocytic senescence-related genes” signature (Fig. 3 I). Astrocytic upregulation of A 2A R favors neuronal excitability. Astrocytes regulate glutamate neurotransmission and synaptic plasticity 62 . Interestingly, the above-mentioned transcriptomic data supported that astrocytic upregulation of A 2A R significantly impacts genes involved in glutamate release, in line with previous data reporting that A 2A R tightly controls (inhibits) glutamate reuptake by astrocytes 5 , 6 . This led us to address the effect of A 2A R upregulation on neuro-astroglial communication and particularly markers of neuronal excitability. We first prepared hippocampal synaptosomes from AAV-GFP and AAV-A2A mice to evaluate the phosphorylation of glutamatergic receptor subunits important for the synaptic trafficking and conductance of NMDA and AMPA receptors, focusing on Y1472 of GluN2B and S831 of GluA1 63,64 . While pY1472 GluN2B/GluN2B ratio did not significantly change in AAV-A2A mice, we observed a significant increase (+ 161.4 ± 94.7%; P = 0.003; Student’s t-test) of the synaptosomal pS831 GluA1/GluA1 ratio as compared to AAV-GFP animals (Fig. 4 A). Then, we used a DREADD chemogenetic tool to determine whether A 2A R upregulation in astrocytes was prone to affect neuronal activation. We jointly expressed the activatory Gq-coupled DREADD receptor (hM3Dq; with an mCherry reporter) in CA1 neurons with A 2A R or GFP in CA1 astrocytes (Fig. 4 B). A co-immunostaining against A 2A R and RFP, in order to amplify and visualize the hM3Dq-mCherry signal, confirmed the astrocytic expression of A 2A R (green; Fig. 4 C) and the neuronal expression of the hM3Dq (red; Fig. 4 C). Two months post-AAV injections, we intraperitoneally injected the exogeneous and synthetic ligand CNO of hM3Dq, or saline as control, to selectively activate hippocampal neurons. Animals were studied 90 min later (Fig. 4 B). We evaluated the mRNA expression of immediate early genes (IEGs) as an indicator of neuronal activation by CNO injection. As expected, our data demonstrated a significant hippocampal increase of Dusp1 , JunB and c-fos levels in CNO-treated (vs. saline) animals (Figs. 4 D-F). Notably, the magnitude of IEG activation was significantly larger in AAV-A2A mice as compared to AAV-GFP animals in CNO-treated condition (Figs. 4 D-F). C-Fos immunohistochemistry confirmed qPCR data, as shown on Figs. 4 G-H (controls are shown in supplementary Fig. 4 ). Altogether these data demonstrated that astrocytic changes elicited by A 2A R upregulation led to an alteration of neuro-astroglial communication towards an exacerbated neuronal response upon DREADD-mediated activation. Astrocytic A 2A R upregulation alters microglial phenotype. Besides the link with neurons and considering that A 2A R upregulation activates a pro-inflammatory signature in astrocytes, we further aimed at determining to which extent such astrocyte-autonomous changes may impact microglial cells. We first performed an immunohistochemistry directed against Iba1, a calcium binding protein increased following microglial activation (Fig. 5 A). Our results showed a significant increase of the Iba1 + staining in AAV-A2A animals without change in the density of Iba1 + cells (Figs. 5 B-C). Like astrocytes, microglial phenotype exhibits a significant heterogeneity and complexity depending on cellular environment 65 . To capture potential changes of microglial morphology, we performed a 3D-analysis from Iba1 immunofluorescence. The Sholl analysis revealed a slight but significant reduction of the microglial complexity in AAV-A2A animals as compared to AAV-GFP controls (P < 0.001; Two-Way ANOVA; Figs. 5 D-E). In addition, we evaluated the level of CD68, a lysosomal marker associated with microglial phagocytosis, essential for eliminating pathogens and misconformed proteins. To do so, co-immunostaining against both Iba1 and CD68, was performed and, after 3D analysis, we observed a decreased level of the CD68 + staining in the Iba1 + cells, suggestive of a reduced microglia phagocytosis in the AAV-A2A animals as compared to the AAV-GFP control (-41.6 ± 32.4%; P = 0.046; Student’s t-test; Figs. 5 F-G). Together these data suggested that the upregulation of A 2A R in astrocytes is sufficient to affect microglial phenotype. Astrocytic A 2A R upregulation in CA1 impairs short-term spatial memory and spatial learning. Finally, to determine whether cell-autonomous and non-cell-autonomous modifications induced by the astrocytic upregulation of A 2A R in the hippocampus were prone to alter memory performances, we performed two hippocampus-dependent spatial memory tests following an Elevated-Plus Maze evaluation. Elevated-Plus Maze revealed no difference in either the locomotor activity or anxiety-like behavior (percentage of the time spent in the open arms) in animals overexpressing astrocytic A 2A R ( Supplementary Figs. 5A-C ). Short-term spatial memory performance was assessed using the Y-maze test (Fig. 6 A). We first observed no difference regarding the distance moved and the velocity ( Supplementary Figs. 5D-E ). A discrimination index, corresponding to the animal preference for the new arm versus the familiar arm during the retention phase, was calculated and showed a preference > 50% i.e. vs. chance for both groups (P < 0.0001; One-Sample Student’s t-test), indicating that AAV-GFP and AAV-A2A mice both had a significant preference for the novel arm (Fig. 6 B). However, the discrimination index was significantly lower in AAV-A2A animals as compared to AAV-GFP controls, indicating reduced short-term spatial memory (P = 0.03; Fig. 6 B). We also evaluated long-term spatial memory performances using the Barnes maze task (Fig. 6 C). During the 4 days of the learning, the AAV-A2A group exhibited significantly higher distance (P < 0.001; Two-Way ANOVA), primary latency (P < 0.001; Two-Way ANOVA) and primary errors (P < 0.001; Two-Way ANOVA) to find the goal box as compared to AAV-GFP animals, suggesting altered spatial learning abilities (Figs. 6 D-F). During the retention stage, 24 h after the learning, the animals did not show any difference regarding the distance moved or the velocity ( Supplementary Figs. 5F-G ). The percentage of time spent in the target quadrant was significantly higher than 25% i.e. than chance (P = 0.021 in AAV-GFP animals; P < 0.0001 in AAV-A2A animals; Student’s t-test) and similar for both groups, indicating intact spatial memory for both groups (Fig. 6 G). Overall, these data highlighted that the multicellular alterations induced by the hippocampal upregulation of A 2A R in astrocytes ultimately lead to impairments of the short-term spatial memory and learning abilities in mice. DISCUSSION The adenosine A 2A receptor is essential to fine tune synaptic plasticity by coordinating neuro-glial communication, in particular in the hippocampus. Indeed, presynaptic A 2A R controls the release of neurotransmitters such as glutamate 66 while it gates its post-synaptic activity, notably by regulating NMDAR and mGluR5 functions 2 , 67 . Astrocytic A 2A R also controls neuronal activity by directly regulating the extracellular levels of both glutamate and GABA in the synaptic cleft. Indeed, A 2A R has been shown to respectively inhibit glutamate and increase GABA uptake by astrocytes 5 – 7 . The importance of astrocytic A 2A R has been particularly emphasized by the compromised hippocampal synaptic plasticity and glutamate homeostasis following conditional deletion of astrocytic A 2A R in mouse models 32 , 68 . Overall, neuronal and astroglial A 2A R are likely to support hippocampal function and memory encoding. Importantly, the level of A 2A R is upregulated in hippocampal neurons and astrocytes in different allostatic and pathological contexts. Neuronal A 2A R upsurge during development has been recently shown to control synapse stability 69 while during aging it favors alterations in plasticity and memory loss 17 , 70 . Neuronal A 2A R also rises in several detrimental situations such as chronic stress 19 as well as in Alzheimer’s Disease (AD) and other tauopathies 16 , 17 . Our recent works in AD models particularly highlighted the instrumental role of neuronal A 2A R dysregulation in the loss of synapses though neuron-microglia miscommunication 16 , 27 . In sharp contrast, although astrocytic dysregulation of A 2A R has been observed in AD and epilepsy 18 , 22 , the outcomes and their underlying mechanisms remain largely ill-defined. The present study tackles, for the first time, the cellular and molecular in vivo mechanisms resulting from A 2A R upregulation in hippocampal astrocytes. Our data demonstrate that the sole upregulation of astrocytic A 2A R is sufficient to induce multi-cellular alterations involving astrocyte-autonomous and non-cell autonomous effects, ultimately interfering with learning and memory processes. Our data show that A 2A R upregulation induces astrocyte-autonomous effects characterized by astrocytic reactivity as well as significant morphological and molecular changes. Indeed, in response to A 2A R upregulation, hippocampal astrocytes adopt a so-called reactive phenotype 50 , 71 , characterized by an increased expression of GFAP, STAT3 and Timp1 , an inhibitor of metalloproteinase, all being particularly identified as markers common to several neurodegenerative conditions, including AD 49 , 52 , 72 . Morphologically, this reactivity translates into a greater number of astrocytic processes, and therefore complexity of astrocytic arborization, that may lead to functional modifications, particularly at the tripartite synapses where astrocytes finely regulate neurotransmitter dynamics and neuronal signaling 73 , 74 . Our data suggest that astrocyte reactivity induced by A 2A R upregulation might promote impairments of the excitatory/inhibitory balance. Indeed, our RNA-Seq data notably highlight a reduction in a signaling pathway related to the astrocytic management of glutamate. This observation is in line with previous data showing an antagonistic interaction between A 2A R and Na + /K + ATPase-α2 controlling GLT-1-dependent glutamate uptake by astrocytes 6 . Our hypothesis is also sustained by two other experimental evidence. First, astrocytic upregulation of A 2A R leads to increased hippocampal levels of phosphorylation at serine 831 of the GluA1 subunit of AMPA receptors, which has been associated with enhanced network activity in epilepsy 75 . Second, we found that A 2A R upregulation by astrocytes exacerbates the response of IEGs to chemogenetic neuronal activation, supporting a link between astrocytic A 2A R and hyperexcitability. Together, our observations fit well with the A 2A R upregulation by hippocampal astrocytes observed in patients presenting with mesial temporal lobe epilepsy or also observed in the kainate mouse model 22 , 76 . This is also coherent with the astrocytic A 2A R upregulation described in the brain of AD patients 18 , where the risk of seizures is at least 3 times higher than in healthy controls 77 . These changes of hippocampal excitability might occur concomitantly with other changes unveiled by our transcriptomic data that also indicate that astrocytic A 2A R upregulation favors miscommunication between astrocytes and neurons by presumably impairing insulin-mediated glucose uptake and astrocytic glycolysis 35 , 78 . The astrocytic transcriptomic signature unveiled numerous processes associated with an aging-like astrocytic phenotype such as oxidative stress, induction of the NF-κB pathway, DNA repair mechanisms as well as a significant reduction in transcriptional processes and cell cycle 79 . Accordingly, we have identified glutamine synthetase (Log2 FC=-1.75; Padj = 2.37 E − 11 ) and the cyclin-dependent kinase cdk6 among the most downregulated genes, previously described to be markers of astrocytic senescence 58 , 80 , 81 . Recent data demonstrated that hippocampal astrocytic senescence associates with a reduction of cdk6 induced by a defective YAP activity, a transcriptional coregulator, in these glial cells 60 . This study also reported that the conditional deletion of YAP in astrocytes is sufficient to promote several hallmarks of astrocyte senescence, including hypertrophic morphology, increased β-galactosidase activity, and upregulation of several senescence-associated genes such as p16, p53 and NF-κB as well as downregulation of Lamin B1 58 . Interestingly, our data also demonstrate a loss of YAP expression in GFAP + astrocytes, as well as a significant reduction of the YAP level in astrocytic nuclei upon upregulation of A 2A R. This fits well with the observation that activation of Gs-Protein Coupled Receptors, like A 2A R, promotes phosphorylation of YAP, preventing its translocation to the nucleus and its action as a transcriptional co-regulator 82 . Further, we identified a significant increase of nuclei size in astrocytes presenting with reduced YAP immunoreactivity, also characteristic of astrocytic senescence 60 . Finally, we found some overlap between our transcriptomic dataset and the signatures of astrocytes isolated from aged mice 61 as well as an "astrocytic senescence-related genes" signature extracted from the literature 54 , 60 , 79 . Overall, our data therefore support that A 2A R upregulation by hippocampal astrocytes implements several cell-autonomous processes associated with astrocyte senescence and aging. Astrocytic senescence is also associated with the upregulation of molecules associated to SASP (senescent-associated secreted phenotype), some of which were unveiled in our transcriptomic dataset such as interleukins (i.e. IL-1, IL-33), metalloproteases and metalloproteases inhibitors (i.e. MMP15, Timp1) 83 . These factors enable paracrine signaling to other astrocytes, but also presumably to surrounding cells such as neurons and microglia. Interestingly, our data show that the sole astrocytic upregulation of A 2A R is sufficient to promote microglial reactivity and morphological changes, i.e. a reduction in the number of processes and therefore of microglial complexity. We also observed a striking reduction of microglial lysosomal activity. Interestingly, these morphological and functional microglial changes have been associated with hippocampal aging and neurodegeneration 84 . Our observations, showing a causal link between astrocytic A 2A R and microglia are in line with data previously obtained in a mouse model of Sandhoff disease, showing an association between astrocytic upregulation of A 2A R and microglial activation 34 . Taken together, our data indicate that the sole upregulation of A 2A R in hippocampal astrocytes promote multicellular impairments that impact the hippocampal neuronal network functionality, the astrocyte-neuron coupling and microglial function all previously identified as mandatory for the regulation of memory processes 78 , 85 , 86 . In accordance, we demonstrate that the upregulation of astrocytic A 2A R in the hippocampus is sufficient to promote spatial learning and memory alterations. This observation is in line with a previous work from Orr et al. (2015) 18 showing that conditional removal of A 2A R in astrocytes improved memory in aged mice while the chemogenetic activation of astrocytic Gs-coupled signaling impaired it. However, these data seem controversial with other reports that rather support the detrimental impact of astrocytic A 2A R deletion in memory settings 32 , 68 . A tight control of astrocytic A 2A R might be necessary to ensure proper cognitive functions, especially in pathological conditions. Considering the clinical interest of A 2A R targeting drugs in AD and epilepsy, this warrants further mechanistic studies. In conclusion, our data demonstrate for the first time the cell-autonomous and non-cell autonomous impact of A 2A R astrocytic upregulation in hippocampal astrocytes and support their key role in the control of hippocampal function. Declarations CONFLICTS OF INTEREST DB is a (non-appointed) member of the scientific advisory board of Marvel Biosciences Corp developing an A 2A R antagonist. But there is no conflict of interest regarding the present work. ACKNOWLEDGEMENTS This project was supported by fundings from ANR ADORASTrAU, ANR JANUS and FONDATION Alzheimer (ADOMEMOTAU). This work was also supported by grants from Programmes d’Investissements d’Avenir LabEx (excellence laboratory) DISTALZ (Development of Innovative Strategies for a Transdisciplinary approach to ALZheimer’s disease), Fondation pour la Recherche Médicale as well as Inserm, CNRS, Université de Lille. AL was supported by PhD grants from Fondation pour la Recherche Médicale (ECO202106013670) and Vaincre Alzheimer (FR-24054T). TG was supported by University of Lille and Fondation Vaincre Alzheimer. VG-M was supported by Fondation Alzheimer and Fondation pour la Recherche Médicale (SPF20160936000). Sequencing was performed by the GenomEast platform, a member of the ‘France Génomique’ consortium (ANR-10-INBS-0009). We thank members of the different facilities of "Plateformes Lilloises en Biologie et Santé (PLBS)” - UAR 2014 - US 41 (SOFP facility, In vivo and functional exploration platform and BiCeL). EA was supported by the Association France Alzheimer. References Cunha RA. 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Additional Declarations The authors have declared there is NO conflict of interest to disclose Supplementary Files FigureSupp1.pdf FigureSupp2.pdf FigureSupp3.pdf FigureSupp4.pdf FigureSupp5.pdf SupplementaryTable1.xlsx SupplementaryTable2.xlsx SupplementaryTables345.xlsx Supplementarymaterialuncroppedgels.pdf Supplementarymethods.docx Cite Share Download PDF Status: Published Journal Publication published 23 Jul, 2025 Read the published version in Molecular Psychiatry → Version 1 posted Editorial decision: revise 31 Oct, 2024 Review # 1 received at journal 22 Oct, 2024 Review # 2 received at journal 04 Oct, 2024 Review # 6 received at journal 02 Oct, 2024 Review # 4 received at journal 02 Oct, 2024 Review # 3 received at journal 02 Oct, 2024 Review # 5 received at journal 26 Sep, 2024 Reviewer # 6 agreed at journal 20 Sep, 2024 Reviewer # 5 agreed at journal 20 Sep, 2024 Reviewer # 4 agreed at journal 20 Sep, 2024 Reviewer # 3 agreed at journal 20 Sep, 2024 Reviewer # 2 agreed at journal 20 Sep, 2024 Reviewer # 1 agreed at journal 20 Sep, 2024 Reviewers invited by journal 19 Sep, 2024 Editor assigned by journal 25 Jul, 2024 Submission checks completed at journal 25 Jul, 2024 First submitted to journal 24 Jul, 2024 Unknown event 24 Jul, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4791082","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":331647328,"identity":"f804fbea-8545-4e41-bcef-43980af5d479","order_by":0,"name":"David Blum","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2ElEQVRIie3OsQrCQAyA4chBp1jngvgGQlwKgvgslUKnio6OguAzKPQhnJwjGVwK3USogyB06qC4FrRQC05XR4f7OQ5y3AcBMJn+MS4PAncBFHP11Lr+RBDA8j5EUSOBimD9s4HYx3jAOVyQTvFD5sVo1l8q664jThzSIYIMKZ3uZLsOhntWaqMjxIEnCFKR9lLI5Y5oF6Mkq0l4FSxeJVFKT84+16S8LW4mzvnGh4gydNKApL32yZUGYieT1T1fXHp26t+eWIzJPa705LPe9/ALMJlMJpO+N43ZUVUge4nGAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0001-5691-431X","institution":"Inserm, U1172","correspondingAuthor":true,"prefix":"","firstName":"David","middleName":"","lastName":"Blum","suffix":""},{"id":331647329,"identity":"44049a2d-63bb-4460-bee5-a308715cb7c6","order_by":1,"name":"Agathe Launay","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Agathe","middleName":"","lastName":"Launay","suffix":""},{"id":331647335,"identity":"71813666-d7e6-4f1a-98ca-58d573d03d65","order_by":2,"name":"Kévin Carvalho","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Kévin","middleName":"","lastName":"Carvalho","suffix":""},{"id":331647340,"identity":"4ac779fb-aeae-4fd7-b102-b1f75b638665","order_by":3,"name":"Thibaut Gauvrit","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Thibaut","middleName":"","lastName":"Gauvrit","suffix":""},{"id":331647342,"identity":"45bcfbef-1d12-4d62-a178-c045c10cb87d","order_by":4,"name":"Victoria Gomez Murcia","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Victoria","middleName":"Gomez","lastName":"Murcia","suffix":""},{"id":331647345,"identity":"e7e67058-258e-4612-b854-13f4843510cd","order_by":5,"name":"Emma Augustin","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Emma","middleName":"","lastName":"Augustin","suffix":""},{"id":331647346,"identity":"d28cf792-5656-4543-a7d2-202a309920e3","order_by":6,"name":"Anaëlle Burgard","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Anaëlle","middleName":"","lastName":"Burgard","suffix":""},{"id":331647348,"identity":"2e194484-69dc-4db0-908d-f60d1c9a5635","order_by":7,"name":"Bryan Thiroux","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Bryan","middleName":"","lastName":"Thiroux","suffix":""},{"id":331647349,"identity":"40e802a6-e0ae-449b-ba02-28a97c42ee70","order_by":8,"name":"Didier Vieau","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Didier","middleName":"","lastName":"Vieau","suffix":""},{"id":331647350,"identity":"3a9b6119-f72a-430d-9f61-1459024b586c","order_by":9,"name":"Alexis-Pierre Bemelmans","email":"","orcid":"https://orcid.org/0000-0001-7605-5225","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Alexis-Pierre","middleName":"","lastName":"Bemelmans","suffix":""},{"id":331647351,"identity":"ee55f8b9-1d94-48ac-b5dd-12529490927e","order_by":10,"name":"Stéphanie LeGras","email":"","orcid":"https://orcid.org/0000-0001-6293-6507","institution":"University of Strasbourg, CNRS UMR7104, Inserm U1258","correspondingAuthor":false,"prefix":"","firstName":"Stéphanie","middleName":"","lastName":"LeGras","suffix":""},{"id":331647352,"identity":"adddcd9d-98f5-4ea3-9a9e-8921a02e2d30","order_by":11,"name":"Luc Buee","email":"","orcid":"https://orcid.org/0000-0002-6261-4230","institution":"Univ. Lille, Inserm, CHU-Lille, UMR-S1172 - JPArc - Centre de Recherche Jean-Pierre AUBERT","correspondingAuthor":false,"prefix":"","firstName":"Luc","middleName":"","lastName":"Buee","suffix":""},{"id":331647353,"identity":"84c9a947-b578-436b-9d06-968ddc1308eb","order_by":12,"name":"Miranda Orr","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Miranda","middleName":"","lastName":"Orr","suffix":""},{"id":331647354,"identity":"ec741523-856e-4e4d-acc9-fbe4b2bbf294","order_by":13,"name":"Anne Laurence Boutillier","email":"","orcid":"https://orcid.org/0000-0002-2317-9280","institution":"LNCA","correspondingAuthor":false,"prefix":"","firstName":"Anne","middleName":"Laurence","lastName":"Boutillier","suffix":""},{"id":331647355,"identity":"477c153c-0f17-46d5-afcc-f49ed5e124d5","order_by":14,"name":"Gilles Bonvento","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Gilles","middleName":"","lastName":"Bonvento","suffix":""},{"id":331647356,"identity":"6dfbb799-743d-4fa3-b6d4-a103ec178128","order_by":15,"name":"Karine Cambon","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Karine","middleName":"","lastName":"Cambon","suffix":""},{"id":331647357,"identity":"42ee91b5-ed52-4b57-8d0c-5e02f2a290c7","order_by":16,"name":"Emilie Faivre","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Emilie","middleName":"","lastName":"Faivre","suffix":""},{"id":331647358,"identity":"ac1229fd-5a13-4ee2-87f9-58e2e8eb9ee3","order_by":17,"name":"Déborah Fourmy","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Déborah","middleName":"","lastName":"Fourmy","suffix":""}],"badges":[],"createdAt":"2024-07-23 20:25:57","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4791082/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4791082/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41380-025-03115-9","type":"published","date":"2025-07-23T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":63235721,"identity":"cb33c6dc-c42c-425e-b56a-479f6509308b","added_by":"auto","created_at":"2024-08-26 03:00:16","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1165697,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAAV-based astrocytic A\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2A\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eR upregulation in the CA1 hippocampus in mice. \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Astrocytic A\u003csub\u003e2A\u003c/sub\u003eR upregulation (or the GFP as control) has been obtained following bilateral hippocampal injection (CA1) of AAV2/9 carrying a transgenic A\u003csub\u003e2A\u003c/sub\u003eR murine (AAV-A2A; blue) or enhanced form of GFP (AAV-GFP; white) genes under the control of the GfaABC1D astrocytic promoter. (\u003cstrong\u003eB\u003c/strong\u003e) Representative images of A\u003csub\u003e2A\u003c/sub\u003eR immunostaining highlighting A\u003csub\u003e2A\u003c/sub\u003eR upregulation in the mouse CA1 hippocampus (scale bar=500 μm, upper picture) and, at higher magnification, the distribution within the \u003cem\u003estratum oriens \u003c/em\u003e(SO), pyramidal layer (PL) and \u003cem\u003estratum radiatum \u003c/em\u003e(SR) sublayers of CA1 (scale bar=60 μm, lower picture). (\u003cstrong\u003eC\u003c/strong\u003e) Representative images of A\u003csub\u003e2A\u003c/sub\u003eR co-immunostainings with the astrocytic markers GFAP (a), Sox2 (b) and S100β (c), neuronal marker NeuN (d) and microglial marker Iba1 (e) highlighting the exclusive expression of A\u003csub\u003e2A\u003c/sub\u003eR in CA1 astrocytes (scale bar=20 μm).\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4791082/v1/7bcb6191526244988e8255d7.png"},{"id":63235099,"identity":"4502cd74-aacd-4747-a714-505ad99d5d44","added_by":"auto","created_at":"2024-08-26 02:52:15","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1047669,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAstrocytic A\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2A\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eR upregulation in hippocampus impacts reactivity and complexity.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e Representative images of GFAP immunostainings in the CA1 of AAV-GFP (left panel) and AAV-A2A (right panel) animals (scale bars=100 μm; magnification in red inserts scale bar=50 μm). \u003cstrong\u003e(B)\u003c/strong\u003e Quantification indicates a significant increase of the GFAP\u003csup\u003e+\u003c/sup\u003e staining in the hippocampal CA1 astrocytes of AAV-A2A mice as compared to AAV-GFP controls (N=5-6 mice/group; **P\u0026lt;0.01 vs. AAV-GFP; Student’s t-test). \u003cstrong\u003e(C)\u003c/strong\u003e Representative images of GFAP (green), STAT3 (red) and DAPI (blue) co-immunostainings in the hippocampal CA1 area of AAV-GFP (upper panel) and AAV-A2A (lower panel) animals (scale bar=50 μm). \u003cstrong\u003e(D)\u003c/strong\u003e A significant increase of the STAT3\u003csup\u003e+\u003c/sup\u003e staining in the GFAP\u003csup\u003e+\u003c/sup\u003e astrocytes of AAV-A2A mice was observed as compared to AAV-GFP controls in the hippocampal CA1 area (N=4-6 mice/group; **P\u0026lt;0.01 vs. AAV-GFP; Student’s t-test). \u003cstrong\u003e(E-F)\u003c/strong\u003e A retro-orbital injection of an AAV-PHP.eB carrying Tomato gene under the control of the GfaABC1D astrocytic promoter in both AAV-GFP and AAV-A2A animals was performed \u003cstrong\u003e(E)\u003c/strong\u003e in order to express Tomato protein in sparse CA1 astrocytes (cytosol and arborization; scale bar=60 μm; \u003cstrong\u003eF\u003c/strong\u003e). \u003cstrong\u003e(G)\u003c/strong\u003e Isolated Tomato\u003csup\u003e+\u003c/sup\u003e astrocytes of the \u003cem\u003estratum radiatum\u003c/em\u003e CA1 sublayer were imaged by high-resolution imaging (a) and 3D-reconstructed using Imaris software (b). \u003cstrong\u003e(H-J)\u003c/strong\u003e An increase in the intersection number, as shown by Sholl analysis (***P\u0026lt;0.0001 vs. AAV-GFP, Two-Way ANOVA; \u003cstrong\u003eH\u003c/strong\u003e) as well as of the number of the astrocytic processes (N=14-17 astrocytes from N=5-6 mice/group; **P\u0026lt;0.01 vs. AAV-GFP, Student’s t-test; \u003cstrong\u003eI\u003c/strong\u003e) was found in the \u003cem\u003estratum radiatum\u003c/em\u003e CA1 astrocytes of AAV-A2A condition as compared to the AAV-GFP controls. That was accompanied by a reduced process length (N=14-17 astrocytes from N=5-6 mice/group; *P\u0026lt;0.05 vs. AAV-GFP; Student’s t-test; \u003cstrong\u003eJ\u003c/strong\u003e) \u003cstrong\u003e(K-L)\u003c/strong\u003e No change in the overall astrocyte volume (P=0.40;\u0026nbsp;\u003cstrong\u003eK\u003c/strong\u003e) or the astrocytic soma volume (P=0.74;\u0026nbsp;\u003cstrong\u003eL\u003c/strong\u003e) could be observed AAV-A2A animals as compared to the AAV-GFP controls (N=14-17 astrocytes from N=5-6 mice/group). Values are represented as mean ± SEM. \u003cem\u003eStratum oriens\u003c/em\u003e (SO); pyramidal layer (PL); \u003cem\u003estratum radiatum\u003c/em\u003e (SR) sublayers.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4791082/v1/67139a38dc3d13207355956a.png"},{"id":63235100,"identity":"5a3002bf-4a32-4874-9d31-c206932acb6a","added_by":"auto","created_at":"2024-08-26 02:52:16","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":832483,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAstrocytic A\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2A\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eR upregulation in hippocampus is associated with an aging-like astrocyte signature. (A)\u003c/strong\u003e The principal component analysis (PCA) performed on RNA-seq datasets shows that the two conditions AAV-GFP (white dots) and AAV-A2A (blue dots) are nicely separated on the first Principal component (PC1). \u003cstrong\u003e(B)\u003c/strong\u003e Volcano plot representing the deregulation of 1127 genes in the astrocytes of AAV-A2A vs. AAV-GFP mice using cut offs of |Log2 fold-change (FC)|\u0026gt;0.32 and adjusted P-value (Padj)\u0026lt;0.05. 916 genes were found downregulated and 211 upregulated, including the \u003cem\u003eAdora2a\u003c/em\u003e gene. \u003cstrong\u003e(C)\u003c/strong\u003e Top 10 genes significantly downregulated (red) (i) and upregulated (green) (ii) in hippocampal astrocytes of AAV-A2A animals vs. AAV-GFP controls with |Log2 fold-change (FC)|\u0026gt;0.32 and Padj\u0026lt;0.05. \u003cstrong\u003e(D-E)\u003c/strong\u003e Functional enrichment analysis of GO Biological Process annotations using DAVID (upper panels) and GSEA analysis (lower panels) associated to \u003cstrong\u003e(D)\u003c/strong\u003e downregulated genes and \u003cstrong\u003e(E)\u003c/strong\u003e upregulated genes.\u003cstrong\u003e (F)\u003c/strong\u003e Representative images of GFAP (green), YAP (red) and DAPI (blue) co-immunostainings in the CA1 of AAV-GFP (upper panel) and AAV-A2A (lower panel) groups (scale bar=50 μm). Inserts represent YAP\u003csup\u003e+\u003c/sup\u003e nuclei (dashed lines) in both groups (scale bar=10 μm\u003cstrong\u003e). (G)\u003c/strong\u003e A significant decrease in the percentage of YAP\u003csup\u003e+\u003c/sup\u003e cells among GFAP\u003csup\u003e+\u003c/sup\u003e astrocytes as well as a reduction of the YAP staining within the nuclei of GFAP\u003csup\u003e+\u003c/sup\u003e astrocytes were observed in the CA1 hippocampal area of AAV-A2A mice vs. AAV-GFP controls (N=4-5 mice/group; *P\u0026lt;0.05, **P\u0026lt;0.01 vs. AAV-GFP, Student’s t-test). \u003cstrong\u003e(H)\u003c/strong\u003e Venn diagram comparing the signatures of A\u003csub\u003e2A\u003c/sub\u003eR-overexpressing astrocytes with signature of aged astrocytes (Pan et al., 2018) and astrocytic senescence-related genes. Common genes are annotated as a, b and c. \u003cstrong\u003e(I)\u003c/strong\u003e Heatmap representing the z-score expression (from RNA-Seq data) of common genes (a, b, c) uncovered by the Venn diagram.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4791082/v1/91f5472e02324dc05c227ce8.png"},{"id":63235102,"identity":"83ef9c52-51b8-45d1-a4e3-a417f94b14c1","added_by":"auto","created_at":"2024-08-26 02:52:16","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1060920,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAstrocytic A\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2A\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eR upregulation in hippocampus alters neuronal activation.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e Phosphorylation of the GluN2B subunit (NMDAR) at Y1472 and of the GluA1 subunit (AMPAR) at pS831 in synaptosomal fraction of AAV-GFP and AAV-A2A hippocampi. Quantification shows a significant increase of pS831 GluA1 in AAV-A2A vs. AAV-GFP mice (*P\u0026lt;0.05; **P\u0026lt;0.01 vs. AAV-GFP; Student’s t-test; N=6/group). \u003cstrong\u003e(B)\u003c/strong\u003e Bilateral injection of an AAV5 carrying the hM3Dq gene under the control of the CAMKII neuronal promoter allows the activatory Gq-coupled DREADD-mCherry receptor expression in hippocampal neurons of AAV-GFP and AAV-A2A animals. 2 months following the AAV co-injections, an i.p administration of the exogeneous and synthetic hM3Dq ligand (clozapine-N-oxyde, CNO; 5 mg/kg), or saline (NaCl; 0.9%) as a control, induced neuronal activation measured by the immediate early gene (IEGs) expression. \u003cstrong\u003e(C) \u003c/strong\u003eRepresentative images of A\u003csub\u003e2A\u003c/sub\u003eR (green), RFP-amplified hM3Dq-mCherry (red) and DAPI (blue) co-immunostainings in CA1 hippocampal area (scale bar=200 μm; magnification: scale bar=10 μm). \u003cstrong\u003e(D-F)\u003c/strong\u003e The mRNA expression of \u003cem\u003eDusp1\u003c/em\u003e \u003cstrong\u003e(D)\u003c/strong\u003e, \u003cem\u003eJunB\u003c/em\u003e \u003cstrong\u003e(E) \u003c/strong\u003eand \u003cem\u003ec-fos\u003c/em\u003e \u003cstrong\u003e(F)\u003c/strong\u003e was found significantly higher in CNO-treated vs. saline-treated animals. Notably, IEG expressions were significantly enhanced in AAV-A2A as compared to AAV-GFP mice in CNO-treated conditions. (N=4-5 mice/group; \u003csup\u003e##\u003c/sup\u003eP\u0026lt;0.01; \u003csup\u003e###\u003c/sup\u003eP\u0026lt;0.001: CNO-treated vs. respective saline-treated animals; \u003csup\u003e+\u003c/sup\u003eP\u0026lt;0.05; \u003csup\u003e+++\u003c/sup\u003eP\u0026lt;0.001 in AAV-A2A + CNO-treated animals vs. AAV-GFP + CNO-treated; One-Way ANOVA followed by Tukey’s post-hoc test). \u003cstrong\u003e(G)\u003c/strong\u003e Representative images of c-Fos immunostaining in the CA1, CA2 and DG regions of hippocampus in AAV-GFP (higher panel) and AAV-A2A (lower panel) animals treated with CNO (scale bar=400 μm). \u003cstrong\u003e(H)\u003c/strong\u003e As expected, the intensity of c-Fos was significantly higher in CNO-treated vs. saline-treated animals. c-Fos immunostaining intensity was found significantly higher in AAV-A2A + CNO as compared to AAV-GFP + CNO controls. (N=4-5 mice/group; \u003csup\u003e##\u003c/sup\u003eP\u0026lt;0.01; \u003csup\u003e###\u003c/sup\u003eP\u0026lt;0.001: CNO-treated vs. respective saline-treated animals; \u003csup\u003e+\u003c/sup\u003eP\u0026lt;0.05 in AAV-A2A + CNO-treated animals vs. AAV-GFP + CNO-treated; One-Way ANOVA followed by Tukey’s post-hoc test). Values are represented as mean ± SEM.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4791082/v1/34ad046a1e3299b3c7de8f61.png"},{"id":63235722,"identity":"4e408b9d-2dcc-4e13-b46e-6badd3876055","added_by":"auto","created_at":"2024-08-26 03:00:16","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":939148,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAstrocytic A\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2A\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eR upregulation in hippocampus alters microglial phenotype. (A)\u003c/strong\u003e Representative images of Iba1 immunostaining in AAV-GFP (left panel) and AAV-A2A (right panel) conditions in the CA1 hippocampal area (scale bar=100 μm; magnifications: scale bar=10 μm). \u003cstrong\u003e(B-C)\u003c/strong\u003e A significant increase of the Iba1\u003csup\u003e+\u003c/sup\u003e staining was observed in the hippocampal astrocytes of AAV-A2A condition as compared to the AAV-GFP control (N=4-6 mice/group; *P\u0026lt;0.05 vs. AAV-GFP; Student’s t-test; \u003cstrong\u003eB\u003c/strong\u003e) with no change in the density of Iba1\u003csup\u003e+\u003c/sup\u003e cells \u003cstrong\u003e(C)\u003c/strong\u003e. \u003cstrong\u003e(D)\u003c/strong\u003e Representative images of Iba1 immunostaining (a) and 3D reconstruction (b) (scale bar=10 μm; magnification: scale bar=2 μm). \u003cstrong\u003e(E)\u003c/strong\u003e Decreased in the intersection number (complexity), as shown by Sholl analysis, was found in the microglia of AAV-A2A mice as compared to AAV-GFP controls (N=10-15 microglia from N=5 mice/group; Two-Way ANOVA). \u003cstrong\u003e(F)\u003c/strong\u003e Representative images of Iba1 (red), CD68 (yellow) and DAPI (blue) co-immunostainings in the CA1 hippocampal area in AAV-GFP (higher panel) and AAV-A2A (lower panel) groups (scale bar=10μm; magnification: scale bar=2 μm). \u003cstrong\u003e(G)\u003c/strong\u003e A significant reduction of the CD68\u003csup\u003e+\u003c/sup\u003e staining in the Iba1\u003csup\u003e+\u003c/sup\u003e cells of the \u003cem\u003estratum radiatum\u003c/em\u003e CA1 sublayer was found in AAV-A2A as compared with AAV-GFP controls (N=5-6 mice/group; *P\u0026lt;0.05 vs. AAV-GFP; Student’s t-test). Values are represented as mean ± SEM.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4791082/v1/20b9decd72f16aec79dad829.png"},{"id":63235104,"identity":"fac07a6b-c07a-4f30-a64f-e20cdc63eb6b","added_by":"auto","created_at":"2024-08-26 02:52:16","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":293487,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAstrocytic A\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2A\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eR upregulation in hippocampus impairs short-term spatial memory and spatial learning.\u003c/strong\u003e \u003cstrong\u003e(A)\u003c/strong\u003e Schematic representation of the Y-Maze experimental paradigm described in the Material and Methods section. \u003cstrong\u003e(B)\u003c/strong\u003e The discrimination index, taken as a measure of the short-term spatial preference for the novel arm in the Y-maze task, was significantly lower for the AAV-A2A animals as compared with AAV-GFP controls (N=9 mice/group; *P\u0026lt;0.05 vs. AAV-GFP; Student’s t-test). \u003cstrong\u003e(C)\u003c/strong\u003e Schematic representation of the Barnes Maze experimental paradigm described in the Material and Methods section. \u003cstrong\u003e(D-F)\u003c/strong\u003e During the learning phase of the Barnes task, the distance \u003cstrong\u003e(D)\u003c/strong\u003e, the primary latency \u003cstrong\u003e(E)\u003c/strong\u003e and the number of primary errors \u003cstrong\u003e(F)\u003c/strong\u003e were found significantly higher for the AAV-A2A animals as compared to AAV-GFP controls (N=11-12 mice/group; ***P\u0026lt;0.001; Two-Way ANOVA). \u003cstrong\u003e(G)\u003c/strong\u003e No change regarding the time spent in the target quadrant during the retention phase was found between AAV-A2A and AAV-GFP animals (N=11-12 mice/group; P\u0026gt;0.05; Student’s t-test). Values are represented as mean ± SEM.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4791082/v1/642202dce794c0e3cff6a380.png"},{"id":87465479,"identity":"e74bf6ab-e546-449d-8a1d-befe8b989184","added_by":"auto","created_at":"2025-07-24 07:16:18","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7138454,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4791082/v1/db55f573-d2a3-411f-989c-3094932512d1.pdf"},{"id":63235106,"identity":"85ac3c6a-7c55-435f-85dc-62fe7063a6dd","added_by":"auto","created_at":"2024-08-26 02:52:16","extension":"pdf","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":159535,"visible":true,"origin":"","legend":"","description":"","filename":"FigureSupp1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4791082/v1/06bf66491a3496fe8e30ef9c.pdf"},{"id":63235107,"identity":"b4490346-3bd1-4484-bed9-c0f25d2df89d","added_by":"auto","created_at":"2024-08-26 02:52:16","extension":"pdf","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":414705,"visible":true,"origin":"","legend":"","description":"","filename":"FigureSupp2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4791082/v1/cf06335bee7d802b56c7e92b.pdf"},{"id":63235108,"identity":"8a59f5bf-8542-407b-96eb-a0ada795152a","added_by":"auto","created_at":"2024-08-26 02:52:16","extension":"pdf","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":1052558,"visible":true,"origin":"","legend":"","description":"","filename":"FigureSupp3.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4791082/v1/e618fe3d95e9048b54d23093.pdf"},{"id":63235114,"identity":"103e7ff9-dfb3-4a31-bbc9-d33ee838e385","added_by":"auto","created_at":"2024-08-26 02:52:17","extension":"pdf","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":342969,"visible":true,"origin":"","legend":"","description":"","filename":"FigureSupp4.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4791082/v1/0b62b1ad9b50a2e97d8c4c8e.pdf"},{"id":63236263,"identity":"7ca2c80f-97e0-488c-8f29-621788966874","added_by":"auto","created_at":"2024-08-26 03:08:16","extension":"pdf","order_by":12,"title":"","display":"","copyAsset":false,"role":"supplement","size":134016,"visible":true,"origin":"","legend":"","description":"","filename":"FigureSupp5.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4791082/v1/24f15a4f3433af79c0c30187.pdf"},{"id":63235112,"identity":"e0a50553-a8a1-4d88-9b7f-f34a742d7213","added_by":"auto","created_at":"2024-08-26 02:52:16","extension":"xlsx","order_by":13,"title":"","display":"","copyAsset":false,"role":"supplement","size":10003,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4791082/v1/1ba66b2f6802192c47c33108.xlsx"},{"id":63235110,"identity":"b314466d-7f8d-4176-9f40-3c10e6ed8ded","added_by":"auto","created_at":"2024-08-26 02:52:16","extension":"xlsx","order_by":14,"title":"","display":"","copyAsset":false,"role":"supplement","size":12830,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4791082/v1/edafaf0d653f21e358969eb4.xlsx"},{"id":63235115,"identity":"07b12d57-9f4a-4322-bce1-1d3e36da13c4","added_by":"auto","created_at":"2024-08-26 02:52:17","extension":"xlsx","order_by":15,"title":"","display":"","copyAsset":false,"role":"supplement","size":107882,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTables345.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4791082/v1/d515c7b905d2cbedb1375cf4.xlsx"},{"id":63235105,"identity":"6e52997b-8f00-4dfe-9ca0-c54a0ecd6a37","added_by":"auto","created_at":"2024-08-26 02:52:16","extension":"pdf","order_by":16,"title":"","display":"","copyAsset":false,"role":"supplement","size":745027,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterialuncroppedgels.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4791082/v1/018be9d2e0b06076b94b77c2.pdf"},{"id":63235723,"identity":"6932cb82-56b0-48b9-8e47-9388cfb3448d","added_by":"auto","created_at":"2024-08-26 03:00:16","extension":"docx","order_by":17,"title":"","display":"","copyAsset":false,"role":"supplement","size":25967,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymethods.docx","url":"https://assets-eu.researchsquare.com/files/rs-4791082/v1/31679bb0b929ad6a1c2a7739.docx"}],"financialInterests":"The authors have declared there is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose","formattedTitle":"\u003cp\u003eUpregulation of adenosine A\u003csub\u003e2A\u003c/sub\u003e receptor by astrocytes is sufficient to trigger hippocampal multicellular dysfunctions and memory deficits.\u003c/p\u003e","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eAdenosine is a widespread modulator of the central nervous system (CNS) involved in a large number of key processes controlling cerebral homeostasis\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. The effects of adenosine in the brain are mediated by the activation of G-protein-coupled adenosine receptors, in particular A\u003csub\u003e1\u003c/sub\u003e (A\u003csub\u003e1\u003c/sub\u003eR) and A\u003csub\u003e2A\u003c/sub\u003e (A\u003csub\u003e2A\u003c/sub\u003eR) receptors. Together, they fine tune synaptic plasticity through neuronal and glial-based mechanisms, by regulating both the pre-synaptic release and neurotransmitter uptake (e.g. glutamate and GABA) as well as post-synaptic activity, notably by affecting the function of other receptors, for instance D2R, mGluR5 or NMDAR\u003csup\u003e\u003cspan additionalcitationids=\"CR2 CR3 CR4 CR5 CR6 CR7 CR8\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. These functions likely explain the modulatory impact of caffeine, a non-selective adenosine receptor antagonist, towards brain network activity, related molecular processes and cognitive functions in various species\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan additionalcitationids=\"CR11 CR12 CR13\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe parenchymal levels of adenosine receptors, especially the A\u003csub\u003e2A\u003c/sub\u003eR subtype, are upregulated in several neurological conditions\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, and particularly in the brain of patients with cognitive pathologies such has Alzheimer\u0026rsquo;s disease, other tauopathies, depression or seizures as well as related experimental models\u003csup\u003e\u003cspan additionalcitationids=\"CR17 CR18 CR19 CR20 CR21 CR22\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Accrediting a detrimental role of such A\u003csub\u003e2A\u003c/sub\u003eR dysregulation, receptor overactivation using a selective agonist\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e or its selective upregulation in neurons\u003csup\u003e\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e have been shown to favor the development of cognitive impairments. In line, normalizing A\u003csub\u003e2A\u003c/sub\u003eR function was found to alleviate behavioral and plasticity impairments in various neurodegenerative models\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan additionalcitationids=\"CR29 CR30\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eBesides neurons, pathological upregulation of A\u003csub\u003e2A\u003c/sub\u003eR also occurs in astrocytes\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e, glial cells highly intertwined with synaptic processes\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Few \u003cem\u003ein vitro\u003c/em\u003e studies demonstrated that A\u003csub\u003e2A\u003c/sub\u003eR upregulation in astrocytes functionally alters several of their known functions such as the recycling of glutamate, the activity of Cx43 gap junction hemichannels and their pro-inflammatory profile\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan additionalcitationids=\"CR37\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eIn vivo\u003c/em\u003e, cell-autonomous and non-cell autonomous effects of such A\u003csub\u003e2A\u003c/sub\u003eR astrocytic dysregulation remain largely ill-defined. To fill this gap, the present study aimed at investigating the functional outcomes of A\u003csub\u003e2A\u003c/sub\u003eR astrocytic upregulation on hippocampal astrocyte biology as well as its consequences on neuronal and microglial biology and, ultimately, memory.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003e\u003cb\u003eAnimals.\u003c/b\u003e Two-months male C57Bl6/J mice (from Charles Rivers) were maintained in groups of 5\u0026ndash;6 in ventilated cages in a SOPF facility (12h/12h light/dark cycle, 22\u0026deg;C), with \u003cem\u003ead libitum\u003c/em\u003e access to food and water. The animals were maintained in compliance with European standards for the care and use of laboratory animals and experimental protocols were approved by the CEEA75 ethical committee (12787-2015101320441671v9).\u003c/p\u003e \u003cp\u003e \u003cb\u003eAstrocytic A\u003c/b\u003e \u003csub\u003e \u003cb\u003e2A\u003c/b\u003e \u003c/sub\u003e \u003cb\u003eR hippocampal overexpression.\u003c/b\u003e Upregulation the murine A\u003csub\u003e2A\u003c/sub\u003eR (mAdora2a), or the enhanced form of the green fluorescence protein (eGFP) as control, were obtained thanks to an adeno-associated virus (AAV) of 2/9 serotype with gene expressions under the control of a GfaABC1D astrocyte-selective promoter (AAV2/9-GfaABC1D-A2A or AAV-A2A; AAV2/9- GfaABC1D-GFP or AAV-GFP, respectively) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e; \u003cb\u003eSupplementary Fig.\u0026nbsp;1\u003c/b\u003e). For the surgical procedure, mice were deeply anesthetised with ketamine (150 mg/kg) and xylazine (10 mg/kg). Buprenorphine (0.05 mg/Kg) was injected subcutaneously 30 min before the beginning of surgery followed by lidocaine (5 mg/kg) applied on the scalp. The AAVs were bilaterally injected in the CA1 hippocampal area of 2 months old C57Bl6/J mice at the following stereotaxic coordinates: -2.5 mm rostro-caudal; +/-1.5 mm medio-lateral axis; -1.7 mm dorso-ventral) with a final concentration of 1.10\u003csup\u003e9\u003c/sup\u003e vg/\u0026micro;l and a total volume of 2 \u0026micro;l per injection site, at a rate of 0.25 \u0026micro;l/min. Before the AAVs injection, the needle was left in place 1 min and 5 min at the end, before being slowly removed. The skin was sutured and mice were allowed to recover. Except for behavioral studies where animals were investigated at the age of 3 to 4 months (i.e. 1 to 2m post-injection), all other evaluations were performed at 4 months of age (i.e. 2m post-injection).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eBehavioral evaluations.\u003c/b\u003e Behavioral experiments as detailed in \u003cb\u003eSupplementary methods\u003c/b\u003e were conducted on animals randomly assigned by experimenters blinded to the injection group.\u003c/p\u003e \u003cp\u003e \u003cb\u003eEuthanasia.\u003c/b\u003e Mice were sacrificed 2 months post-AAVs injection. They were deeply anesthetized with Dolethal (200 mg/kg, intraperitoneal injection, i.p) and lidocaine was applied on the skin and ribs before transcardially perfusion with cold NaCl (0.9%). Brains were removed and post-fixed in 4% paraformaldehyde (PFA) (in PBS; pH 7.4) for 24h at 4\u0026deg;C and transferred into sucrose 30% overnight for saturation before being frozen. Coronal brains sections (35 \u0026micro;m) were cut using a Leica cryostat and stored in a PBS-azide (0,2%) at 4\u0026deg;C until immunohistochemical analysis (A\u003csub\u003e2A\u003c/sub\u003eR, GFP, Iba1, GFAP, STAT-3, YAP). Immunohistological and quantification procedures related to these samples are described below.\u003c/p\u003e \u003cp\u003e \u003cb\u003eTranscriptomic analysis of hippocampal astrocytes.\u003c/b\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eTissue preparation.\u003c/b\u003e The cell sorting method was performed using the Neural Tissue Dissociation Kit (#130-092-628; Miltenyi Biotec; \u003cb\u003eSupplementary Fig.\u0026nbsp;2A\u003c/b\u003e). To do so, left and right hippocampi were dissected out following cervical dislocation and directly collected in HBSS buffer (1X; #55021C; sigma) and kept at 4\u0026deg;C. Hippocampi were then cut using dissecting scissors and incubated for 15 min at 37\u0026deg;C to facilitate enzymatic digestion. Additional cell dissociation was performed using polished Pasteur pipettes with three different diameters (from the largest 5\u0026ndash;6 mm, to the medium 3\u0026ndash;4 mm, to the smallest 1\u0026ndash;2 mm). Homogenates were slowly aspirated 10 times in each pipette and then were incubated at 37\u0026deg;C for 10 min under slow and continuous rotation. Samples were then centrifuged (1249 rpm; 10min at room temperature, RT) in an HBSS solution (1X; #55037C; Sigma) and the supernatants discarded.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMyelin removal with MS column.\u003c/b\u003e The pellet was resuspended in an enzymatic solution from the kit before being incubated with the magnetic myelin removal beads (#130-096-731; Miltenyi Biotec) for 15 min at 4\u0026deg; C. Cells were then washed by using Buffer myelin removal (BMR) solution and then centrifuged (1249 rpm; 10 min at RT). The pellet was re-suspended in BMR solution and loaded onto the MS column, placed in the magnetic field separator. The eluted solution (unlabeled cells) was collected, containing cells without the myelin, while the magnetically labeled cells (cells with myelin) were retained on the MS column. The eluted solution was centrifuged in order to eliminate dead cells (1249 rpm; 5 min at RT).\u003c/p\u003e \u003cp\u003e \u003cb\u003eAnti-ACSA-2 Microbeads labeling with MS column.\u003c/b\u003e The pellet was re-suspended in BMR solution with FcR blocking solution. Samples were incubated 10 min at 4\u0026deg;C before addition of the anti-ACSA-2 (Astrocyte Cell Surface Antigen 2; \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e Microbeads (#130-097-679; Miltenyi Biotec) and the solution was re-incubated for 15 min at 4\u0026deg;C then centrifugated (1249rpm; 10 min at 4\u0026deg;C). The pellet was re-suspended in BMR solution and loaded onto the MS column, placed in the magnetic field of the MACS separator. The magnetically labeled ACSA-2 cells were retained within the column, while the unlabeled cells ran through and were eluted. To increase purity, the positively selective fraction was separated over a second MS column. In order to perform the sequencing analysis, RNA was extracted from the ACSA-2 positive cell suspension corresponding of astrocytic cells and \u003cb\u003eRNAseq\u003c/b\u003e performed as indicated in the \u003cb\u003eSupplementary methods\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003e3D astrocytic morphology.\u003c/b\u003e At the time of AAV-A2A and AAV-GFP stereotaxic injections, a dedicated group of mice also received a retro-orbital (RO) venous sinus delivery of an AAV-PHP.eB at 5.10\u003csup\u003e10\u003c/sup\u003e vg in 50 \u0026micro;l total volume using a U100 insulin syringe (BD micro-fine 0.3 mL, 30-gauge needle). This AAV with a PHP.eB capsid (AAV-PHP.eB-GfaABC1D-Tomato or AAV-PHP.eB), known to cross the blood-brain barrier after intravenous injection\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e, was used to express the fluorescent Tomato protein in sparse astrocytes to allow 3D imaging afterwards (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE-G). For these mice, brains were removed and post-fixed in 4% PFA (in PBS; pH 7.4) for 24 h at 4\u0026deg;C and stored into PBS-azide until process. They were cut using a Leica vibratome (parameters: 0.12 speed and amplitude of 2 \u0026micro;m) at a thickness of 100 \u0026micro;m according to the coronal axis. The astrocytic 3D-reconstructions were performed thanks to the confocal microscope and the imaging software Imaris.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eAstrocytic 3D-reconstruction and analysis.\u003c/b\u003e The 100 \u0026micro;m mouse floating vibratome sections were directly mounted on slides and coverslips (High performance thickness 1\u003csup\u003e1/2\u003c/sup\u003e and diameter 0.170 \u0026plusmn; 0.005mm; Zeiss). Tomato-positive astrocytes of the CA1 area were imaged on a Zeiss LSM-710 confocal microscope taking-up to 50 stacks at 0.43\u0026micro;m steps with a x63 oil objective (optimal frame size of 1348). The complex arborization of the CA1 astrocytes was 3D reconstructed using the \u0026ldquo;filament tracer\u0026rdquo; rendering function in Imaris (Bitplane) and 3D analyzed using the Imaris plug-in \u0026ldquo;X-tension\u0026rdquo; (4\u0026ndash;6 astrocytes/mice). Then the 3D cell and soma volumes as well as the number, the length of the processes and the Sholl intersections were analyzed. Sholl intersections are the number of times a cell extension in a 2D representation intersects the concentric circles, or radius, positioned at a 1 \u0026micro;m interval and starting from the cell soma.\u003c/p\u003e \u003cp\u003e \u003cb\u003e3D Microglia 3D-reconstruction and analysis.\u003c/b\u003e After performing Iba1 immunofluorescence on the 35 \u0026micro;m mouse floating sections, Iba1\u003csup\u003e+\u003c/sup\u003e cells of the CA1 area were imaged using a Zeiss Spinning Disk high-resolution microscope taking-up to 15 z-stacks at 1.5 \u0026micro;m steps with a 40x oil objective. The complex arborization of the CA1 microglia was 3D reconstructed using the \u0026ldquo;filament tracer\u0026rdquo; rendering function in Imaris and 3D analyzed using the Imaris plug-in \u0026ldquo;X-tension\u0026rdquo; (10\u0026ndash;15 microglia/mice). Then the Sholl intersections were analyzed.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDetermination of Neuronal excitability using DREADD.\u003c/b\u003e In order to evaluate the neuronal excitability in animals with astrocytic A\u003csub\u003e2A\u003c/sub\u003eR upregulation, a serotype 5 AAV carrying hM3Dq DREADD receptor (Designer Receptor Exclusively Activated by Designer Drugs) and mCherry genes, under the control of the neuronal CaMKII promoter, was used (AAV5-CAMKII-hM3Dq-mCherry or AAV-hM3Dq). The AAV-hM3Dq was bilaterally co-injected with the AAV-A2A or AAV-GFP in the CA1 hippocampus area at the concentration of 1.10\u003csup\u003e9\u003c/sup\u003e vg/\u0026micro;l with a total volume of 2 \u0026micro;l per injection site at a rate of 0.25 \u0026micro;l/min (N\u0026thinsp;=\u0026thinsp;5/group). Two months following the AAV stereotaxic injections, mice were i.p. administrated with clozapine-N-oxide (CNO; 5 mg/kg), an hM3Dq exogeneous and synthetic ligand, or saline (NaCl; 0,9%) as a control, 90 min before sacrifice (as described above) to acutely activate the hM3Dq-DREADD receptor and evaluate the immediate early gene (IEG) expression by mRNA and immunohistochemical analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). To validate the neuronal expression of hM3Dq-DREADD receptor, an RFP immunostaining was performed to amplify and visualize the mCherry signal. RNA extractions and RT-qPCR from whole hippocampi were performed as described previously\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Sequences of primers used in this study are given in \u003cb\u003eSupplementary Table\u0026nbsp;1\u003c/b\u003e. Cyclophilin A was used as a reference housekeeping gene for normalization. Immunohistological and analysis procedures regarding these samples are described in the \u003cb\u003eSupplementary methods\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003ePreparation of synaptosomes.\u003c/b\u003e One hippocampus per mouse was homogenized in 200\u0026micro;l of Tris buffer (pH 7.4) containing 10% sucrose using a Potter-Elvehjem on ice. Protein concentrations were quantified using the BCA assay (Pierce) and diluted in Tris buffer (pH 7.4) containing 10% sucrose to obtain a final concentration of 4ug/\u0026micro;l. 200\u0026micro;l of Syn-PER\u0026trade; Synaptic Protein Extraction Reagent (#87793; Thermofisher, France) was then added to 100\u0026micro;l of the protein homogenates. The samples were homogenized again using a Potter-Elvehjem and then centrifuged (1200g; 10 min at 4\u0026deg;C). Cell debris were discarded, supernatants centrifuged (15000g; 20min at 4\u0026deg;C) and the resulting pellets resuspended in 100\u0026micro;l of Syn-PER\u0026trade; solution. These samples were stored at -20\u0026deg;C until use. Related Western blot analysis is described in the \u003cb\u003eSupplementary methods\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eStatistical analysis.\u003c/b\u003e The results were expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Differences between groups were determined using either Student's t-test, One-Way ANOVA followed by a Tukey\u0026rsquo;s post-hoc test or by two-way ANOVA. All statistical analyzes were performed with GraphPad Prism version 8 software. A p-value (P) of less than 0.05 was considered significant.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e \u003cb\u003eAstrocytic upregulation of A\u003c/b\u003e \u003csub\u003e \u003cb\u003e2A\u003c/b\u003e \u003c/sub\u003e \u003cb\u003eR in the mouse CA1.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo determine the impact of astrocytic A\u003csub\u003e2A\u003c/sub\u003eR upregulation in the mouse hippocampus, we bilaterally injected dedicated adeno-associated viral (AAV) vectors allowing the selective expression of A\u003csub\u003e2A\u003c/sub\u003eR (AAV-A2A) or GFP (AAV-GFP), taken as a control, in the CA1 astrocytes of 2 months-old C57Bl6/J mice and animals studied 2 months post-injection (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Immunohistochemistry using antibodies against A\u003csub\u003e2A\u003c/sub\u003eR or GFP allowed to visualize their hippocampal levels in the different CA1 sublayers i.e. \u003cem\u003estratum oriens\u003c/em\u003e (SO), pyramidal layer (PL) and \u003cem\u003estratum radiatum\u003c/em\u003e (SR) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB; \u003cb\u003eSupplementary Fig.\u0026nbsp;1\u003c/b\u003e). Co-immunostainings using antibodies directed against astrocytic (GFAP, Sox2, S100β), neuronal (NeuN) and microglial (Iba1) markers showed an exclusive expression of A\u003csub\u003e2A\u003c/sub\u003eR by astrocytes, as expected (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003cb\u003eCell-autonomous impact of astrocytic A\u003c/b\u003e \u003csub\u003e \u003cb\u003e2A\u003c/b\u003e \u003c/sub\u003e \u003cb\u003eR upregulation on reactivity and morphological complexity.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAstrocytes can adopt a reactive phenotype which is notably characterized by an increased expression of GFAP, an intermediate filament protein. We found a significant increase of the GFAP\u003csup\u003e+\u003c/sup\u003e staining in the CA1 of AAV-A2A mice as compared to the AAV-GFP control (+\u0026thinsp;100.2 \u0026plusmn; 38.3%; P\u0026thinsp;=\u0026thinsp;0.0034; Student\u0026rsquo;s t-test; Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-B). Recent studies have also identified the JAK2/STAT3 signaling pathway as a central player in the induction of the astrocytic reactive state in pathological conditions such as Alzheimer's disease and Huntington's disease\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. Accordingly, we performed a GFAP/STAT3 co-immunostaining and observed a significant increase of STAT3\u003csup\u003e+\u003c/sup\u003e staining within the GFAP\u003csup\u003e+\u003c/sup\u003e astrocytes of the CA1 area in the AAV-A2A condition as compared to the AAV-GFP control (+\u0026thinsp;68.3 \u0026plusmn; 35.9%; P\u0026thinsp;=\u0026thinsp;0.0024; Student\u0026rsquo;s t-test; Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-D). Moreover, astrocytes continually adapt to their environment and exhibit morphological changes eventually associated with their reactive state\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. In order to assess more finely the morphological changes of astrocytes upon A\u003csub\u003e2A\u003c/sub\u003eR upregulation, a 3D-reconstruction of the latter was performed, following a retro-orbital injection of an AAV-PHP.eB allowing the selective expression of tdTomato in sparse CA1 astrocytes (cytosol and arborization), of both AAV-A2A and AAV-GFP-injected mice (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE-F). We thus imaged isolated tdTomato\u003csup\u003e+\u003c/sup\u003e astrocytes in the CA1 \u003cem\u003estratum radiatum\u003c/em\u003e sublayer by high-resolution imaging and performed their 3D-reconstruction using Imaris software (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG). Our results indicated a significant higher number of intersections, as shown by Sholl analysis, consistent with a higher number of the astrocytic processes in AAV-A2A vs. AAV-GFP mice (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH-I). A decreased astrocytic processes length was also observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ) without change of the overall astrocyte or soma volumes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eK-L). Together, these results support that astrocytic A\u003csub\u003e2A\u003c/sub\u003eR upregulation promotes astrocyte reactivity and complexity.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAging-like molecular signature induced by astrocytic A\u003c/b\u003e \u003csub\u003e \u003cb\u003e2A\u003c/b\u003e \u003c/sub\u003e \u003cb\u003eR upregulation.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo gain molecular insights on the cell-autonomous impact of astrocytic A\u003csub\u003e2A\u003c/sub\u003eR upregulation, we performed a RNA sequencing analysis from hippocampal astrocytes of AAV-A2A and AAV-GFP-injected mice. To do so, we used magnetic cell sorting thanks to ACSA-2 (Astrocyte Cell Surface Antigen 2\u003csup\u003e39\u003c/sup\u003e; \u003cb\u003eSupplementary Fig.\u0026nbsp;2A\u003c/b\u003e) magnetic microbeads in order to isolate astrocytes from freshly dissected hippocampi. Following RNA sequencing, and using an available database\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e, we first validated our enrichment procedure by comparison of the expression of known genes specifically expressed by astrocytes with genes expressed by other cell types (\u003cb\u003eSupplementary Fig.\u0026nbsp;2B\u003c/b\u003e). The first Principal Composant (PC1), which explained 40.24% of the variance, nicely separated our experimental conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). We found 1127 differentially expressed genes (DEG) in the AAV-A2A condition as compared to the AAV-GFP control, 916 being downregulated and 211 upregulated (|Log2 fold-change (FC)|\u0026gt;0.32; Adjusted P-value (Padj)\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eB; list of DEG available on \u003cb\u003eSupplementary Table\u0026nbsp;3\u003c/b\u003e). The top ten of downregulated genes is given on Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u003cb\u003eCi\u003c/b\u003e. Functional enrichment analysis provided by DAVID and GSEA analysis indicated that downregulated astrocytic genes in AAV-A2A mice are associated with several metabolic processes such as insulin signaling as well as glucose and glutamate metabolisms. We also observed a significant impact on transcriptional processes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eD; \u003cb\u003eSupplementary Fig.\u0026nbsp;3\u003c/b\u003e). Regarding upregulated genes, as expected, we found A\u003csub\u003e2A\u003c/sub\u003eR (\u003cem\u003eAdora2a\u003c/em\u003e). We also observed the upregulation of \u003cem\u003eTimp1\u003c/em\u003e (Log2 FC\u0026thinsp;=\u0026thinsp;3.19; Padj\u0026thinsp;=\u0026thinsp;1.59\u003csup\u003eE-07\u003c/sup\u003e), a gene encoding a metalloproteinase inhibitor, being involved in astrocyte reactivity as well as in neurodegenerative conditions\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e,\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u003cb\u003eCii\u003c/b\u003e). GSEA analysis supported that upregulated genes were associated with inflammatory processes (NFκB, TNFɑ and Il1) as well as oxidative stress and DNA repair pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eE; \u003cb\u003eSupplementary Fig.\u0026nbsp;3\u003c/b\u003e), all previously linked to cellular processes associated with aging\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e,\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e. One of the hallmarks of aging, cellular senescence, has been particularly linked with astrocytes in AD\u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e,\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. Interestingly, it has been recently demonstrated that astrocytic senescence associates with a reduction of cdk6 through an impairment of the activity of YAP (Yes1 Associated Transcriptional Regulation)\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. Accordingly, our data highlighted significant reductions in the expressions of \u003cem\u003ecdk6\u003c/em\u003e (Log2 FC=-3.03; Padj\u0026thinsp;=\u0026thinsp;8.85\u003csup\u003eE-31\u003c/sup\u003e; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003e\u003cb\u003eCi\u003c/b\u003e) and \u003cem\u003eyap1\u003c/em\u003e, coding YAP (Log2 FC=-1.12; Padj\u0026thinsp;=\u0026thinsp;6.93\u003csup\u003eE-05\u003c/sup\u003e). In order to validate impairment of YAP, instrumental to astrocyte senescence\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e, we performed co-immunostainings against YAP and GFAP (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Our data showed a significant reduction in the percentage of YAP\u003csup\u003e+\u003c/sup\u003e cells among GFAP\u003csup\u003e+\u003c/sup\u003e cells in AAV-A2A vs. AAV-GFP mice (-25.0 \u0026plusmn; 11.2%; P\u0026thinsp;=\u0026thinsp;0.002; Student\u0026rsquo;s t-test) as well as a reduction of the nuclear YAP staining of GFAP\u003csup\u003e+\u003c/sup\u003e astrocytes (-45.0 \u0026plusmn; 11.5%; P\u0026thinsp;=\u0026thinsp;0.0183; Student\u0026rsquo;s t-test) in the CA1 of AAV-A2A vs. AAV-GFP mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eG). Further, we also found that the volume of astrocyte nuclei was significantly enlarged in YAP\u003csup\u003e+\u003c/sup\u003e GFAP\u003csup\u003e+\u003c/sup\u003e of A\u003csub\u003e2A\u003c/sub\u003eR expressing astrocytes as compared to GFP controls (+\u0026thinsp;22.8 \u0026plusmn; 8.0%; P\u0026thinsp;=\u0026thinsp;0.0344; Student\u0026rsquo;s t-test; 6\u0026ndash;8 nuclei per animals; N\u0026thinsp;=\u0026thinsp;4\u0026ndash;5/group; not shown), another hallmark of senescence\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e,\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. Together, these data suggested that the upregulation of A\u003csub\u003e2A\u003c/sub\u003eR in hippocampal astrocytes is sufficient to elicit an aged and senescent-like phenotype. In this sense, we compared our transcriptomic data with the signature of astrocytes isolated from aged mice\u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e but also to a list of \u0026ldquo;astrocytic senescence-related genes\u0026rdquo; extracted from the literature (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eH; \u003cb\u003eSupplementary Table\u0026nbsp;4\u003c/b\u003e; \u003cb\u003eSupplementary Table\u0026nbsp;5\u003c/b\u003e). We found an overlap with 33 genes (out of 358) varying similarly in the AAV-A2A animals when compared the \u0026ldquo;aged\u0026rdquo; signature. Same applied regarding 13 genes (out of 62) belonging the \u0026ldquo;astrocytic senescence-related genes\u0026rdquo; signature (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eI).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eAstrocytic upregulation of A\u003c/b\u003e \u003csub\u003e \u003cb\u003e2A\u003c/b\u003e \u003c/sub\u003e \u003cb\u003eR favors neuronal excitability.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAstrocytes regulate glutamate neurotransmission and synaptic plasticity\u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e. Interestingly, the above-mentioned transcriptomic data supported that astrocytic upregulation of A\u003csub\u003e2A\u003c/sub\u003eR significantly impacts genes involved in glutamate release, in line with previous data reporting that A\u003csub\u003e2A\u003c/sub\u003eR tightly controls (inhibits) glutamate reuptake by astrocytes\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. This led us to address the effect of A\u003csub\u003e2A\u003c/sub\u003eR upregulation on neuro-astroglial communication and particularly markers of neuronal excitability. We first prepared hippocampal synaptosomes from AAV-GFP and AAV-A2A mice to evaluate the phosphorylation of glutamatergic receptor subunits important for the synaptic trafficking and conductance of NMDA and AMPA receptors, focusing on Y1472 of GluN2B and S831 of GluA1\u003csup\u003e63,64\u003c/sup\u003e. While pY1472 GluN2B/GluN2B ratio did not significantly change in AAV-A2A mice, we observed a significant increase (+\u0026thinsp;161.4 \u0026plusmn; 94.7%; P\u0026thinsp;=\u0026thinsp;0.003; Student\u0026rsquo;s t-test) of the synaptosomal pS831 GluA1/GluA1 ratio as compared to AAV-GFP animals (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Then, we used a DREADD chemogenetic tool to determine whether A\u003csub\u003e2A\u003c/sub\u003eR upregulation in astrocytes was prone to affect neuronal activation. We jointly expressed the activatory Gq-coupled DREADD receptor (hM3Dq; with an mCherry reporter) in CA1 neurons with A\u003csub\u003e2A\u003c/sub\u003eR or GFP in CA1 astrocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). A co-immunostaining against A\u003csub\u003e2A\u003c/sub\u003eR and RFP, in order to amplify and visualize the hM3Dq-mCherry signal, confirmed the astrocytic expression of A\u003csub\u003e2A\u003c/sub\u003eR (green; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eC) and the neuronal expression of the hM3Dq (red; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Two months post-AAV injections, we intraperitoneally injected the exogeneous and synthetic ligand CNO of hM3Dq, or saline as control, to selectively activate hippocampal neurons. Animals were studied 90 min later (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). We evaluated the mRNA expression of immediate early genes (IEGs) as an indicator of neuronal activation by CNO injection. As expected, our data demonstrated a significant hippocampal increase of \u003cem\u003eDusp1\u003c/em\u003e, \u003cem\u003eJunB\u003c/em\u003e and \u003cem\u003ec-fos\u003c/em\u003e levels in CNO-treated (vs. saline) animals (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-F). Notably, the magnitude of IEG activation was significantly larger in AAV-A2A mice as compared to AAV-GFP animals in CNO-treated condition (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-F). C-Fos immunohistochemistry confirmed qPCR data, as shown on Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eG-H (controls are shown in \u003cb\u003esupplementary Fig.\u0026nbsp;4\u003c/b\u003e). Altogether these data demonstrated that astrocytic changes elicited by A\u003csub\u003e2A\u003c/sub\u003eR upregulation led to an alteration of neuro-astroglial communication towards an exacerbated neuronal response upon DREADD-mediated activation.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAstrocytic A\u003c/b\u003e \u003csub\u003e \u003cb\u003e2A\u003c/b\u003e \u003c/sub\u003e \u003cb\u003eR upregulation alters microglial phenotype.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eBesides the link with neurons and considering that A\u003csub\u003e2A\u003c/sub\u003eR upregulation activates a pro-inflammatory signature in astrocytes, we further aimed at determining to which extent such astrocyte-autonomous changes may impact microglial cells. We first performed an immunohistochemistry directed against Iba1, a calcium binding protein increased following microglial activation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Our results showed a significant increase of the Iba1\u003csup\u003e+\u003c/sup\u003e staining in AAV-A2A animals without change in the density of Iba1\u003csup\u003e+\u003c/sup\u003e cells (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB-C). Like astrocytes, microglial phenotype exhibits a significant heterogeneity and complexity depending on cellular environment\u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e. To capture potential changes of microglial morphology, we performed a 3D-analysis from Iba1 immunofluorescence. The Sholl analysis revealed a slight but significant reduction of the microglial complexity in AAV-A2A animals as compared to AAV-GFP controls (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Two-Way ANOVA; Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD-E). In addition, we evaluated the level of CD68, a lysosomal marker associated with microglial phagocytosis, essential for eliminating pathogens and misconformed proteins. To do so, co-immunostaining against both Iba1 and CD68, was performed and, after 3D analysis, we observed a decreased level of the CD68\u003csup\u003e+\u003c/sup\u003e staining in the Iba1\u003csup\u003e+\u003c/sup\u003e cells, suggestive of a reduced microglia phagocytosis in the AAV-A2A animals as compared to the AAV-GFP control (-41.6 \u0026plusmn; 32.4%; P\u0026thinsp;=\u0026thinsp;0.046; Student\u0026rsquo;s t-test; Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF-G). Together these data suggested that the upregulation of A\u003csub\u003e2A\u003c/sub\u003eR in astrocytes is sufficient to affect microglial phenotype.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eAstrocytic A\u003c/b\u003e \u003csub\u003e \u003cb\u003e2A\u003c/b\u003e \u003c/sub\u003e \u003cb\u003eR upregulation in CA1 impairs short-term spatial memory and spatial learning.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eFinally, to determine whether cell-autonomous and non-cell-autonomous modifications induced by the astrocytic upregulation of A\u003csub\u003e2A\u003c/sub\u003eR in the hippocampus were prone to alter memory performances, we performed two hippocampus-dependent spatial memory tests following an Elevated-Plus Maze evaluation. Elevated-Plus Maze revealed no difference in either the locomotor activity or anxiety-like behavior (percentage of the time spent in the open arms) in animals overexpressing astrocytic A\u003csub\u003e2A\u003c/sub\u003eR (\u003cb\u003eSupplementary Figs.\u0026nbsp;5A-C\u003c/b\u003e). Short-term spatial memory performance was assessed using the Y-maze test (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). We first observed no difference regarding the distance moved and the velocity (\u003cb\u003eSupplementary Figs.\u0026nbsp;5D-E\u003c/b\u003e). A discrimination index, corresponding to the animal preference for the new arm versus the familiar arm during the retention phase, was calculated and showed a preference\u0026thinsp;\u0026gt;\u0026thinsp;50% i.e. vs. chance for both groups (P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; One-Sample Student\u0026rsquo;s t-test), indicating that AAV-GFP and AAV-A2A mice both had a significant preference for the novel arm (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). However, the discrimination index was significantly lower in AAV-A2A animals as compared to AAV-GFP controls, indicating reduced short-term spatial memory (P\u0026thinsp;=\u0026thinsp;0.03; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). We also evaluated long-term spatial memory performances using the Barnes maze task (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). During the 4 days of the learning, the AAV-A2A group exhibited significantly higher distance (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Two-Way ANOVA), primary latency (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Two-Way ANOVA) and primary errors (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Two-Way ANOVA) to find the goal box as compared to AAV-GFP animals, suggesting altered spatial learning abilities (Figs.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD-F). During the retention stage, 24 h after the learning, the animals did not show any difference regarding the distance moved or the velocity (\u003cb\u003eSupplementary Figs.\u0026nbsp;5F-G\u003c/b\u003e). The percentage of time spent in the target quadrant was significantly higher than 25% i.e. than chance (P\u0026thinsp;=\u0026thinsp;0.021 in AAV-GFP animals; P\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 in AAV-A2A animals; Student\u0026rsquo;s t-test) and similar for both groups, indicating intact spatial memory for both groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG). Overall, these data highlighted that the multicellular alterations induced by the hippocampal upregulation of A\u003csub\u003e2A\u003c/sub\u003eR in astrocytes ultimately lead to impairments of the short-term spatial memory and learning abilities in mice.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe adenosine A\u003csub\u003e2A\u003c/sub\u003e receptor is essential to fine tune synaptic plasticity by coordinating neuro-glial communication, in particular in the hippocampus. Indeed, presynaptic A\u003csub\u003e2A\u003c/sub\u003eR controls the release of neurotransmitters such as glutamate\u003csup\u003e\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e while it gates its post-synaptic activity, notably by regulating NMDAR and mGluR5 functions\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e. Astrocytic A\u003csub\u003e2A\u003c/sub\u003eR also controls neuronal activity by directly regulating the extracellular levels of both glutamate and GABA in the synaptic cleft. Indeed, A\u003csub\u003e2A\u003c/sub\u003eR has been shown to respectively inhibit glutamate and increase GABA uptake by astrocytes\u003csup\u003e\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. The importance of astrocytic A\u003csub\u003e2A\u003c/sub\u003eR has been particularly emphasized by the compromised hippocampal synaptic plasticity and glutamate homeostasis following conditional deletion of astrocytic A\u003csub\u003e2A\u003c/sub\u003eR in mouse models\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e. Overall, neuronal and astroglial A\u003csub\u003e2A\u003c/sub\u003eR are likely to support hippocampal function and memory encoding.\u003c/p\u003e \u003cp\u003eImportantly, the level of A\u003csub\u003e2A\u003c/sub\u003eR is upregulated in hippocampal neurons and astrocytes in different allostatic and pathological contexts. Neuronal A\u003csub\u003e2A\u003c/sub\u003eR upsurge during development has been recently shown to control synapse stability\u003csup\u003e\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e while during aging it favors alterations in plasticity and memory loss\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e. Neuronal A\u003csub\u003e2A\u003c/sub\u003eR also rises in several detrimental situations such as chronic stress\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e as well as in Alzheimer\u0026rsquo;s Disease (AD) and other tauopathies\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Our recent works in AD models particularly highlighted the instrumental role of neuronal A\u003csub\u003e2A\u003c/sub\u003eR dysregulation in the loss of synapses though neuron-microglia miscommunication\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. In sharp contrast, although astrocytic dysregulation of A\u003csub\u003e2A\u003c/sub\u003eR has been observed in AD and epilepsy\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, the outcomes and their underlying mechanisms remain largely ill-defined. The present study tackles, for the first time, the cellular and molecular \u003cem\u003ein vivo\u003c/em\u003e mechanisms resulting from A\u003csub\u003e2A\u003c/sub\u003eR upregulation in hippocampal astrocytes. Our data demonstrate that the sole upregulation of astrocytic A\u003csub\u003e2A\u003c/sub\u003eR is sufficient to induce multi-cellular alterations involving astrocyte-autonomous and non-cell autonomous effects, ultimately interfering with learning and memory processes.\u003c/p\u003e \u003cp\u003eOur data show that A\u003csub\u003e2A\u003c/sub\u003eR upregulation induces astrocyte-autonomous effects characterized by astrocytic reactivity as well as significant morphological and molecular changes. Indeed, in response to A\u003csub\u003e2A\u003c/sub\u003eR upregulation, hippocampal astrocytes adopt a so-called reactive phenotype\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e,\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/sup\u003e, characterized by an increased expression of GFAP, STAT3 and \u003cem\u003eTimp1\u003c/em\u003e, an inhibitor of metalloproteinase, all being particularly identified as markers common to several neurodegenerative conditions, including AD\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e,\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e,\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u003c/sup\u003e. Morphologically, this reactivity translates into a greater number of astrocytic processes, and therefore complexity of astrocytic arborization, that may lead to functional modifications, particularly at the tripartite synapses where astrocytes finely regulate neurotransmitter dynamics and neuronal signaling\u003csup\u003e\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e,\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eOur data suggest that astrocyte reactivity induced by A\u003csub\u003e2A\u003c/sub\u003eR upregulation might promote impairments of the excitatory/inhibitory balance. Indeed, our RNA-Seq data notably highlight a reduction in a signaling pathway related to the astrocytic management of glutamate. This observation is in line with previous data showing an antagonistic interaction between A\u003csub\u003e2A\u003c/sub\u003eR and Na\u003csup\u003e+\u003c/sup\u003e/K\u003csup\u003e+\u003c/sup\u003eATPase-α2 controlling GLT-1-dependent glutamate uptake by astrocytes\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. Our hypothesis is also sustained by two other experimental evidence. First, astrocytic upregulation of A\u003csub\u003e2A\u003c/sub\u003eR leads to increased hippocampal levels of phosphorylation at serine 831 of the GluA1 subunit of AMPA receptors, which has been associated with enhanced network activity in epilepsy \u003csup\u003e\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e\u003c/sup\u003e. Second, we found that A\u003csub\u003e2A\u003c/sub\u003eR upregulation by astrocytes exacerbates the response of IEGs to chemogenetic neuronal activation, supporting a link between astrocytic A\u003csub\u003e2A\u003c/sub\u003eR and hyperexcitability. Together, our observations fit well with the A\u003csub\u003e2A\u003c/sub\u003eR upregulation by hippocampal astrocytes observed in patients presenting with mesial temporal lobe epilepsy or also observed in the kainate mouse model\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e,\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e\u003c/sup\u003e. This is also coherent with the astrocytic A\u003csub\u003e2A\u003c/sub\u003eR upregulation described in the brain of AD patients\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, where the risk of seizures is at least 3 times higher than in healthy controls\u003csup\u003e\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e\u003c/sup\u003e. These changes of hippocampal excitability might occur concomitantly with other changes unveiled by our transcriptomic data that also indicate that astrocytic A\u003csub\u003e2A\u003c/sub\u003eR upregulation favors miscommunication between astrocytes and neurons by presumably impairing insulin-mediated glucose uptake and astrocytic glycolysis\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe astrocytic transcriptomic signature unveiled numerous processes associated with an aging-like astrocytic phenotype such as oxidative stress, induction of the NF-κB pathway, DNA repair mechanisms as well as a significant reduction in transcriptional processes and cell cycle\u003csup\u003e\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e\u003c/sup\u003e. Accordingly, we have identified \u003cem\u003eglutamine synthetase\u003c/em\u003e (Log2 FC=-1.75; Padj\u0026thinsp;=\u0026thinsp;2.37\u003csup\u003eE\u0026thinsp;\u0026minus;\u0026thinsp;11\u003c/sup\u003e) and the \u003cem\u003ecyclin-dependent kinase cdk6\u003c/em\u003e among the most downregulated genes, previously described to be markers of astrocytic senescence\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e,\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e,\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e\u003c/sup\u003e. Recent data demonstrated that hippocampal astrocytic senescence associates with a reduction of cdk6 induced by a defective YAP activity, a transcriptional coregulator, in these glial cells\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. This study also reported that the conditional deletion of YAP in astrocytes is sufficient to promote several hallmarks of astrocyte senescence, including hypertrophic morphology, increased β-galactosidase activity, and upregulation of several senescence-associated genes such as p16, p53 and NF-κB as well as downregulation of Lamin B1\u003csup\u003e58\u003c/sup\u003e. Interestingly, our data also demonstrate a loss of YAP expression in GFAP\u003csup\u003e+\u003c/sup\u003e astrocytes, as well as a significant reduction of the YAP level in astrocytic nuclei upon upregulation of A\u003csub\u003e2A\u003c/sub\u003eR. This fits well with the observation that activation of Gs-Protein Coupled Receptors, like A\u003csub\u003e2A\u003c/sub\u003eR, promotes phosphorylation of YAP, preventing its translocation to the nucleus and its action as a transcriptional co-regulator\u003csup\u003e\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e\u003c/sup\u003e. Further, we identified a significant increase of nuclei size in astrocytes presenting with reduced YAP immunoreactivity, also characteristic of astrocytic senescence\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. Finally, we found some overlap between our transcriptomic dataset and the signatures of astrocytes isolated from aged mice\u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e as well as an \"astrocytic senescence-related genes\" signature extracted from the literature\u003csup\u003e\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e,\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e,\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e\u003c/sup\u003e. Overall, our data therefore support that A\u003csub\u003e2A\u003c/sub\u003eR upregulation by hippocampal astrocytes implements several cell-autonomous processes associated with astrocyte senescence and aging.\u003c/p\u003e \u003cp\u003eAstrocytic senescence is also associated with the upregulation of molecules associated to SASP (senescent-associated secreted phenotype), some of which were unveiled in our transcriptomic dataset such as interleukins (i.e. IL-1, IL-33), metalloproteases and metalloproteases inhibitors (i.e. MMP15, Timp1)\u003csup\u003e\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e\u003c/sup\u003e. These factors enable paracrine signaling to other astrocytes, but also presumably to surrounding cells such as neurons and microglia. Interestingly, our data show that the sole astrocytic upregulation of A\u003csub\u003e2A\u003c/sub\u003eR is sufficient to promote microglial reactivity and morphological changes, i.e. a reduction in the number of processes and therefore of microglial complexity. We also observed a striking reduction of microglial lysosomal activity. Interestingly, these morphological and functional microglial changes have been associated with hippocampal aging and neurodegeneration\u003csup\u003e\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e\u003c/sup\u003e. Our observations, showing a causal link between astrocytic A\u003csub\u003e2A\u003c/sub\u003eR and microglia are in line with data previously obtained in a mouse model of Sandhoff disease, showing an association between astrocytic upregulation of A\u003csub\u003e2A\u003c/sub\u003eR and microglial activation\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTaken together, our data indicate that the sole upregulation of A\u003csub\u003e2A\u003c/sub\u003eR in hippocampal astrocytes promote multicellular impairments that impact the hippocampal neuronal network functionality, the astrocyte-neuron coupling and microglial function all previously identified as mandatory for the regulation of memory processes\u003csup\u003e\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e,\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e,\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e\u003c/sup\u003e. In accordance, we demonstrate that the upregulation of astrocytic A\u003csub\u003e2A\u003c/sub\u003eR in the hippocampus is sufficient to promote spatial learning and memory alterations. This observation is in line with a previous work from Orr et al. (2015)\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e showing that conditional removal of A\u003csub\u003e2A\u003c/sub\u003eR in astrocytes improved memory in aged mice while the chemogenetic activation of astrocytic Gs-coupled signaling impaired it. However, these data seem controversial with other reports that rather support the detrimental impact of astrocytic A\u003csub\u003e2A\u003c/sub\u003eR deletion in memory settings\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e. A tight control of astrocytic A\u003csub\u003e2A\u003c/sub\u003eR might be necessary to ensure proper cognitive functions, especially in pathological conditions. Considering the clinical interest of A\u003csub\u003e2A\u003c/sub\u003eR targeting drugs in AD and epilepsy, this warrants further mechanistic studies. In conclusion, our data demonstrate for the first time the cell-autonomous and non-cell autonomous impact of A\u003csub\u003e2A\u003c/sub\u003eR astrocytic upregulation in hippocampal astrocytes and support their key role in the control of hippocampal function.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eCONFLICTS OF INTEREST\u003c/h2\u003e \u003cp\u003eDB is a (non-appointed) member of the scientific advisory board of Marvel Biosciences Corp developing an A\u003csub\u003e2A\u003c/sub\u003eR antagonist. But there is no conflict of interest regarding the present work.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eACKNOWLEDGEMENTS\u003c/h2\u003e \u003cp\u003eThis project was supported by fundings from ANR ADORASTrAU, ANR JANUS and FONDATION Alzheimer (ADOMEMOTAU). This work was also supported by grants from Programmes d\u0026rsquo;Investissements d\u0026rsquo;Avenir LabEx (excellence laboratory) DISTALZ (Development of Innovative Strategies for a Transdisciplinary approach to ALZheimer\u0026rsquo;s disease), Fondation pour la Recherche M\u0026eacute;dicale as well as Inserm, CNRS, Universit\u0026eacute; de Lille. AL was supported by PhD grants from Fondation pour la Recherche M\u0026eacute;dicale (ECO202106013670) and Vaincre Alzheimer (FR-24054T). TG was supported by University of Lille and Fondation Vaincre Alzheimer. VG-M was supported by Fondation Alzheimer and Fondation pour la Recherche M\u0026eacute;dicale (SPF20160936000). Sequencing was performed by the GenomEast platform, a member of the \u0026lsquo;France G\u0026eacute;nomique\u0026rsquo; consortium (ANR-10-INBS-0009). We thank members of the different facilities of \"Plateformes Lilloises en Biologie et Sant\u0026eacute; (PLBS)\u0026rdquo; - UAR 2014 - US 41 (SOFP facility, In vivo and functional exploration platform and BiCeL). EA was supported by the Association France Alzheimer.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCunha RA. How does adenosine control neuronal dysfunction and neurodegeneration? J Neurochem 2016; 139: 1019\u0026ndash;1055.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRebola N, Lujan R, Cunha RA, Mulle C. Adenosine A2A receptors are essential for long-term potentiation of NMDA-EPSCs at hippocampal mossy fiber synapses. 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J Neurochem 2019; 151: 676\u0026ndash;688.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSuzuki A, Stern SA, Bozdagi O, Huntley GW, Walker RH, Magistretti PJ \u003cem\u003eet al.\u003c/em\u003e Astrocyte-neuron lactate transport is required for long-term memory formation. Cell 2011; 144: 810\u0026ndash;823.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou Z, Okamoto K, Onodera J, Hiragi T, Andoh M, Ikawa M \u003cem\u003eet al.\u003c/em\u003e Astrocytic cAMP modulates memory via synaptic plasticity. Proc Natl Acad Sci U S A 2021; 118: e2016584118.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"molecular-psychiatry","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"mp","sideBox":"Learn more about [Molecular Psychiatry](http://www.nature.com/mp/)","snPcode":"41380","submissionUrl":"https://mts-mp.nature.com/cgi-bin/main.plex","title":"Molecular Psychiatry","twitterHandle":"@molpsychiatry","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-4791082/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4791082/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAdenosine is an ubiquitous neuromodulator that ensures cerebral homeostasis. It exerts numerous functions through the activation of G-protein-coupled adenosine receptors (ARs), in particular A\u003csub\u003e1\u003c/sub\u003e (A\u003csub\u003e1\u003c/sub\u003eR) and A\u003csub\u003e2A\u003c/sub\u003e (A\u003csub\u003e2A\u003c/sub\u003eR) receptors. Interestingly, A\u003csub\u003e2A\u003c/sub\u003eR levels are upregulated in cortical and hippocampal regions in several pathological conditions such as Alzheimer\u0026rsquo;s disease, tauopathies or epilepsia. Such abnormal upregulations have been particularly reported in astrocytes, glial cells that play a key role in regulating synaptic plasticity. However, the overall impact and the underlying mechanisms associated with increased A\u003csub\u003e2A\u003c/sub\u003eR in astrocytes remain poorly understood. In the present study, we induced the upregulation of A\u003csub\u003e2A\u003c/sub\u003eR in hippocampal astrocytes using dedicated AAVs and comprehensively evaluated the functional consequences in 4 months-old C57Bl6/J mice. Our results show that A\u003csub\u003e2A\u003c/sub\u003eR upregulation promotes cell-autonomous alterations of astrocyte reactivity, morphology and transcriptome, with a link to aging-like phenotype as well as non-cell autonomous impairments of neuronal excitability and microglial phenotype. These changes driven by a restricted A\u003csub\u003e2A\u003c/sub\u003eR upregulation in hippocampal astrocytes were sufficient to induce impairments of short-term spatial memory (Y-Maze task) and spatial learning (Barnes Maze task). This study highlights the impact of astrocytic A\u003csub\u003e2A\u003c/sub\u003eR upregulation, as seen in various neurological conditions, on the development of a detrimental multicellular response associated with memory alterations and provides an additional proof-of-concept for the value of targeting this receptor in different neurodegenerative conditions.\u003c/p\u003e","manuscriptTitle":"Upregulation of adenosine A2A receptor by astrocytes is sufficient to trigger hippocampal multicellular dysfunctions and memory deficits.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-26 02:52:11","doi":"10.21203/rs.3.rs-4791082/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2024-10-31T11:43:49+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-10-22T13:32:11+00:00","index":1,"fulltext":"This content is not 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