Astrocytic adenosine receptor A2A and Gs-coupled signaling regulate memory.
Astrocytes express a variety of G protein-coupled receptors and might influence cognitive functions, such as learning and memory.However, the roles of astrocytic Gs-coupled receptors in cognitive function are not known.Together, these findings establish a regulatory role for astrocytic Gs-coupled receptors in memory and suggest that AD-linked increases in astrocytic A2A receptor levels contribute to memory loss.
Affiliation: 1] Gladstone Institute of Neurological Disease, San Francisco, California, USA.  Department of Neurology, University of California, San Francisco, California, USA.
Astrocytes express a variety of G protein-coupled receptors and might influence cognitive functions, such as learning and memory. However, the roles of astrocytic Gs-coupled receptors in cognitive function are not known. We found that humans with Alzheimer's disease (AD) had increased levels of the Gs-coupled adenosine receptor A2A in astrocytes. Conditional genetic removal of these receptors enhanced long-term memory in young and aging mice and increased the levels of Arc (also known as Arg3.1), an immediate-early gene that is required for long-term memory. Chemogenetic activation of astrocytic Gs-coupled signaling reduced long-term memory in mice without affecting learning. Like humans with AD, aging mice expressing human amyloid precursor protein (hAPP) showed increased levels of astrocytic A2A receptors. Conditional genetic removal of these receptors enhanced memory in aging hAPP mice. Together, these findings establish a regulatory role for astrocytic Gs-coupled receptors in memory and suggest that AD-linked increases in astrocytic A2A receptor levels contribute to memory loss.
- Gene Expression Regulation/physiology*
- Memory, Long-Term/physiology*
- Receptor, Adenosine A2A/genetics/metabolism*
- Receptors, Serotonin, 5-HT4/genetics/metabolism*
- Signal Transduction/physiology*
- Alzheimer Disease/pathology
- Animals, Newborn
- Cytoskeletal Proteins/genetics/metabolism
- Exploratory Behavior/drug effects/physiology
- Glial Fibrillary Acidic Protein/genetics/metabolism
- Maze Learning/physiology
- Mice, Inbred C57BL
- Mice, Transgenic
- Nerve Tissue Proteins/genetics/metabolism
- Recognition (Psychology)/drug effects/physiology
- Serotonin Antagonists/pharmacology
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Figure 4: Rs1 ligand increases Gs-coupled signaling in Rs1-expressing astrocytes(a) Relative cAMP levels in primary astrocyte cultures established from non-transgenic mice and transduced (Lenti-Rs1) or not (Con) with a lentivirus encoding Rs1. Following 15-min treatment with vehicle (V) or the Rs1 ligand GR-125487 (GR, 1 μM), cultures were analyzed for intracellular cAMP levels. Forskolin (FSK, 30 μM) and prostaglandin E2 (PGE2, 40 μM) served as positive controls. cAMP levels were normalized to the average levels in vehicle-treated cells within each transduction condition. Two-way ANOVA: F(1, 35) = 81.75, P < 0.0001 for interaction effect. One-way ANOVA: F(3, 38) = 729.7, P < 0.0001. n = 15 Con/V, 3 Con/FSK, 16 Con/PGE2, 8 Con/GR, 8 Lenti-Rs1/V, and 8 Lenti-Rs1/GR wells from three independent experiments. ***P < 0.001 vs. leftmost bar (Dunnett’s test) or as indicated by bracket (Bonferroni test). (b) Dose-response curve for relative cAMP levels after GR treatment in Lenti-Rs1-transduced astrocytes. n = 8 (1e-14 M), 7 (1e-10 M), 6 (1e-9 M), 6 (1e-8 M), 9 (1e-7 M), and 8 (1e-6 M) wells from 2–3 independent experiments. R2 = 0.866. (c) Calcium levels in primary astrocyte cultures transduced as in (a). Cells were treated with GR (10 μM) or ATP (100 μM) and assayed for intracellular calcium levels with Calcium-5, a calcium-sensitive fluorescent dye. Fluorescence intensities were normalized to the average baseline intensity within each well. The maximum intensities per well were compared between groups. Two-way ANOVA: F(1, 20) = 0.14, P = 0.72 for interaction effect, F(1, 20) = 14.57, P = 0.0011 for agonist effect, F(1, 20) = 0.16, P = 0.69 for Lenti-Rs1 effect. n = 6 wells per condition from two independent experiments. (d–e) Proteins regulated by Gs-coupled signaling were quantified in Lenti-Rs1-transduced astrocytes by western blot analysis after treating cultures with vehicle or GR (1 μM) for the indicated durations. Representative cropped western blots (d) and densitometric quantification of western blot signals (e). Blots were probed for phosphorylated (p) and total (t) CREB and ERK. Ratios of phosphorylated/total protein were normalized to the average ratio in vehicle-treated cells. n = 5 pCREB/V-10, 7 pCREB/GR-10, 6 pCREB/GR-30, 3 pCREB/GR-60, 6 pERK/V-10, 7 pERK/GR-10, 6 pERK/GR-30, and 3 pERK/GR-60 wells from two independent experiments. One-way ANOVA: F(3, 17) = 6.61, P = 0.0037 (pCREB); F(3, 18) = 5.66, P = 0.0065 (pERK); **P < 0.01 vs. V (Dunnett’s test). Full-length blots are presented in Supplementary Figure 13. (f) GR concentration in whole hemibrains of 3–4-month-old non-transgenic mice determined by mass spectrometry-HPLC following a single i.p. injection of GR (1 mg/kg) and transcardial perfusion at the indicated times post-injection. n = 3 mice per time point. (g) Hippocampal and cortical pERK levels in 2–4-month-old GFAP-tTA (Con) and GFAP-Rs1 mice determined by western blot analysis after a single injection of vehicle or GR (3 mg/kg, i.p.). pERK/tERK ratios were normalized to the average ratios in saline-treated mice within each genotype and brain region. Two-way ANOVA: Hippocampus, F(2, 32) = 8.06, P = 0.0015 for genotype by treatment interaction; Cortex, F(2, 28) = 0.09, P = 0.91 for genotype by treatment interaction; n = 5 HP/Con/V-1h, 10 HP/Con/GR-1h, 4 HP/Con/GR-3h, 5 HP/GFAP-Rs1/V-1h, 10 HP/GFAP-Rs1/GR-1h, 4 HP/GFAP-Rs1/GR-3h, 4 HP/GFAP-Rs1/GR-6h, 5 CTX/Con/V-1h, 10 CTX/Con/GR-1h, 4 CTX/Con/GR-3h, 3 CTX/GFAP-Rs1/V-1h, 8 CTX/GFAP-Rs1/GR-1h, 4 CTX/GFAPRs1/GR-3h, and 4 CTX/GFAP-Rs1/GR-6h mice. **P < 0.01, ***P < 0.001 vs. HP/GFAP-Rs1/V (Bonferroni test). Values are means ± s.e.m.
To determine if activation of astrocytic Gs-coupled receptors affects learning and memory, we used the synthetic Rs1 ligand GR-125487, which activates Rs1 but not wildtype 5HT4 receptors. In Rs1-expressing cultured astrocytes, GR-125487 increased the levels of cAMP and activated cAMP-linked downstream mediators, including ERK and CREB, but did not affect calcium levels (Fig. 4a–e), confirming that GR-125487 induces Gs- but not Gq-coupled signaling. GR-125487 increased cAMP to similar levels as stimulation of endogenous Gs-coupled prostaglandin receptors with PGE2 (Fig. 4a), suggesting that Rs1 activation increases Gs-coupled signaling within a physiological range. In complementary in vivo experiments, we established that GR-125487 crossed the blood-brain barrier within minutes after intraperitoneal (i.p.) injection, achieved maximal concentrations in the brain within 30 min and exhibited a short half-life (Fig. 4f). In GFAP-Rs1 mice, but not in controls, phosphorylated ERK levels were increased in the hippocampal formation, but not cortex, one hour after ligand injection and returned to baseline within six hours (Fig. 4g).