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.
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We thank P.E. Sanchez for advice on behavioral experiments, R. Ponnusamy for comments on the manuscript, J.-F. Chen (Boston University School of Medicine) for providing loxP-Adora2a transgenic mice, N. Déglon (Lausanne University Hospital) for providing the lentiviral vector and packaging plasmids, B. Khakh (University of California, Los Angeles) for providing the plasmid encoding the membrane-targeting region of lck, S.-W. Min and L. Gan (Gladstone Institute of Neurological Disease) for providing brain tissue from Tau-P301S mice, L. Leung and Y. Huang (Gladstone Institute of Neurological Disease) for providing brain tissue from ApoE3-KI and ApoE4-KI mice, A. Lundquist, G. Shlager, C. Wang and E. Whiting for technical support, R. Craft, T. Hamto, I. Lo and B. Masatsugu of the Gladstone Behavioral Core for advice and technical support, and M. Dela Cruz and A. Cheung for administrative assistance. This study was supported by US National Institutes of Health grants AG039220 to A.G.O., AR056299 to E.C.H., AG034531 to D.B.D. HL60664 and HL100406 to B.R.C., and AG022074 and NS065780 to L.M., and a MetLife Foundation Award to L.M. The Gladstone Institutes received support from a National Center for Research Resources Grant RR18938.
The authors declare no competing financial interests.
Integrated supplementary information
Supplementary Figure 1 Transgenic mouse lines used to alter astrocytic gene expression; minimal microglial A2A receptor expression in the human brain.
(a) Schematic of the GFAP-Cre/loxP-Adora2a transgenic system. Cre, Cre recombinase. (b) Schematic of the GFAP-tTA/TetO-Rs1 transgenic system. (c) Table of Rs1 expression and signaling in GFAP-Rs1 mice. (d) Representative photomicrographs of hippocampal sections co-immunolabeled for the A2A receptor (green) and the microglial marker Iba1 (red) from a control (Con) and an AD case. n = 3 cases per disease condition. Scale bar: 50 µm.
(a) Primary astrocyte cultures from wild-type mice were or were not (Con) transduced with lentiviral constructs encoding mCherry (mCher) or the mouse A2A receptor (A2A). Representative cropped western blots show A2A receptor expression in A2A-transduced astrocytes, but not in mCher-transduced or Con astrocytes. The quantity of lentiviral particles (Lenti) used in the cultures is indicated as ng of p24 protein per well. GFAP served as a loading control. n = 2 or more wells per condition from 1–3 independent experiments. (b) Relative cAMP levels in primary astrocyte cultures established from wild-type mice and transduced or not (Con) with lentivirus encoding mCherry (mCh) or the A2A receptor. Cultures were treated for 15 min with vehicle, adenosine (10 µM), or the A2A-selective agonist CGS-21680 (CGS, 10 µM). cAMP levels were normalized to the average levels in vehicle-treated cells within each transduction condition. Two-way ANOVA: F(4, 96) = 22.74, P < 0.0001 for interaction effect. n = 14 Con/Vehicle, 6 Con/Adenosine, 14 Con/CGS, 14 mCh/Vehicle, 6 mCh/Adenosine, 14 mCh/CGS, 16 A2A/Vehicle, 6 A2A/Adenosine, and 15 A2A/CGS wells from two independent experiments. ##P < 0.01, ###P < 0.001 vs. vehicle (Bonferroni test). (c) Intracellular calcium levels. Con or A2A-transduced astrocytes were treated with CGS (10 µM) or ATP (100 µM) as a positive control, and assayed for intracellular calcium levels with Calcium-5. Fluorescence intensities were normalized to the average baseline intensity within each well. Peak intensities were compared between groups. Two-way ANOVA: F(1, 26) = 0.02, P = 0.88 for interaction effect, F(1, 26) = 28.51, P < 0.0001 for agonist effect. n = 8 Con/ATP, 7 Con/CGS, 7 A2A/ATP, and 8 A2A/CGS wells from two independent experiments. (d–e) Phosphorylated (p) and total (t) CREB and ERK levels in primary wild-type astrocytes transduced (d) or not (e) with a lentivirus encoding the A2A receptor and treated with vehicle, CGS (10 µM, 10 min), or forskolin (FSK, 30 µM, 30 min), an activator of adenylate cyclase. Representative cropped western blots and densitometric quantification of western blot signals are shown. Ratios of phosphorylated/total protein were normalized to the average ratios in vehicle-treated cells. Student's t test with Welch's correction (vs. vehicle of matching protein): (d) P = 0.0006 (pCREB), P = 0.042 (pERK). n = 6 Vehicle/pCREB, 10 CGS/pCREB, 6 Vehicle/pERK, and 10 CGS/pERK wells from two independent experiments. (e) P = 0.0008 (pCREB), P = 0.0004 (pERK). n = 7 Vehicle/pCREB, 8 FSK/pCREB, 5 Vehicle/pERK, and 6 FSK/pERK wells from two independent experiments. *P < 0.05, ***P < 0.001 vs. Vehicle (Student's t test). Full-length blots are presented in Supplementary Figure 13. Data are presented as mean ± s.e.m.
Supplementary Figure 3 Preservation of neuronal A2A receptor expression in GFAP-Cre/loxP-Adora2a mice.
(a) Representative photomicrographs of striatal and cortical sections from 12–13-month-old GFAP-Cre/loxP-Adora2a mice and 2-month-old global homozygous A2A receptor knockout mice (A2A-gKO) immunostained for the A2A receptor. Insets (i–iv): magnified views of the boxed regions. Scale bar: 500 µm. n = 3 mice per genotype. (b–c) Representative confocal micrographs (top) and quantitative histological analyses (bottom) of hippocampal CA1 sections from 12–13-month-old wild-type controls (Control) and A2A-cKO mice, and 2-month-old A2A-gKO mice co-immunostained for the A2A receptor (red) and an astrocyte marker (GFAP, green) or a neuronal marker (NeuN). Cell nuclei were labeled with DAPI (blue). Scale bar: 10 µm. One-way ANOVA: (b) F(2, 11) = 8.17, P = 0.0067; (c) F(2, 11) = 83.95, P < 0.0001. n = 5 Con, 6 A2A-cKO and 3 A2A-gKO mice. Data are presented as mean ± s.e.m.
Supplementary Figure 4 Conditional ablation of A2A receptors in astrocytes does not affect swim speed or anxiety behavior.
(a–b) Swim speeds of GFAP-Cre/loxP-Adora2a mice during Morris water maze probe trials corresponding to (a) Figure 2b and (b) Figure 2h. One-way ANOVA: (a) F(2, 43) = 1.83, P = 0.17, n = 12 cWT, 18 cHET, and 17 cKO mice; (b) F(2, 30) = 2.67, P = 0.086, n = 7 cWT, 15 cHET, and 11 cKO mice. P > 0.05 vs. cWT (Dunnett's test). (c–d) GFAP-Cre/loxP-Adora2a mice were assessed in the open field at 2–4 months of age. Average ratios of activity in the center of the field to total activity in the field are shown for trials corresponding to (c) Figure 2c and (d) Figure 2e. Repeated measures two-way ANOVA: (c) F(12, 270) = 0.65, P = 0.80 for interaction effect, F(2, 45) = 0.84, P = 0.44 for genotype effect. n = 12 cWT, 18 cHET, and 17 cKO mice. (d) F(6, 129) = 0.87, P = 0.52 for interaction effect, F(2, 43) = 0.12, P = 0.89 for genotype effect. n = 12 cWT, 18 cHET, and 16 cKO mice. Data are presented as mean ± s.e.m.
Supplementary Figure 5 Conditional ablation of A2A receptors in astrocytes increases baseline Arc levels.
Mice resting in their home cages during their daytime sleep cycle were killed rapidly as described in Methods. (a–b) Number of cells per section (a) and total area per section (b) immunoreactive for Arc in the granular layer of the dentate gyrus (DGgl) of GFAP-Cre/loxP-Adora2a mice at 3–22 months of age. Con: singly transgenic loxP-Adora2a and GFAP-Cre control mice. One-way ANOVA: (a) F(2, 31) = 3.68, P = 0.0367; (b) F(2, 31) = 4.83, P = 0.015. n = 12 Con, 12 cHET, and 10 cKO mice. *P < 0.05, **P < 0.01 vs. Con (Dunnett's test). (c) Levels of Arc mRNA in the visual cortex of GFAP-Cre/loxP-Adora2a mice determined by qRT-PCR at 9–22 months of age. Gapdh mRNA served as a loading control. One-way ANOVA: F(2, 32) = 5.59, P = 0.0083. n = 8 Con, 19 cHET, and 8 cKO mice. **P < 0.01 vs. Con (Dunnett's test). (d–e) Ages of mice shown in a–b (d) and c (e). One-way ANOVA: (d) F(2, 31) = 0.23, P = 0.80; (e) F(2, 32) = 4.24, P = 0.023. P > 0.05 vs. Con (Dunnett's test). Data are presented as mean ± s.e.m.
Supplementary Figure 6 Rs1 expression, normal body weight and unaltered behavioral functions in GFAP-Rs1 mice.
(a–b) Representative photomicrographs of FLAG-immunostained brain sections depicting the hippocampal formation (a) and thalamus (b) of 2–4-month-old TetO-Rs1, GFAP-tTA, and GFAP-Rs1 mice. In contrast to GFAP-Rs1 doubly transgenic mice, TetO-Rs1 or GFAP-tTA singly transgenic mice did not show Rs1 expression. Continuous maintenance of GFAP-Rs1 mice on DOX before and after birth prevented FLAG-Rs1 expression. Scale bars: 200 µm. Insets (i–ii): magnified views of the boxed regions. n = 3 TetO-Rs1, 9 GFAP-tTA, 15 GFAP-Rs1 and 3 GFAP-Rs1/DOX mice. (c–d) Representative photomicrographs of FLAG-immunostained sections depicting the hippocampal formation (c) and thalamus (d) of 20–22-month-old GFAP-Rs1 mice. Scale bar: 200 µm. Insets (iii–iv): magnified views of the boxed regions. n = 7 mice. (e–f) Weight of GFAP-tTA (Con) and GFAP-Rs1 mice at 2–9 (e) and 15–18 (f) months of age. Student's t test (vs. Con of matching sex): (e) P = 0.69 (Females), P = 0.30 (Males); (f) P = 0.71 (Females), P = 0.92 (Males). n = 7 Con females, 9 GFAP-Rs1 females, 8 Con males, and 8 GFAP-Rs1 males (e); n = 28 Con females, 27 GFAP-Rs1 females, 23 Con males, and 19 GFAP-Rs1 males (f). (g–k) Behavioral assessment of GFAP-tTA (Con) and GFAP-Rs1 mice at 2–3 months of age. (g) Rota-Rod test (16 rpm). n = 13 Con and 13 GFAP-Rs1 mice. (h) Open-field test (15-min duration). n = 13 Con and 13 GFAP-Rs1 mice. (i) Hot-plate test. n = 12 Con and 13 GFAP-Rs1 mice. (j–k) Elevated plus maze at 3–5 (j) or 18–19 (k) months of age. n = 13 Con and 13 GFAP-Rs1 mice (j) or 14 Con and 11 GFAP-Rs1 mice (k). Student's t test (vs. Con): (g) P = 0.90; (h) P = 0.34 (Total activity), P = 0.73 (Center/total activity); (i) P = 0.84; (j) P = 0.93; (k) P = 0.50. Data are presented as mean ± s.e.m.
(a–b) Swim speeds of GFAP-tTA (Con), TetO-Rs1 and GFAP-Rs1 mice during probe trials corresponding to (a) Figure 5c and (b) Figure 5i and Supplementary Figure 7e. Student's t test (vs. Saline of matching genotype): (a) P = 0.17 (Con); P = 0.23 (GFAP-Rs1). (b) P = 0.91 (GFAP-tTA); P = 0.54 (TetO-Rs1), P = 0.49 (GFAP-Rs1). n = 27 Con/saline, 28 Con/GR, 18 GFAP-Rs1/saline, and 20 GFAP-Rs1/GR mice (a); n = 19 GFAP-tTA/saline, 18 GFAP-tTA/GR, 14 TetO-Rs1/saline, 18 TetO-Rs1/GR, 16 GFAP-Rs1/saline, and 16 GFAP-Rs1/GR mice (b). (c–e) Rs1 ligand did not impair the performance of TetO-Rs1 singly transgenic mice in the Morris water maze at 2–11 months of age. (c) Experimental design for data in (d–e). TetO-Rs1 mice were trained for two days to locate the hidden platform and injected with saline or GR-125487 (GR, 3 mg/kg, i.p.) one day after training. Memory of the platform location was tested in a probe trial two days after training. (d) Distance traveled to reach the hidden platform during training. Two-way ANOVA: F(1, 30) = 0.01, P = 0.93 for interaction effect; F(1, 30) = 0.02, P = 0.88 for agonist effect. n = 14 TetO-Rs1/saline and 18 TetO-Rs1/GR mice. (e) Probe. Left, crossings. Student's t test with Welch's correction (Target vs. Other): P = 0.047 (Saline), P = 0.0042 (GR). Right, latency to reach target location. One-tailed Student's t test: P = 0.14; n = 14 TetO-Rs1/saline and 18 TetO-Rs1/GR mice. (f–g) Rs1 ligand did not acutely impair memory retrieval in Con and GFAP-Rs1 mice in the Morris water maze at 2–11 months of age. (f) Experimental design for data in (g). Mice were trained for two days to locate the hidden platform. Memory of the platform location was tested in a probe trial two days after training. Mice received an injection of saline or GR-125487 (GR, 3 mg/kg, i.p.) 4 h before the probe trial. (g) Probe. Left, crossings. Two-way ANOVA: F(1, 72) = 0.16, P = 0.69 for genotype by treatment interaction. Student's t test with Welch's correction (Target vs. Other): P = 0.0003 (Con/Saline), P = 0.033 (Con/GR), P < 0.0001 (GFAP-Rs1/Saline), P = 0.008 (GFAP-Rs1/GR). Right, latency to reach target location. Two-way ANOVA: F(1, 72) = 0.35, P = 0.56 for genotype by treatment interaction. n = 21 Con/saline, 20 Con/GR, 18 GFAP-Rs1/saline, and 17 GFAP-Rs1/GR mice. *P < 0.05, **P < 0.01, ***P < 0.001 (Student's t test with Welch's correction). Data are presented as mean ± s.e.m.
(a–d) Rs1 expression increases constitutive Gs-coupled signaling in primary astrocytes. Primary astrocyte cultures from wild-type mice were transduced or not (Con) with lentiviral constructs encoding mCherry (mCher) or FLAG-Rs1 (Rs1). None of the cultures were treated with GR-125487. (a) Rs1 expression was confirmed by immunoprecipitation with antibodies to human 5HT4b receptor followed by western blotting with antibodies to FLAG. A cropped western blot is shown from one experiment. (b) Intracellular cAMP levels were measured and normalized to the average level in Con cells. One-way ANOVA: F(2, 19) = 20.02, P < 0.0001. n = 7 Con, 8 mCher, and 7 Rs1 wells per condition from three independent experiments. ***P < 0.001 vs. Con (Dunnett's test). (c) Intracellular calcium levels were measured using Calcium-5, a calcium-sensitive fluorescent dye. Fluorescence intensity was normalized to the average intensity in Con wells. Student's t test: P = 0.02. n = 32 wells per condition from two independent experiments. *p < 0.05. (d) Representative cropped western blots (top) and densitometric quantification (bottom) of phosphorylated (p) and total (t) CREB. tCREB served as a loading control and pCREB/tCREB ratios were normalized to the average ratio in Con cells. Student's t test with Welch's correction: P = 0.0017. n = 6 wells per condition from two independent experiments. **p < 0.01. (e) Untreated GFAP-tTA (Con) and GFAP-Rs1 mice showed no target preference in a probe trial conducted 15 days after training in the Morris water maze. Untreated 5–7-month-old Con and GFAP-Rs1 mice were trained in the Morris water maze as shown in Figure 6a. Three probe trials were conducted before this probe, as shown in Figures 6b–e. Mice were guided to the platform location after completion of each probe trial. One-tailed Student's t test with Welch's correction (Target vs. Other of matching genotype): P = 0.17 (Con), P = 0.47 (GFAP-Rs1). n = 16 Con mice and 16 GFAP-Rs1 mice. (f–g) Untreated 3.5–6-month-old Con and GFAP-Rs1 mice were impaired in a novel object-recognition test. (f) During training, mice were presented for 10 min with one of two sets of identical objects (AA or BB) placed in the right (R) or left (L) half of the arena. One-tailed Student's t test (vs. A/R of matching genotype): P = 0.72 (Con A/L), 0.69 (Con B/R), 0.97 (Con B/L), 0.2 (GFAP-Rs1 A/L), 0.65 (GFAP-Rs1 B/R), and 0.47 (GFAP-Rs1 B/L). n = 12 Con A/R, 12 Con A/L, 5 Con B/R, 5 Con B/L, 8 GFAP-Rs1 A/R, 8 GFAP-Rs1 A/L, 12 GFAP-Rs1 B/R, and 12 GFAP-Rs1 B/L mice. (g) Two days after training, mice were presented for 10 min with a new object and one of the objects they were exposed to during training ('AB'). The average time spent exploring the new object relative to the familiar object is shown. Student's t test: P = 0.0021. n = 26 Con and 24 GFAP-Rs1 mice. **p < 0.01 (Student's t test). (h) Gfap mRNA levels in the cortex of young (2–4 months) and old (19–22 months) GFAP-tTA and GFAP-Rs1 mice. Gapdh served as a loading control. Gfap/Gapdh ratios were normalized to the average ratio in young GFAP-tTA mice. Two-way ANOVA: F(1, 25) = 25.16, p < 0.0001 for age effect. n = 5 Con/young, 10 GFAP-Rs1/young, 5 Con/old, and 9 GFAP-Rs1/old mice. ##p < 0.01 vs. young mice of matching genotype (Bonferroni test). (i–j) Untreated GFAP-Rs1 mice showed normal working memory in the Y maze. Con and GFAP-Rs1 mice were tested in the Y maze at 7–9 (i) and 18–19 (j) months of age. Student's t test (vs. Con): P = 0.76 (i), 0.42 (j). n = 21 Con and 22 GFAP-Rs1 mice (i); 13 Con and 10 GFAP-Rs1 mice (j). Full-length blots are presented in Supplementary Figure 13. Data are presented as mean ± s.e.m.
Supplementary Figure 9 In aging hAPP mice, A2A receptor expression is most prominent in astrocytes adjacent to amyloid plaques, but is not detected in microglia.
Representative photomicrographs of immunostained hippocampal sections are shown. (a) Aβ (6E10 antibody, green) and GFAP/astrocyte (red) staining in a 13-month-old hAPP mouse. Cell nuclei were labeled with DAPI (blue). Scale bar: 50 µm. (b–c) A2A receptor (green) and Iba1/microglia (red) staining in a 13-month-old hAPP mouse. Scale bars: 50 µm (b), 25 µm (c). (d–e) Thioflavin-S staining of amyloid plaques (green) and A2A labeling (red) in the hippocampus of an 18-month-old hAPP mouse. Insets show magnified views of the boxed regions. Scale bars: 50 µm. n = 3 (a), 2 (b–c), 4 (d–e) hAPP mice. DGgl: dentate gyrus granular layer.
Supplementary Figure 10 Astrocytic A2A receptor levels are increased in aging but not young hAPP mice.
(a–b) Representative photomicrographs of hippocampal sections from young (a) and older (b) nontransgenic (NTG) and hAPP mice immunostained for the A2A receptor (green) and GFAP/astrocytes (red). Ages of the mice are indicated in months (m, left). Insets (i–v) show magnified views of the boxed regions. DGgl: dentate gyrus granular layer; DGmol: dentate gyrus molecular layer. n = 4 NTG/3 m, 5 hAPP/3 m, 8 NTG/14 m, 7 hAPP/14 m, and 8 hAPP/18 m mice. Scale bars: 50 µm (a), 100 µm (b).
Supplementary Figure 11 Astrocytic A2A receptor levels are increased in young wild-type mice after kainate administration.
Representative photomicrographs of hippocampal sections from 2–3-month-old wild-type FVBN/J mice immunostained for the A2A receptor (green) and GFAP (red). Mice received a single i.p. injection of saline (Con) or kainate (KA, 30 mg/kg) and were perfused at the indicated time points after injection. Cell nuclei were labeled with DAPI (blue). Insets (i-iv) show magnified views of the boxed regions. DG: dentate gyrus; CA1: cornu ammonis area 1. Scale bar: 200 µm. n = 2 Con, 3 KA-day 1, 3 KA-day 3, and 3 KA-day 7 mice.
Supplementary Figure 12 Conditional ablation of astrocytic A2A receptors does not reduce behavioral abnormalities in young hAPP mice.
(a–d) hAPP mice with or without conditional ablation of astrocytic A2A receptors were assessed in the elevated plus maze at 2–4 months of age (a), the open field at 3–5 months of age (b), and the Morris water maze at 7–9 months of age (c–d). (a) Distance (left) and time spent (right) in the open arms of the elevated plus maze. Con: singly transgenic loxP-Adora2a control mice. One-way ANOVA: F(2, 38) = 4.72, P = 0.015 (left); F(2, 38) = 5.75, P = 0.0066 (right); n = 14 Con, 14 hAPP, and 13 hAPP/A2A-cKO mice. *P < 0.05, **P < 0.01 vs. Con (Dunnett's test). (b) Open-field habituation test. Mice were habituated to the open field in 5-min trials (1–2 trials per day) for 4 days. Graph shows the extent of dishabituation during a test trial 13 days later (test trial relative to the last training trial). One-way ANOVA: F(2, 35) = 7.29, P = 0.0023. n = 14 Con, 12 hAPP, and 12 hAPP/A2A-cKO mice. *P < 0.05, **P < 0.01 vs. Con (Dunnett's test). (c) Distance traveled to reach the platform during hidden platform training in the Morris water maze (two trials per session, two sessions per day for six days). Repeated measures two-way ANOVA revealed a significant difference between Con and hAPP mice (F(1, 20) = 48.36, P < 0.0001), but not between hAPP and hAPP/A2A-cKO mice (F(1, 16) = 0.37, P = 0.55). n = 13 Con, 9 hAPP, and 9 hAPP/A2A-cKO mice. (d) Probe trial conducted one day after training in the Morris water maze. Left, durations in target and non-target (other) quadrants. One-way ANOVA: F(2, 28) = 6.05, P = 0.0065; one-sample t test vs. chance duration of 15 s with FDR correction for multiple comparisons (Target vs. chance): P = 0.0003 (Con), 0.13 (hAPP), 0.15 (hAPP/ A2A-cKO). #P < 0.05, ##P < 0.01 (Dunnett's test); ***P < 0.001 vs. chance duration of 15 s. Right, latency to reach target location. One-way ANOVA: F(2, 29) = 3.78, P = 0.035. oP = 0.066, *P < 0.05 vs. Con (Dunnett's test). n = 13 Con, 10 hAPP, and 8 hAPP/A2A-cKO mice. Data are presented as mean ± s.e.m.
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Orr, A., Hsiao, E., Wang, M. et al. Astrocytic adenosine receptor A2A and Gs-coupled signaling regulate memory. Nat Neurosci 18, 423–434 (2015). https://doi.org/10.1038/nn.3930
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