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Restoring metabolism of myeloid cells reverses cognitive decline in ageing

Abstract

Ageing is characterized by the development of persistent pro-inflammatory responses that contribute to atherosclerosis, metabolic syndrome, cancer and frailty1,2,3. The ageing brain is also vulnerable to inflammation, as demonstrated by the high prevalence of age-associated cognitive decline and Alzheimer’s disease4,5,6. Systemically, circulating pro-inflammatory factors can promote cognitive decline7,8, and in the brain, microglia lose the ability to clear misfolded proteins that are associated with neurodegeneration9,10. However, the underlying mechanisms that initiate and sustain maladaptive inflammation with ageing are not well defined. Here we show that in ageing mice myeloid cell bioenergetics are suppressed in response to increased signalling by the lipid messenger prostaglandin E2 (PGE2), a major modulator of inflammation11. In ageing macrophages and microglia, PGE2 signalling through its EP2 receptor promotes the sequestration of glucose into glycogen, reducing glucose flux and mitochondrial respiration. This energy-deficient state, which drives maladaptive pro-inflammatory responses, is further augmented by a dependence of aged myeloid cells on glucose as a principal fuel source. In aged mice, inhibition of myeloid EP2 signalling rejuvenates cellular bioenergetics, systemic and brain inflammatory states, hippocampal synaptic plasticity and spatial memory. Moreover, blockade of peripheral myeloid EP2 signalling is sufficient to restore cognition in aged mice. Our study suggests that cognitive ageing is not a static or irrevocable condition but can be reversed by reprogramming myeloid glucose metabolism to restore youthful immune functions.

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Fig. 1: PGE2 EP2 receptor regulates myeloid metabolism and inflammation in ageing.
Fig. 2: Myeloid knockdown of the EP2 receptor prevents cognitive ageing.
Fig. 3: EP2-directed glycogen synthesis regulates macrophage bioenergetics and immune responses.
Fig. 4: EP2 blockade reverses age-associated inflammation and spatial memory loss.

Data availability

Raw data for nanostring analysis are available from the Gene Expression Omnibus under accession code GSE155992. Untargeted metabolomics data are provided in the source data file for Extended Data Fig. 3dSource data are provided with this paper.

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Acknowledgements

This work was supported by RO1AG048232 (K.I.A.), RF1AG058047 (K.I.A.), R21NS087639 (K.I.A.), American Heart Association 19PABH134580007 (K.I.A.), 1P50 AG047366 (K.I.A.), 1P30 AG066515 (K.I.A.), Bright Focus (K.I.A.), the Paul and Daisy Soros Fellowship for New Americans (P.S.M.), the Gerald J. Lieberman Fellowship (P.S.M.), DP1DK113643 (M.R.M., L.L. and J.D.R.), the HHMI Hanna H. Gray Fellows Program (M.R.M.), the Burroughs Wellcome Fund PDEP (M.R.M.), the Stanford Innovation Fund (A.U.J. and D.M.-R.), the Takeda Pharmaceuticals Science Frontier Fund (D.M.-R.), the Ludwig Cancer Foundation (I.L.W.), NIH/NCI F30 CA228215 (J.Q.H), NIH/NCI R35CA220434 (I.L.W.), the Japan Science and Technology Agency’s Exploratory Research for Advanced Technology (JST-ERATO) Suematsu Gas Biology Project (Y.S. and M.S.), the Scully Family Initiative (F.M.L.), the Taube Family Foundation (F.M.L.) and the Jean Perkins Foundation (F.M.L.). The authors thank J. Perrino at the Stanford Cell Sciences Imaging Facility (supported by NIH 1S10RR02678001) and the Stanford Human Immune Monitoring Center.

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Authors

Contributions

P.S.M., Q.W., A.U.J., E.G., J.Q.H., A.S.D., C.W., M.L. and P.K.M. designed and performed experiments and analysed the data. M.R.M., L.L. and J.D.R. performed targeted metabolomics and quantification of isotope labelling and analysed the data. A.L.-H. and F.M.L. performed electrophysiology experiments and analysed the data. A.R. performed initial experiments of the effects of EP2 on mitochondria. Y.S. and M.S. carried out prostaglandin measurements. R.M., I.L.W. and D.M.-R. supervised experiments. P.S.M. and K.I.A. conceived and supervised the project, designed experiments, interpreted the data and wrote the manuscript.

Corresponding author

Correspondence to Katrin I. Andreasson.

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The authors declare no competing interests.

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Peer review information Nature thanks Shuh Narumiya, Jonas Neher and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Fig. 1 PGE2 regulates macrophage energy metabolism via the EP2 receptor.

Data are mean ± s.e.m. unless otherwise specified. a, The PGE2 synthetic pathway. Arachidonic acid is converted to the prostaglandin precursor PGH2 by the action of constitutive COX-1 and/or inducible COX-2; this step is inhibited by non-steroidal anti-inflammatory drugs (NSAIDs). PGE2 synthase converts PGH2 to PGE2 which can bind to four distinct G-protein-coupled receptors (EP1–EP4 receptors). b, Top, representative immunoblot of two independent experiments quantifying COX-2 and PGE2 synthase in young and aged human MDMs. Bottom, quantification shows higher COX-2 and mPGE2 synthase levels in aged (above 65 years) compared to young (below 35 years) human MDMs (n = 6 donors per group; **P = 0.0030, *P = 0.0106, Student’s two tailed t-test). c, Top, representative immunoblot of two independent experiments quantifying EP1–EP4 in young and aged human MDMs; last lane, HEK cells transfected with EP receptor cDNA as a positive control. Bottom, quantification shows increased EP2 in aged (over 65 years) compared to young (below 35 years) human MDMs (n = 6 donors per group; ****P < 0.0001, Student’s two tailed t-test). df, Quantification of basal respiration and ECAR of human MDMs treated with ascending doses of the EP1 agonist iloprost (d), the EP3 agonist AE248 (e) and the EP4 agonist AE1329 (f) for 20 h (n = 5 donors per group). g, h, Dose response of the brain-impermeant EP2 antagonist PF-04418948 (g) and the brain-penetrant EP2 antagonist C52 (h) in human MDMs at 20 h. Data are represented as box plots (5–95 percentile); one-way ANOVA for OCR and ECAR, P < 0.0001; Tukey’s post hoc test for PF-04418948: **P = 0.0083, ***P = 0.0005, #P < 0.0001; Tukey’s post hoc test for C52: #P < 0.0001 (n = 5 donors per group). i, LC–MS quantification of PGE2 levels in the plasma and cerebral cortex of aged (18–20 months) and young (3–4 months) mice. ****P < 0.0001 by two-tailed Student’s t-test (n = 6 mice per group) j, LC–MS quantification of PGD2, PGF2a and prostacyclin (measured as 6-keto-PGF1a) in the cerebral cortex of young and aged mice (n = 5–6 mice per group). k, Left, Representative immunoblot of EP1–EP4 levels in mouse peritoneal macrophages isolated from young (3–4 months) and aged (18–20 months) mice; last lane, HEK cells transfected with EP receptor cDNAs as a positive control (n = 6 mice per group). Right, EP2 is selectively upregulated in aged mouse peritoneal macrophages, similar to aged human MDMs in c; **P = 0.0018 (n = 6 mice per group). Source data

Extended Data Fig. 2 Myeloid EP2 receptor knockdown prevents age-associated inflammation.

Data are mean ± s.e.m. unless otherwise specified. a, MFI quantification from three independent experiments for the anti-inflammatory markers CD71 and EGR2 and pro-inflammatory markers CD80 and CD86 in young (3–4 months) and aged (20–23 months) Cd11bCre and Cd11bCre;EP2lox/lox mice (n = 10,000–20,000 cells per point; n = 3 mice per group). Two-way ANOVA: effects of genotype and age P < 0.0001; Tukey’s post hoc test **P < 0.01, ***P < 0.001, ****P < 0.0001. b, c, Quantification of immune factors in plasma (b) and hippocampus (c) comparing aged Cd11bCre and aged Cd11bCre;EP2lox/lox mice; *P < 0.05, **P < 0.01 by two-tailed Student’s t-test (n = 6 mice per group). d, Representative immunoblot and quantification from two independent experiments comparing EP2 levels in 6-month-old Cd11bCre and wild-type mice (n = 6 per group). e, Primary latency in the Barnes maze for the five learning trials (n = 10 (young), n = 11 (aged Cd11bCre), n = 7 (aged Cd11bCre;EP2lox/lox). f, Representative immunoblot and quantification of hippocampal presynaptic proteins synapsin and SNAP-25 and postsynaptic proteins PSD95 and CamKIIα; #P = 0.0005, ***P = 0.0006, ****P < 0.0001 by two-tailed Student’s t-test (n = 6 mice per group; 20–23-month-old mice). g, Input/output curves as a measure of basal synaptic transmission in the CA1 region of the hippocampus (n = 8 slices, 3 mice per group). h, PGE2 activation of the EP2 receptor activates AKT signalling through phosphorylation of Ser473. Activated pAKT (Ser473) inactivates GSK3β through inhibitory phosphorylation of Ser9. Inactivation of GSK3β leads to constitutive activity of glycogen synthase 1 (GYS1) and glycogen synthesis. Conversely, deletion or antagonism of EP2 receptor signalling leads to downstream inhibitory phosphorylation of Ser641, Ser645 and Ser649 on GYS1 by activated, non-phosphorylated GSK3β. i, Quantification of EP2, pAKT (Ser 473)/total AKT, pGSK3β (Ser9)/total GSK3β and pGYS1 (Ser 641, 645, 649)/GYS1 levels in peritoneal macrophages from 6-month-old Cd11bCre and Cd11bCre;EP2lox/lox mice; ***P = 0.0002, ****P < 0.0001 by two-tailed Student’s t-test (n = 6 mice per group). Source data

Extended Data Fig. 3 Macrophage EP2 signalling increases glycogen synthesis.

Data are mean ± s.e.m. unless otherwise specified. a, Representative immunoblots and quantification of the EP2–AKT–GSK3β–GYS1 signalling pathway in mouse peritoneal macrophages isolated from 6-month-old wild type C57B6/J mice treated with EP2 antagonist C52 (100 nM), EP2 agonist butaprost (100 nM) or PGE2 (100 nM) for 20 h. b, Quantification of a; P < 0.0001 by one-way ANOVA; Tukey’s post hoc test **P = 0.0072, ***P < 0.001, ****P < 0.0001 (n = 6 mice per group). c, Quantification of glycogen levels in human MDMs (age (mean ± s.e.m.) 43.9 ± 3.451 years) treated with EP2 agonist butaprost (100 nM) or EP2 antagonist C52 (100 nM) for 20 h. P < 0.001 by one-way ANOVA; Tukey’s post hoc test ****P < 0.0001 (n = 9 per group). d, LC–MS analysis of human MDMs (age (mean ± s.e.m.) 43.9 ± 3.451 years) treated with C52 (100 nM, 20 h) demonstrates upregulation of proximal glycolytic pathway (G6P, F6P, F-1,6-BP) and GSH and downregulation of UDPG and GSSG. Red circles represent metabolites with fold change >1.5, blue circles with fold change <1.5; q-value < 0.05 with false discovery rate (FDR) correction (n = 6 donors per group). e, Enrichment pathway analysis of d using MetaboAnalyst. f, Schematic depicting U-13C-glucose metabolism via glucose-1P (G1P) and UDP-glucose to glycogen synthesis (yellow shaded box) versus flux towards the pentose phosphate pathway (green shaded box), glycolysis or lactate, and the TCA cycle (blue shaded box) with associated mass-labelled molecules. Glucose is labelled at all 6 carbons (in green; M+6) and is converted via the glycolytic pathway to two molecules of pyruvate (or M+3). Pyruvate is transported into the mitochondria and undergoes oxidative decarboxylation to yield acetyl-CoA (M+2) which enters the TCA cycle. Successive additions of labelled acetyl-CoA through the TCA cycle yield M+2, M+4 and M+6. g, Isotope tracing of U-13C-glucose metabolism was performed in human MDMs (age (mean ± s.e.m.) 42.13 ± 3.674 years) treated with EP2 agonist butaprost (100 nM, 20 h) or EP2 inhibitor C52 (100 nM, 20 h; n = 6 donors per group). Activation of EP2 signalling with butaprost increases incorporation of heavy glucose in glycogen precursors G1P and UDP-glucose and reduces labelling of glycolytic intermediates (F-1,6-BP and pyruvate) as well as TCA cycle intermediates (citrate and succinate); inhibition of EP2 with C52 conversely reduces synthesis of glycogen precursors and increases glycolytic and TCA cycle intermediates. h, Schematic depicting changes in glucose metabolism in Fig. 2h, i. i, Representative flow cytometry histograms and corresponding MFI quantification of human MDMs with or without the EP2 inhibitor C52 (100 nM, 20 h) from three independent experiments. Surface levels of anti-inflammatory markers CD206 and CD163 increase with inhibition of EP2 signalling whereas levels of pro-inflammatory markers CD86 and CD64 decrease. **P < 0.01, ***P = 0.002 by two-tailed Student’s t-test (n = 30,000 – 40,000 cells per point; n = 3 donors per group). j, Quantification of phagocytosis of fluorescent E. coli particles in human MDMs treated with EP2 inhibitor C52 (100 nM, 20 h) from two independent experiments. ***P = 0.0002 by two-tailed Student’s t-test (n = 9 donors per group). Source data

Extended Data Fig. 4 Knockdown of GYS1 promotes macrophage glucose metabolism and anti-inflammatory polarization.

Data are mean ± s.e.m. unless otherwise specified. af, Human MDMs are from donors (age (mean ± s.e.m.) 47.2 ± 1.582 years); gi, Human MDMs are from young (below 35 years) and aged (over 65 years) donors. a, Representative immunoblots and quantification of human MDMs transfected with two different shRNAs to human GYS1 at 8 h. P < 0.0001 by one-way ANOVA; Tukey’s post hoc test ****P < 0.0001 (n = 6 donors per group). b, Quantification of glycogen levels in human MDMs transfected with shRNAs to GYS1 at 8 h. P < 0.0001 by one-way ANOVA; Tukey’s post hoc test ****P < 0.0001 (n = 6 donors per group). c, Representative traces and quantification of OCR and ECAR for three independent experiments in human MDMs transfected with shRNAs for GYS1 at 8 h (n = 5 donors per group). P < 0.0001 by one-way ANOVA; Tukey’s post hoc test ****P < 0.0001 (n = 6 donors per group). Black arrows represent addition of oligomycin (1 μM), FCCP (2 µM), and rotenone/antimycin (500 nM), respectively, at time points indicated. d, Hierarchical clustering of targeted metabolomics for glycolysis, pentose phosphate shunt and TCA cycle metabolites in human MDMs transfected with shRNA to GYS1 at 8h (n = 5 donors per group). e, Isotope tracing of U-13C-glucose in human MDMs transfected with shRNA to GYS1 at 8 h reveals a decreased labelling in the glycogen precursor UDP-glucose and an increase in glycolytic intermediates F-1,6-BP and pyruvate (n = 6 donors per group). f, Representative flow cytometry histograms of three independent experiments for the pro-inflammatory markers CD86 and CD64 and anti-inflammatory markers CD206 and CD163 in human MDMs treated with or without shRNA to GYS1. Bottom, quantification of MFI, two-tailed Student’s t-test, *P < 0.05, **P < 0.01 (n = 20,000–40,000 cells per point, n = 3 donors per group). g, Representative trace of real-time changes in OCR from three independent experiments of young (below 35 years) and aged (over 65 years) human MDMs treated with or without shRNA to GYS1 (n = 5 young, n = 6 aged donors). h, Representative histograms of anti- and pro-inflammatory surface markers in young and aged human MDMs treated with or without shRNA to GYS1 (n = 3 donors per group). i, Representative traces of real-time changes in OCR from two independent experiments in young (below 35 years) and aged (over 65 years) human MDMs transfected with shRNA to GYS1 and treated 8 h later with butaprost (100 nm) for 20 h. OCR in GYS1-deficient aged human MDMs does not change with activation of the EP2 receptor by butaprost (n = 4 donors per group). Source data

Extended Data Fig. 5 EP2 blockade restores AKT–GSK3β–GYS1 signalling, glycolysis and mitochondrial respiration to youthful levels.

Data are mean ± s.e.m. unless otherwise specified. a, Quantification of PGE2 levels in young (below 35 years) and aged (over 65 years) human MDMs treated with or without C52 (100 nM, 20 h). Effects of age and treatment P < 0.0001 by two-way ANOVA; Tukey’s post hoc test ****P < 0.0001 (n = 5 donors per group). b, c, Representative immunoblots (b) and quantification (c) of effects of C52 treatment (100 nM, 20 h) in human MDMs from young (below 35 years) and aged (over 65 years) donors. Two-way ANOVA, age and treatment, P < 0.0001; Tukey’s post hoc test **P = 0.002, ***P = 0.0005, ****P < 0.0001 (n = 6 donors per group). d, Real-time changes in OCR from three independent experiments of young and aged human MDMs treated with or without C52 (100 nM, 20 h; n = 5 donors per group). e, Representative immunoblots of effects of EP2 inhibition on mitochondrial protein levels in young and aged human MDMs (n = 6 donors per group; OMM, outer mitochondrial membrane; IMM, inner mitochondrial membrane). See Fig. 3h for quantification. f, Membrane potential (TMRE) in young and aged human MDMs treated with or without C52 (100 nM, 20 h). Two-way ANOVA: age P = 0.0009 and treatment P = 0.0333; Tukey’s post hoc test, ***P = 0.0006, **P = 0.0088 (n = 6 donors per group). g, ROS in young and aged human MDMs treated with or without C52 (100 nM, 20 h). Two-way ANOVA: age P = 0.0022 and treatment P = 0.0055; Tukey’s post hoc test **P = 0.0045, ##P = 0.0084 (n = 7 donors per group). h, Young and aged human MDMs were incubated with U-13C-glucose for 20 h with or without C52. Aged human MDMs showed increased labelling in glycogen precursors (G1P and UDP-glucose) and decreased labelling in TCA cycle intermediates. This was prevented with EP2 inhibition; n = 3 donors per group. i, Quantification of TCA cycle metabolites from Fig. 3i. Note normalization of citrate, α-ketoglutarate (α-KG), succinate, fumarate and malate in aged human MDMs with EP2 inhibition. Also note that itaconate, which is increased in models of acute macrophage stimulation with LPS, is not changed with ageing (n = 3 donors per group). Two-way ANOVA with Tukey’s multiple comparisons test; *P < 0.05, **P < 0.01, ***P < 0.001. j, k, Representative traces of real-time changes in OCR (j) and quantification of basal respiration and ECAR (k) from two independent experiments in peritoneal macrophages isolated from young (3–4 months) and aged (18–20 months) mice treated with COX-2 inhibitor SC236 (100 nM, 20 h). Two-way ANOVA: age and treatment, P < 0.0001 and P = 0.004, respectively; Tukey’s post hoc test, ****P < 0.0001 (n = 5 mice per group). Source data

Extended Data Fig. 6 EP2 blockade does not alter LPS-mediated glucose metabolism reprogramming, and aged macrophages lose the capacity to use alternative fuel sources.

Data are mean ± s.e.m. unless otherwise specified. a, Human MDMs with or without LPS treatment (100 ng ml−1, 20 h; n = 4 (veh), n = 4 (veh + C52), n = 5 (LPS), n = 7 (veh + LPS) donors per group, age (mean ± s.e.m.) 42.3 ± 8.212 years) were stimulated with or without C52 (100 nM, 20 h). Blockade of EP2 does not rescue OCR and ECAR in LPS-treated human MDMs. b, 13C-glucose isotope tracing of human MDMs with or without LPS (100 ng ml−1, 20 h) and with or without C52 (100 nM, 20 h), demonstrates reprogramming of the TCA cycle towards increased production of itaconate with LPS stimulation that is not reversed with EP2 inhibition (n = 3 donors per group). c, Hierarchical clustering of glycolytic and TCA cycle metabolites in human MDMs with or without LPS (100 ng ml−1, 20 h) and with or without C52 (100 nM, 20 h) (n = 3 donors per group). d, Model highlighting differences in glucose metabolism and the TCA cycle in LPS-stimulated macrophages versus aged macrophages. LPS stimulation upregulates glycolysis and lactate production and increases itaconate production from aconitate by cis-aconitate decarboxylase, the protein product of Irg1, which is highly induced in settings of inflammation; increased itaconate suppresses SDH. In aged human MDMs, glucose is diverted from glycolysis to glycogen, reducing glucose flux into the TCA cycle; ageing is characterized by low SDH activity arising from deficient SIRT3 deacetylation of complex II subunits as a result of declining NAD+ levels17. In both cases, lower succinate dehydrogenase activity leads to accumulation of the pro-inflammatory TCA cycle intermediate succinate, which enhances pro-inflammatory gene expression by stabilizing the transcription factor HIF-1α. e, Diagram illustrating fuel substrates for the TCA cycle and mitochondrial OXPHOS. Glucose and pyruvate/lactate can feed into the TCA cycle via acetyl-CoA; glutamine is metabolized via anaplerosis to α-KG, a TCA cycle intermediate. f, Young (below 35 years) and aged (over 65 years) human MDMs received U-13C-glucose [M+6] for 20 h. Isotope tracing reveals young macrophages can incorporate significantly higher 13C from glucose into the TCA cycle compared to aged macrophages (P = 0.0026 by Student’s two-tailed t-test with Welch’s correction for 13C citrate[M+2] proportion in young versus aged cells; n = 3 donors per group). g, Young (below 35 years) and aged (over 65 years) human MDMs received U-13C-pyruvate [M+3] for 20 h. Isotope tracing reveals young macrophages are able to incorporate significantly higher 13C from pyruvate into the TCA cycle compared to aged macrophages (P < 0.0001 by Student’s two-tailed t-test with Welch’s correction for 13C -citrate[M+2] in young versus aged cells; n = 3 donors per group). h, Young (below 35 years) and aged (over 65 years) human MDMs received U-13C-lactate [M+3] for 20 h. Isotope tracing reveals young macrophages are able to incorporate significantly higher 13C from lactate into the TCA cycle than aged macrophages (P = 0.0083 by Student’s two-tailed t-test with Welch’s correction for 13C citrate[M+2] in young versus aged cells; n = 3 donors per group). i, Young (below 35 years) and aged (over 65 years) human MDMs received U-13C-glutamine [M+5] for 20 h. Isotope tracing reveals young macrophages are able to incorporate significantly higher 13C from glutamine into the TCA cycle than aged macrophages (P < 0.0001 by Student’s two-tailed t-test with Welch’s correction for13C-α-KG [M+5] in young versus aged cells; n = 3 donors per group). j, Fuel flexibility was assayed by inhibiting fatty acid oxidation, glutamine metabolism, and pyruvate transport into the mitochondria. Representative trace of real-time changes in OCR from two independent experiments demonstrating increased dependence on glucose and reduced capacity by aged mouse macrophages (20–23 months) for oxidative phosphorylation (n = 5 donors per group). UK5099: mitochondrial pyruvate carrier inhibitor; BPTES: glutaminase inhibitor; etomoxir: inhibitor of carnitine palmitoyltransferase 1 (CPT1) which transports fatty acids into mitochondrial matrix. For dependency traces cells received UK5099 in first injection and BPTES/Etomoxir in second injection as indicated on figure. For capacity traces cells received BPTES/Etomoxir in first injection and UK5099 in second injection. k, Quantification of j; n = 5 donors per group; ****P < 0.0001 by two-tailed Student’s t-test. Source data

Extended Data Fig. 7 EP2 is expressed in hippocampal CA1 microglia.

Data are mean ± s.e.m. unless otherwise specified. Scale bars, 40 μm. a, b, Images are from the CA1 region of the hippocampus. a, Colocalization of EP2 immunofluorescence with IBA1 in microglia (18-month-old mice). b, Microglial EP2 is not detected in RosaCreERT2;EP2lox/lox mice (15–16-month-old mice). c, d, Images are from the cerebral cortex. c, Colocalization of EP2 immunofluorescence with IBA1 in microglia (18-month-old mice). d, Microglial EP2 is not detected in RosaCreERT2;EP2lox/lox mice (15–16-month-old mice). e, f, Quantification of immune factors in the plasma (e) and hippocampus (f) from young (3–4 months) and aged (22–24 months) mice with or without C52 treatment (10 mg/kg/d for 1 month; n = 3 mice/ young + veh group; n = 4 mice per group for all other groups). g, Quantification of percent CD68+/IBA1+ cells in CA3 hippocampus from young and aged mice with or without C52 treatment (10 mg/kg/day, 1 month); effect of age and treatment P < 0.0001 by two-way ANOVA; Tukey’s post hoc test ****P < 0.0001 (n = 8 slices, n =4 mice per group). h, Quantification of microglial numbers from young and aged mice with or without C52 treatment (10mg/kg/day, 1 mo; n = 8 slices, n =3 young and n =4 aged mice per group). i, Representative immunoblot of the pre- and postsynaptic proteins synapsin, PSD95, SNAP25 and CamKIIa in young and aged hippocampi in mice with or without C52 treatment (10 mg/kg/day, 1 month); n =3 young and n =4 aged mice per group. j, Quantification of i. Synapsin: effects of age (P = 0.0685) and treatment (P = 0.0072) by two-way ANOVA; Tukey’s post hoc test **P = 0.0069, ***P = 0.0010. PSD95: effects of age (P = 0.0019) and treatment (P = 0.0009) by two-way ANOVA; Tukey’s post hoc test **P = 0.0020, ***P = 0.0007. SNAP25: effects of age (P = 0.1930) and treatment (P = 0.0463) by two-way ANOVA; Tukey’s post hoc test *P = 0.0121. CamKIIα: effects of age (P = 0.9210) and treatment (P = 0.0025) by two-way ANOVA; Tukey’s post hoc test *P = 0.0132 (n = 3 young and 4 aged mice per group). Source data

Extended Data Fig. 8 Effects of in vivo EP2 inhibition on macrophage metabolism, synaptic mitochondria and mitochondrial morphology in young and aged mice.

Data are mean ± s.e.m. unless otherwise specified. a, Peritoneal macrophage metabolite labelling following in vivo U-13C-Glucose isotope tracing (n = 6 mice per group). b, Quantification of per cent time in the target quadrant of the Barnes maze. Two-way ANOVA, effect of age or treatment ****P < 0.0001, Tukey’s post hoc test ****P < 0.0001 (n = 10 mice per group). c, Long-term potentiation (LTP) in the CA1 hippocampal region over a 120-min recording interval. Acute administration of C52 (100 nM, 1 h) before TBS does not alter LTP in aged mice (20-22 mo mice, n = 3 mice per group). d, Synaptic mitochondria were isolated from synaptosome fractions prepared from 16–month-old mice treated with or without C52 (10 mg/kg/d, 10 days). Coupling between the mitochondrial electron transport chain and oxidative phosphorylation (using succinate as the substrate) was determined using the Seahorse XFe24 analyser. Basal respiration (state II) and ADP-supplemented respiration (state III) reflect both electron transport and ATP generation and increased by twofold with EP2 blockade. Maximal respiration (state IIIu) after application of the H+ gradient uncoupler FCCP was also higher with EP2 inhibition, and state IVo, reflecting blockade of ATP synthase with oligomycin, was unchanged. *P < 0.05, two-tailed Student’s t-test (n = 7 mice per group). e, Coupling assay trace of synaptic mitochondria from aged mice treated with or without C52 for 10 days. Rates of basal complex II respiration as well as states III (ADP stimulated respiration), IV (oligomycin) and IIIu (FCCP) were consecutively measured (n = 7 mice per treatment group). f, TEM images at 5,000× magnification of microglia in the CA3 region of the hippocampus from young (3–4 months) and aged (22–24 months) mice treated with or without C52 (10 mg/kg/d, 1 month). Aged mice exhibit abnormal, non-electron-dense mitochondria; these features are rescued with C52 treatment. Arrows (black for vehicle, white for C52) point to mitochondria within microglia; blue shaded areas are non-microglial cells. Nu, nuclei; white scale bars, 1 μm. g, Higher-power representative TEM images of aged (22–24 months) mice treated with or without C52 (10 mg/kg/d, 1 month) showing differences in cristae and electron density. White scale bars, 500 nm. h, Quantification of per cent abnormal mitochondria in microglia of young (3–4 months) and aged (22–24 months) mice treated with or without C52 (10 mg/kg/d, 1 month); n = 66 mitochondria per group, 2-way ANOVA with Tukey’s post hoc test, ****P < 0.001. Source data

Extended Data Fig. 9 Peripheral EP2 blockade restores youthful inflammatory profile and cognitive status.

Data are mean ± s.e.m. unless otherwise specified. a, Average speed (inches per second) and percentage of time spent in the inner zone in young (3–4 months) and aged (20–23 months) Cd11bCre;EP2lox/lox mice. There are no significant differences between age-matched Cd11bCre and Cd11bCre;EP2lox/lox mice (n = 10 mice (young); n = 7 mice (aged Cd11bCre;EP2lox/lox); n = 11 mice (aged Cd11bCre)). b, Average speed (inches/second) and percentage of time spent in the inner zone in young (3–4 months) and aged (22–24 months) mice treated with C52 compound (10 mg/kg/d, 1 month). There are no significant differences between age-matched vehicle and C52-treated mice (n = 8 mice for young + veh; n = 9 mice per group for all other groups). c, Average speed (inches per second) and percentage of time spent in the inner zone in young (3–4 months) and aged (20–22 months) mice treated with PF compound (2.5 mg/kg/d, 6 weeks.) There are no significant differences between age-matched vehicle or PF-treated mice (n = 7 mice for young groups; n = 6 mice for aged groups). d, Real-time changes in OCR and quantification of basal respiration and ECAR of three independent experiments on mouse hippocampal neurons treated with PGE2 (100 nM, 20 h), butaprost (100 nM, 20 h) and C52 (100 nM, 20 h) (n = 6 (butaprost), n = 7 (all others) biologically independent samples per group). Black arrows represent addition of oligomycin (1 µM), FCCP (2 μM), and rotenone/antimycin (500 nM), respectively, at time points indicated. e, Real-time changes in OCR and quantification of basal respiration and ECAR of three independent experiments on mouse astrocytes treated with PGE2 (100 nM, 20 h), butaprost (100 nM, 20 h) and C52 (100 nM, 20 h) (n = 7 biologically independent samples per group). Black arrows represent addition of oligomycin (1 μM), FCCP (2 µM), and rotenone/antimycin (500 nM), respectively, at time points indicated. f, LC–MS analysis of plasma and brain levels of PF-04418948 (2.5 mg/kg/d, 6 weeks). PF-04418948 was not detected in whole-brain lysates of treated mice (n = 5–6 mice per group, 20–22 mo). gk, Young (3–4 months) and aged (20–22 months) mice were treated with vehicle or PF-04418948 at 2.5 mg/kg/d for 6 weeks. g, Quantification of significantly regulated immune factors in hippocampi (n = 3 young mice; n = 5 aged mice per group). h, Quantification of significantly regulated immune factors in plasma (n = 3 young mice; n = 5 aged mice per group). i, Primary latency in the Barnes maze for the five learning trials (n = 7 young mice; n = 6 aged mice per group). j, Representative traces of paths taken to the target hole (green) on the day of testing in the Barnes maze comparing aged mice with or without PF-04418948. k, Input/output curves as a measure of basal synaptic transmission in the CA1 region of the hippocampus (n = 8 slices, 3 mice per group). l, Microglia were isolated from brains of young (2–3 months) and aged (20–22 months) mice and assayed for EP2 receptor, pAKT and total AKT levels. Left, representative western blot; right, quantification. Two-tailed Student’s t-test, ***P < 0.001, **** P < 0.0001 (n = 6 mice per group). m, Microglia were isolated from brains of aged (20–24 months) mice treated with vehicle, PF-04418948 (10 mg/kg/day for 10 days) or C52 (10 mg/kg/day for 10 days). Left, representative western blot; right, quantification with two-tailed Student’s t-test, **** P < 0.0001 (n = 6 mice per group). Source data

Extended Data Fig. 10 Transcriptomics of primary peritoneal macrophages from young and aged mice treated with vehicle or with the non-brain-penetrant EP2 inhibitor PF-04418948.

Data are mean ± s.e.m. unless otherwise specified. a, Heat map of 1448 genes from Nanostring assay reveals that aged mice treated with PF-04418948 cluster with young mice treated with vehicle or PF-04418948. b, Volcano plot of peritoneal macrophages collected from aged versus young vehicle-treated mice. Red dots indicate genes that are absolute-value[log2(FC)] ≥ 2 and FDR < 0.05 by t-test with Benjamini–Hochberg correction. c, Volcano plot of peritoneal macrophages collected from aged + PF-04418948-treated versus aged + vehicle-treated mice. Red dots indicate genes that are absolute-value[log2(FC)] ≥ 1.5 and FDR < 0.05 by t-test with Benjamini–Hochberg correction. d, Top 10 signalling pathways from the Reactome pathway database for FDR < 0.05 genes comparing aged +veh versus young + v eh mice (top) and aged + PF-04418948 versus aged + veh mice (bottom). e, Hierarchical clustering of top differentially regulated chemokine and cytokines transcripts (FDR < 0.05) demonstrates that PF-04418948 treatment shifts expression towards young macrophage levels. f, Nanostring analysis of significantly regulated genes (FDR < 0.05) demonstrates differential expression of bioenergetic transcripts in peritoneal macrophages isolated from young (3–4 months) and aged (20–22 months) mice with or without PF-04418948 (2.5 mg/kg/d for 6 weeks). Aged peritoneal macrophages exhibit suppressed expression of genes that encode critical glycolytic enzymes, including the rate-limiting enzyme phosphofructokinase-1 (PFK-1) as well as the rate-setting TCA cycle enzyme, citrate synthase. Peripheral myeloid EP2 inhibition with PF-04418948 corrects the age-associated suppression of myeloid glycolytic and TCA cycle gene expression.

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Minhas, P.S., Latif-Hernandez, A., McReynolds, M.R. et al. Restoring metabolism of myeloid cells reverses cognitive decline in ageing. Nature 590, 122–128 (2021). https://doi.org/10.1038/s41586-020-03160-0

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