The prostaglandin E2 EP2 receptor accelerates disease progression and inflammation in a model of amyotrophic lateral sclerosis

Abstract

Objective: Inflammation has emerged as an important factor in disease progression in human and transgenic models of amyotrophic lateral sclerosis (ALS). Recent studies demonstrate that the prostaglandin E(2) EP2 receptor is a major regulator of inflammatory oxidative injury in innate immunity. We tested whether EP2 signaling participated in disease pathogenesis in the G93A superoxide dismutase (SOD) model of familial ALS.

Methods: We examined the phenotype of G93A SOD mice lacking the EP2 receptor and performed immunocytochemistry, quantitative reverse transcriptase polymerase chain reaction, and Western analyses to determine the mechanism of EP2 toxicity in this model.

Results: EP2 receptor is significantly induced in G93A SOD mice in astrocytes and microglia in parallel with increases in expression of proinflammatory enzymes and lipid peroxidation. In human ALS, EP2 receptor immunoreactivity was upregulated in astrocytes in ventral spinal cord. In aging G93A SOD mice, genetic deletion of the prostaglandin E(2)EP2 receptor improved motor strength and extended survival. Deletion of the EP2 receptor in G93A SOD mice resulted in significant reductions in levels of proinflammatory effectors, including cyclooxygenase-1, cyclooxygenase-2, inducible nitric oxide synthase, and components of the NADPH oxidase complex. In alternate models of inflammation, including the lipopolysaccharide model of innate immunity and the APPSwe-PS1DeltaE9 model of amyloidosis, deletion of EP2 also reduced expression of proinflammatory genes.

Interpretation: These data suggest that prostaglandin E(2) signaling via the EP2 receptor functions in the mutant SOD model and more broadly in inflammatory neurodegeneration to regulate expression of a cassette of proinflammatory genes. Inhibition of EP2 signaling may represent a novel strategy to downregulate the inflammatory response in neurodegenerative disease.

Figure 1

Aging G93A superoxide dismutase (SOD) mice demonstrate increased oxidative stress in neurons and induced expression of proinflammatory enzymes. (A) Gas chromatography mass spectrometric quantification of lipid peroxidation in 4-month-old male G93A SOD and wild-type (WT) mice demonstrates a selective increase in F₄-neuroprostanes (neuroPs) but not F₂-isoprostanes (isoPs) in cerebral cortex (*p < 0.05). (B–D) Lumbar spinal cord messenger RNA of 1- and 3.3-month-old male G93A SOD and WT mice was assayed for expression of inducible nitric oxide synthase (iNOS) (B), cyclooxygenase-1 (COX-1) and COX-2 (C), and NADPH oxidase subunits gp91^phox, p22^phox, p47^phox, and p67^phox. (D) Significant increases occur in 3.3-month-old but not 1-month-old G93A SOD mice relative to WT age-matched littermates for iNOS, COX-2, and NADPH oxidase subunits (n = 6–10 per age per genotype; *p < 0.05 in 3-month-old WT vs mutant SOD mice). There is a developmental downregulation of p67^phox between 1 and 3.3 months of age in WT spinal cord (*p < 0.01).

Figure 2

EP2 is induced in astrocytes and microglia in G93A superoxide dismutase (SOD) mice. (A) EP2 receptor colocalizes with the astrocytic marker glial fibrillary acidic protein (GFAP) in 3.3-month-old male G93A SOD but not wild-type (WT) lumbar cord (vertical arrows). (B) EP2 receptor colocalizes with the microglial marker Iba1 in SOD but not WT lumbar cord (horizontal arrows). (C) EP2 colocalizes with NeuN in motor neurons in both SOD and WT lumbar cord. Scale bar = 10μm. (D) EP2 is induced in SOD as compared with WT lumbar ventral horn. Left graph represents percentage EP2-immunopositive Iba1 microglial cells; right graph represents percentage EP2-immunopositive GFAP astrocytes. There is a significant increase in ratio of EP2-Iba1/Iba1 cells and EP2-GFAP/GFAP cells in SOD versus WT spinal cord (n = 4 per genotype with three sections counted 150μm apart; ***p < 0.01).

Figure 3

EP2 is induced in human amyotrophic lateral sclerosis (ALS) spinal cord in astrocytes. (A) Low magnification (40X) view of ventral horn (vh) and lateral corticospinal tract (cst) in control human thoracic cord demonstrates EP2 expression in large motor neurons. (B) Low-magnification view (original magnification X40) of same region in ALS thoracic cord demonstrates motor neuron expression of EP2, as well as EP2 staining of the hypercellular infiltrate in corticospinal tract and ventral horn. (C) Higher magnification (original magnification, X400) view shows perinuclear staining of EP2 in motor neurons in ventral horn. (D) Similar view of ALS ventral horn shows a degenerating EP2-positive motor neuron (asterisk) and also EP2-immunopositive cells (arrows) that are morphologically consistent with astrocytes. (E, F) EP2 is expressed in astrocytes (arrow); EP2 immunoreactivity (brown) is prominently expressed in a glial fibrillary acidic protein staining astrocyte (purple) in ALS (F) as compared with control ventral horn (E).

Figure 4

Phenotypes of G93A superoxide dismutase (SOD) mice lacking the EP2 receptor. (A) Deletion of the EP2 receptor resulted in increased survival in G93A SOD mice (p < 0.02) (n = 11 and 22 female G93A SOD mice in EP2^−/− [green lines] and EP2^+/+ and EP2^+/− [red lines] backgrounds). (B) Deletion of the EP2 receptor in aging G93A SOD mice resulted in improved grip strength. Two-way analysis of variance with repeated measures (time in weeks and genotype) between weeks 10 and 19 showed a significant effect of genotype [F(1,19) = 5.32; p < 0.05]. (C) No differences were observed in weight.

Figure 5

Expression levels of proinflammatory enzymes are reduced in G93A superoxide dismutase (SOD) mice lacking the EP2 receptor. (A) Reverse transcriptase polymerase chain reaction (RT-PCR) quantification of cyclooxygenase-1 (COX-1) and COX-2 messenger RNA (mRNA) in lumbar spinal cord of female G93A SOD mice in EP2^+/+ and EP2^−/− backgrounds. Significant decreases are observed in the EP2^−/− background for both COX-1 and COX-2 (n = 5 for each genotype; *p < 0.05). (B) Protein levels of COX-2 were assayed in cervical cord lysates of female G93A SOD mice in EP2^+/+ and EP2^−/− backgrounds, and show a significant reduction by quantitative Western analysis in EP2^−/− background, quantified in (C) (n = 4–5 samples/genotype; *p < 0.05). (D) RT-PCR quantification shows significant decreases in expression for both inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) in the EP2^−/− background (n = 5 for each genotype; p < 0.05). (E) Protein levels of iNOS in cervical cord lysates are reduced by quantitative Western analysis in G93A SOD mice lacking the EP2 receptor and are quantified in (F) (p < 0.05). (G) Levels of six NADPH oxidase subunits were assayed by RT-PCR of lumbar spinal cord of G93A SOD mice in the EP2^−/− and EP2^+/+ backgrounds. Significant decreases occur in the EP2^−/− background for the membrane-bound subunit p22^phox, and for the cytoplasmic subunits p40^phox, p47^phox, and p67^phox (n = 5 for each genotype; p < 0.05). (H) Protein levels of p67^phox in cervical spinal cord lysates are reduced by quantitative Western analysis in G93A SOD mice in the EP2^−/− background (first four lanes) but do not vary in wild-type versus EP2^−/− mice (last four lanes). (I) Quantification of p67^phox immunoreactivity (**p < 0.01; n = 4–5 samples/genotype). (J) Gas chromatography mass spectrometry quantification of lipid peroxidation in cerebral cortex shows a trend toward decreased levels of F₄-neuroprostanes (NeuroPs) in the EP2^−/− background (n = 4–5 per genotype). IsoPs = isoprostanes.

Figure 6

EP2 deletion decreases expression of proinflammatory genes in the lipopolysaccharide (LPS) model of innate immunity and the APPSwe-PS1ΔE9 model of familial Alzheimer’s disease (AD). (A) By quantitative reverse transcriptase polymerase chain reaction (RT-PCR), expression of cyclooxygenase-2 (COX-2), p47^phox, p67^phox, gp91^phox, and inducible nitric oxide synthase (iNOS) are induced 24 hours after intracerebroventricular (ICV) LPS in wild-type mice (*p < 0.05: vehicle vs LPS; n = 4–6 mice/genotype), but in EP2^−/− mice, this induction is blocked (*p < 0.05: EP2 wild type vs EP2^−/− with LPS). Gray bars represent EP2^−/−; black bars represent EP2^+/+. (B) Representative quantitative Western analysis demonstrates induction of p67^phox in cytosolic and membrane fractions with ICV LPS; the induction of p67^phox is inhibited with deletion of EP2. (C) Quantitative RT-PCR demonstrates a significant increase in EP2 receptor expression in 12-month-old APPSwe-PS1ΔE9 mice (n = 7–9 male twelve-month-old mice per genotype; **p < 0.01). (D) By RT-PCR, levels of COX-2 and p67^phox are increased in 12-month-old APPSwe-PS1ΔE9 mice, but (E) this increase is abrogated with deletion of the EP2 receptor (*p < 0.05; n = 4–9 twelve-month-old male mice per genotype). (D) White bars represent wild type; black bars represent APPS. (E) Black bars represent APPS:EP2^+/+; gray bars represent APPS:EP2^−/−. (F) Model of proinflammatory mechanism of prostaglandin E2 (PGE₂) EP2 signaling: PGE₂ signaling through the EP2 receptor on a glial cell (astrocyte or microglia) upregulates expression of proinflammatory proteins including subunits of the NADPH oxidase complex, COX-1 and COX-2, and iNOS, which are capable of producing ROS that damage neurons (yellow arrow), as evidenced by increased neuronal F₄-neuroprostanes (see Fig 1). Because COX-1 and COX-2 catalyze the first committed step in prostaglandin synthesis leading to further PGE₂ production, regulation of COX-1/2 expression by EP2 creates a feed-forward cycle in which further PGE₂ synthesis perpetuates and amplifies EP2 upregulation of proinflammatory proteins. Inhibition of COX activity with NSAIDs would block this feed-forward pathway.