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. 2007 Jan 4:4:2.
doi: 10.1186/1742-2094-4-2.

Prostaglandin E2 receptor subtype 2 (EP2) regulates microglial activation and associated neurotoxicity induced by aggregated alpha-synuclein

Jinghua Jin  1 , Feng-Shiun Shie, Jun Liu, Yan Wang, Jeanne Davis, Aimee M Schantz, Kathleen S Montine, Thomas J Montine, Jing Zhang
Affiliations

Prostaglandin E2 receptor subtype 2 (EP2) regulates microglial activation and associated neurotoxicity induced by aggregated alpha-synuclein

Jinghua Jin et al. J Neuroinflammation. .

Abstract

Background: The pathogenesis of idiopathic Parkinson's disease (PD) remains elusive, although evidence has suggested that neuroinflammation characterized by activation of resident microglia in the brain may contribute significantly to neurodegeneration in PD. It has been demonstrated that aggregated alpha-synuclein potently activates microglia and causes neurotoxicity. However, the mechanisms by which aggregated alpha-synuclein activates microglia are not understood fully.

Methods: We investigated the role of prostaglandin E2 receptor subtype 2 (EP2) in alpha-synuclein aggregation-induced microglial activation using ex vivo, in vivo and in vitro experimental systems.

Results: Results demonstrated that ablation of EP2(EP2-/-) significantly enhanced microglia-mediated ex vivo clearance of alpha-synuclein aggregates (from mesocortex of Lewy body disease patients) while significantly attenuating neurotoxicity and extent of alpha-synuclein aggregation in mice treated with a parkinsonian toxicant 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Furthermore, we report that reduced neurotoxicity by EP2-/- microglia could be attributed to suppressed translocation of a critical cytoplasmic subunit (p47-phox) of NADPH oxidase (PHOX) to the membranous compartment after exposure to aggregated alpha-synuclein.

Conclusion: Thus, it appears that microglial EP2 plays a critical role in alpha-synuclein-mediated neurotoxicity.

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Figures

Figure 1
Figure 1
Lack of EP2 enhanced microglial clearance of α-synuclein aggregates. A and B). Tissue sections obtained from patients with dementia with Lewy body disease were incubated with WT and EP2-/- microglia for 48 hrs. Residual α-synuclein aggregates were determined by Western blotting (two bands corresponding to dimers and trimers of α-synuclein). *: p < 0.05 for amount of residual synuclein aggregates in tissue sections treated with EP2-/- vs. WT microglia. C) Tissue sections were stained with CD11b antibody 48 hrs after incubation with microglia. The image demonstrates an activated microglial cell in ex vivo-cultured slides in close proximity to aggregated α-synuclein. Red, CD11b for microglia showing a macrophage-like microglia; green, antibody against human α-synuclein, demonstrating α-synuclein aggregates; and blue, DAPI nuclear staining).
Figure 2
Figure 2
Lack of EP2 suppressed loss of striatal dopamine in MPTP-treated mice. Mice were treated with either a chronic (panel A) or sub-chronic regimen of MPTP (panel B). In the chronic protocol, mice were treated with MPTP (30 mg/kg × 10) and the adjuvant probenecid (250 mg/kg) on a five-week schedule with an interval of 3.5 days between consecutive doses. In the sub-chronic model, mice were treated with MPTP (30 mg/kg*day) or vehicle for five days. Remaining DA in the striatum of mice was measured by HPLC 5 weeks or 5 days post-final treatment in the chronic and subchronic models, respectively. Data are expressed as % control where control mice were the corresponding genotype treated with vehicle. *, **: p < 0.05 and p < 0.01 comparing EP2-/- with WT mice, respectively.
Figure 3
Figure 3
Lack of EP2 attenuated formation of NP40-insoluble α-synuclein aggregates in both SN and striatum. SN and striatum were dissected at 5 weeks after the last treatment and fractionated into NP40-soluble and NP40-insoluble/SDS-soluble fractions, followed by assessment by Western blot. Data is expressed as NP-40 insoluble (SDS-soluble) α-synuclein aggregates as a fraction of total (NP40- and SDS-) soluble α-synuclein. *: p < 0.05 comparing WT-MPTP vs. WT-control (con) and EP2-/--MPTP vs. WT-MPTP, respectively, in the SN. **: p < 0.01 comparing EP2-/--MPTP vs. WT-MPTP in the striatum (n = 8).
Figure 4
Figure 4
Lack of EP2 reduced translocation of p47-phox but not p67-phox subunit from cytoplasm to membrane in microglia after α-synuclein oligomer treatment. WT or EP2-/- microglia were seeded in 6-well plates overnight and then treated with aged α-synuclein or vehicle for 30 min. The cells were homogenized and fractionated into cytoplasm (cyt) and membrane fractions (mem). The relative distribution of p67-phox (panel A) and p47-phox (panel B) in the two fractions was analyzed with Western blot in order to assess translocation from the cytoplasm to the membrane (expressed as ratio of relative amount in membrane fraction to membrane + cytoplasm). *: p < 0.05, comparing EP2-/- with WT microglia. **: P < 0.01, comparing α-synuclein treatment with control.
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