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. 2024 May 9:18:1382465.
doi: 10.3389/fncel.2024.1382465. eCollection 2024.

Reactive spinal glia convert 2-AG to prostaglandins to drive aberrant astroglial calcium signaling

Klaudia Dócs  1 , Anita Balázs  2 , Ildikó Papp  1 , Peter Szücs  1   3 , Zoltán Hegyi  1
Affiliations

Reactive spinal glia convert 2-AG to prostaglandins to drive aberrant astroglial calcium signaling

Klaudia Dócs et al. Front Cell Neurosci. .

Abstract

The endogenous cannabinoid 2-arachidonoylglycerol (2-AG) influences neurotransmission in the central nervous system mainly by activating type 1 cannabinoid receptor (CB1). Following its release, 2-AG is broken down by hydrolases to yield arachidonic acid, which may subsequently be metabolized by cyclooxygenase-2 (COX-2). COX-2 converts arachidonic acid and also 2-AG into prostanoids, well-known inflammatory and pro-nociceptive mediators. Here, using immunohistochemical and biochemical methods and pharmacological manipulations, we found that reactive spinal astrocytes and microglia increase the expression of COX-2 and the production of prostaglandin E2 when exposed to 2-AG. Both 2-AG and PGE2 evoke calcium transients in spinal astrocytes, but PGE2 showed 30% more efficacy and 55 times more potency than 2-AG. Unstimulated spinal dorsal horn astrocytes responded to 2-AG with calcium transients mainly through the activation of CB1. 2-AG induced exaggerated calcium transients in reactive astrocytes, but this increase in the frequency and area under the curve of calcium signals was only partially dependent on CB1. Instead, aberrant calcium transients were almost completely abolished by COX-2 inhibition. Our results suggest that both reactive spinal astrocytes and microglia perform an endocannabinoid-prostanoid switch to produce PGE2 at the expense of 2-AG. PGE2 in turn is responsible for the induction of aberrant astroglial calcium signals which, together with PGE2 production may play role in the development and maintenance of spinal neuroinflammation-associated disturbances such as central sensitization.

Keywords: 2-AG; CB1; COX-2; astrocyte; calcium signaling; cannabinoid; prostaglandin; reactive astrocyte.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Immunohistochemical localization and its histogram profile of DGL-α, CB1 and COX-2 in control and CFA-treated animals. Following a CFA treatment, DGL-α immunostaining showed an unaltered intensity in the SDH of mice (A–C). CFA decreased CB1 (D–F) but increased COX-2 (G–I) expression in the SDH (A–F). n = 9 spinal dorsal horns. Box plot properties: center: median; edges: 25th–75th percentiles; whiskers: extrema; outliers plotted individually. Statistical comparison: Kolmogorov–Smirnov test. Scale bar: 100 μm.
Figure 2
Figure 2
Astrocytes and microglial cells in the spinal dorsal horn increase the expression of COX-2 following a CFA treatment. Micrographs of single 1-μm-thick confocal optical sections showing the co-localization between astrocytes (GFAP-positive profiles, red) or microglial cells (Iba1-positive profiles, blue) and COX-2 immunostained spots (green) in the SDH of naive (A) and CFA-treated mice (G). Z-projection of 15 1-μm-thick confocal optical sections to show the density of COX-2 immunoreactive puncta, GFAP and Iba1-immunostained profiles in naïve (B,C,E, respectively) and CFA-treated animals (H,I,K, respectively). Note that the number of pixels corresponding to COX-2 immunostaining (M) and their co-localization with GFAP or Iba1 positive profiles are significantly increased in the SDH of CFA-treated mice (D vs. J,F vs. L,N). n = 9 spinal dorsal horns. Box plot properties: center: median; edges: 25th–75th percentiles; whiskers: extrema; outliers plotted individually. Statistical comparison: two-tailed non-parametric Mann–Whitney U test. Scale bar: 5 μm.
Figure 3
Figure 3
The expression of COX-2 is increased in spinal astrocyte-microglia co-culture (A,B). A representative image of a capillary Western blot (WES) analysis of COX-2 expression in control and LPS-treated astrocyte-microglia co-cultures (A,B). n = 9 cultures. Box plot properties: center: median; edges: 25th–75th percentiles; whiskers: extrema; outliers plotted individually. Statistical comparison: two-way Anova with Bonferroni correction.
Figure 4
Figure 4
Micrographs of immunostained control and LPS-treated spinal astrocytes-microglia co-cultures illustrating that reactive spinal astrocytes and microglia increase the expression of COX-2 following an LPS stimulus. Both astrocytes (GFAP, red, A) and microglia (Iba1, red, C) increase the expression of COX-2 (green, A,C) following a treatment with LPS (B,D). n = 18 cells. Box plot properties: center: median; edges: 25th–75th percentiles; whiskers: extrema; outliers plotted individually. Statistical comparison: two-tailed non-parametric Mann–Whitney U test. Scale bar: 5 μm.
Figure 5
Figure 5
PGE2 production is increased in spinal astrocyte-microglia co-cultures in the presence of 2-AG in both control and LPS-treated cultures. Application of 2-AG does not alter PGE2 production in the presence of the COX-2 inhibitor nimesulide. n = 9 cultures. Box plot properties: center: median; edges: 25th–75th percentiles; whiskers: extrema; outliers plotted individually. Statistical comparison: two-way Anova with Bonferroni correction. n.s: not significant. ***p < 0.001.
Figure 6
Figure 6
Both 2-AG and PGE2 induce calcium transients in spinal cultured astrocytes. Equimolar concentration of PGE2 is more potent and efficacious than 2-AG at inducing calcium transients (A,B). n = 18 cells.
Figure 7
Figure 7
2-AG induced exaggerated calcium signals in reactive astrocytes are dependent on COX-2 activity (A–J). Calcium transients following the application of 2-AG are fully prevented by the inhibition of CB1, but not affected by the inhibition of COX-2 in control astrocytes (A,C,E,G,I,J). Inhibition of CB1 only partially, but inhibition of COX-2 almost fully prevents the induction of calcium signals in LPS-treated astrocytes (B,D,F,H,I,J). n = 18 cells. Box plot properties: center: median; edges: 25th–75th percentiles; whiskers: extrema; outliers plotted individually. Statistical comparison: two-way Anova with Bonferroni correction. n.s, not significant. *p < 0.05; **p < 0.005; ***p < 0.001.
Figure 8
Figure 8
Clustering of calcium transients of control and LPS-treated astrocytes following the inhibition of CB1 or COX-2. Clusters and confidence ellipses were identified using K-means cluster analysis. The left bottom cluster indicates that the effects of 2-AG are abolished by the CB1 antagonist AM251 in resting cells. The top right cluster shows that, in LPS-treated cells, effects of 2-AG are abolished by the COX-2 inhibitor nimesulide, but not AM251.
Figure 9
Figure 9
2-AG-induced calcium transients in normal astrocytes are mediated by CB1. Reactive glia convert 2-AG and its hydrolytic metabolite arachidonic acid into PGE2 which evokes aberrant calcium signals in reactive astrocytes.
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Grants and funding

The author(s) declare that financial support was received for the research, authorship, and/or publication of this article. This work was supported by the Hungarian Brain Research Program (2017–1.2.1-NKP-2017-00002, PS) and a Bridging Fund from the Faculty of Medicine, University of Debrecen (ZH).