Combined Therapy of A1AR Agonists and A2AAR Antagonists in Neuroinflammation

Abstract

Alzheimer's, Parkinson's, and multiple sclerosis are neurodegenerative diseases related by neuronal degeneration and death in specific areas of the central nervous system. These pathologies are associated with neuroinflammation, which is involved in disease progression, and halting this process represents a potential therapeutic strategy. Evidence suggests that microglia function is regulated by A₁ and A_2A adenosine receptors (AR), which are considered as neuroprotective and neurodegenerative receptors, respectively. The manuscript's aim is to elucidate the role of these receptors in neuroinflammation modulation through potent and selective A₁AR agonists (N⁶-cyclopentyl-2'- or 3'-deoxyadenosine substituted or unsubstituted in 2 position) and A_2AAR antagonists (9-ethyl-adenine substituted in 8 and/or in 2 position), synthesized in house, using N13 microglial cells. In addition, the combined therapy of A₁AR agonists and A_2AAR antagonists to modulate neuroinflammation was evaluated. Results showed that A₁AR agonists were able, to varying degrees, to prevent the inflammatory effect induced by cytokine cocktail (tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and interferon (IFN)-γ), while A_2AAR antagonists showed a good ability to counteract neuroinflammation. Moreover, the effect achieved by combining the two most effective compounds (1 and 6) in doses previously found to be non-effective was greater than the treatment effect of each of the two compounds used separately at maximal dose.

Keywords: A1AR agonist; A2AAR antagonist; combination therapy; cytokine; neuroinflammation; neuroprotection.

Figure 1

Double immunofluorescence staining of N13 cell cultures. Cells were treated with A₁ adenosine receptor (AR) polyclonal antibody Alexa Fluor^® 488-conjugated (green) and adenosine A_2A receptor antibody Alexa Fluor^® 594-conjugated (red). (A) Negative staining performed in Chinese Hamster Ovary (CHO) wild-type (WT) cells not expressing the adenosine receptors; (B) staining of A₁- and A_2AAR with Alexa Fluor antibodies (10× magnification); (C) staining of A₁- and A_2AAR with Alexa Fluor antibodies (60× magnification).

Figure 2

Effects of CCPA and compounds 1–3 on N13 cells. Percentage of cell viability after treatment with different ligand concentrations for 15 or 30 min of incubation. Results represent the average of 3–5 independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 (one-way ANOVA followed by Dunnett’s multiple comparison test of treated cells against control).

Figure 3

Effects of ZM241385 and compounds 4–6 on N13 cells. Percentage of cell viability after treatment with different ligand concentrations for 15 or 30 min of incubation. Results represent the average of 3–5 independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 (one-way ANOVA followed by Dunnett’s multiple comparison test of treated cells against control).

Figure 4

Protective effects of CCPA and compounds 1–3 against cytokine (CK) aggression on N13 cells. Percentage of cell viability after treatment with the CK cocktail for 48 h. Results represent the average of 3–5 independent experiments. ** p < 0.01, *** p < 0.001 (one-way ANOVA followed by Dunnett’s multiple comparison test of treated cells against CK).

Figure 5

Restoring effects of ZM241385 and compounds 4–6 against CK aggression on N13 cells. Results represent the average of 3–5 independent experiments. ** p < 0.01, *** p < 0.001 (one-way ANOVA followed by Dunnett’s multiple comparison test of treated cells against CK).

Figure 6

Protective (compound 1) or restoring (compound 6) effects after or before incubation of cells with CK. Bar graphs presents the percentage cell viability, and results are presented as the average of 3–5 independent experiments. *** p < 0.001 (one-way ANOVA followed by Dunnett’s multiple comparison test of treated cells against CK).

Figure 7

Co-incubation of N13 cells with the A₁AR antagonist DPCPX and the selective A_2AAR agonist CGS21680 in combination with compounds 1 and 6 or CCPA and ZM241385, respectively. * p < 0.05, *** p < 0.001 (one-way ANOVA followed by Dunnett’s multiple comparison test of treated cells against CK).

Figure 8

Co-incubation of N13 cells with compounds 1 and 6 or with reference compounds CCPA and ZM241385. Results represent the average of 3–5 independent experiments. ** p < 0.01, *** p < 0.001 (one-way ANOVA followed by Dunnett’s multiple comparison test of treated cells against control).

Figure 9

Co-incubation of N13 cells with compounds 1 and 6 or with reference compounds CCPA and ZM241385. Results represent the average of 3–5 independent experiments. *** p < 0.001 (one-way ANOVA followed by Dunnett’s multiple comparison test of treated cells against control).

Figure 10

Example of an image obtained through Hoechst assay. In the picture, arrows point to apoptotic cells (40× magnification).

Figure 11

Cell area and circularity after the pre- and post-treatment with compound 1 and compound 6, respectively, in presence of the CK cocktail. Results represent the average of three independent experiments. ** p < 0.01, *** p < 0.001 (one-way ANOVA followed by Dunnett’s multiple comparison test of treated cells against CK).

Figure 12

(1) Annexin V/PI double staining assay of N13 cells treated for 48 h with CK cocktail (A,B) and CK cocktail plus compound 1 (C,D) in comparison with untreated control (E,F) and solvent control (G,H). Each group of pictures (10× magnification) is composed of contrast phase and florescence imagines, which underline the anti-apoptotic effect of compound 1. (2) Percentage of apoptotic cells after treatment with CK cocktail or CK plus compound 1 or 6, in comparison with the untreated control and solvent control. Each bar represents mean ± S.E. *** p < 0.001 (one-way ANOVA followed by Dunnett’s multiple comparison test of treated cells or controls against CK).