Adenosine Receptors as Neuroinflammation Modulators: Role of A1 Agonists and A2A Antagonists

Abstract

The pathological condition of neuroinflammation is caused by the activation of the neuroimmune cells astrocytes and microglia. The autacoid adenosine seems to be an important neuromodulator in this condition. Its main receptors involved in the neuroinflammation modulation are A₁AR and A_2AAR. Evidence suggests that A₁AR activation produces a neuroprotective effect and A_2AARs block prevents neuroinflammation. The aim of this work is to elucidate the effects of these receptors in neuroinflammation using the partial agonist 2'-dCCPA (2-chloro-N⁶-cyclopentyl-2'-deoxyadenosine) (C1 K_iA₁AR = 550 nM, K_iA_2AAR = 24,800 nM, and K_iA₃AR = 5560 nM, α = 0.70, EC₅₀A₁AR = 832 nM) and the newly synthesized in house compound 8-chloro-9-ethyl-2-phenethoxyadenine (C2 K_iA_2AAR = 0.75 nM; K_iA₁AR = 17 nM and K_iA₃AR = 227 nM, IC₅₀A_2AAR = 251 nM unpublished results). The experiments were performed in in vitro and in in vivo models of neuroinflammation. Results showed that C1 was able to prevent the inflammatory effect induced by cytokine cocktail (TNF-α, IL-1β, and IFN-γ) while C2 possess both anti-inflammatory and antioxidant properties, counteracting both neuroinflammation in mixed glial cells and in an animal model of neuroinflammation. In conclusion, C2 is a potential candidate for neuroinflammation therapy.

Keywords: A1AR agonist; A2AAR antagonist; LPS; cytokine; glia; neuroinflammation.

Figure 1

Compound 1 and 2 structures.

Scheme 1

Drug application protocol.

Figure 2

Double immunofluorescence staining in mixed glial cell cultures. (A) Cells were treated with Adenosine A₁ Receptor Polyclonal Antibody ALEXA FLUOR^® 488 Conjugated (green). (B) Higher magnification displaying A₁AR expression on the plasma membrane. (C) Adenosine A_2A Receptor antibody ALEXA FLUOR^® 594 Conjugated (red). (D) Higher magnification displaying A_2AAR expression on the plasma membrane.

Figure 3

Effects of 2-chloro-N⁶-cyclopentyladenosine (CCPA) and C1 on mixed glial cell culture viability. Percentage of cell viability after treatment with different ligand concentrations (2–30 μM) for 15, 30, or 60 min of incubation. Results represent the average of 3–5 independent experiments. * p < 0.05 of treated cells against control.

Figure 4

Effects of ZM241385 and C2 on mixed glial cell culture viability. Percentage of cell viability after treatment with the ligand after 15, 30, or and 60 min of incubation. Results represent the average of 3–5 independent experiments. * p < 0.05, ** p < 0.01 of treated cells against control.

Figure 5

Protective effects of CCPA and C1 against CK aggression on mixed glial cell culture. Percentage of cell viability after treatment with the CK cocktail for 48 h. Results represent the average of 3–5 independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 of treated cells against CK.

Figure 6

Restoring effects of ZM241385 and C2 against CK aggression on mixed glial cell culture. Results represent the average of 3–5 independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 of treated cells against CK.

Figure 7

Protective (A) and restoring (B) effects reference compounds and compounds 1 and 2 against CK aggression on mixed glial cell culture and neurons. Percentage of cell viability after treatments. Control, CK, and reference compound results were obtained in glial cell culture. Results represent the average of 3–5 independent experiments. ** p < 0.01, *** p < 0.001 of treated cells against CK.

Figure 8

Protective (A) and restoring (B) effects of C1 and C2, respectively, in presence or absence of A₁AR (DPCPX) or A_2AAR (SCH58261) antagonists in presence or in absence of CK. Percentage of cell viability after treatments. Results represent the average of 3–5 independent experiments. * p < 0.05, ** p < 0.01 of treated cells against control.

Figure 9

Antioxidant effects of ZM241385 and C2 on mixed glial cell culture. Ratio of NO¬¬¬¬₂¯ production after treatments. Results represent the average of 3–5 independent experiments. *** p < 0.001 of treated cells against control.

Figure 10

Antioxidant effects of ZM241385 and C2 after pre-treatment with CK cocktail on mixed glial cell culture. Ratio of NO¬¬¬¬₂¯ production after treatments. Results represent the average of 3–5 independent experiments. *** p < 0.001 of treated cells against CK.

Figure 11

Example of an image obtained through Hoechst assay. In the picture is pointed the chromatin condensation.

Figure 12

Cell area and circularity after the pre and post treatment with CCPA or C1 and ZM241385 or C2 respectively in presence of the CK cocktail in mixed glial cell cultures. The results represent the average of 3–5 independent experiments. ** p < 0.01 of treated cells against CK.

Figure 13

Effects of treatment in distance and velocity performed by rats in Open Field. Saline (sal), Vehicle (veh, vehicle used for the ligand dilution).

Figure 14

Effects of treatment in permanence time in the outside area (O) and inside area (I) in Open Field. Saline (sal), Vehicle (veh, vehicle used for the ligand dilution). *** p < 0.001 of inside area against outside area. Inside areas did not demonstrate to be statistically different among them.

Figure 15

Effects of treatment in time spent in novel (N) and old (O) arms. Saline (sal), Vehicle (veh, vehicle used for the ligand dilution). ** p < 0.05, ** p < 0.01, *** p < 0.001 of novel area against old area.