Dual CD73/A2AR blockade modulates the neurotoxic astrocyte phenotype without disrupting core inflammatory signaling

Introduction Excessive activation of the adenosine A 2A receptor (A 2A R) contributes to chronic neuroinflammation, in part through spatial coupling with the adenosine-generating enzyme CD73, which enables localized adenosine signaling. Coordinated regulation of Nt5e and Adora2a across neuropathological conditions supports dual targeting of the CD73/A 2A R axis to constrain maladaptive inflammatory signaling. Methods Primary rat astrocytes were exposed to TNF-α, IL-1α, and C1q (TIC) to induce a neurotoxic reactive astrocyte (nRA) substate. Concomitant pharmacological inhibition of CD73 (APCP, 100 μM) and A 2A R (istradefylline, 10 μM) was applied. Morphological, redox, inflammatory, and functional outcomes were assessed, including CD73 expression and activity, CD73/A 2A R spatial proximity, cytokine release, and astrocyte-mediated neurotoxicity. Results Dual CD73/A 2A R blockade attenuated key features of the nRA phenotype, including astrocyte hypertrophy, oxidative stress, and impaired antioxidant capacity. These effects were associated with normalization of CD73 expression and activity, reduced spatial proximity between CD73 and A 2A R, suppression of IL-1β release and complement- and immune cell-recruiting effector programs (C3, VCAM1), and modulation of redox-sensitive pathways ( Nos2 /NO, NRF2). Notably, IL-6- and TNFα-driven core inflammatory signaling remained preserved. Functionally, dual blockade shifted astrocytes toward a less neurotoxic phenotype, reducing their impact on neuronal Ca 2+ homeostasis and improving neuronal viability. Discussion These findings demonstrate that dual CD73/A 2A R blockade selectively reconfigures astrocyte inflammatory networks under the tested conditions, without broadly suppressing inflammatory or homeostatic functions at the examined time point. This supports the CD73/A 2A R axis as a promising therapeutic target for limiting chronic astrocyte-driven neurotoxicity.