Expression of functionally distinct ecto-5′-nucleotidase/CD73 glycovariants in reactive astrocytes in experimental autoimmune encephalomyelitis and neuroinflammatory conditions in vitro

Abstract

Ecto-5′-nucleotidase/CD73 (eN/CD73) is a membrane-bound enzyme involved in extracellular production of adenosine and a cell adhesion molecule involved in cell–cell interactions. In neuroinflammatory conditions such as experimental autoimmune encephalomyelitis (EAE), reactive astrocytes occupying active demyelination areas significantly upregulate eN/CD73 and express additional eN/CD73 variants. The present study investigated whether the different eN/CD73 variants represent distinct glycoforms and the functional consequences of their expression in neuroinflammatory states. The study was performed in animals at different stages of EAE and in primary astrocyte cultures treated with a range of inflammatory cytokines. Upregulation at the mRNA, protein, and functional levels, as well as the appearance of multiple eN/CD73 glycovariants were detected in the inflamed spinal cord tissue. At the peak of the disease, eN/CD73 exhibited higher AMP turnover and lower enzyme-substrate affinity than the control group, which was attributed to altered glycosylation under neuroinflammatory conditions. A subsequent in vitro study showed that primary astrocytes upregulated eN/CD73 and expressed the multiple glycovariants upon stimulation with TNFα, IL-1β, IL-6, and ATP, with the effect occurring at least in part via induction of JAK/STAT3 signaling. Experimental removal of glycan moieties from membrane glycoproteins by PNGaseF decreased eN/CD73 activity but had no effect on the enzyme's involvement in astrocyte migration. Our results suggest that neuroinflammatory states are associated with the appearance of functionally distinct eN/CD73 glycovariants, which may play a role in the development of the reactive astrocyte phenotype.