Astrocytes play several functions in the brain: they give structural and metabolic support to neurons and modulate synaptic transmission by the release of gliotransmitters including ATP. Here we asked whether astrocytes also have a "dark side", specifically contributing to HSV-1 infection of neurons. HSV-1 (1 MOI) was applied for 1 h (adsorption time) to cocultures of mouse cortical neurons and astrocytes to allow virus binding and entry into cells. After 24 h, infection was assessed by immunocytochemistry and Western blot analysis of viral proteins. We observed that astrocytes were more susceptible than neurons to HSV-1 infection, in agreement with our finding that astrocytes exhibit 4-fold greater expression of Heparan Sulfate Proteoglycans (binding sites for viral glicoproteins C and B) than neurons (p<0.05). The probability to find HSV-1-infected (HSV-1+) neurons was significantly higher in proximity (<100 ?m) of virus-infected astrocytes. At such distance about 78±6% of neurons were HSV-1+ vs. only 11±6% of neurons far from HSV-1+ astrocytes (p<0.05). When co-cultures were challenged with HSV-1 after metabolic inhibition of astrocytes by fluorocitrate (FC, 200 ?M), neither astrocytes nor neurons were HSV-1+, thus suggesting that the presence of functional astrocytes is necessary for neuronal virus infection. HSV-1 binding to astrocytic plasma membrane triggered IP3-dependent intracellular Ca2+ transients followed by ATP release, whose concentration in the culture medium, assessed by HPLC, was up to 4-fold higher than that of control cultures (44.1±11.3 vs. 10.3±1.2 nM; p<0.05). ATP released from astrocytes diffused locally and activated purinergic P2 receptors (P2R) on neurons and astrocytes themselves that sustained and amplified Ca2+ transients. Application of the P2R blocker suramin (200 ?M) during the adsorption time prevented HSV-1 infection, thus suggesting that ATP released from astrocytes and the consequent P2R-dependent Ca2+ transients are necessary for virus infection of both astrocytes and neurons. Accordingly, if exogenous ATP (100 ?M) was applied to FC-treated co-cultures during the adsorption time more than 70% of neurons was HSV-1+ after 24 h. ATP-induced P2R-mediated Ca2+ transients caused activation of the Glicogen Synthase Kinase (GSK)-3 by phosphorylation at Y216 in both astrocytes and neurons, and GSK-3 inhibition by SB216763 (10 ?M) during the adsorption time completely prevented HSV-1 infection. In conclusion, we found that ATP released from astrocytes after HSV-1 binding diffuses locally and causes P2R-dependent activation of GSK-3 in both neurons and astrocytes, likely involved in HSV-1 infection.
ATP released from astrocytes mediates Herpes Simplex Virus type 1 (HSV-1) infection in neurons
G DE CHIARA;
2018
Abstract
Astrocytes play several functions in the brain: they give structural and metabolic support to neurons and modulate synaptic transmission by the release of gliotransmitters including ATP. Here we asked whether astrocytes also have a "dark side", specifically contributing to HSV-1 infection of neurons. HSV-1 (1 MOI) was applied for 1 h (adsorption time) to cocultures of mouse cortical neurons and astrocytes to allow virus binding and entry into cells. After 24 h, infection was assessed by immunocytochemistry and Western blot analysis of viral proteins. We observed that astrocytes were more susceptible than neurons to HSV-1 infection, in agreement with our finding that astrocytes exhibit 4-fold greater expression of Heparan Sulfate Proteoglycans (binding sites for viral glicoproteins C and B) than neurons (p<0.05). The probability to find HSV-1-infected (HSV-1+) neurons was significantly higher in proximity (<100 ?m) of virus-infected astrocytes. At such distance about 78±6% of neurons were HSV-1+ vs. only 11±6% of neurons far from HSV-1+ astrocytes (p<0.05). When co-cultures were challenged with HSV-1 after metabolic inhibition of astrocytes by fluorocitrate (FC, 200 ?M), neither astrocytes nor neurons were HSV-1+, thus suggesting that the presence of functional astrocytes is necessary for neuronal virus infection. HSV-1 binding to astrocytic plasma membrane triggered IP3-dependent intracellular Ca2+ transients followed by ATP release, whose concentration in the culture medium, assessed by HPLC, was up to 4-fold higher than that of control cultures (44.1±11.3 vs. 10.3±1.2 nM; p<0.05). ATP released from astrocytes diffused locally and activated purinergic P2 receptors (P2R) on neurons and astrocytes themselves that sustained and amplified Ca2+ transients. Application of the P2R blocker suramin (200 ?M) during the adsorption time prevented HSV-1 infection, thus suggesting that ATP released from astrocytes and the consequent P2R-dependent Ca2+ transients are necessary for virus infection of both astrocytes and neurons. Accordingly, if exogenous ATP (100 ?M) was applied to FC-treated co-cultures during the adsorption time more than 70% of neurons was HSV-1+ after 24 h. ATP-induced P2R-mediated Ca2+ transients caused activation of the Glicogen Synthase Kinase (GSK)-3 by phosphorylation at Y216 in both astrocytes and neurons, and GSK-3 inhibition by SB216763 (10 ?M) during the adsorption time completely prevented HSV-1 infection. In conclusion, we found that ATP released from astrocytes after HSV-1 binding diffuses locally and causes P2R-dependent activation of GSK-3 in both neurons and astrocytes, likely involved in HSV-1 infection.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
