Abstract W P221: Argonaute 2 Mediates Sildenafil-Promoted Axonal Outgrowth

Molecular mechanisms are not fully known for the inhibitory effect of chondroitin sulfate proteoglycans (CSPGs) on axonal outgrowth. The present study investigated the effect of CSPGs on miRNA expression and the role of Argonaute 2 (Ago2) in mediating Sildenafil-induced axonal outgrowth under CSPG conditions. Primary rat embryonic cortical neurons were cultured in a microfluidic chamber which separates somata and axons. Selective application of CSPGs within the axonal compartment markedly inhibited axonal outgrowth (567±15 μm vs 688 ± 21 μm in control, n=5, p<0.05). Axonal application of Sildenafil, a PDE5 inhibitor, completely reversed the inhibitory effect of CSPG on axonal outgrowth (671±17 μm, n=5). Analysis of miRNA PCR array showed that axonal application of CSPGs robustly changed miRNA profiles not only in axons but also in somata, indicating that axonal signals regulate soma miRNAs. Sildenafil inversely altered miRNA up- and down-regulated by CSPGs, including miR-29c, miR-9a, miR-20b, miR-298 and miR-34a. Attenuation of Ago2, a regulator of miRNA activity, in neurons by siRNA abolished Sildenafil-increased axonal outgrowth under CSPG conditions, suggesting that miRNAs are required for Sildenafil-enhanced axonal outgrowth. One of putative targets of miR-29c is integrin β1. Western blot analysis showed that CSPGs and Sildenafil reduced and increased, respectively, axonal integrin β1 and its downstream protein, focal adhesion kinase, suggesting that miR-29c regulates these protein levels. To examine cause-effect of integrin β1, we applied a neutralizing antibody against integrin β1 into the axonal compartment and found that the antibody attenuated the Sildenafil-reversed inhibitor effect of CSPGs on axonal outgrowth (523±34 μm vs 671±17 μm in Sildenafil, n=5, p<0.01). In conclusion, the present study indicates that axonal signals induced by CSPGs and Sildenafil change miRNA profiles in the entire neuron and that Sildenafil abolishes the CSPG inhibitory effect on axons via Ago2.