Heparan Sulfated Glypican-4 is Released from Astrocytes Predominantly by Proteolytic Shedding

Abstract

Astrocytes provide neurons with diffusible factors that promote synapse formation and maturation. In particular, glypican-4/GPC4 released from astrocytes promotes the maturation of excitatory synapses. Unlike other secreted factors, GPC4 contains the C-terminal GPI-anchorage signal. However, the mechanism by which membrane-tethered GPC4 is released from astrocytes is unknown. Using primary astrocyte cultures and a quantitative luciferase-based release assay, we show that GPC4 is expressed on the astrocyte surface exclusively via a GPI-anchorage. Soluble GPC4 is robustly released from the astrocytes predominantly by proteolytic shedding and, to a lesser extent, by GPI-anchor cleavage, but not by vesicular release. Pharmacological, overexpression, and loss of function screens showed that ADAM9 in part mediates the release of GPC4 from astrocytes. The released GPC4 contains the heparan sulfate side chain, suggesting that these release mechanisms provide the active form that promotes synapse maturation and function. Overall, our studies identified the release mechanisms and the major releasing enzyme of GPC4 in astrocytes and will provide insights into understanding how astrocytes regulate synapse formation and maturation. Significance StatementAstrocyte-derived diffusible factors regulate synapse development and function. However, the regulatory mechanism underlying the release of astrocyte-derived factors is poorly understood. Noting that, unlike many other secreted factors, glypican-4/GPC4 is GPI-anchored, we characterized the release mechanism of GPI-anchored GPC4 from astrocytes and identified the releasing enzyme. Heparan sulfated GPC4 is robustly released from the astrocytes predominantly by proteolytic shedding. In particular, ADAM9 in part mediates the release of GPC4 from astrocytes. Our study provides an enzymatic mechanism for releasing GPC4 from astrocytes and will provide a novel opportunity to understand the regulatory mechanism of neuron-glia communication for synaptogenesis.