UC San Diego

Reactive astrogliosis, a process by which astrocytes undergo several molecular and morphological changes, is a major response to CNS injury. In severe cases of CNS insult, hyperproliferative and migrating astrocytes form a glial scar around the site of injury that can have both protective and deleterious effects on surrounding tissues. In astrocytes, cyclic-AMP signaling underlies several physiological changes during development and in response to injury.Previously, soluble adenylyl cyclase (sAC), a unique source of cyclic-AMP, was linked to transendothelial migration and proliferation of some cancers. These data suggest that sAC may play a role in the proliferative and migratory response of additional cell types, such as reactive astrocytes. Based on this understanding, we hypothesized that soluble adenylyl cyclase signaling in astrocytes promotes proliferation and migration in response to injury. Using pharmacological inhibition of sAC with a selective small molecule inhibitor KH7 which is specific to soluble at concentrations up to 100uM, we demonstrate that sAC plays a significant role in promoting the proliferative and migratory response of reactive astrocytes. Inhibition of sAC function decreased these cellular responses in vitro. Furthermore, we lay down the foundation for future genetic knockdown experiments with siRNA. A qPCR protocol to analyze change in sAC gene expression was optimized using a cDNA dilution and subsequent gel electrophoresis to confirm reaction efficiency and primer accuracy. Time- and concentration-dependent knockdown of sAC with siRNA was observed and validated our experimental system. Using qPCR, optimum siRNA concentrations and cell harvest time points were determined to achieve maximal knockdown for future loss-of-function experiments.