Investigating the Role of NADPH Oxidase in Ischemic Stroke Injury: An mRNA Knockdown Approach

Ischemic stroke occurs when a blood vessel in the brain is blocked, depriving the affected area of oxygen and nutrients. This causes extensive cell death and tissue damage. As a result of ischemic injury, AMPA receptors (AMPARs), the major excitatory receptor in the brain, undergo a critical compositional switch becoming GluA2-lacking AMPARs. GluA2 is an essential component as it provides AMPARs the ability to be impermeable to calcium ions, protecting the cells from damage. The GluA2-lacking AMPARs, which are prevalent post-ischemia are permeable to calcium and, therefore, play a major role in mediating delayed cell death. In the current study, we are testing the hypothesis that the activation of an enzyme complex, NADPH oxidase, triggers the AMPAR compositional switch to GluA2-containing to GluA2-lacking AMPARs. mRNA knockdown is a technique that utilizes shRNA sequences, which destabilize and degrade specific mRNAs. By using this method, we can decrease the activity of the NADPH oxidase complex by decreasing the expression of a crucial component of this enzyme, p67phox. To do this, we utilize viruses as a delivery system to transport shRNA sequences into neuronal cells. Three shRNA sequences are currently being tested for their ability to infect cells and how effectively they decrease the activity of NADPH oxidase and knock down the protein levels of p67phox. Future studies will test the GluA2 subunit levels in cells subjected to ischemia to determine if inactivating NADPH oxidase mitigates the loss of GluA2 from AMPARs. We predict that, because NADPH oxidase will be inactive, the AMPAR switch will not occur. Our results will determine if NADPH oxidase does indeed play a role in the AMPAR switch and thus perpetuate tissue damage during stroke