Abstract TP268: Regulation of oxidative stress mediated cell cycle progression, apoptosis, and antioxidant defense mechanisms in neuronal cells

Background: Cerebral ischemia/reperfusion is strongly linked to excessive reactive oxygen species (ROS) that worsens brain damage. Molecular mechanisms of ROS-induced neuronal cell death and brain damage needs further understanding. Dual oxidase 1 (Duox1) generates H 2 O 2 /ROS. We have identified that focal cerebral ischemia and reperfusion in a rat model induces the expression of Duox1 in endothelial and neuronal cells in the ischemic brain. Here, we examined the direct effects of Duox1 on neuronal cells toxicity. Methods: We established a neuronal cell culture model over-expressing Duox1. The effects of Duox1 on neuronal ROS, cell viability, cell cycle progression, and mitochondrial function were examined using DCF-DA, live/dead cell assay, flow cytometry, and Mito-Tracker-FM. Molecular targets of Duox1 were identified by whole transcriptome analysis using RNA-sequencing. Cell viability, inflammatory, and Oxidative Stress (OS) response genes were identified using pathway specific PCR arrays. Highlighted genes were validated by real-time PCR and immunofluorescence staining. Results: Neuronal cells over-expressing Duox1 exhibited morphological changes including cell size and clumping. Duox1 over-expression lead to increased ROS, decreased cell viability, evoked significant G 2 /M arrest, and decreased mitochondrial function. Mechanistically, Duox1 over-expression increased the expression of pro-apoptotic genes including TNF-related apoptosis-inducing ligand TNFSF10, and OS genes including heat shock protein HSPA1A. Importantly, Duox1 increase was associated with decrease in the expression of genes involved in antioxidant system, including prostaglandin reductase 1 (PTGR1), glutathione peroxidase 4 (GPX4), and solute carrier family 7 member (SLC7A11), a precursor for glutathione (GSH). Neuroprotective neuropeptide-Y (NPY) and immunoglobulin superfamily member 1 (IGSF1) were also decreased in Duox1 over-expressing neuronal cells. Conclusions: Increased Duox1 contributes to decreased neuronal cell viability, mitochondrial dysfunction, dysregulated cell cycle progression, and reduced antioxidant defense mechanisms. Duox1-mediated ROS may ultimately exacerbate brain damage. Duox1 may be a novel therapeutic target for stroke, which warrants further investigation.