NCOA7-AS IS REQUIRED FOR PREVENTION OF OXIDATIVE STRESS IN MOUSE ASTROCYTES

Background: NCOA7-AS is a member of the OXR1 family of oxidation resistance genes that play critical roles in a cell’s response to oxidative damage caused by reactive oxygen species (ROS). ROS are byproducts of aerobic metabolism that damage DNA and are associated with the induction of cancer, aging, and a number of neurodegenerative diseases. NCOA7-AS has antioxidant activity and expression of this gene in bacterial cells increases cellular resistance to oxidative stress. Methods: We employed CRISPR-Cas9 to knock out the unique exon 10a of NCOA7-ASin mouse astrocytes, an abundant cell type in the mammalian central nervous system. We were able to isolate a cell line (CV110) that was deleted for exon 10a and its upstream promoter. We employed propidium iodide and fluorescence activated cell sorting to assess ROS sensitivity in mutant and wild type cell lines. Results: Cells deficient in NCOA7-ASwere predicted to be sensitive to ROS relative to wild type cells, however, when we assayed CV110 cells for sensitivity to the ROS-producing agents hydrogen peroxide, glucose oxidase, or tert-butyl peroxide, we found that these mutant cells were consistently more resistant than wild type astrocytes. When RNA expression levels were assayed in untreated cells using microarrays and qRT-PCR, we found several ROS-associated genes, including catalase and aldehyde dehydrogenase, induced over two-fold compared to wild type cells. Conclusions: These phenotypes suggest that a deficiency in NCOA7-AS leads cells to increase expression of oxidative DNA damage responses, likely due to the higher than normal endogenous levels of ROS. Induction of NCOA7-AS by interferon beta suggests that it may allow cells to tolerate elevated levels of ROS produced during an immune response. The observation that oxidative stress response genes are induced in an NCOA7-AS deletion mutant suggest that even its low, uninduced expression is required in astrocytes to reduce levels of ROS even in the absence of an immune response.