SELENON congenital myopathy is a rare pediatric muscle condition that prevents Selenoprotein-N production, which is an endoplasmic reticulum membrane reductase that regulates cellular redox homeostasis. Patients with SELENON congenital myopathy present with muscle weakness, spinal rigidity, and respiratory insufficiency, with no current non-palliative treatments. While prior lab results have measured oxidative stress in SELENON-deficient cells using fluorescence-based redox readouts, these assays are not suited for mid-throughput drug screening. The purpose of this project is to develop a reproducible flow cytometry based assay to measure oxidative stress at the single cell level to discriminate between SELENON-deficient and wild-type human and mouse cells. We hypothesize that oxidative stress levels will be elevated in the SELENON-knockout cells compared to the wild-type, and that this change will be robust enough to test small molecules.
Assay development was performed with mouse immortalized C2C12 myoblasts as well as human immortalized myoblasts using wild-type and two SELENON-knockout cell lines. Wild-type and SELENON-knockout cells were treated with fluorescent probes: CellROX reported total cellular reactive oxygen species (ROS), MitoSOX reported mitochondria expressing ROS, and SYTOX nuclear stain was used to exclude nonviable cells from analysis. Fluorescence intensity from ROS was compared between wild-type and SELENON-knockouts.
The SELENON-knockout exhibited increased redox fluorescence relative to wild-type, producing two population peaks distinguishing knockout from healthy samples. This flow cytometry assay will allow candidate drugs to be screened by how fully they reduce the SELENON-deficient redox fluorescence levels down towards the wild-type reference. Results from these experiments will advance early-stage therapeutic testing for SELENON congenital myopathy.