A CRISPR knockout EGR1 model in the neuronal cell line SH-SY5Y

The transcription factor EGR1 is an immediate-early gene often used as an indirect marker to measure neuronal activity. It has been hypothesized to have a role in many biological processes in neuronal cells, such as neuronal plasticity and development of learning and memory. The expression of EGR1 is often altered in neurodegenerative diseases and psychiatric disorders including Alzheimer’s disease and schizophrenia. However, the role of EGR1 and the transcriptional programs under its control in neuronal development are not fully understood. In this regard, we established a neuronal cell model with a defective EGR1 gene. We generated a knockout cell (KO) line for EGR1 gene using one of the most popular neuronal cell line, the SH-SY5Y, that can be differentiated to a more mature neuron-like phenotype using retinoic acid (RA). EGR1-KO cells show clear morphological differences compared to WT cells, particularly evident for neurite arborization. In addition, EGR1-KO cells show higher proliferation and migration rate compared to WT cells. Under different stress conditions, such as serum withdrawal and H2O2-induced oxidative stress, KO cells have higher survival rate than WT cells. A preliminary molecular analysis demonstrated that KO cells react to stress-induced conditions likely involving autophagy process in contrast to WT cells. Surprisingly, EGR1-KO cells undergo cell death after RA treatment in contrast to WT cells that undergo neuronal differentiation. Molecular analyses of the EGR1-KO model show a dysregulation of many differentiation markers, including those related to neurite maturation and synaptogenesis. In fact, the western blotting analysis shows a higher level of GAP43 and SYT1 proteins in KO cells compared to WT. Therefore, EGR1 knockout cell model obtained by CRISPR/Cas9 method may represent an interesting model for studying the molecular mechanisms regulated by EGR1 for the proper development, differentiation, and survival of neurons.