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BACKGROUND: The role of RE1-Silencing Transcription Factor (REST) in human neurogenesis remains unexplored. This study aims to investigate the expression patterns and possible targets of REST during the early stages of human neurogenesis. Methods: Human neural stem cells and neurons from human induced pluripotent stem cells (hiPSCs) were generated through established protocols. hiPSCs and derived neurons were characterized by immunocytochemistry (SOX2, OCT4, TUJ-1) and qPCR for pluripotency and neuronal marker expression. REST expression and the effect of REST inhibition using X5050 at different stages of development were evaluated using immunostaining. Further, proteomic analysis was performed to identify the key molecules and signalling pathways impacted by the inhibition of REST during neurogenesis. Western blotting and quantitative PCR were used to validate the key molecule targeted by REST inhibition. Results: Immunocytochemistry and qPCR examinations have affirmed the promising expression of pluripotency markers (SOX2, OCT4, Nanog) in hiPSCs and the neuronal marker TUJ-1 in differentiated neurons. REST expression is seen in hiPSCs, immature neurons, and mature neurons derived from hiPSCs. In immature neurons, REST proteins are seen in the soma and axons, while in mature neurons, REST is seen in the soma and is absent in the axons. The inhibition of REST in hiPSCs, NSCs and neuronal precursor cells with X5050 significantly reduced REST levels. Reduced REST levels in NSCs led to significant downregulation of essential proteins involved in neurogenesis, including SOX2, a key regulator of neural stem cell proliferation. REST inhibition by X5050 disrupted pivotal neurogenic signaling axis, including MAPK and WNT pathways, and reduced the mRNA expression of NESTIN, β-catenin (CTNNB1), and MAPK3, indicating perturbation of neural stem cell identity and key regulatory mechanisms. Conclusion: REST is a crucial regulator of human neurogenesis. REST is essential to drive neurogenesis as it controls the SOX2 levels during this stage. The role of REST in regulating neurogenic pathways offers novel perspectives on its potential as a target for therapy of neurodevelopmental diseases. Graphical abstract.
Nassar et al. (Tue,) studied this question.