MiR-124 and small molecules synergistically regulate the direct generation of neuronal cells from rat cortical reactive astrocytes

Abstract

Background：Irreversible neuron loss caused by central nervous system injuries usually lead to persistent neurological dysfunction. Reactive astrocytes, because of their high proliferative capacity, proximity to neuronal lineage, and significant involvement in glial scarring, are ideal starting cells for neuronal regeneration. Having previously identified several small molecules as important regulators of astrocyte-to-neuron reprogramming, our aim in this study was to explore whether other small molecules and miR-124, a key neural differentiation mediator, could co-regulate reactive astrocyte-to-neuron conversion.Methods: MiR-124, ruxolitinib, SB203580, and forskolin were used to induce postnatal rat cortex reactive astrocytes, and the neuronal phenotype of the induced cells was characterised. To understand the genetic changes, RNA-sequencing analyses were performed on reactive astrocytes, induced neurons, and rat neurons, and the mechanisms underlying the regulatory role of miR-124 during the neuronal conversion was explored.Results：MiR-124, ruxolitinib, SB203580, and forskolin could co-convert rat cortical reactive astrocytes into neurons. The induced cells had reduced astroglial properties, displayed typical neuronal morphologies, and expressed neuronal markers, reflecting 25.9% of cholinergic neurons. Gene analysis revealed that induced neuron gene expression patterns were more similar to that of primary neurons than of initial reactive astrocytes. On the molecular level, miR-124-driven neuronal differentiation of reactive astrocytes was via targeting of the SOX9-NFIA-HES1 axis to inhibit HES1 expression.Conclusions：Providing a novel approach for inducing endogenous rat cortical reactive astrocytes into neurons by co-regulation involving miR-124 and three small molecules, our research has potential implications for inhibiting glial scar formation and promoting neuronal regeneration after central nervous system injury or disease.