Papel do cluster MIR-17~92 no crescimento e regeneração de neuritos em cultura primária de neurônios corticais

Abstract

Neurodegenerative disorders (ND) are a group of conditions, including chronic neurodegenerative diseases and acute traumatic injuries, that lead to neurodegeneration and permanent neurological deficits. In ND, axons are affected early, lose their structural function and suffer axonal degeneration. Furthermore, mature neurons when injured do not regenerate their axons, which leads to permanent deficits. Therefore, it is necessary to seek new therapies to contain axonal degeneration and promote its regeneration, with the aim of restoring neuronal function. MicroRNAs (miRNAs) are promising targets to promote axonal protection and regeneration. The cluster miR-17~92, target of this study, regulates different genes involved in neurodegenerative and regenerative processes. For this reason, miR-17~92 can be considered a promising genetic modulation tool for ND. The aim of this study was to evaluate the effect of the overexpression of the miR-17~92 cluster, mediated by adeno-associated viral (AAV) vectors, on the growth and regeneration of neurites and axons in the central nervous system (CNS) using in vitro models. The experiments involving animals were carried out with the approval of the ethics committee for animal experimentation at UFMG (237/2018). First, cultures of primary cortical neurons were prepared from 18-day-old Wistar rat embryos. Cultures were transduced with AAV vectors, the control vector expressing only the mCherry reporter gene (AAV.CTRL) and the experimental vector consisting of the mCherry reporter gene and the miR-17~92 cluster (AAV.miR-17~92). After different time points, immunocytochemical analyses were performed to evaluate neurite growth, branching and regeneration, as well as axonal regeneration in neuronal cultures. The AAV.miR-17~92 and AAV.CTRL vectors were able to efficiently transduce primary cortical neurons in vitro, as assessed by mCherry fluorescence in these neurons, without induce differential cytotoxicity. Transduction with AAV.miR-17~92 increases neurite outgrowth and branching compared to AAV.CTRL. Preliminary analysis showed a trend toward increased neurite regeneration induced by AAV.miR-17~92, as well as increased axonal regeneration, but further experiments are necessary to confirm these effects. Bioinformatic analysis identified 251 target mRNAs of the miR- 17~92 cluster. A gene ontology analysis of the genes that codified the target mRNAs showed that those genes could be involved in neurite growth and regeneration. In conclusion, this study showed that the miR-17~92 cluster is able to promote neurite outgrowth and branching in primary cortical neurons, suggesting that this miRNA could be beneficial to promote regeneration in the CNS. This work may provide a better understanding of the role of the miR-17~92 cluster in regeneration after CNS damage, which could be important to the development of new treatments to DN.