Análise do papel do miR-22 na degeneração e regeneração de neurônios em modelos in vitro

Abstract

The central nervous system (CNS) of adult mammals is characterized by having intrinsic and extrinsic properties that inhibit axon regeneration. When an injury occurs, neuronal cells can have their functions permanently compromised, as observed in neurodegenerative disorders. In this sense, it is essential to search for strategies that mitigate or reverse this damage, such as promoting neuroprotection and axonal regeneration. To achieve this, it is possible to adopt techniques that modulate intrinsic neuronal capacity, a mechanism associated with gene expression and widely investigated in the development of gene therapies. A therapeutic alternative consists of regulating gene expression using microRNAs, small non-coding RNAs that act as post-transcriptional silencers, through the use of viral vectors. In this work, we investigated the role of miR-22, an important regulator of neuronal survival, in axonal degeneration and regeneration after CNS injury. For this, recombinant adeno-associated virus (rAAV) vectors expressing miR-22 and the fluorescent protein mCherry (rAAV.miR-22) and a control vector expressing only the mCherry protein (rAAV.CTRL) were used. First, cell viability and transduction efficiency assays of viral vectors were carried out in primary culture of cortical neurons. Next, we verified whether the rAVV.miR-22 would be capable of modulating neurite arborization, growth and regeneration. We also evaluated, using microfluidic chambers, whether the rAVV.miR-22 would be able to promote axonal regeneration and protecting neurons from degeneration. Finally, bioinformatics analyzes to identify the predicted and validated targets of miR-22 and functional analyzes were performed. From the results obtained, it was observed that the rAAV vectors presented high neuronal transduction efficiency and there was no differential cytotoxicity between the rAAV.CTRL and the rAVV.miR-22. For the analyzes of neurite outgrowth and arborization, no significant differences were observed between rAAV.CTRL and rAAV.miR-22. Neurite regeneration assays, in contrast, revealed that rAAV.miR-22 increases neurite regeneration. When evaluating axonal regeneration, it was observed that overexpression of miR-22 also promotes axonal regeneration. Axonal degeneration analyses, however, showed that rAAV.miR-22 had no significant effect on this process. Finally, bioinformatics analyzes revealed that miR-22 has 34 validated targets and 330 predicted targets, which, according to functional analyses, are associated with important processes in neurodegeneration. Therefore, this study allows a better understanding of the role of miR-22 and points to it as a potential therapeutic target for promoting axonal regeneration and CNS repair.