Superexpressão do MicroRNA-26a como estratégia promotora de crescimento e regeneração de neuritos in vitro

Abstract

Damage to the central nervous system (CNS) of adult mammals, whether caused by neurodegenerative diseases or traumatic mechanical injuries, results in irreparable neurological deficits in patients, mainly due to the low regenerative capacity of neurons. This regenerative incapacity is due in part to the inhibitory environment generated after injuries and to factors intrinsic to neurons, in which anti-regenerative gene expression programs are activated to the detriment of pro-regenerative genes. Therefore, studying molecules that control gene expression, such as microRNAs (miRNA), may be important for the development of new regenerative therapies for neurodegenerative disorders, based on gene therapy. An interesting miRNA target is miR-26a, which has been shown to regularly promote neurite outgrowth in the CNS and axonal regeneration in the nervous system. However, the role of miR-26a in the regeneration of CNS neurons is still not very well understood. This study proposes the application of gene therapy mediated by overexpression of miR-26a conducted by adeno-associated virus (AAV) vectors in cortical neurons in vitro. For this purpose, an AAV vector overexpressing miR-26a (AAV.miR-26a) and the fluorescent protein EGFP was developed. As a control, a vector expressing only EGFP (AAV.CTRL) was developed. To validate the efficiency of the proposed gene therapy, primary cultures of embryonic cortical neurons from Wistar rats were used, through transduction with AAV.CTRL and AAV.miR-26a. Initially, the transduction efficiency of these vectors was tested and cytotoxicity to neurons was observed, with a transduction efficiency close to 90%, without triggering differential cellular cytotoxicity between them. Next, neurite growth and arborization (dendrites and axons collectively) were analyzed, and no statistically significant differences were found between neurons transduced with the AAV.CTRL and AAV.miR-26a vectors in either experiment. The next analyses consisted of evaluating the potential of AAV.miR-26a to promote neurite regeneration in cortical neurons after scratch injury, with a statistically significant increase in neurite regeneration being observed. Finally, in an attempt to evaluate the possible mechanisms by which miR-26a promotes neurite regeneration, a bioinformatics analysis was performed to identify the target mRNAs of miR-26a. To this end, I used the miRWalk platform using the TargetScan algorithm and found a total of 242 target mRNAs for miR-26a, of which 164 had not been validated and 78 had already been validated. Among them, mRNAs of genes involved in neurite regeneration were found. These results indicate a possible use of miR-26a as a promising regenerative strategy for CNS neurons. However, additional experiments are needed to confirm the regenerative effect of miR-26a and its efficiency in regenerative gene therapies for the CNS.