Development of molecular tools and cellular models for the characterization of regulatory mechanisms of Tau protein alternative splicing

The human MAPT gene encodes Tau, a microtubule-stabilizing protein essential for cytoskeletal organization. Of the 16 MAPT exons, six of them are subjected to alternative splicing, generating a plethora of different Tau protein isoforms. The alternative splicing of exon 10 has been extensively studied over the years and, in particular, the imbalance in the expression of exon 10 isoforms has been associated with the development of Alzheimer’s disease. Additionally, Tau isoforms containing exon 6 have attracted research interest due to their ability to i) block Tau polymerization and ii) promote neural differentiation in vitro. Therefore, understanding the Tau exons 6 and 10 splicing mechanisms could contribute to the identification of potential therapeutic strategies to fight neurodegenerative disorders. The aim of this work was to develop molecular tools and advanced cellular models suitable to study Tau alternative splicing. To develop novel molecular tools to study Tau alternative splicing, FIMO bioinformatic analysis was applied to find genomic regions surrounding exons 6 and 10 enriched in binding sites for RNA-binding proteins (RBPs). After identifying such regions, they were cloned into minigene vectors and transfected them in conjunction with vectors overexpressing two RBP of interest, PTBP1 and RBM20, to analyze changes in Tau exons 6 and 10 splicing pattern caused by these proteins. RT-PCR and RT-qPCR analysis showed a statistical reduction of exons 6 and exon 10 inclusion in the minigene model after overexpressing RBM20 or PTBP1, suggesting a role for these RBPs in modulating Tau alternative splicing. RNA-binding protein immunoprecipitation (RIP) assay supported the direct binding between PTBP1 and Tau mRNA and RBM20 and Tau mRNA. Finally, to take advantage of these molecular tools to study the importance of cis-acting sequences on Tau exon 6 alternative splicing, site-directed mutagenesis was employed to disrupt specific regions in intron 5 and intron 6. Interestingly, a specific 129 bp deletion localized from +43 to +171 of intron 6 led to exclusive Tau exon 6 skipping. In silico structural modeling suggested that this deletion led to the disruption of a short hairpin structure in the mRNA, potentially involved in the splicing mechanism of Tau exon 6. Overall, these results highlighted how minigenes can be a valuable tool to identify novel trans-acting factor able to influence Tau splicing and cis-acting sequences important for such mechanisms. To develop advanced cellular models suitable to study Tau alternative splicing, the StemDiff™ Cerebral Organoid Kit was used to differentiate iPSC (induced pluripotent stem cells) line XF-iPS into 3D brain organoids. After verifying the insurgence of neural phenotype in the differentiated organoids at early timepoints (20 and 40 days of differentiation), RT-qPCR was used to assess changes in RBP and Tau expression during neural development. A statistically significant decrease in PTBP1 expression and an increase in RBM20 expression were detected when comparing undifferentiated cells with differentiated brain organoids. Furthermore, Tau expression was regulated during organoid differentiation, recapitulating in vivo Tau expression. These results suggest the feasibility of using the brain organoid technology to study Tau alternative splicing during neural development. These innovative 3D cellular models could be applied to the study and characterization of pathological processes taking place in Tau-related pathologies. This understanding could be pivotal to develop Tau-targeted therapeutics for these diseases.