Mechanisms of post-transcriptional regulation of genes involved in FTDP-17

MicroRNAs (miRNAs) are small non coding RNAs of 18-25 nt, capable of regulating mRNA translation and gene expression at post-transcriptional level. Alteration of miRNAs expression is often associated with human diseases, such as cancers and neurodegenerative pathologies. The main objective of this study is an analysis of the post-transcriptional regulation played by miRNAs of two important genes, MAPT and GRN, involved in Frontotemporal Dementia with Parkinsonism linked to chromosome 17 (FTDP-17). This is one of the major degenerative dementia syndromes, characterized by atrophy of the prefrontal and anterior temporal lobes. Several studies identified 44 pathogenic mutations in MAPT, which encodes for microtubule associated tau protein. In the brain tau has important functions in the assembly and stability of microtubules, that are fundamental for neuronal integrity and function. Disruption of tau role due to tau aggregations cause devastating effects that trigger neurodegeneration. To date 69 different mutations were found in GRN in the presence of FTD. GRN encodes a secreted precursor protein called progranulin that is expressed in neurons, microglia and represents an important growth factor involved in the regulation of multiple processes. The study identified miRNAs that can bind to GRN and MAPT mRNAs, affecting the translation efficiency of these transcripts, with a consequently reduction of the protein production. The project combined standard bioinformatic tools with a novel capture affinity assay, called miR-CATCH, in order to identify the best putative miRNAs. The assay was performed with specific biotinylated anti-sense oligonucleotides for the pull-down of GRN and MAPT mRNAs in different neuroblastoma cell lines. The binding of selected miRNAs on the 3’UTR regions of these genes, was validated using over-expressing vectors and reporter constructs to perform luciferase assays. Regulation of selected miRNAs over-expression on progranulin and tau proteins was investigated in a neuroblastoma cell line, using western blot. This effect was further analyzed with Real Time PCR reactions and polysomal analysis to understand if the selected miRNAs control progranulin and tau expression through a mechanism of translational repression or transcripts degradation. Two of the analyzed candidates, miR-659-3p and miR-608 were found important for the regulation of both genes, GRN and MAPT implicated in the disease, with a subsequent control on the endogenous progranulin and tau production in neuroblastoma cell line. Whereas miR-939-5p and miR-615-5p are only involved in the regulation of GRN expression. In addition we identified several SNPs located on miRNAs binding sites that are predicted to increase or decrease miRNAs binding. Since neurological disorders are strongly influenced by common genetic variability, SNPs or variations that overlapped miRNA-binding sites, could represent potential and important risks factors for generation of FTD.