miRNA Dysregulation Drives Neuronal Intracellular Chloride Accumulation in Down Syndrome

Down syndrome (DS) is caused by the presence of an extra-copy of chromosome 21 and is the most frequent genetic cause of mental retardation. DS cognitive disabilities primary arise from the triplication of dosage-sensitive genes on chromosome 21. However, a global dysregulation in the expression of extra-chromosome 21 genes greatly complicates the understanding of the underling pathological mechanisms. We have recently found that cognitive impairment in the Ts65Dn mouse model of DS depends on the upregulation of a non-triplicated gene encoding for the chloride importer NKCC1, which we found increased also in brain tissue from individuals with DS. Consequently, the intracellular chloride concentration is increased and GABAergic signaling, through chloride-permeable GABAA receptors, is depolarizing rather than hyperpolarizing. Here, we aimed at addressing the molecular mechanisms responsible for NKCC1 overexpression in DS. Real-time qPRC and Western Blot analysis showed that NKCC1 overexpression in trisomic neurons does not derive from greater mRNA transcription or decreased protein turnover but rather from a diminished translational repression exerted on the 3’ untranslated region (3'UTR) of the gene. As 3’ UTRs are the preferred sites of action of microRNAs (miRs), we applied a combination of bioinformatics prediction tools and gene expression screening to identify candidate miRs downregulated in trisomic samples that could mediate NKCC1 overexpression. Our results show that different candidates miRs interact with NKCC1 3’UTR and repress its expression. Additionally, overexpression of the same miRs can normalize NKCC1 levels and intracellular chloride concentration in trisomic neurons, restoring GABAergic inhibitory signaling at the network level as shown by Multi-Electrode Array recordings. Our findings will help to elucidate molecular pathways dysregulated in DS and suggest possible targets for future therapeutic intervention.