Regulation of human PAX6 expression by miR-7

The paired box gene 6 (PAX6) is a powerful mediator of eye and brain organogenesis whose spatiotemporal expression is exquisitely controlled by multiple mechanisms, including post-transcriptional regulation by microRNAs (miRNAs). In the present study, we use bioinformatic predictions to identify three candidate microRNA-7 (miR-7) target sites in the human PAX6 3′ untranslated region (3′-UTR) and demonstrate that two of them are functionally active in a human cell line. Furthermore, transient transfection of cells with synthetic miR-7 inhibits PAX6 protein expression but does not alter levels of PAX6 mRNA, suggesting that miR-7 induces translational repression of PAX6. Finally, a comparison of PAX6 3′-UTRs across species reveals that one of the functional miR-7 target sites is conserved, whereas the second functional target site is found only in primates. Thus, the interaction between PAX6 and miR-7 appears to be highly conserved; however, the precise number of sites through which this interaction occurs may have expanded throughout evolution

Neuronal methylome reveals CREB-associated neuro-axonal impairment in multiple sclerosis

BACKGROUND: Due to limited access to brain tissue, the precise mechanisms underlying neuro-axonal dysfunction in neurological disorders such as multiple sclerosis (MS) are largely unknown. In that context, profiling DNA methylation, which is a stable and cell type-specific regulatory epigenetic mark of genome activity, offers a unique opportunity to characterize the molecular mechanisms underpinning brain pathology in situ. We examined DNA methylation patterns of neuronal nuclei isolated from post-mortem brain tissue to infer processes that occur in neurons of MS patients. RESULTS: We isolated subcortical neuronal nuclei from post-mortem white matter tissue of MS patients and non-neurological controls using flow cytometry. We examined bulk DNA methylation changes (total n = 29) and further disentangled true DNA methylation (5mC) from neuron-specific DNA hydroxymethylation (5hmC) (n = 17), using Illumina Infinium 450K arrays. We performed neuronal sub-type deconvolution using glutamate and GABA methylation profiles to further reduce neuronal sample heterogeneity. In total, we identified 2811 and 1534 significant (genome-wide adjusted P value < 0.05) differentially methylated and hydroxymethylated positions between MS patients and controls. We found striking hypo-5mC and hyper-5hmC changes occurring mainly within gene bodies, which correlated with reduced transcriptional activity, assessed using published RNAseq data from bulk brain tissue of MS patients and controls. Pathway analyses of the two cohorts implicated dysregulation of genes involved in axonal guidance and synaptic plasticity, with meta-analysis confirming CREB signalling as the most highly enriched pathway underlying these processes. We functionally investigated DNA methylation changes of CREB signalling-related genes by immunohistofluoresence of phosphorylated CREB in neurons from brain sections of a subcohort of MS patients and controls (n = 15). Notably, DNA methylation changes associated with a reduction of CREB activity in white matter neurons of MS patients compared to controls. CONCLUSIONS: Our data demonstrate that investigating 5mC and 5hmC modifications separately allows the discovery of a substantial fraction of changes occurring in neurons, which can escape traditional bisulfite-based DNA methylation analysis. Collectively, our findings indicate that neurons of MS patients acquire sustained hypo-5mC and hyper-5hmC, which may impair CREB-mediated neuro-axonal integrity, in turn relating to clinical symptoms

MiRNAs in early brain development and pediatric cancer

The size and organization of the brain are determined by the activity of progenitor cells early in development. Key mechanisms regulating progenitor cell biology involve miRNAs. These small noncoding RNA molecules bind mRNAs with high specificity, controlling their abundance and expression. The role of miRNAs in brain development has been studied extensively, but their involvement at early stages remained unknown until recently. Here, recent findings showing the important role of miRNAs in the earliest phases of brain development are reviewed, and it is discussed how loss of specific miRNAs leads to pathological conditions, particularly adult and pediatric brain tumors. Let-7 miRNA downregulation and the initiation of embryonal tumors with multilayered rosettes (ETMR), a novel link recently discovered by the laboratory, are focused upon. Finally, it is discussed how miRNAs may be used for the diagnosis and therapeutic treatment of pediatric brain tumors, with the hope of improving the prognosis of these devastating diseases.A.P. was funded by a predoctoral fellowship from Fundación Tatiana Pérez de Guzmán el Bueno. Work in the lab was supported by grants from the European Research Council (309633) and the Spanish State Research Agency (PGC2018-102172-B-I00, as well as through the “Severo Ochoa” Programme for Centers of Excellence in R&D, ref. SEV-2017-0723).Peer reviewe