Periodic Reporting for period 1 - EpiMIRgen (A comprehensive analysis of microRNA-124 regulated gene networks and its role in epilepsy.) Periodo di rendicontazione: 2016-07-01 al 2018-06-30 Sintesi del contesto e degli obiettivi generali del progetto EpiMIRgen is focussed on understanding the mechanisms of gene expression and gene expression regulation in both the normal healthy brain and in epilepsy. It is particularly focussed on a small group of molecules called microRNAs which can regulate gene expression post-transcriptionally. these small molecules can have profound effects on the gene expression profiles of a cell and influence of the function of the cell in different ways. Little is known about how these molecules interact with other regulators which influence gene expression and whether they "talk to each other" is unclear. EpiMIRgen will try to identify whether this cross-talk exists between microRNAs and a group of proteins which influence gene expression by interacting directly with genes. This interaction may be affected in epilepsy and this will also be investigated.Epilepsy is a chronic neurological disorder characterised by spontaneous recurrent seizures. It affects about 55 million people worldwide, exacts an enormous toll on human potential and is estimated to cost about Euro 20 billion per year in Europe alone. Additionally, current anti-seizure medications fail about one third of patients so there is a real unmet clinical need to develop novel therapeutics which treat the underlying causes of the disease. There are also a number of comorbidities associated with the disease, for example people who suffer from epilepsy are also more likely to experience anxiety, depression and sudden unexpected death from epilepsy. While some epilepsies are genetic in cause, many are caused by trauma to the brain. These epilepsy-inciting events initiate processes in the brain which then give rise to the spontaneous seizures, these processes include things like neuroinflammation, cell death and reorganisation of surviving neurons. Large scale changes in gene expression and gene expression regulation likely give rise to the common pathological mechanisms of epilepsy development. MicroRNAs are a strong candidate as regulators of this process.The overall objectives of this project are to explore the role of miRNAs in healthy brain and in epilepsy. We hope to identify the gene networks governed by miRNA and then target these molecules or one/some of its targets to try to block the development of epilepsy in pre-clinical models. We will also assess their biomarker potential as there is also a real need for the development of simple diagnostic tests which can identify patients at risk of developing epilepsy.Conclusion of the action: Transgenic and reporter mouse models are critical for intensive molecular interrogation of microRNA function in healthy and diseased brain. MicroRNAs have been shown to regulate many cellular processes and neuronal and glial activity but at present there is a dearth of knowledge about the expression profiles of microRNAs in vivo and particularly within complex tissues such as brain. The Ago2-flag-Cre mice generated in this project now allow us to profile miRNA expression within neurons and glia in healthy and diseased brain, specifically changes within these cell types in epilepsy. Second, the diagnosis of epilepsy and determining whether neonates have experienced a seizure remains fraught with complications requiring EEG. This requires significant resources which are often unavailable in remote regions and in less well developed countries. As such there is a significant need for the development of a molecular, non-invasive biomarker which can accurately predict the development of epilepsy in adults or can identify whether a neonate has experienced a real seizure. MiRNAs possess many physical characteristics which render them excellent potential biomarkers. Human umbilical cord blood plasma could potentially contain molecular signatures useful for the diagnosis of disease or traumatic event such as hypoxic ischemic encephalopathy. For the first time we profiled miRNAs in umbilical cord blood plasma from healthy and diseased babies and found that there are inherent sex differences in cord blood make up as well as editing differences and these may be exploited to stratify babies who have undergone mild, moderate and severe hypoxia and may be useful for the prediction of negative outcome during development. Lavoro eseguito dall’inizio del progetto fino alla fine del periodo coperto dalla relazione e principali risultati finora ottenuti The overall scientific goals of the project were achieved. We generated a novel mouse line which allows us to analyse microRNA expression and function in discrete populations of cells within key brain regions. We are currently expanding this and performing these analyses in a genome wide manner to identify full microRNA expression in control brain but also in epileptic brain. This will guide future studies which attempt to block or prevent epilepsy development.We developed and adapted small-RNA-Seq for the identification and profiling of miRNAs in umbilical cord blood plasma for the first time. We also analysed RNA editing in this biofluid. We identified sex differences in miRNA editing in umbilical cord blood plasma and are establishing a reference dataset for umbilical cord blood miRNAs in healthy children. This baseline database allows us to compare miRNA profiles from babies with more complicated births such as hypoxia, perinatal asphyxia etc.During the course of this project additional opportunities arose and we profiled miRNAs from peripheral blood samples to test whether we could identify signatures of microRNAs in plasma which may be indicative of disease development. The further development of these findings is a critical objective for us going forward as at present there is no way to predict which patients will develop epilepsy even after similar head traumas or neurological events like stroke.The work completed as a Marie Sklodowska Curie fellow has to date resulted in 3 journal articles, 4 conference talks and 3 additional journal articles are currently in preparation.(Some details of results have been omitted due to confidentiality) Progressi oltre lo stato dell’arte e potenziale impatto previsto (incluso l’impatto socioeconomico e le implicazioni sociali più ampie del progetto fino ad ora) The work in EpiMIRgen has resulted in the development of novel transgenic mouse models which allow us to analyse microRNAs in discrete cell populations of the brain. This has enormous potential as a research tool not just for epileptologists but also those studying other neurological disorders, development and ageing and normal brain function. We will continue to develop the model to further enhance our understanding of the nuanced role of individual cell types in epilepsy. Over 30% of patients with epilepsy are resistant to medication and there is a real unmet clinical need to develop drugs which target the underlying physiology of the disorder. It is only by gaining a deep understanding of the mechanisms which lead to the development of epilepsy that we will identify pathways which can have disease modifying effects.This has the potential to improve the quality of life for millions worldwide. Additionally the profiling of umbilical cord blood plasma from healthy controls provides a database which will be freely accessible by researchers to compare healthy baby umbilical cord blood to that from complicated births. This may aid the development of miRNA biomarkers to predict negative developmental outcome as a result of gestational or neonatal complications.Stratifying children likely to thrive and those faced with poor neurodevelopmental outcome will enable targeted therapies and strategies to help improve outcome for these children. jpg-final-summary.jpg
