WP2 Ethical approval and clinical trial protocols were generated for the human work in WP2. Recruitment and brain and biofluid samples from patients were obtained and sample analysis began. 

WP3 Ethical approval was obtained for the animal work and sample production progressed well. A new EEG system was introduced and tested for the long-term monitoring. Ethics for the tVNS and deep brain stimulation in epilepsy patients was obtained and clinical trial protocols were completed.

WP4 Reporter assays to validate targets of novel miRNAs were established and miRNA profiles generated on a set of potential cell lines for use. 

WP5 Ethical approval was obtained from each of the centres donating DNA, inclusion/exclusion criteria agreed and sample collection is at an advanced stage. The sequence capture kit was designed and sample sequencing began.

WP6 Work focused mainly on overcoming technical aspects of RNAseq analysis. BC Platforms started the installation process on the EpimiRNA server with a package that supports several analysis tools and an easy to use web interface.

WP7 Computational model of a hippocampal neurons have been established that incorporate effects on dendritic spine changes and calcium effects along with single cell imaging methods to quantify and validate predictions. 

WP8 Preliminary work for this WP, set to start later in the project, included testing ways to up-regulate miRNAs in in the brain. 

WP9 Work on a medium-high throughput cell assay progressed well with development of a 3’UTR reporter highly sensitive to miR-134 which will be used for screening. Initial screens with the reporter in rat hippocampal neurons were performed using a chemical library of plant compounds with putatively anti-epileptic properties, assembled by 14 Bicoll. Two of the test compounds reproducibly reduced miR-134 dependent repression indicating the potential of the screening assay to identify miR-134 targeting small molecules.

WP2 Ethics approved and patient recruitment at all three clinical sites has been very successful. High-throughput qPCR platform analysis of miRNA in peri-ictal blood sampled from TLE patients at the centre in Germany is completed and is well-advanced in Ireland. The collection of hippocampus from TLE patients after neurosurgery progressed well. A prototype of the device for combined intracortical EEG depth-electrode and microdialysis was manufactured and clinical trial planning progressed.

WP3 Production of brain tissue is completed for the rat model of epileptic tolerance. The brain samples have been processed for quantitative analysis of miRNAs for this animal model, including both whole hippocampus and the individual subfields. Ethics for the tVNS and brain stimulation in epilepsy patients was obtained at all three sites and the tVNS clinical trial began.

WP4 miRNA-target analysis identification efforts progressed well with quantitative proteomic analysis of miRNA effects in cells undertaken. A number of potential targets of novel epilepsy-associated miRNAs were identified and the cell phenotypes expressing these miRNAs were identified using in situ hybridization and cell-based assay.

WP5 DNA sample number targets were achieved for this WP with >850 TLE+HS and over 1000 sporadic (other) epilepsy samples from carefully phenotyped patients in place, as well as over 1700 controls. Sequencing of miRNA and variation in the biogenesis enzymes and targets of miRNAs is nearly complete for all controls and more than half-way completed for patient samples.

WP6 Work progressed very well with analysis of over 130 brain samples from across the three animal models. Results show outstanding depth and quality of data. BC Platforms completed the installation of the EpimiRNA server and has populated with numerous annotated data from AGO2 sequencing and biomarker datasets from animal models and patient samples.

WP7 Computational modelling of the effects of miRNA manipulation on single hippocampal pyramidal neuron electrophysiology and function were validated and pathway maps of the known targets were developed for key miRNAs linked to epilepsy. The model was validated experimentally to correctly predict changes in neuronal excitability in response to a miRNA manipulation.

WP9 Using the miRNA-based medium-high throughput cell assay we have performed extensive screening of the chemical library of plant compounds, assembled by partner #14 Bicoll. Neuroprotective compounds were also identified using a second cell-based assay.

Within WP10 a database of all published miRNA research in epilepsy was made publicly available (www.EpimiRBase.eu), a number of publications were submitted as well as patents filed. The EpimiRNA website continued to be populated and a new factsheet was developed. Social media was used including a Twitter account (@Epilepsylab) to generate interest in the project. Press features on the project and multiple presentations of the project at scientific meetings and exhibitions were performed.