miR-134 in human tissue (Nov 2019 - March 2019; August 2021-Nov 2021): I established from scratch a new laboratory facility at Beaumont Hospital Dublin, for human brain tissue work. I created a collaborative network between neurosurgeons, neurologists, pathologists and scientists and wrote SOPs to facilitate this work. I then performed a series of trial experiments, in collaboration with J Cryan in neuropathology, to develop a method to transport live tissue to the research laboratory, without impacting neuropathological readouts. Using this technique, I began to create acute slice preparations of human neocortical tissues, incubating them for 24 hrs in either ant-134 or scr control. I used this tissue to test the molecular effects of ant-134 in human neocortex, and to begin to explore it's biophysical impacts on the tissue. Main results achieved: 1) Set-up of new lab facility and training of other users (A Lacey, A Sanfeliu). 2) Established method to transport viable human tissues in artificial CSF at Beaumont Hospital. 3) Bioanalyser analysis showed that samples processed in this way have far greater RNA quality than those transported with no solutions (as previously). 4) Incubation of slices for 24 hrs in 1 uM or 3 uM ant-134 inhibits functional miR-134 expression in human neocortex in a dose-dependent manner., as measured using qPCR 5) Despite the reduction in miR-134 expression, I saw no evidence for de-repression of key miR-134 targets, including LIMK1, DCX and CREB. 6) I performed initial work to explore the network biophysical impact of ant-134 in human neocortex. I established a method to induce seizure-like activity in human brain tissue treated with antimiRs. Dissemination: A manuscript is being prepared for dissemination of this work in an open access peer-reviewed journal. I authored review articles detailing the use of human tissues in brain research (
https://doi.org/10.14573/altex.2007082(si apre in una nuova finestra)) and prospects for using antimirs in neurological diseases (
https://doi.org/10.1016/j.tips.2021.04.007(si apre in una nuova finestra)).
miR-134 in rodent epilepsy models: I also performed pre-clinical experiments to interrogate the therapeutic potential of ant-134 in a number of rodent epilepsy models. Work in a P21 mouse model of epilepsy showed for the first time that ant-134 is a viable treatment for epilepsies in juveniles (
https://doi.org/10.1038/s41598-020-79350-7(si apre in una nuova finestra)). Building on this, work in mouse models of Dravet syndrome and Angelman syndrome, both genetic epilepsy syndromes) generated promising data showing a novel use for ant-134 to treat genetic epilepsies (both publications under review).
Other microRNA targets in epilepsy: As the molecular mechanisms of miR-134 were not conserved in human tissues, I also explored other potential therapeutic microRNA dysregulations in epilepsy models. Notably, my work showed that rational correction of upregulated microRNAs in TLE is therapeutic (
http://dx.doi.org/10.1073/pnas.1919313117(si apre in una nuova finestra)). More recent work identified miR-335 as a novel microRNA dysregulation in epilepsy. Unlike other upregulated miRs, miR-335 inhibition actually exacerbates seizure phenotypes. Through combined biophysical and molecular analysis, we showed that miR-335 likely acts via VGSC transcripts, and that miR-335 overexpression may be therapeutic.
