An investigation into the expression, content and function of exosomes in an experimental model of epilepsy

Epilepsy is a serious neurological disease characterised by recurrent unprovoked seizures affecting 65 million people worldwide. Current anti-epileptic therapies target only symptomatic seizures and are not fully effective in ameliorating disease pathophysiology. Temporal lobe epilepsy is the most common form of epilepsy in adults and a third of temporal lobe epilepsy patients are drug refractory, and the underlying pathophysiology remains to be fully elucidated. Recent work revealed microRNAs serve important functions in cells, in post-transcriptional regulation of genes associated with altered neuronal structure, inflammation and cell death. All of which are pathological hallmarks of epilepsy. While the site of these effects is assumed to be intracellular, exosomes have recently emerged as carriers of microRNAs between cells. Exosome-carried microRNAs may represent a novel mechanism of cell-to-cell communication within the brain, both in health and disease states. The motivation for the studies was to identify whether status epilepticus and/or chronic epilepsy is associated with the formation of unique exosome profiles that are distinct from controls. Here, the intra-amygdala kainic acid model of status epilepticus was used to carry out most studies in mice. Messenger RNA and protein levels of various genes involved in exosome biogenesis pathway were analysed at key timepoints in our mouse model of epilepsy. This analysis revealed a time and brain region specific regulation of exosome biogenesis components in epilepsy. In particular, significant changes were found in the dentate gyrus of the hippocampus. The results suggested a role for this process in pathogenesis and maintenance of the epileptic state. To investigate this, two protocols were established within our team for isolating exosomes from brain tissue; a filtration and centrifugation protocol, and one using a commercial precipitation kit. Successful exosome-enriched fractions were confirmed by protein markers, zetasizer and electron microscopy. Furthermore, RNA Sequencing was carried out to characterise microRNA content in control versus mouse epileptic tissue. A set of microRNAs were found common to exosomes within the brain regardless of method of extraction. Detected were both neuron- and glia- enriched microRNAs within isolated exosomes. We determined that several miRNAs within exosome-enriched fractions were differentially expressed during epileptogenesis and experimental epilepsy such as miR-21a-3p, miR-21a-5p and miR-146a-5p. By comparing to data already obtained within our team, differential expression highlighted in these exosome-enriched fractions mainly reflected what was happening at the whole hippocampal level at 24 hours and at 2 weeks. In addition, results show successful attenuation of exosome production in vivo as judged by reduction in surrogate markers of extracellular vesicles (Alix and Flotillin1). This reduction in protein by exosome production-inhibitors GW4869 and cambinol was brought forward to final functional study to investigate if exosome release following status epilepticus plays a causal role in the pathogenesis of experimental temporal lobe epilepsy and subsequent development of recurrent seizures. In summary, the thesis was an investigation into the expression, content and function of exosomes in an experimental model of epilepsy. We identified time and region specific changes in the biogenesis pathway of exosomes and demonstrated that exosome-enriched fractions contain microRNAs which are differentially expressed in epileptogenesis and in epilepsy. Finally, we managed to attenuate expression levels of exosome-related proteins in vivo in order to later examine the effect on the epileptic state.</div

Brain cell-specific origin of circulating microRNA biomarkers in experimental temporal lobe epilepsy

The diagnosis of epilepsy is complex and challenging and would benefit from the availability of molecular biomarkers, ideally measurable in a biofluid such as blood. Experimental and human epilepsy are associated with altered brain and blood levels of various microRNAs (miRNAs). Evidence is lacking, however, as to whether any of the circulating pool of miRNAs originates from the brain. To explore the link between circulating miRNAs and the pathophysiology of epilepsy, we first sequenced argonaute 2 (Ago2)-bound miRNAs in plasma samples collected from mice subject to status epilepticus induced by intraamygdala microinjection of kainic acid. This identified time-dependent changes in plasma levels of miRNAs with known neuronal and microglial-cell origins. To explore whether the circulating miRNAs had originated from the brain, we generated mice expressing FLAG-Ago2 in neurons or microglia using tamoxifen-inducible Thy1 or Cx3cr1 promoters, respectively. FLAG immunoprecipitates from the plasma of these mice after seizures contained miRNAs, including let-7i-5p and miR-19b-3p. Taken together, these studies confirm that a portion of the circulating pool of miRNAs in experimental epilepsy originates from the brain, increasing support for miRNAs as mechanistic biomarkers of epilepsy