Molecular mechanisms of biofluid microRNA transfer in human and experimental epilepsy

Epilepsy is a common neurological disease characterised by recurring seizures. Diagnosing epilepsy is both difficult and expensive. A molecular biomarker to aid with diagnosis would be a major advance in the field. Plasma microRNAs (miRNAs) could provide a potential solution as a molecular biomarker. MiRNAs are small non-coding RNAs that regulate protein expression. MiRNAs are enriched in the brain and are also found in biofluids; typically bound to the Ago2 protein and encapsulated in exosomes. Studies have reported dysregulated levels of brain-enriched miRNAs in blood samples in human and experimental epilepsy. Although it is assumed that circulating miRNAs originate in the brain, no study to date has proven this.

Two central questions were addressed in this thesis: In epilepsy, which brain-enriched miRNAs circulate bound to Ago2 and which encapsulated in exosomes? Which brain cells release these miRNAs into circulation?

This thesis investigated the molecular carriage and brain-cell-type origin of circulating miRNAs in epilepsy. Together, mechanistic links were enhanced between the biomarker and disease, and the sensitivity and specificity were improved. Human and mouse model plasma was collected. A combination of biochemical techniques to separate plasma fractions, small RNASeq, bioinformatics, RT-qPCR and in vivo inducible Cre/loxP technology was used.

In Results I, RNASeq on Ago2 and exosomes from epilepsy patient plasma was performed. The Ago2 fraction provided superior discrimination between epilepsy and controls. Let-7i-5p and miR-19b were identified as plausible brain-enriched-miRNA biomarkers. Taken together, these studies represent the first characterisation of miRNA in multiple sub-fractions of human plasma in patients with epilepsy.

In Results II, parallel studies were performed using mouse plasma. Experimental epilepsy was induced and samples collected at time-points

covering different phases of epilepsy development. Upon analysing patterns of miRNAs, the studies revealed that the release mechanism differed depending on the molecular carriage.

In Results III, new transgenic mice expressing an epitope-tagged-Ago2 in neurons and microglia (using inducible Thy1 and CX3cr1 driven cre-recombinase) were developed and characterised. These mice were subjected to SE and plasma collected at two timepoints. Let-7i-5p, miR-19b-3p and miR-320a were bound to cre-Thy1-FLAG-Ago2 in the plasma of mice 2 wk-post-SE. These results provide strong proof that a part of the circulating miRNA pool in plasma has originated from neurons.

In summary, these data indicate that specific brain-enriched miRNA are present in the Ago2 fraction of plasma in epilepsy. Furthermore, these specific miRNA were conclusively determined to be released from neurons in chronic epilepsy. Based on these findings, let-7i-5p, miR-19b-3p and miR-320a are proposed as novel, mechanistic molecular biomarkers of epilepsy.