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.</p

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