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.

Mesial temporal lobe epilepsy (mTLE) is a chronic disease characterized by recurrent seizures that originate in the temporal lobes of the brain. Anti-epileptic drugs (AEDs) are the standard treatment for managing seizures in mTLE patients, but are frequently ineffective. Resective surgery is an option for some patients, but does not guarantee a postoperative seizure-free period. Therefore, further insight is needed into the pathogenesis of mTLE to enable the design of new therapeutic strategies. Circular RNAs (circRNAs) have been identified as important regulators of neuronal function and have been implicated in epilepsy. However, the mechanisms through which circRNAs contribute to epileptogenesis remain unknown. Here, we determine the circRNA transcriptome of the hippocampus and cortex of mTLE patients by using RNA-seq. We report 333 differentially expressed (DE) circRNAs between healthy individuals and mTLE patients, of which 23 circRNAs displayed significant adjusted p-values following multiple testing correction. Interestingly, hippocampal expression of circ_Satb1, a circRNA derived from special AT-rich sequence binding protein 1 (SATB1), is decreased in both mTLE patients and in experimental epilepsy. Our work shows that circ_Satb1 displays dynamic patterns of neuronal expression in vitro and in vivo. Further, circ_Satb1-specific knockdown using CRISPR/CasRx approaches in hippocampal cultures leads to defects in dendritic spine morphology, a cellular hallmark of mTLE. Overall, our results identify a novel epilepsy-associated circRNA with disease-specific expression and previously unidentified cellular effects that are relevant for epileptogenesis.

The porcine brain closely resembles the human brain in aspects such as development and morphology. Temporal miRNA profiling in the developing embryonic porcine cortex revealed a distinct set of miRNAs, including miR-34c and miR-204, which exhibited a highly specific expression profile across the time of cortical folding. These miRNAs were found to target Doublecortin (DCX), known to be involved in neuron migration during cortical folding of gyrencephalic brains. In vivo modulation of miRNA expression in mouse embryos confirmed that miR-34c and miR-204 can control neuronal migration and cortical morphogenesis, presumably by posttranscriptional regulation of DCX.