Oligonucleotide therapies offer precision treatments for a variety of neurological diseases, including epilepsy, but their deployment is hampered by the blood-brain barrier (BBB). Previous studies showed that intracerebroventricular injection of an antisense oligonucleotide (antagomir) targeting microRNA-134 (Ant-134) reduced evoked and spontaneous seizures in animal models of epilepsy. In this study, we used assays of serum protein and tracer extravasation to determine that BBB disruption occurring after status epilepticus in mice was sufficient to permit passage of systemically injected Ant-134 into the brain parenchyma. Intraperitoneal and intravenous injection of Ant-134 reached the hippocampus and blocked seizure-induced upregulation of miR-134. A single intraperitoneal injection of Ant-134 at 2 h after status epilepticus in mice resulted in potent suppression of spontaneous recurrent seizures, reaching a 99.5% reduction during recordings at 3 months. The duration of spontaneous seizures, when they occurred, was also reduced in Ant-134-treated mice. In vivo knockdown of LIM kinase-1 (Limk-1) increased seizure frequency in Ant-134-treated mice, implicating de-repression of Limk-1 in the antagomir mechanism. These studies indicate that systemic delivery of Ant-134 reaches the brain and produces long-lasting seizure-suppressive effects after systemic injection in mice when timed with BBB disruption and may be a clinically viable approach for this and other disease-modifying microRNA therapies.

No abstract

Repetitive or prolonged seizures (status epilepticus) can damage neurons within the hippocampus, trigger gliosis, and generate an enduring state of hyperexcitability. Recent studies have suggested that microvesicles including exosomes are released from brain cells following stimulation and tissue injury, conveying contents between cells including microRNAs (miRNAs). Here, we characterized the effects of experimental status epilepticus on the expression of exosome biosynthesis components and analyzed miRNA content in exosome-enriched fractions. Status epilepticus induced by unilateral intra-amygdala kainic acid in mice resulted in acute subfield-specific, bidirectional changes in hippocampal transcripts associated with exosome biosynthesis including up-regulation of endosomal sorting complexes required for transport (ESCRT)dependent and-independent pathways. Increased expression of exosome components including Alix were detectable in samples obtained 2 weeks after status epilepticus and changes occurred in both the ipsilateral and contralateral hippocampus. RNA sequencing of exosome-enriched fractions prepared using two different techniques detected a rich diversity of conserved miRNAs and showed that status epilepticus selectively alters miRNA contents. We also characterized editing sites of the exosomeenriched miRNAs and found six exosome-enriched miRNAs that were adenosine-toinosine (ADAR) edited with the majority of the editing events predicted to occur within miRNA seed regions. However, the prevalence of these editing events was not altered by status epilepticus. These studies demonstrate that status epilepticus alters the exosome pathway and its miRNA content, but not editing patterns. Further functional studies will be needed to determine if these changes have pathophysiological significance for epileptogenesis.

30RNA therapies such as oligonucleotides (OGNs) offer precision treatments for a variety of 31 neurological diseases, including epilepsy but their deployment is hampered by the blood brain 32 barrier (BBB). Here we used brain imaging and assays of serum proteins and tracer 33 extravasation, to determine that BBB disruption occurring after status epilepticus in mice was 34 sufficient to permit passage of systemically-injected antisense OGNs targeting microRNA-134 35 (Ant-134) into the brain parenchyma. A single intraperitoneal injection of Ant-134 two hours 36 after status epilepticus in mice resulted in potent suppression of spontaneous recurrent 37 seizures, reaching a 99.5% reduction during recordings at three months. The duration of 38 spontaneous seizures, when they occurred, was also reduced in Ant-134-treated mice. These 39 studies indicate that systemic delivery of Ant-134 reaches the brain and produces disease-40 modifying effects after systemic injection in mice when timed with BBB disruption and may be 41 a clinically-viable approach for this and other disease-modifying microRNA therapies. 42 43 44 45

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 Ago2-bound miRNAs in plasma samples collected from mice subject to status epilepticus (SE) 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.