Abstracts

Rationale: Cntnap2 encodes contactin-associated protein-like 2 and plays a vital role in neuron-glia interaction and action potential propagation. Mutations in Cntnap2 are associated with epilepsy and autism. Cntnap2 knockout (KO) mice display deficits in social and repetitive behavior, alterations in neuronal excitability at early stages and develop spontaneous recurrent seizures later in life. However, the mechanisms of Cntnap2 deletion leading to altered neuronal excitability and epilepsy and potential therapeutic targets are unknown. MicroRNAs (miRNAs) regulate post-transcriptional expression of mRNA and can be targeted with sense or antisense oligonucleotides to regulate gene expression. Several microRNAs have been implicated in epilepsy. Inhibition of miR-324-5p, for example, leads to reduced seizure activity, whereas miR-218-5p may increase seizure susceptibility in mouse models of epilepsy. So far, it is unknown if microRNA-induced silencing plays a role in Cntnap2-associated epilepsy. Here, we used antisense oligonucleotides (antagomirs) to explore the effect of inhibition of miR-324-5p and miR-218-5p on seizure susceptibility and interictal spike activity in adult and older Cntnap2 KO mice.

Methods: Mice were genotyped and investigated at adult (4-6 months) and older (12-16 months) age points to investigate spike and seizure development. Seizures were confirmed using the tail-suspension assay and cortical EEG recordings using a wireless EEG system to confirm seizures and spike occurrence. Post confirmation of seizures, older mice were injected with miR-324-5p (n=7), miR-218-5p (n=9) or scrambled (n=8) antagomirs, respectively, and EEG monitored for a week. Younger mice underwent a kainic acid seizure onset challenge. At the conclusion of the experiments, mouse brains were perfused and were collected for future histological analyses.

Results: Younger mice displayed increased spike activity, but no spontaneous or handling-induced seizures were observed in the EEG. Older mice displayed both epileptiform spikes and spontaneous and handling-induced seizures. At younger and older age point EEG analyses showed on average increased numbers of seizures (non-significant) in miR-218 antagomir-injected mice compared to 324-5p or scrambled control (one-way ANOVA, p >0.05) but no neuroprotective effect of miR-324-5p inhibition was observed. EEG waveform and spike analysis showed no significant difference in EEG waveforms between antagomir treatment and SCR control (preliminary data). No difference in the average spike count or spike trains were observed (one-way ANOVA, p >0.05). An increase in seizure associated mortality post 218-antagomir injection was observed.

Conclusions: This study demonstrated the potential for microRNA-mediated regulation of seizures in Cntnap2 KO mice. Ongoing studies are focused on tissue analysis using immunohistochemistry, sleep-wake patterns in EEG activity, and seizure development and susceptibility in heterozygous mice.

Funding: Please list any funding that was received in support of this abstract.: NIH grant R01NS092705.