Abstracts

Rationale: The voltage-gated potassium channel Kv4.2 has a crucial role in modulating neuronal excitability in the brain. Recent studies have correlated reduced expression of the Kv4.2 channel protein and its auxiliary subunits, such as Dipeptidyl peptidase-like protein (DPPs) and Potassium Channel Interacting Proteins (KChIPs) with increased dendritic excitability and epilepsy. MicroRNAs (miRNAs) are small non-coding RNAs that regulate post-transcriptional expression of protein-coding mRNAs. Emerging evidence suggests miRNAs as crucial regulators of genes involved in epilepsy. This study investigates the regulation of Kv4.2 and its auxiliary proteins using candidate miRNAs. Methods: Luciferase reporter assays and western blotting was used to investigate the regulation of Kv4.2 and associated subunits by candidate miRNAs in cultured cell lines and primary neurons. Immunoprecipitation was used to study recruitment of Kv4.2 to the RNA induced silencing complex (RISC). For in-vivo investigation, 6-8 weeks C57BL/6 male mice were treated with intracerebroventricular injections of antagomirs (miRNA inhibitors) specific to Kv4.2 targeting microRNAs and scrambled antagomirs (control) to study the effect on seizure severity and onset in kainic acid model of Status epilepticus. ICV antagomirs injection was also performed in mouse from C57BL/6 x FVB breeding's using pilocarpine mouse model. Results: Luciferase reporter assays and western blot analysis showed that Kv4.2 and its auxiliary subunits are regulated by specific microRNAs (n=10, p < 0.05 and n=6, p < 0.05). Immunoprecipitation analysis showed recruitment of Kv4.2 mRNA to the RNA induced silencing complex post kainic acid treatment (n=5, p < 0.05), suggesting that Kv4.2 mRNA is regulated by microRNA-induced silencing in response to neuronal hyperactivity. Inhibition of microRNA-induced silencing of Kv4.2 by intracerebroventricular injection of antagomirs targeting Kv4.2 microRNAs reduced seizure severity and excitotoxicity and significantly delayed kainic acid-induced seizure onset (n=14, p < 0.05). Moreover, our experiments using a mouse model of temporal lobe epilepsy suggest that blocking a Kv4.2-targeting microRNA reduces seizure frequency in chronically seizing mice. Conclusions: Overall these results suggest that our candidate microRNA-mediated silencing of Kv4.2 plays an important role in seizure development and could be explored as therapeutic target in epilepsy in the future. Funding: Trustee Award-Cincinnati Research Foundation (C.G), NIH grant NS092705 (C.G), MH085617 (G.J.B)