Abstracts

Rationale: The need for new treatments in epilepsy remains currently significant, emphasizing the importance of developing new relevant disease models. Organotypic hippocampal slices culture (OHSC) is considered a relevant model of epileptogenesis and drug-resistant epileptic seizures. The model was pharmacologically characterized by testing the effect of standard antiseizure medications (ASMs) on epileptic markers. For new target identification, we performed a transcriptomic analysis to identify gene clusters changing during the development of epileptiform activity. To validate targets identified via transcriptomic study or other sources, we used several modalities: small molecules or biologics such as neutralizing antibodies, genetic manipulation with viral vectors or antisense oligonucleotides (ASOs).


Methods: Hippocampal slices were prepared from 8-day-old rats and cultured on multi-electrodes arrays (MEAs) during 21 days-in-vitro. The phenotypic epileptic readout was measured with local field potentials. Spontaneous ictal-like activity was quantified through parameters of ictal-like events. Bulk RNAseq analysis was performed on individual slices at different time points. Treatments were directly applied in the medium.


Results: ASMs demonstrated different levels of efficacy in the OHSC model. They reduced the severity of epileptiform activity without completely suppressing ictal-like activity confirming the relevance of OHSC model for drug resistant epilepsies. Transcriptomic analysis using gene coexpression profiles identified differential expression of coregulated genes. Interestingly, modules identified in the in vitro OHSC model were conserved in temporal lobe epilepsy (TLE) datasets. From one of the gene modules, we identified the tyrosine kinase receptor CSF1R as potential target and showed that a treatment with a Csf1R inhibitor decreases ictal event duration. Similarly, incubation with a neutralizing antibody against tumor necrosis factor-alpha (TNFa), also identified as a potential target, decreased the incidence of ictal events. We virally over-expressed a target gene A identified to be down-regulated in OHSC model and demonstrated a significant decrease of ictal-like activity, suggesting that gene A down-regulation plays an important role in ictal activity generation. Finally, a preliminary study using an ASO against a target B showed a significant downregulation of the protein, indicating ASOs as a suitable modality to selectively silence genes of interest in OHSC model.


Conclusions: We confirmed that spontaneous epileptic discharges developing in the OHSC model show features of drug resistance to standard ASMs. The transcriptomic analysis unveiled major changes in gene regulatory networks that are relevant for TLE. Inhibition of identified targets activity with small molecules or biologics had an impact on the epileptic phenotype. In addition, modulation of protein expression with genetic tools impacted the epileptic phenotype as expected. The OHSC model can be considered as a valuable model for the discovery of new targets for drug-resistant epilepsies.


Funding: UCB Pharma-sponsored study. All authors are employees of UCB Pharma.