Abstracts

Rationale: Newly generated granule cells after epileptic seizures show structural abnormalities including persistent basal dendrites and mossy fiber sprouting, in addition to ectopic migration to the hilus of the dentate gyrus. In contrast, seizure-experiencing mature granule cells have only been reported to exhibit a reduction in spine density, without other gross morphological changes. However, molecular signature of mature granule neurons after acute seizures is still unknown.Methods: To identify actively translated mRNAs in mature granule cells after status epilepticus (SE) compared to sham granule neurons, we used neurotrophin-3 (NTF3) driven BAC-TRAP (bacterial artificial chromosome-translating ribosome affinity purification) transgenic mice. These mice express eGFP-tagged ribosomal protein L10a in mature dentate granule neurons, allowing mRNA isolation associated with polysomes by eGFP immunoprecipitation (IP). Seven-week-old male and female NTF3 BAC-TRAP mice were administered scopolamine and terbutaline (i.p., 2 mg/kg), followed by pilocarpine injection (i.p., 200 mg/kg for males, 230 mg/kg for females). SE was evaluated by a modified Racine’s scale and diazepam was administered to terminate acute seizures at 3 h from SE onset. At 8 days after pilocarpine or saline injection, mice were sacrificed and allocated into RNA-seq or histology groups. For RNA-seq, 8 hippocampi isolated from 2 males and 2 females were pooled for further steps. After eGFP immunoprecipitation, purified mRNAs were subjected to the library generation for RNA-seq, which was sequenced with Illumina platform. For histology, immunohistochemistry was performed.Results: We confirmed that eGFP was expressed in the granule cells of sham- and pilocarpine-injected mice, without a colocalization of eGFP and doublecortin, suggesting that eGFP was expressed only in embryo-generated mature granule cells in the dentate gyrus. Quantitative PCR analysis using eGFP and Aldh1a1 primers showed that IP fraction was enriched with eGFP and depleted with Aldh1a1, a representative astrocytic gene, compared to unbound fraction. Based on this data indicating that isolated mRNAs are mainly derived from eGFP-positive mature granule neurons, we performed RNA-seq to identify differentially expressed genes in seizure-subjected granule cells compared to sham. Pathway analysis showed that reelin signaling in neurons, axonal guidance, mTOR signaling, actin cytoskeleton signaling, and growth hormone signaling were altered in mature granule cells after acute seizures. As a next step, we plan to validate candidate genes up-regulated or down-regulated by pilocarpine injection and their functional impact on epileptogenesis.Conclusions: We identified mature granule cell-specific transcripts after acute seizures using TRAP technology. We also showed key signaling pathways altered by pilocarpine-induced SE. We hope to address how mature granule cells after acute seizures contribute to the development of epilepsy and their critical molecular mechanisms to control epileptogenesis, which may provide new treatment options for preventing epilepsy.