Abstracts

Rationale: Alterations in subunit expression and composition that affect the localization, function, and pharmacology of GABAA receptors have been demonstrated during primary epileptogenesis. Among various subunits, alpha 1 (GABRA1) and alpha 4 (GABRA4) have been found to be regulated by brain derived neurotrophic factor (BDNF)-induced signaling. For example, seizure-induced BDNF activates the Janus kinase (JAK)/ Signal Transducer and Activators of Transcription (STAT) pathway that in turn controls the synthesis of ICER, a repressor of GABRA1 transcription. BDNF is also the endogenous signal that induces early growth response factor 3 (Egr3) synthesis and increased transcription of GABRA4 via protein kinase C (PKC) and mitogen activated protein kinase (MAPK). In addition, Egr3 may play an important role in neuronal morphogenesis, p75 neurotrophin expression and regulation of NMDA receptor levels. Further, Egr3 is an important transcriptional regulator of cellular proliferation and differentiation. Egr3 has been shown to mediate axogenesis and dentritogenesis, and also modulates the Notch signaling pathway which has been implicated in the regulation of neurogenesis. Taken together, these findings suggest that Egr3 regulates a number of processes that are important in epileptogenesis. Methods: In this pilot study, we use RNA interference technique to assess the function of Egr3 in dentate granule neurons in the pilocarpine-induced SE model. Small interfering RNA (siRNA) can trigger sequence-specific gene silencing in mammalian cells. We used lentiviral vectors for delivery of short hairpin RNA (shRNA), a precursor of siRNA, into dentate gyrus to suppress Egr3 gene expression. The design of our viral vector are alpha 4 promotor-GFP-Erg3 snRNA. The Alpha 4 promoter was chosen in order to reliably activate Egr3 siRNA transcription in the dentate gyrus after SE. Lentivirus was injected via miropump into the dentate gyrus. SE was induced selectively one week after microinjection of Lentivirus vector. The hippocampi were examined 24 hours and 3 weeks after the SE, and we assessed the distribution of GFP-positive neurons in dentate gyrus. Results: One week after microinjection of Lentivirus vector, the GFP-positive neurons were detected in the inner granular cell layer. The number of GFP-positive neurons became more prominent 4 weeks after microinjection. By using double and triple staining techniques, we identified that some GFP-positive neurons became mature granular cells (NeuN-positive and DCX-negative). By using BrdU injection 3-5 days after SE, it is evident that new GFP-positive neurons continuously proliferate, suggesting the Lentivirus transduces progenitor cells. We next assessed the effect of Egr3 siRNA on neurogenesis in dentate gyrus after SE. The number of GFP-positive neurons were noted to be less in Egr3 siRNA-transgene injected dentate gyrus compared to the scrambled siRNA controls. Compared with the blank control (no virus injection but with SE), the immature granular (DCX-positive) cells were also less prominent in Egr3 siRNA-transgene injected dentate gyrus. Aberrant BrdU-positive granule cells were also less prominent in the hippocampus when Egr3 siRNA was present. Conclusions: These preliminary findings suggest that Egr3 siRNA may suppress dentate gyrus neurogenesis after SE. Additional studies are required to fully validate and characterize the role Egr3 may play in regulation of neurogenesis after SE. Funding: NIH R01 NS051710 C.U.R.E AWD-101849 C2D2 AWD-112299