Abstracts

Rationale: X-linked Infantile Spasms Syndrome (ISSX) is a catastrophic epilepsy syndrome. Few treatments exist that effectively reduce spasms, prevent epilepsy and improve developmental outcomes in ISSX. The Arx(GCG)10+7 mouse model carries the most common mutation in ISSX and exhibits many phenotypic features of ISSX including epilepsy, motor spasms in early life, and loss of GABAergic interneurons. We have shown that treatment with 17߭Estradiol (E2) in Arx(GCG)10+7 neonates (P3-10) reduces neonatal spasms, seizures in adults, and rescues interneuron subtypes. However, adult treatment (P33-40) had no effect on these phenotypes suggesting the antiepileptogenic effect of E2 is developmentally confined. In order to effectively translate this therapy to the clinic, it is crucial to further investigate the temporal boundary for E2 efficacy in Arx(GCG)10+7. In this work, we examined the effect of delaying treatment initiation by several days, until the second week of life. Little is understood about the cellular mechanisms underlying the loss of GABAergic interneuron subtypes in Arx(GCG)10+7 mutants or why there is a critical window for neonatal E2 treatment. In mice, GABAergic interneurons undergo apoptotic pruning during the first week of life and as ARX is predominantly expressed in this cell type, we speculated that Arx(GCG)10+7 mutation may affect this developmental apoptosis. Methods: Delayed E2 Treatment: 17߭Estradiol (40ng/g and 80ng/g) or vehicle (vegetable or sesame oil) was administered daily from P7 to P13 to Arx(GCG)10+7 males by subcutaneous injection. Mice were surgically implanted for video-EEG at P40 and EEG was recorded from P45-59. Immunohistochemistry: Arx(GCG)10+7 mice were crossed to the VGAT-Venus transgenic line, which expresses venus fluorescent protein in all VGAT-positive GABAergic interneurons. Animals were sacrificed at P7 and brain tissue was collected. Apoptotic interneurons were quantified in neocortex and hippocampus using cleaved caspase-3 (CC3) antibody. Results: Delaying E2 treatment until the second postnatal week (P7-13), using either 40ng/g/day or 80ng/g/day, had no effect on seizures and interictal spike frequency (a second measure of cortical hyperexcitability). At P7, Arx(GCG)10+7 mutation had no effect on the number of Venus-positive cells that express CC3 in Arx(GCG)10+7;VGAT-Venus mice as compared to WT littermate controls. Conclusions: These data suggest a critical developmental window for the antiepileptogenic effect of E2 in the Arx(GCG)10+7 model of ISSX. Furthermore, we found that within this critical widow, Arx(GCG)10+7 mutation has no effect on GABAergic interneuron cell death marked by CC3 in Arx(GCG)10+7;VGAT-Venus suggesting that cell death is likely not the mode by which GABAergic interneuron subtypes are reduced in Arx(GCG)10+7 mutants. Future work will further examine the effect of Arx(GCG)10+7 mutation on neonatal apoptotic pruning of GABAergic interneurons and the effect of E2 on neonatal brain apoptosis during this critical window. Funding: This work was supported by CURE Infantile Spasms Research Initiative.