Abstracts

Rationale: X-linked Infantile Spasms Syndrome (ISSX) is a catastrophic epilepsy syndrome. Few treatments exist that effectively reduce infantile 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. Recent work has shown that treatment with 17β-Estradiol (E2) in Arx(GCG)10+7 neonates (P3-10) reduced neonatal spasms and seizures in adults. However, E2 treatment in adult mice (P33-40) had no effect on these phenotypes. This suggests that the antiepileptogenic effect of E2 in Arx(GCG)10+7 is developmentally confined and 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. Little is understood about the molecular and cellular mechanisms behind the developmental window for E2 treatment and as E2 is known to have neuroprotective and anti-apoptotic effects in the brain, we tested whether E2 may reduce apoptotic markers in the Arx(GCG)10+7 neonatal brain.Methods: Delayed E2 Treatment: 17β-Estradiol (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. Spasms were monitored daily from P8-P11. Pups were placed in chambers, allowed to habituate then video recorded for 30min. Abdominal flexions and extensions were scored. Mice were surgically implanted for video-EEG at P40 and EEG was recorded from P45-59. Immunohistochemistry: Arx(GCG)10+7 and WT males were treated with 40ng/g/day E2 from P3-7. Animals were sacrificed at P7 and brain tissue was collected. Apoptotic cells 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 neonatal spasm frequency, seizure and interictal spike frequency (a second measure of cortical hyperexcitability). As E2 is known to have anti-apoptotic effects, we examined expression of the apoptotic marker CC3 in neonates. We found that E2 treatment (40ng/g/day) from P3-7 reduced total CC3+ cells at P7 in Arx(GCG)10+7 neocortex but had no effect on CC3+ cell number in WT neocortex.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, E2 reduces total apoptotic cells marked by CC3 expression in Arx(GCG)10+7 neocortex. However, this effect was not observed in WT suggesting E2 does not reduce general apoptosis in the neonatal brain and Arx(GCG)10+7 mutation may perturb developmental apoptosis. Future work will examine if Arx(GCG)10+7 exhibits aberrant apoptosis during development and how neonatal E2 treatment modifies disease phenotypes via cell death pathways.