Abstracts

Rationale: The most common human mutation in X-linked infantile spasms syndrome (ISSX), a developmental epileptic encephalopathy, is a triplet repeat GCG expansion in ARX. The mouse model of this mutation, Arx(GCG)10+7, exhibits neonatal spasms, seizures, and reduced cortical GABAergic interneurons. Treating Arx(GCG)10+7 with 17β-estradiol (E2) during the first postnatal week prevents spasms and epilepsy, while later treatment is ineffective, suggesting a developmental window for therapeutic intervention, however, the pathogenic cellular defects targeted by treatment are unknown. As Arx expansion mutations dysregulate apoptotic factors in developing brain, we explored whether programmed cell death is affected in neonatal Arx(GCG)10+7 cortex. Since ACTH, the frontline clinical therapy for IS, is a potent anti-inflammatory drug, we searched for evidence of inflammation as a key component of ISSX pathophysiology. Methods: Immunohistochemistry & mRNA profiling: Arx(GCG)10+7 and WT males were treated daily with 40ng/g/day E2, 4IU/kg/day ACTH(1-24), or vehicle from P3-7 and sacrificed at P7. Cortical apoptotic cells and interneurons were quantified using cleaved caspase3 (CC3), Arx antibodies, and TUNEL assay. Inflammation was profiled using antibodies to GFAP, IBA-1, CD68 and qPCR and Nanostring analysis of cytokines. Spasms & Video EEG: Arx(GCG)10+7 males were treated with 2IU/kg/day or 4IU/kg/day ACTH(1-24) from P3-10. Spasms were recorded from P7-11, mice were implanted at P35, and EEG recorded weekly from P45-72. Results: We identified a transient wave of elevated apoptosis in neocortex during the first week of life in Arx(GCG)10+7 mutants. This excess cell death was non-cell autonomous and did not involve mutant Arx+ interneurons. Surprisingly, we found no astrogliosis, microglial invasion, or aberrant cytokine expression in neonatal mutant cortex, and treatment with E2 and ACTH elevated ARX+ cell density but did not reduce apoptosis, suggesting that inflammation is neither the cause or target of these treatments. Conclusions: We conclude that the efficacy of early hormone stimulation in the Arx(GCG)10+7 model of ISSX is likely due to direct neuroprotective actions rather than anti-inflammatory activity in immature brain. Funding: This work was supported by Citizens United for Research in Epilepsy (CURE)