Analysis of Treatment Efficacy and Molecular and Cellular Outcomes in Mouse Models of Congenital Epilepsy and Intellectual Disability

Abstract

Children with severe intellectual disability have an increased prevalence of refractory seizures. Steroid treatment may improve seizure outcomes, but the mechanism remains unknown. Here we demonstrate that short term, daily delivery of an exogenous steroid 17β-estradiol (40 ng/g) in early postnatal life significantly reduced the number and severity of seizures, but did not improve behavioural deficits, in mice modelling mutations in the Aristaless-related homeobox gene (ARX), expanding the first (PA1) or second (PA2) polyalanine tract. ARX polyalanine expansion mutations in children cause intellectual disability and developmental delay, frequently presenting with comorbidities such as autism, dystonia, and refractory seizures and infantile spasms. Frequency of observed seizures on handling (n = 14/treatment/genotype) were significantly reduced in PA1 (32% reduction) and more modestly reduced in PA2 mice (14% reduction) with steroid treatment compared to vehicle. Spontaneous seizures were assessed (n = 7/treatment/genotype) at 7 weeks of age coinciding with a peak of seizure activity in untreated mice. PA1 mice treated with steroids no longer present with the most severe category of prolonged myoclonic seizures, while treated PA2 mice had a complete absence of any seizures during this analysis. Despite the reduction in seizures, 17β-estradiol treated mice showed no improvement in behavioural or cognitive outcomes in adulthood. For the first time we show that these deficits due to mutations in Arx are already present before seizure onset and do not worsen with seizures. ARX is a transcription factor and Arx PA mutant mice have deregulated transcriptome profiles in the developing embryonic brain. At postnatal day 10, treatment completion, RNAseq identified 129 genes significantly deregulated (log2FC >± 0.5, Pvalue< 0.05) in the frontal cortex of mutant compared to wild-type mice. This list reflects genes deregulated in disease and was particularly enriched for known genes in neurodevelopmental disorders and those involved in signalling and developmental pathways. 17β-estradiol treatment of mutant mice significantly deregulated 295 genes, with only 23 deregulated genes overlapping between vehicle and steroid treated mutant mice. Furthermore, when we investigated populations of inhibitory interneurons in the cortex of PA mice immediately following estradiol treatment at P10, we saw no improvement to cell density, despite seeing a marked change in the expression of interneuron associated genes both due to disease, and changing with administration of estradiol at the same developmental timepoint. We conclude that 17β- estradiol treatment recruits processes and pathways to reduce the frequency and severity of seizures in the Arx PA mutant mice but does not precisely correct the deregulated transcriptome, cellular deficits, mortality, or behavioural and cognitive deficits. Our outcomes show that although estradiol may not represent the ideal therapeutic option for PA patients currently, our data provides insights into developing novel drugs by broadening our understanding of the mechanisms of disease caused by Arx PA mutations, particularly by uncovering pathways that may overlap with other neurodevelopmental disorders and present convergent targets for future treatment options.