A polyalanine tract expansion in Arx forms intranuclear inclusions and results in increased cell death

A growing number of human disorders have been associated with expansions of a tract of a single amino acid. Recently, polyalanine (polyA) tract expansions in the Aristaless-related homeobox (ARX) protein have been identified in a subset of patients with infantile spasms and mental retardation. How alanine expansions in ARX, or any other transcription factor, cause disease have not been determined. We generated a series of polyA expansions in Arx and expressed these in cell culture and brain slices. Transfection of these constructs results in nuclear protein aggregation, filamentous nuclear inclusions, and an increase in cell death. These inclusions are ubiquitinated and recruit Hsp70. Coexpressing Hsp70 decreases the percentage of cells with nuclear inclusions. Finally, we show that expressing mutant Arx in mouse brains results in neuronal nuclear inclusion formation. Our data suggest expansions in one of the ARX polyA tracts results in nuclear protein aggregation and an increase in cell death; likely underlying the pathogenesis of the associated infantile spasms and mental retardation

Targeted loss of Arx results in a developmental epilepsy mouse model and recapitulates the human phenotype in heterozygous females

Mutations in the X-linked aristaless-related homeobox gene (ARX) have been linked to structural brain anomalies as well as multiple neurocognitive deficits. The generation of Arx-deficient mice revealed several morphological anomalies, resembling those observed in patients and an interneuron migration defect but perinatal lethality precluded analyses of later phenotypes. Interestingly, many of the neurological phenotypes observed in patients with various ARX mutations can be attributed, in part, to interneuron dysfunction. To directly test this possibility, mice carrying a floxed Arx allele were generated and crossed to Dlx5/6CRE-IRES-GFP(Dlx5/6CIG) mice, conditionally deleting Arx from ganglionic eminence derived neurons including cortical interneurons. We now report that Arx−/y;Dlx5/6CIG (male) mice exhibit a variety of seizure types beginning in early-life, including seizures that behaviourally and electroencephalographically resembles infantile spasms, and show evolution through development. Thus, this represents a new genetic model of a malignant form of paediatric epilepsy, with some characteristics resembling infantile spasms, caused by mutations in a known infantile spasms gene. Unexpectedly, approximately half of the female mice carrying a single mutant Arx allele (Arx−/+;Dlx5/6CIG) also developed seizures. We also found that a subset of human female carriers have seizures and neurocognitive deficits. In summary, we have identified a previously unrecognized patient population with neurological deficits attributed to ARX mutations that are recapitulated in our mouse model. Furthermore, we show that perturbation of interneuron subpopulations is an important mechanism underling the pathogenesis of developmental epilepsy in both hemizygous males and carrier females. Given the frequency of ARX mutations in patients with infantile spasms and related disorders, our data unveil a new model for further understanding the pathogenesis of these disorders