Microtubule organization and splicing switches are altered in Arx animal models for neurodevelopmental disorders

The correct development of mammalian brain requires a fine-tuning orchestration of several homeotic transcription factors (TFs), the deregulation of which may cause neurodevelopmental diseases (NDDs). Aristaless-related homeobox is an X-linked gene encoding a bifunctional TF required for proper brain morphogenesis whose mutations cause allelic disorders as X-linked lissencephaly with abnormal genitalia (XLAG), developmental epileptic encephalopathy-1 (DEE1) and intellectual disability. We report an extensive proteomics analysis on the neonatal brain of Arx knockout ArxKO/Y and knock-in Arx(GCG)7/Y mice that are respectively models for XLAG and DEE1. Gene ontology and protein-protein interaction analysis revealed that cytoskeleton and splicing control are deregulated in an allelic-dependent manner. Decreased ?-tubulin content in Arx mice and Arx/alr-1(KO) C. elegans animals and disorganized neurite network in murine primary neurons were consistent with an allelic-dependent secondary tubulinopathy. Allelic-dependent differences were also established in alternative splicing (AS) regulated by PUF60 and SAM68. Abnormal AS repertoires in Neurexin-1, a gene encoding multiple pre-synaptic organizers implicated in autism, were detected in Arx/alr-1(KO) animals and in Arx(GCG)7/Y epileptogenic brain areas and depolarized cortical neurons. Consistent with a conserved role of ARX in modulating AS, we propose an allelic-dependent secondary synaptopathy resulting from aberrant Neurexin-1 repertoire. Overall, our data reveal alterations mirroring the overlapping and variant effects caused by distinct Arx mutations. The identification of these effects can aid in the design of pathway-guided therapy for ARX-endophenotypes and NDDs with overlapping comorbidities.