Understanding the Role of Neurexin1 in Early Human Neurodevelopment: Implications for Autism Spectrum Disorder

Background: Autism spectrum disorders (ASD) are a heterogenous group of neurodevelopmental disorders exhibiting complex genetic and neurobiological aetiology. Despite this complexity, genetic risk-factors often converge to influence neurodevelopmental processes e.g. the development of neuronal cellular morphology. In vitro, key stages of this morphogenesis are recapitulated via neuroepithelium formation and neurite outgrowth. Cell adhesion proteins are critical regulators of neurite outgrowth, particularly at the growth cone leading edge. Gene mutations in the neurexin-neuroligin adhesion complex are frequently associated with ASD pathogenesis, particularly, neurexins (NRXN). However, the role this protein super-family and their ASD-associated counterparts play during early neurodevelopment remains unclear.

Objectives: To characterize the expression profile of NRXN1 isoforms, cellular and subcellular distribution in neurodevelopment, and to investigate the role of this protein at key stages of neurodevelopment.

Methods: Six patient-derived induced pluripotent stem cell (hiPSC) lines were generated; three containing no known genetic mutations with no psychiatric diagnoses (typically developing individuals), one hiPSC line generated from an individual with a large 200 kB deletion spanning chr2:50,579,853-50,786,547 and with a diagnosis of ASD; two hiPSC lines generated from a mother and son pair, both harboring identical deletions in NRXN1 spanning chr2:50,661,714-50,720,591. The mother has no diagnosis, and thus is considered to be typically developing, whereas the son has a diagnosis of ASD and microcephaly. All NRXN1 lines have deletions within the NRXN1 gene at an overlapping site. HiPSC lines were neuralized via dual-SMAD inhibition to generate neural progenitor cells (NPC). Terminally differentiated neurons were obtained by the addition of 10µM DAPT to NPC cultures, resulting in the generation of immature cortical neurons. Cells were assessed at two time points during neurodevelopment coinciding with the generation of the neuroepithelium and immature neurons. Cells were assessed for NRXN1 isoform expression using RT-qPCR and immunocytochemistry at both time points. Neural stem cell markers were assessed via immunocytochemistry at neuroepithelial stage staining for Nestin, Zeb2, Pax6, Tuj1, and FoxG1. Immature neuron morphology was assessed by high-content screening and automated quantification staining for mature cortical neuronal markers MAP2 and Tuj1.

Results: Characterization of all six lines via immunocytochemistry revealed all lines were pluripotent and can generate NPCs as determined by expression of pluripotency markers in iPSCs (Oct4, Nanog, SSEA4, TRA-1-81) and NPC markers in neuroepithelial cells (Nestin, Zeb2, Pax6, Tuj1, FoxG1). Significant differences in TRA-1-81 and FoxG1 expression was observed between control and patient iPSCs. Significant differences in Oct4, Nestin, and Zeb2 expression were observed between control and patient neuroepithelial cells. RT-qPCR revealed the patient lines have altered isoform specific expression of NRXN1 alpha at neuroepithelial stage. Further analysis of immature cortical neurons revealed altered neurite outgrowth phenotypes in patient lines compared to control cells.

Conclusions: These data indicate NRXN1 may play a role in early human neurodevelopment, particularly in neuroepithelium formation and the establishment of early neuronal morphology. Further research will focus on comparing these results to isogenic controls and understanding the mechanisms underlying these phenotypes.