Identifying NRXN1 Function during Neural Differentiation of iPSC

Background: Neurexins are a family of presynaptic adhesion molecules that interact with post-synaptic proteins to regulate synapsis formation and maintenance. NRXN1 mutations have been strongly associated with a range of neurodevelopmental conditions including autism, attention deficit and schizophrenia (Kasem et al., 2017; Shehhi et al., 2018). Specific mutations such us NRXN1 determine autism likelihood but any particular phenotype is an emergent property of regulatory interactions that combines genetic and epigenetic regulation in a functional genome (Karlebach & Shamir, 2008; Huang et al. 2009; Gomez et al. 2011; Vashishtha et al., 2015). For instance, it has been demonstrated that inhibition of NRXN1 in neuronal progenitors derived from iPSC leads changes in the expression of genes enriched that regulate cell biological functions such as cell adhesion and neuronal differentiation, suggesting a potential role for NRXN1 during early neural development (Zeng et al., 2013). Hence, to unravel the molecular mechanism that lead to the autism phenotype in individuals with NRXN1 mutations, it is necessary to identify NRXN1 protein interactions and the cell biological processes regulated by these gene modules during neuronal development.

Objectives:

• To use induced pluripotent stem cells (iPSC) as an in vitro system to recapitulate the regulatory interactions underlying the transition from pluripotency to neural competence in humans.
• To capture the early molecular differences of neurotypical and NRXN1 mutated iPSC lines as they undergo during neuronal differentiation.

Methods: We used iPSC lines derived from 3 neurotypical and 3 autistic individuals with NRXN1 deletions and induced neural differentiation by dual SMAD inhibition (2i). RNA was extracted at 6 time points during the initial 12 days of neural induction and cDNA was synthesized to obtain transcriptome data. The expression patterns of NRXN1a, NRXN1b and genes that have been associated with NRXN1 was analysed in control and NRXN1 mutated lines trough real time PCR

Results: NRXN1 is expressed during early neural induction, suggesting a potential role during neural development. We identify a number of genes that are expressed during this stage that might interact with NRXN1 including neuroligins and other adhesion molecules. We detect systematic changes in the expression levels of genes that have been related with NRXN1 in the mutated cell lines compared with control lines undergoing neuronal differentiation. We identify potential cell biological functions regulated by these sets of genes including cell adhesion and neural rosettes formation.

Conclusions: iPSCs provide a novel approach to study the impact of NRXN1 mutations during neural differentiation in humans, highlighting the utility of iPSC model in understanding the functional role of specific mutations that are associated with autism risk. We show that NRXN1 gene mutations can impact biological networks that are important for neural differentiation.