Investigating the Role of NRXN1 in Autism Using iPSC-Derived Neurons

Background: Hundreds of genes contribute to autism likelihood. Of particular interest are mutations in genes encoding trans-synaptic signalling molecules, such as neurexins and their binding partners which have been implicated in autism. The Neurexin-1 gene (NRXN1) encodes for the presynaptic adhesion molecule neurexin-1, which binds to postsynaptic cell-adhesion molecules. While the role of NRXN1 has been established in regulating typical synaptic function and physiology, the role of its different isoforms and their contribution to the aetiology of autism is only now emerging and our knowledge remains patchy.

Objectives: Introduce the optimised inducible transgene (NGN2) over-expression for forward programming patient derived-iPSC lines and isogenic NRXN1-KO lines to investigate neuronal phenotypes, synaptic and functional changes caused by NRXN1 mutations

Methods: We generated iPSC-lines derived from individuals with autism carrying mutations in NRXN1-α (n= 2, 1 male, 1 female) and healthy controls (n=2, 1 male, 1 female (carrier))and 2 isogenic pair of iPSCs using eSpCAS9 to create NRXN1 knock-out via non-homologous end-joining. We aimed to target exon 4-5 (for NRXN1-α / KO) and exon 19 (shared by both isoforms). We systematically optimised inducible transgene over-expression (OPTi-OX) in the generated lines using a dual genomic safe harbour gene-targeting strategy to overcome silencing of transgenes and allow forward programming of iPSCs into mature functional neurons. We then examined the effect of those mutations on gene expression levels and protein using synaptic genes-specific primers and western blot antibodies. We also investigated neuron morphology using a combination of live cell imagine and immunocytochemistry for synaptic protein markers. Neuronal synaptic activity was recorded using multi-electrode array (MEA) as well as calcium imaging.

Results: The neurons reprogrammed from the generated patient-derived lines as well as the isogenic pair showed high expression of MAP2, SYN1, VGLUT1 and other neuronal markers. Q-PCR show varied expression levels of NRXN and NRXN associated genes in the autism and control lines:while the mutation lies in the NRXN1-α it not only affected this isoform, but also affected NRXN1-bas well as CASK. The neurons were then tested for electrophysiological properties using multi-electrode array.

Conclusions: We have successfully developed a highly consistent and efficient human cell model to study NRXN1 defects in the induced neurons. This model shows accelerated generation of excitatory neurons that express pan-neuronal and glutamatergic markers. The multielectrode array recordings show that those neurons are electrophysiologically functional with synchronised firing starting at Day 21 as compared to Day 100 using traditional differentiation methods.