Stem cell modelling for the role of NRXN1 deletion in Autism Spectrum Disorder

Autism spectrum disorder (ASD), a multi-factorial disease, often has co-morbidity with epilepsy, which is associated with excessive neuronal firing. The pre-synaptic protein Neurexin1 (NRXN1) signals bi-directionally through both excitation and inhibition, by forming synaptic complexes with post-synaptic Neuroligins, GABAergic or Glutamatergic receptors, and the scaffold proteins SHANKs. Deletions and/or mutations of the NRXN1 gene have been implicated in a number of neurodevelopmental diseases including ASD. However, patient-derived disease models are lacking. Induced pluripotent stem cells (iPSCs) have the potential to revolutionize human disease modelling in vitro and to target unmet clinical needs. We hypothesize that NRXN1α gene deletion may dysregulate the balance of synaptic excitation and inhibition. Using skin biopsies from ASD patients with NRXN1α deletion and healthy donors, we converted dermal fibroblasts into iPSCs by reprogramming. Their pluripotency was validated by the expression of stem cell markers (OCT4, SOX2, NANOG, SSEA4, TRA-1-60, TRA-1-81). The iPSCs were directionally differentiated into cortical glutamatergic neurons using a dual SMAD inhibition method. Neural stem cells (NSCs) derived from the iPSCs were shown to express the neural progenitor markers of NESTIN, FOXG1 and PAX6. The 100-day-old neurons were shown to express markers of neuronal maturity (MAP2) and synapses (SYN1), as well as ion channels and transporters, at both the RNA and protein levels. They also transcribed pre- and post-synaptic interaction partners of NRXN1, including CASK, MINT, MUNC18-1 PSD-95, NLGNs and Shanks. There was no significant difference between the control and NRXN1α deletion groups in the proliferation of iPSCs and NSCs, neuronal differentiation or maturation, suggesting that NRXN1α deletion may not affect early neurogenesis. Neuronal function was investigated using single cell patch clamping, and the 100-day neurons with NRXN1α deletion displayed higher potassium and sodium currents, with selectively impaired depolarization and repolarization characteristics. The action potential amplitude was significantly increased, whereas the action potential threshold was decreased in NRXN1α deletion neurons. The repolarization slope was significantly increased and consequently, the repolarization duration was decreased. Moreover, we have carried out live cell calcium imaging on the 100-day neurons with Fluo4-AM, and neuronal networks displayed inherent spontaneous firing activity with a significant increase in the frequency and duration of calcium transients in NRXN1α deletion neurons. The whole genome transcriptome analyses have demonstrated substantial up-regulation in ion channels and transporter activity, with voltage-gated calcium channels (VGCCs), voltage-gated potassium channels (VGKCs) and voltage-gated sodium channels (VGSCs) being mostly enriched among the differentially expressed genes. In addition, the KEGG pathway analyses have revealed further impairments in calcium signalling, vesicle exocytosis, synaptic transmission and MAPK pathways. Our results show for the first time that heterozygous deletions of NRXN1α gene directly impair the non-synaptic function of human neurons, in addition of their calcium transients, illustrating the value of this patient-derived iPSC model with NRXN1α deletion for studying ASD disease phenotypes

Funder

Science Foundation Ireland

Publisher

NUI Galway

Rights

Attribution-NonCommercial-NoDerivs 3.0 Ireland

cbn

Collections

University of Galway Theses (PhD Theses)