Conference Contribution Details
Mandatory Fields
S. Avazzadeh, J. Reilly, S. Boomkamp, V. McInerney, J. Krawczyk, J. Fitzgerald, E. Forman, S. A. Lynch, S. Ennis, N. Cosemans, P. Dockery, L. R. Quinlan, L. Gallagher and S. Shen
International Society for Autism
Oral Presentation
Montreal Canada
Oral Presentation
Optional Fields
02-MAY-18
02-MAY-18
Background: Autism spectrum disorder (ASD) has a high co-morbidity with epilepsy clinically. Genetically it is associated with hundreds of rare risk factors, and NRXN1 gene is among the most common ones in ASD, schizophrenia, intellectual disability, epilepsy and developmental delay. The pre-synaptic protein Neurexin1 (NRXN1) signals bi-directionally through both excitation and inhibition. Previous studies suggest that the short presynaptic NRXN1β is primarily involved in excitation, whereas the long NRXN1α regulates both excitation and inhibition, via differential splicing and interactions with postsynaptic Neuroligins, GABAergic or Glutamatergic receptors and SHANKs. Deletions and/or mutations of the NRXN1 gene have been implicated in a number of neurodevelopmental diseases including ASD. However, the functional consequences of NRXN1 lesions are unknown, due to the lack of patient-derived disease models. Induced pluripotent stem cells (iPSCs) have the potential to revolutionize human disease modelling in vitro and to target unmet clinical needs. Objectives: NRXN1 protein is expressed in both excitatory and inhibitory synapses, and deletion of either heterozygous and homozygous NRXN1 has been implicated in altering the excitatory and inhibitory postsynaptic transmission. Furthermore, Neurexins are essential for coupling voltage-gated calcium channels to the release machinery. We hypothesize that NRXN1α+/- gene deletion may dysregulate the balance of synaptic excitation and inhibition and disturb the electrical electrical firing and calcium signalling of neurons. The aim of this study was to investigate the impact of NRXN1α+/- deletion on iPSC-derived neurons and uncover the functional phenotypes and molecular pathways. Methods: Using skin biopsies from 3 ASD patients with NRXN1a+/- deletion and 5 healthy donors, we derived induced pluripotent stem cells (iPSCs). The iPSCs were differentiated into 100-day cortical excitatory neurons using dual SMAD inhibition. Neuronal function were investigated using single cell patch clamping and calcium imaging. Furthermore, RNA sequencing was performed to investigate the underlying molecular mechanism. Results: 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. live cell calcium imaging on the 100-day neurons with Fluo4-AM showed neuronal networks displayed inherent spontaneous firing activity with a significant increase in the frequency and duration of calcium transients in NRXN1α+/- deletion neurons. The 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 signaling, vesicle exocytosis and synaptic transmission. Conclusions: Our results show for the first time that deletions of NRXN1α+/- gene impair the electrical firing 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. The NRXN1α+/- iPSCs may be offered as a human model with translatable phenotype for drug screening and testing of ASD.
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