Increased Ca2+ signaling in NRXN1α+/− neurons derived from ASD induced pluripotent stem cells

Abstract

BackgroundAutism spectrum disorder (ASD) is a neurodevelopmental disorder with a high co-morbidity of epilepsy and associated with hundreds of rare risk factors. NRXN1 deletion is among the commonest rare genetic factors shared by ASD, schizophrenia, intellectual disability, epilepsy, and developmental delay. However, how NRXN1 deletions lead to different clinical symptoms is unknown. Patient-derived cells are essential to investigate the functional consequences of NRXN1 lesions to human neurons in different diseases.MethodsSkin biopsies were donated by five healthy donors and three ASD patients carrying NRXN1α+/− deletions. Seven control and six NRXN1α+/− iPSC lines were derived and differentiated into day 100 cortical excitatory neurons using dual SMAD inhibition. Calcium (Ca2+) imaging was performed using Fluo4-AM, and the properties of Ca2+ transients were compared between two groups of neurons. Transcriptome analysis was carried out to undercover molecular pathways associated with NRXN1α+/− neurons.ResultsNRXN1α+/− neurons were found to display altered calcium dynamics, with significantly increased frequency, duration, and amplitude of Ca2+ transients. Whole genome RNA sequencing also revealed altered ion transport and transporter activity, with upregulated voltage-gated calcium channels as one of the most significant pathways in NRXN1α+/− neurons identified by STRING and GSEA analyses.ConclusionsThis is the first report to show that human NRXN1α+/− neurons derived from ASD patients’ iPSCs present novel phenotypes of upregulated VGCCs and increased Ca2+ transients, which may facilitate the development of drug screening assays for the treatment of ASD.

Human in vitro models for understanding mechanisms of autism spectrum disorder

“…Several genetic forms of ASD show a decrease in the number of neurons and synapses, including Timothy syndrome-in which there was a decrease in the fraction of neurons expressing lower layer markers [142] and 22q11.2 deletion, which showed a reduced number of neurons accompanied by an increase in the number of astrocytes [124]. Three studies on NRXN1 mutations also found evidence for a decrease in neuronal maturation [134,135,137], a finding which was not replicated in a different study [136]. Similar results (downregulation of neuronal processes) were indirectly observed using transcriptomic analysis from neurons in which CHD8 was either knocked down [132] or heterozygously deleted [131].…”

“…Two kinds of genetic modeling have been performed using cells either from individuals whose genetic contributions are unknown or undefined, so called idiopathic [59,60,[112][113][114][115][116][117][118][119][120], or from individuals harboring major effect mutations that are presumed causal or which have been engineered to carry these mutations. These mutations include ASD-associated CNVs such as 15q11q13 deletion (Angelman syndrome) [121] and duplication (Dup15q syndrome) [122], 22q11.2 deletion (DiGeorge syndrome) [123,124], 16p11.2 deletion and duplication [125], and 15q13.3 deletion [126], as well as single-gene mutations including SHANK3 [127][128][129][130], CHD8 [131,132], NRXN1 [133][134][135][136][137], NLGN4 [138], EHMT1 (Kleefstra syndrome) [139], PTCHD1-AS [140], UBE3A (Angelman's syndrome) [141], and CACNA1C (Timothy syndrome) [142] (summarized in Table 1). In this review, we will not discuss fragile X syndrome, Rett's syndrome, and tuberous sclerosis-related autism as they have all been extensively reviewed previously [148][149][150][151][152][153][154].…”

ASDmiR: A Stepwise Method to Uncover miRNA Regulation Related to Autism Spectrum Disorder

“…As for the identified hub miRNA sponges, functional enrichment analysis results indicate that they are significantly enriched in SMAD binding (GO: 0046332). A previous study ( Avazzadeh et al, 2019 ) has demonstrated that SMAD binding is closely related to ASD. Altogether, the above functional enrichment analysis results imply that the identified hub genes are closely associated with the occurrence and development of ASD.…”

Human stem cell-based models for studying autism spectrum disorder-related neuronal dysfunction

“…Different results have been communicated regarding the functional consequences of mutations in NRXN1 , which encodes for a presynaptic membrane cell adhesion protein. Avazzadeh et al demonstrated that glutamatergic cortical neurons derived from NRXN1α +/− iPSC lines exhibit increased frequency, duration and amplitude of calcium transients in comparison to control neurons derived from healthy donors [ 78 ]. On the other hand, neurons differentiated from patient iPSCs with biallelic NRXN1α deletion displayed a decreased amplitude and slower rise time of depolarization-evoked calcium transients, as well as a reduction in action potential amplitude [ 79 ].…”