CNTN5^−/+orEHMT2^−/+iPSC-Derived Neurons from Individuals with Autism Develop Hyperactive Neuronal Networks

Abstract

25Induced pluripotent stem cell (iPSC)-derived cortical neurons are increasingly 26 used as a model to study developmental aspects of Autism Spectrum Disorder 27 (ASD), which is clinically and genetically heterogeneous. To study the complex 28 relationship of rare (penetrant) variant(s) and common (weaker) polygenic risk 29 variant(s) to ASD, "isogenic" iPSC-derived neurons from probands and family-30 based controls, for modeling, is critical. We developed a standardized set of 31 procedures, designed to control for heterogeneity in reprogramming and 32 differentiation, and generated 53 different iPSC-derived glutamatergic neuronal 33 lines from 25 participants from 12 unrelated families with ASD (14 ASD-affected 34 individuals, 3 unaffected siblings, 8 unaffected parents). Heterozygous de novo 35 (7 families; 16p11.2, NRXN1, DLGAP2, CAPRIN1, VIP, ANOS1, THRA) and 36 rare-inherited (2 families; CNTN5, AGBL4) presumed-damaging variants were 37 characterized in ASD risk genes/loci. In three additional families, functional 38 candidates for ASD (SET), and combinations of putative etiologic variants 39 (GLI3/KIF21A and EHMT2/UBE2I combinations in separate families), were 40 modeled. We used a large-scale multi-electrode array (MEA) as our primary high-41 throughput phenotyping assay, followed by patch clamp recordings. Our most 42compelling new results revealed a consistent spontaneous network hyperactivity 43 3 in neurons deficient for CNTN5 or EHMT2. Our biobank of iPSC-derived neurons 44 and accompanying genomic data are available to accelerate ASD research. 45 46 47 of them show specificity for ASD alone (Malhotra and Sebat, 2012). These 65 genetic alterations are rare in the population (<1% population frequency), and in 66 some individuals, combinations of rare genetic variants affecting different genes 67 can be involved (Devlin and Scherer, 2012), including more complex structural 68 alterations of chromosomes (Brandler et al., 2018, Marshall et al., 2008.

Control iPSC lines with clinically annotated genetic variants for versatile multi-lineage differentiation

“…When we looked for overall novel genomic variation, 917 new single nucleotide variants and one intergenic 32 kb deletion (chr18:12137685-12169689) were found (Table 1). None of these variants were likely pathogenic, similar to our other reported gene edited lines (Deneault et al, 2018, 2019; Zaslavsky et al, 2019).…”

“…At the 7 seven-week time point, we observed synchronous firing across multiple electrodes (minimum 8/16 electrodes) within wells, indicative of neural circuit formation as measured by network burst frequencies. Neurons displayed weighted Mean Firing Rates (wMFR) ranging from 5 to 7.5 Hz and network burst frequencies ranging from 0.1 to 0.35 Hz, which were comparable to or more active (∼5 Hz and ∼0.1 Hz respectively) than our previously published MEA results from NGN2-derived neurons (Deneault et al, 2018, 2019). To confirm that recorded activity was due to synaptic transmission from glutamatergic excitatory neurons, we treated cells with an α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor inhibitor—6-cyano-7-nitroquinoxaline-2,3-dione (CNQX)—which abolished network bursting (Fig.…”

“…To evaluate PGPC iPSC-derived neurons, we infected PGPC lines and a previously published control iPSC line (WT37) (Cheung et al, 2011) with lentivirus bearing doxycycline-inducible NEUROG2 to generate homogenous populations of excitatory cortical neurons (Zhang et al, 2013). Neurons were induced with doxycycline for one week and selected with puromycin and Ara-C (Deneault et al, 2018, 2019) in 6-well plates then re-seeded to 24-well dishes for morphological analysis in co-cultures with mouse astrocytes over seven weeks (Fig. 1A).…”

“…To investigate activity of the variant-preferred PGPC3 neurons, we collected weekly recordings from 48-well MEA plates (Axion BioSystems) for extracellular electrophysiology measurements (Deneault et al, 2018, 2019) over 6 weeks (weeks 2-7). Representative raster plots of PGPC3_75 showed progression of spontaneous activity at three weeks compared to development of network bursts at 5 and 7 weeks (Fig.…”

Systematic phenomics analysis of ASD-associated genes reveals shared functions and parallel networks underlying reversible impairments in habituation learning

“…Indeed, these Drosophila and C. elegans results likely reflect a behavioral outcome of circuit-level hyperexcitability recently discovered in several human iPSC derived neuronal culture models of a number of monogenic ASD risk factors. 68,139 The results also provide a plausible explanation for inconsistent reports of impaired habituation in humans, which variably employ diverse response metrics most often without genetic stratification of patient populations. 69 While these shared phenotypes are exciting, our results also reveal a remarkable diversity and modularity in phenotypic disruptions, and thus clearly indicate that single phenotype in vivo functional validation and characterization efforts will be insufficient to capture the complex multi-facetted phenotypic disruptions that stem from inactivating mutations in ASD-associated genes.…”