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Excitatory dysfunction drives network and calcium handling deficits in 16p11.2 duplication schizophrenia iPSC-derived neurons

  • Author Footnotes
    † Authors contributed equally
    Euan Parnell
    Footnotes
    † Authors contributed equally
    Affiliations
    Department of Neuroscience, Northwestern University Feinberg School of Medicine, Chicago, 60611 IL, USA

    Northwestern University Cecnter for Autism and Neurodevelopment, Chicago IL 60611, USA
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  • Author Footnotes
    † Authors contributed equally
    Lorenza Culotta
    Footnotes
    † Authors contributed equally
    Affiliations
    Department of Neuroscience, Northwestern University Feinberg School of Medicine, Chicago, 60611 IL, USA

    Northwestern University Cecnter for Autism and Neurodevelopment, Chicago IL 60611, USA
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  • Marc P. Forrest
    Affiliations
    Department of Neuroscience, Northwestern University Feinberg School of Medicine, Chicago, 60611 IL, USA

    Northwestern University Cecnter for Autism and Neurodevelopment, Chicago IL 60611, USA
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  • Hiba A. Jalloul
    Affiliations
    Department of Neuroscience, Northwestern University Feinberg School of Medicine, Chicago, 60611 IL, USA

    Northwestern University Cecnter for Autism and Neurodevelopment, Chicago IL 60611, USA
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  • Blair L. Eckman
    Affiliations
    Department of Neuroscience, Northwestern University Feinberg School of Medicine, Chicago, 60611 IL, USA

    Northwestern University Cecnter for Autism and Neurodevelopment, Chicago IL 60611, USA
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  • Daniel D. Loizzo
    Affiliations
    Department of Neuroscience, Northwestern University Feinberg School of Medicine, Chicago, 60611 IL, USA

    Northwestern University Cecnter for Autism and Neurodevelopment, Chicago IL 60611, USA
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  • Katherine K.E. Horan
    Affiliations
    Department of Neuroscience, Northwestern University Feinberg School of Medicine, Chicago, 60611 IL, USA

    Northwestern University Cecnter for Autism and Neurodevelopment, Chicago IL 60611, USA
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  • Marc Dos Santos
    Affiliations
    Department of Neuroscience, Northwestern University Feinberg School of Medicine, Chicago, 60611 IL, USA

    Northwestern University Cecnter for Autism and Neurodevelopment, Chicago IL 60611, USA
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  • Nicolas H. Piguel
    Affiliations
    Department of Neuroscience, Northwestern University Feinberg School of Medicine, Chicago, 60611 IL, USA

    Northwestern University Cecnter for Autism and Neurodevelopment, Chicago IL 60611, USA
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  • Derek J.C. Tai
    Affiliations
    Program in Medical and Population Genetics, Broad Institute of MIT and Harvard, Cambridge, MA, USA

    Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, USA
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  • Hanwen Zhang
    Affiliations
    Center for Psychiatric Genetics, NorthShore University HealthSystem, Evanston, IL 60201, USA

    Department of Psychiatry and Behavioral Neurosciences, The University of Chicago, Chicago, IL 60637, USA
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  • Tracy S. Gertler
    Affiliations
    Division of Neurology, Department of Pediatrics, Ann and Robert H Lurie Childrens Hospital of Chicago; Department of Pharmacology, Northwestern University Feinberg School of Medicine, Chicago, 60611 IL, USA
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  • Dina Simkin
    Affiliations
    Ken and Ruth Davee Department of Neurology, Northwestern University Feinberg School of Medicine, Chicago, 60611 IL, USA

    Department of Neuroscience, Northwestern University Feinberg School of Medicine, Chicago, 60611 IL, USA
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  • Alan R. Sanders
    Affiliations
    Center for Psychiatric Genetics, NorthShore University HealthSystem, Evanston, IL 60201, USA

    Department of Psychiatry and Behavioral Neurosciences, The University of Chicago, Chicago, IL 60637, USA
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  • Michael E. Talkowski
    Affiliations
    Program in Medical and Population Genetics, Broad Institute of MIT and Harvard, Cambridge, MA, USA

    Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, USA
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  • Pablo V. Gejman
    Affiliations
    Center for Psychiatric Genetics, NorthShore University HealthSystem, Evanston, IL 60201, USA

    Department of Psychiatry and Behavioral Neurosciences, The University of Chicago, Chicago, IL 60637, USA
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  • Evangelos Kiskinis
    Affiliations
    Ken and Ruth Davee Department of Neurology, Northwestern University Feinberg School of Medicine, Chicago, 60611 IL, USA

    Department of Neuroscience, Northwestern University Feinberg School of Medicine, Chicago, 60611 IL, USA
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  • Jubao Duan
    Affiliations
    Center for Psychiatric Genetics, NorthShore University HealthSystem, Evanston, IL 60201, USA

    Department of Psychiatry and Behavioral Neurosciences, The University of Chicago, Chicago, IL 60637, USA
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  • Peter Penzes
    Correspondence
    Corresponding Author: Peter Penzes, 303 E. Chicago Ave, Feinberg School of Medicine, Ward 7-176, Chicago, IL 60611, Tel: +1 312 503 5379,
    Affiliations
    Department of Neuroscience, Northwestern University Feinberg School of Medicine, Chicago, 60611 IL, USA

    Northwestern University Cecnter for Autism and Neurodevelopment, Chicago IL 60611, USA

    Division of Neurology, Department of Pediatrics, Ann and Robert H Lurie Childrens Hospital of Chicago; Department of Pharmacology, Northwestern University Feinberg School of Medicine, Chicago, 60611 IL, USA
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  • Author Footnotes
    † Authors contributed equally
Open AccessPublished:November 09, 2022DOI:https://doi.org/10.1016/j.biopsych.2022.11.005

      Background

      Schizophrenia (SCZ) is a debilitating psychiatric disorder with a large genetic contribution; however, its neurodevelopmental substrates remain largely unknown. Modeling pathogenic processes in SCZ using human iPSC-derived neurons (iNs) has emerged as a promising strategy. Copy number variations (CNV) confer high genetic risk for SCZ, with duplication of the 16p11.2 locus increasing risk 14.5 fold.

      Methods

      To dissect the contribution of excitatory (iEN) versus GABAergic (iGN) neurons to SCZ pathophysiology, we induced iNs from CRISPR-Cas9 isogenic and SCZ patient-derived iPSCs, and analyzed SCZ-related phenotypes in iEN monocultures and iEN/iGN cocultures.

      Results

      In iEN/iGN cocultures, neuronal firing and synchrony was reduced at later, but not earlier, stages of in vitro development. These were fully recapitulated in iEN monocultures, indicating a primary role for excitatory neurons. Moreover, isogenic iENs showed reduced dendrite length and deficits in calcium handling. iENs from 16p11.2 duplication-carrying SCZ patients displayed overlapping deficits in network synchrony, dendrite arborization and calcium handling. Transcriptomic analysis of both iEN cohorts revealed molecular markers of disease related to the glutamatergic synapse, neuroarchitecture and calcium regulation.

      Conclusions

      Our results indicate the presence of 16p11.2 duplication-dependent alterations in SCZ patient derived iEN. Transcriptomics and cellular phenotyping reveal overlap between isogenic and patient-derived iENs, suggesting a central role of glutamatergic, morphological and calcium dysregulation in 16p11.2 duplication-mediated pathogenesis. Moreover, excitatory dysfunction during early neurodevelopment is implicated as the basis of SCZ pathogenesis in 16p11.2 duplication carriers. Our results support network synchrony and calcium handling as outcomes directly linked to this genetic risk variant.

      Keywords

      Methods and Materials

      Cell lines

      Isogenic CRISPR-Cas9 modified 16p11.2 duplication cells were generated from GM08330 iPSCs (Control, Coriell Institute) and provided for this study by Dr Talkowski (Boston, MA, USA). The generation and characterization of these CRISPR-modified and control lines has been outlined previously (
      • Tai D.J.
      • Ragavendran A.
      • Manavalan P.
      • Stortchevoi A.
      • Seabra C.M.
      • Erdin S.
      • et al.
      Engineering microdeletions and microduplications by targeting segmental duplications with CRISPR.
      ). Patient iPSCs – SCZ patient (02C12626, 04C27671, 06C58821) and age/ethnicity matched CPLs (06C52982, 05C51897, 05C44627) were sourced from the MSG1/2 schizophrenia clinical cohorts (
      • Shi J.
      • Levinson D.F.
      • Duan J.
      • Sanders A.R.
      • Zheng Y.
      • Pe'er I.
      • et al.
      Common variants on chromosome 6p22.1 are associated with schizophrenia.
      ,
      • Levinson D.F.
      • Duan J.
      • Oh S.
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      Copy number variants in schizophrenia: confirmation of five previous findings and new evidence for 3q29 microdeletions and VIPR2 duplications.
      ,
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      The Internet-based MGS2 control sample: self report of mental illness.
      ), study Accessions: phs000021.v2.p1 and phs000167.v1.p1. Derivation, culture and polygenic risk scoring (PRS) are described in Supplementary Methods.

      iN Differentiation

      iEN (
      • Zhang Y.
      • Pak C.
      • Han Y.
      • Ahlenius H.
      • Zhang Z.
      • Chanda S.
      • et al.
      Rapid single-step induction of functional neurons from human pluripotent stem cells.
      ) and iGN (
      • Yang N.
      • Chanda S.
      • Marro S.
      • Ng Y.H.
      • Janas J.A.
      • Haag D.
      • et al.
      Generation of pure GABAergic neurons by transcription factor programming.
      ) were generated by Ngn2 and Ascl1/Dlx2 induction, respectively. Full protocol available in Supplemental Methods.

      Statistical Analyses

      Statistical analyses were performed using GraphPad Prism v8.0.1 (GraphPad Software Inc.). For details of clone and patient replication, see Supplementary Methods. All data were assessed for Gaussian distribution by Shapiro-Wilk test – based on the results, parametric or non-parametric two-tailed unpaired t-tests were performed with P-value thresholds; *P<0.05, **P<0.01, ***P<0.005, ****P<0.001. A single round of outlier detection (ROUT Q=1%) was performed for each complete data set, and figures represents the cleaned data.

      Data Availability

      Full Methods and Supplementary information is available at BP’s website. Transcriptomic data sets are available at GEO (https://www.ncbi.nlm.nih.gov/geo/, accession #GSE215183).

      Introduction

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      )), the etiological basis of SCZ in early neurodevelopment remains largely unknown.
      The clearest insights into SCZ pathogenesis are from patient studies, which have revealed neuronal deficits likely implicated in disease etiology and pathophysiology. Genome-wide association studies (GWAS) have identified synaptic plasticity as a key molecular pathway disrupted in SCZ, with a strong enrichment of variants within synaptic proteins and voltage gated calcium channels (VGCC) (
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      ). Post mortem studies in SCZ patients have also identified consistent alterations in cortical neuron morphology; SCZ neurons display a dramatic reduction in dendritic length and complexity (
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      )). These findings are consistent with alterations in neuronal network activity observed within the cortex of SCZ patients (
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      Despite these alterations to neuronal circuitry in SCZ, little is known about the early neurodevelopmental deficits that may contribute to SCZ pathogenesis. This is important as SCZ is largely viewed as a neurodevelopmental disorder, despite the typical SCZ age of onset at 15-30 (
      • Birnbaum R.
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      Cross-Disorder Group of the Psychiatric Genomics Consortium. Electronic address pmhe, Cross-Disorder Group of the Psychiatric Genomics C (2019): Genomic Relationships, Novel Loci, and Pleiotropic Mechanisms across Eight Psychiatric Disorders. Cell. 179:1469-1482 e1411.

      ,
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      ) and altered gamma-oscillations within the cortex (
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      ) are observed before the onset of psychoses and formal SCZ diagnoses. Together, these observations support the etiological roots of SCZ in early neurodevelopment. Therefore, appropriate paradigms are required to address the etiology of SCZ within this developmental period; human induced pluripotent stem cells (iPSCs) have emerged as a powerful tool in the study of complex genetic neurodevelopmental disorders (NDD), including SCZ (reviewed in (
      • Ardhanareeswaran K.
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      )). iPSC-derived neurons (iNs) provide a faithful representation of early prenatal neurodevelopment and allow the impact of SCZ risk genes to be evaluated at this critical time point. Therefore, iNs provide an unrivalled approach to dissect early neurodevelopmental dysfunction (
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      ).
      In order to accurately model early SCZ, appropriate iPSC-derived models are required. The recently developed ability to direct the differentiation of iPSCs into pure subpopulations of glutamatergic (iENs, (
      • Zhang Y.
      • Pak C.
      • Han Y.
      • Ahlenius H.
      • Zhang Z.
      • Chanda S.
      • et al.
      Rapid single-step induction of functional neurons from human pluripotent stem cells.
      )) and GABAergic neurons (iGNs, (
      • Yang N.
      • Chanda S.
      • Marro S.
      • Ng Y.H.
      • Janas J.A.
      • Haag D.
      • et al.
      Generation of pure GABAergic neurons by transcription factor programming.
      )) provides a unique means to isolate neuronal subtype-specific deficits that may contribute to excitatory/inhibitory (E/I) imbalance and SCZ susceptibility (
      • Gao R.
      • Penzes P.
      Common mechanisms of excitatory and inhibitory imbalance in schizophrenia and autism spectrum disorders.
      ). However, the heterogenetic nature of SCZ necessitates large patient cohorts to identify conserved markers of neuronal impairment – a major hurdle in identifying neuronal pathogenesis in SCZ. One powerful approach is the study of genetically defined cohorts (
      • Brennand K.J.
      • Landek-Salgado M.A.
      • Sawa A.
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      ), allowing the assessment of shared large effect size genetic variants that confer strong risk to SCZ, and the manner in which they alter neurodevelopment and contribute to SCZ pathogenesis.
      Copy number variants (CNVs) are a type of genetic variant that have the largest effect size in SCZ (
      • Marshall C.R.
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      ). CNVs at the 16p11.2 locus contribute considerable risk for NDDs (
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      ). Whereas both deletion and duplication confer risk for ASD and intellectual disability (
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      ), only duplications are associated with SCZ and psychosis (
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      ), and bipolar disorder (
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      ). Isogenic iPSC and animal models cannot present the full complex interplay of common low effect-size genetic factors observed to be a significant contributing factor in SCZ patients (
      • Ripke S.
      • O'Dushlaine C.
      • Chambert K.
      • Moran J.L.
      • Kahler A.K.
      • Akterin S.
      • et al.
      Genome-wide association analysis identifies 13 new risk loci for schizophrenia.
      ). For these reasons, studying the effect of 16p11.2 duplication in SCZ patient-derived iPSCs may be required to reveal mechanisms that contribute to SCZ progression. The relatively common incidence of 16p11.2 duplication (∼1:4000 live births (
      • Gillentine M.A.
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      )) facilitates the generation of SCZ-16p11.2 duplication patient-derived cohorts. Combined with the large contribution to SCZ risk, 16p11.2 duplication SCZ patient-derived iPSCs provide an attractive model to assess the etiological roots of SCZ. However, to date, no SCZ patient-derived iN studies have been performed on the 16p11.2 duplication risk factor.
      We therefore set out to investigate the role of 16p11.2 duplication in SCZ pathogenesis using iNs. Due to phenotypic noise associated with diverse patient genetic backgrounds, these analyses were first performed employing Crispr-Cas9 modified isogenic lines, where the 16p11.2 duplication was introduced into an iPSC line derived from a healthy individual. To determine the contribution of excitatory versus inhibitory populations, we assessed phenotypic outcomes in iEN/iGN cocultures and observed reduced neuronal network firing rate and synchrony at later stages of in vitro development. These deficits were fully reproduced in iEN monocultures, indicating a primary role for excitatory neurons. In addition, isogenic 16p11.2 duplication iENs showed reduced dendrite length and deficits in calcium handling. In concert, we report the first analyses of SCZ patient iNs harboring the 16p11.2 duplication. Analysis 16p11.2 duplication-carrying SCZ patient iENs revealed overlapping deficits in network synchrony, dendrite arborization and calcium handling, supporting the SCZ relevance of these 16p11.2 duplication endo-phenotypes. Interestingly, “Homophilic cell adhesion”, “Neuron projection”, “Glutamatergic synapse” and “Calcium ion binding” gene sets were found to be altered in isogenic, patient-derived and previous 16p11.2 duplication neuronal studies, indicating them to be strong markers of 16p11.2 duplication-mediated dysfunction in line with observed cellular phenotypes.
      Taken together, our comparative analysis supports excitatory neuronal dysfunction early in neurodevelopment as a basis for SCZ pathogenesis, and identifies synaptic circuit and calcium handling deficits directly linked to this genetic variant. Moreover, these observations are closely related to phenotypes reported in idiopathic SCZ iN (
      • Brennand K.J.
      • Simone A.
      • Jou J.
      • Gelboin-Burkhart C.
      • Tran N.
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      ) and have strong correlates to patient intermediate phenotypes (
      • Uhlhaas P.J.
      • Singer W.
      Abnormal neural oscillations and synchrony in schizophrenia.
      ). Our studies also identified phenotypic differences between isogenic and patient-derived neurons, potentially linked to sex or genetic background.

      Results

      Deficits in neuronal network development in 16p11.2 duplication iEN/iGN cocultures

      The study of large CNVs, such as the 16p11.2 duplication, has been hindered by the inability to generate faithful genetic models to accurately determine molecular and neuronal dysfunction. However, the recent generation of a CRISPR-Cas9 edited isogenic 16p11.2 duplication iPSC model (
      • Tai D.J.
      • Ragavendran A.
      • Manavalan P.
      • Stortchevoi A.
      • Seabra C.M.
      • Erdin S.
      • et al.
      Engineering microdeletions and microduplications by targeting segmental duplications with CRISPR.
      ) provides the means to identify phenotypes arising directly from the 16p11.2 duplication CNV, independently of variability from diverse genetic backgrounds. Thus, we used isogenic 16p11.2 duplication (DUP) and corresponding control (Ctrl) iPSC lines for the generation of iNs. These isogenic cell lines were previously subject to extensive quality control, including RNAseq, chromosomal microarray and western blot analysis (
      • Tai D.J.
      • Ragavendran A.
      • Manavalan P.
      • Stortchevoi A.
      • Seabra C.M.
      • Erdin S.
      • et al.
      Engineering microdeletions and microduplications by targeting segmental duplications with CRISPR.
      ), as well as pluripotency, karyotype analysis and iPSC expression of 16p11.2 genes (
      • Sundberg M.
      • Pinson H.
      • Smith R.S.
      • Winden K.D.
      • Venugopal P.
      • Tai D.J.C.
      • et al.
      16p11.2 deletion is associated with hyperactivation of human iPSC-derived dopaminergic neuron networks and is rescued by RHOA inhibition in vitro.
      ) confirming the presence of the 16p11.2 duplication and the absence of additional chromosomal abnormalities. Because both excitatory and inhibitory neurons have been implicated in SCZ, we differentiated iPSCs (
      • Tai D.J.
      • Ragavendran A.
      • Manavalan P.
      • Stortchevoi A.
      • Seabra C.M.
      • Erdin S.
      • et al.
      Engineering microdeletions and microduplications by targeting segmental duplications with CRISPR.
      ) into excitatory (iEN (
      • Zhang Y.
      • Pak C.
      • Han Y.
      • Ahlenius H.
      • Zhang Z.
      • Chanda S.
      • et al.
      Rapid single-step induction of functional neurons from human pluripotent stem cells.
      ), Fig 1a) and GABAergic (iGN (
      • Yang N.
      • Chanda S.
      • Marro S.
      • Ng Y.H.
      • Janas J.A.
      • Haag D.
      • et al.
      Generation of pure GABAergic neurons by transcription factor programming.
      ), Fig 1b) lineages. iEN/iGN cocultures were generated by combining iENs and iGNs at an 80/20% ratio (Fig 1c), reflecting the neuronal content of the cortex, and matured over a 7 week in vitro (WIV) period on multi-electrode arrays (MEAs, Fig 1d). Cocultured iEN/iGN (Fig S1a), iEN (Fig S1b) and iGN (Fig 1c) cultures were observed to express neuronal markers MAP2 and Synapsin (Syn1), and iGNs expressed the GABAergic marker, GABA, confirming the presence of mature neuronal cultures suitable for the assessment of 16p11.2 duplication phenotypes. GFP-labeled iENs (Fig S1b), iGNs (Fig S1c), and cocultured GFP/RFP iENs/iGNs (Fig S1a) were assessed in terms of neuronal induction efficiency. >98% of GFP/RFP iNs in mono- or co-cultures expressed the neuronal marker MAP2 (Fig S1d-f) indicating successful and highly homogenous neuronal induction with no altered differentiation efficiency between genotypes. Within cocultures, assessment of the iEN/iGN ratio confirmed no differences in seeding density or iEN/iGN viability between genotypes at 7WIV (Fig S1g).
      Figure thumbnail gr1
      Figure 1Modeling 16p11.2 duplication using isogenic 16p11.2 duplication iN. a) Schematic overview of iEN induction. b) Schematic overview of iGN induction. c) Schematic overview of iEN/iGN coculture model. d) Representative MEA plate well of cocultured iENs (GFP) and iGNs (RFP, at 20x, transmission at 10x). Developmental time course of activity (e) and network synchrony (f). g) Representative raster plot of cocultured iENs/iGNs electrical activity at 7WIV. Each well electrode is shown (rows) vs time (x-axis). Mean firing rate (h), synchrony index (i) and network burst frequency (j) at 7WIV (N=6, 2 clones per condition from 3 independent differentiations, n = 24 wells per condition). Ctrl cocult = control iENs/iGNs, DUP cocult = 16p11.2 duplication isogenic iENs/iGNs.
      In both Ctrl and DUP, iEN/iGN network activity developed over 7WIV in terms of firing rate (Fig 1e) and synchronous (simultaneous events across multiple electrodes, Fig S2a) firing (Fig 1f), indicating the presence of a mature and functional network. 7WIV DUP cocultures displayed a significant decrease in mean firing rate (Fig 1e) and synchrony index (Fig 1f) compared to Ctrl lines. Firing rate and network properties were assessed in depth at this time point (Fig 1g). In addition to reduced mean firing rate (Fig 1h), DUP neurons exhibited reduced synchrony (Fig 1i) and network burst frequency (Fig 1j, bursts occurring over multiple electrodes simultaneously, see Fig S2b) and low clonal variability was observed (Fig S2c-e). Together these results reveal highly dysregulated electrical activity and neuronal network formation as a direct result of 16p11.2 duplication.

      16p11.2 duplication iEN monocultures recapitulate iEN/iGN coculture network deficits

      To delineate iGN-mediated versus intrinsic iEN deficits, we took advantage of the ability to culture iENs independently of GABAergic input and compared phenotypes in DUP and Ctrl iEN monocultures. We seeded iEN on MEA (Fig 2a) and matured them over 7WIV. DUP iENs were observed to have significantly lower firing rate (Fig 2b) and synchrony (Fig 2c). At 7WIV, iENs recapitulated all activity and network (Fig 2d-h) deficits observed in iEN/iGN and these effects were not due to clonal variation (Fig S2f-h). As expected, TTX totally abolished all events detected by MEA (Fig S3). Application of ion channel antagonists at 4WIV indicate ∼40% of activity was due to synaptic AMPAr, and 20% due to NMDAr activity (Fig S3a,c). However, at 7WIV, NBQX inhibited 90% and APV blocked ∼50% of activity (Fig S3b,d), suggesting maturation of synaptic connectivity between 4 and 7WIV coinciding with the presentation of network synchrony, and of DUP phenotypes. Moreover, these results confirm a pronounced effect of 16p11.2 duplication on excitatory neuronal function and network formation, independently of iGNs. These results were not due to the lack of integrity of iGNs within the iENs/iGNs cocultures as transcriptomic analyses indicate the presence of interneuron markers and MEA analyses demonstrate the functionality of iGNs in altering the activity, network formation and longitudinal development of cultures (Fig S4).
      Figure thumbnail gr2
      Figure 2Analysis of neuronal network activity and transcriptome of isogenic 16p11.2 duplication iEN monoculture. a) Representative MEA well of monocultured iENs, Scale bar - 50 μm. Developmental time course of iEN activity (b) and network synchrony (c). d) Representative raster plot of iENs activity at 7WIV. Well and plate averages of firing rate (e), synchrony index (f) and network burst frequency (g) at 7WIV (N=6, 2 clones from 3 independent differentiations, n=30 wells per condition). Ctrl = control lines, DUP = 16p11.2 duplication. h) Volcano plot of DEGs identified in isogenic DUP lines (FDR<0.1, blue = upregulated, red = downregulated, 16698 expressed with mean read counts>5) with top 10 non-16p11.2 genes highlighted (black). i) volcano plot and j) heatmap of 16p11.2 gene expression (fold change over mean control). C1_A=clone1, sample A, etc. k) Significantly enriched GO terms within 16p11.2 duplication iENs. Volcano plot of top GO terms; “Homophilic cell adhesion”, green (i), “Glutamatergic Synapse”, purple (m) and “Calcium ion binding”, yellow (n) and heatmap of aggregated DEGs from these terms (o).
      To guide further analyses of cellular phenotypes, we employed an unbiased approach to determine altered molecular pathways that may contribute to iEN dysfunction. Control and DUP iENs were matured to 7WIV before RNA extraction and sequencing. 62 genes were found to be upregulated, and 133 downregulated within DUP iEN (Fig 2h, Table S1). As expected, expressed 16p11.2 genes were significantly upregulated 1.2-1.7 fold, consistent with the presence of a third copy of each gene (Fig 2i-j). Gene ontology (GO) analyses were performed on significantly altered up and downregulated gene sets independently (excluding 16p11.2 genes, false discovery rate normalized P-value (FDR)<0.1) to identify molecular pathways that could direct cellular phenotyping (Table S2). GO terms associated with significantly down-regulated DEGs included “Homophilic cell adhesion”, “Neuron projection development”, “glutamatergic synapse” and “Calcium Ion Binding” (Fig 2k). Few GO terms were associated with up-regulated DEGs, with the strongest term being “Regulation of cell shape” (FDR=0.44). 16p11.2 duplication is a risk factor for numerous neurodevelopmental disorders, such as ASD, ID, epilepsy as well as SCZ (
      • Niarchou M.
      • Chawner S.
      • Doherty J.L.
      • Maillard A.M.
      • Jacquemont S.
      • Chung W.K.
      • et al.
      Psychiatric disorders in children with 16p11.2 deletion and duplication.
      ). To assess the DUP transcriptome in terms of disease profile, enrichment of risk genes was ascertained from exome sequencing and GWAS in SCZ, epilepsy, ASD and ID (
      • Fromer M.
      • Pocklington A.J.
      • Kavanagh D.H.
      • Williams H.J.
      • Dwyer S.
      • Gormley P.
      • et al.
      De novo mutations in schizophrenia implicate synaptic networks.
      ,
      • Howrigan D.P.
      • Rose S.A.
      • Samocha K.E.
      • Fromer M.
      • Cerrato F.
      • Chen W.J.
      • et al.
      Exome sequencing in schizophrenia-affected parent-offspring trios reveals risk conferred by protein-coding de novo mutations.
      ,
      • Genovese G.
      • Fromer M.
      • Stahl E.A.
      • Ruderfer D.M.
      • Chambert K.
      • Landen M.
      • et al.
      Increased burden of ultra-rare protein-altering variants among 4,877 individuals with schizophrenia.
      ,
      Schizophrenia Working Group of the Psychiatric Genomics C
      Biological insights from 108 schizophrenia-associated genetic loci.
      ,
      • Rees E.
      • Han J.
      • Morgan J.
      • Carrera N.
      • Escott-Price V.
      • Pocklington A.J.
      • et al.
      De novo mutations identified by exome sequencing implicate rare missense variants in SLC6A1 in schizophrenia.
      ). We found significant enrichment exclusively in SCZ exome-wide de novo variants (Fig S5a) and SCZ GWAS loci (Fig S5b), as well as the presence of multiple high confidence SCZ risk genes (Fig S5C, (
      • Wang D.
      • Liu S.
      • Warrell J.
      • Won H.
      • Shi X.
      • Navarro F.C.P.
      • et al.
      Comprehensive functional genomic resource and integrative model for the human brain.
      )). These results support the suitability of isogenic 16p11.2 duplication iENs to model molecular pathways relevent to SCZ. Moreover, SCZ-associated risk genes (Fig S5d) were observed within the observed GO terms, such as PCDHA2 (Fig 2l), Shank1 (Fig 2m) and NCAN (Fig 2n) and DEGs were highly consistent between clones/replicates (Fig 2o).
      To assess synaptic DEG function relating to the “Glutamatergic synapse”, synapse-specific GO analyses (SynGO, Table S3) were performed using combined up/down gene sets and including 16p11.2 genes. An enrichment in synaptic (Fig S5d-e) and “Presynaptic vesicle release” genes (Fig S5e, blue) was observed, supporting previous observations of deregulated synaptic transmission in SCZ (
      • Mudge J.
      • Miller N.A.
      • Khrebtukova I.
      • Lindquist I.E.
      • May G.D.
      • Huntley J.J.
      • et al.
      Genomic convergence analysis of schizophrenia: mRNA sequencing reveals altered synaptic vesicular transport in post-mortem cerebellum.
      ,
      • Wu J.Q.
      • Wang X.
      • Beveridge N.J.
      • Tooney P.A.
      • Scott R.J.
      • Carr V.J.
      • et al.
      Transcriptome sequencing revealed significant alteration of cortical promoter usage and splicing in schizophrenia.
      ). Protein-protein interaction (PPI) analysis (Fig S5f) revealed that DEG products formed into a network with 121 nodes and 211 edges associated with neuroarchitecture terms (Fig S5g,h, Table S4). These findings suggest the presence of numerous molecular alterations that may contribute to activity and network phenotypes.

      Deficits in dendritic length and calcium homeostasis in 16p11.2 duplication iENs

      Downregulation of “Neuron projection development”, “Dendrite” and “Growth cone” DEGs suggest potential deficits in DUP iEN architecture. We therefore analyzed dendritic morphology in 7WIV iENs after staining for MAP2. Sholl (Fig 3a) and branch point analysis (Fig S6a) revealed no alteration in dendritic arbor complexity. However, we observed a significant decrease in total neurite length (Fig 3b,c) and the length of the longest neurite (Fig S6b) consistent with previous observations (
      • Tai D.J.C.
      • Razaz P.
      • Erdin S.
      • Gao D.
      • Wang J.
      • Nuttle X.
      • et al.
      Tissue- and cell-type-specific molecular and functional signatures of 16p11.2 reciprocal genomic disorder across mouse brain and human neuronal models.
      ,
      • Deshpande A.
      • Yadav S.
      • Dao D.Q.
      • Wu Z.Y.
      • Hokanson K.C.
      • Cahill M.K.
      • et al.
      Cellular Phenotypes in Human iPSC-Derived Neurons from a Genetic Model of Autism Spectrum Disorder.
      ). As MAP2 stains only the dendritic arbor, but not axons (Fig S6d), this alteration in neurite length can be ascribed to a reduction in dendritic length, consistent with SCZ post-mortem observations (
      • Kalus P.
      • Muller T.J.
      • Zuschratter W.
      • Senitz D.
      The dendritic architecture of prefrontal pyramidal neurons in schizophrenic patients.
      ,
      • Black J.E.
      • Kodish I.M.
      • Grossman A.W.
      • Klintsova A.Y.
      • Orlovskaya D.
      • Vostrikov V.
      • et al.
      Pathology of layer V pyramidal neurons in the prefrontal cortex of patients with schizophrenia.
      ). We employed the presynaptic marker Syn1 to assess gross alterations to presynaptic number. DUP iENs showed no alterations in the size (Fig S6c) or number of presynaptic puncta (Fig 3d,e) as previously reported (
      • Urresti J.
      • Zhang P.
      • Moran-Losada P.
      • Yu N.K.
      • Negraes P.D.
      • Trujillo C.A.
      • et al.
      Cortical organoids model early brain development disrupted by 16p11.2 copy number variants in autism.
      ). Results were highly consistent between clones (Fig S6e-i).
      Figure thumbnail gr3
      Figure 3Deficits in neuroarchitecture and calcium handling in isogenic 16p11.2 duplication iENs at 7 weeks in vitro. a) Sholl analysis of control and 16p11.2 duplication lines. b) Total neurite length (2 clones, 2 independent differentiations, n=∼100 cells). c) Representative traces of control (blue) and 16p11.2 duplication (tan) iENs (scale bar-50 μm). d) Representative image of MAP2 (green) and Syn1 (magenta) stained iENs. e) Synapsin1 puncta density (total normalized to cell number per field of view, (2 clones, 2 independent differentiations, n= 20 images per condition). f) Average calcium peak half width maximum (duration of event normalized to control per independent experiment). g) Representative time course of a single spontaneous calcium event (SE). h) Representative trace of control (blue) and 16p11.2 duplication (tan) calcium event (average trace of five independent calcium events from 10 cells from 3 coverslips per condition). i) Calcium event frequency (normalized to control condition within experiment). Representative calcium trace of control (j, blue) and 16p11.2 duplication (k, tan). l) Synchronous calcium network event frequency (normalized to control condition within each independent experiment). m) Raster plot of representative control (Ctrl, blue) and 16p11.2 duplication (Dup, red) coverslips (points indicate the time (x-axis) of calcium events for each cell within the coverslip (y-axis). n) Representative heatmap of pairwise correlation of each cell to each other cell within a field of view (no correlation between a pair = red, 50% of spontaneous event activity correlated in a pairwise fashion = yellow). o) Pairwise correlation coefficient (normalized to control condition within each independent experiment). N=6; 2 clones, 3 independent differentiations, n=20-24 coverslips.
      Calcium signaling is a major driver of synaptic plasticity and “Calcium-ion binding” DEGs were observed within DUP iENs. Therefore, dysregulated calcium homeostasis may contribute to electrical and network phenotypes in DUP iENs. To assess this, calcium handling and activity was observed by incubating 7WIV iENs with Cal520, a calcium-sensitive fluorescent probe. Observed calcium transients were dependent on neuronal activity, confirmed by their responsiveness to synaptic and sodium channel blockers (Fig S7). At the single cell level, we observed a reduction in the duration of calcium events within DUP iENs (Fig 3f,g). Analysis of the average calcium peak profile revealed a pronounced enhancement in the half-maximal recovery time to baseline (Fig 3h), but no difference in event amplitude (Fig S8a) or resting calcium levels (Fig S8b). At the network level, we observed a decrease in the frequency of spontaneous (Fig 3i-k) and synchronous network calcium events (Fig 3l,m) in DUP iENs. Moreover, the correlation in activity of each cell to each other cell within the network (pairwise correlation, Fig 3n,o) was impaired, further supporting a deficit in the formation of functional networks. Observed phenotypes were not driven by clonal differences (Fig S8c-k). These results confirm alterations in activity and network properties observed by MEA and reveal a pronounced disruption to calcium handling in DUP iENs.

      Abnormalities in network activity and transcriptomic profile of iENs derived from SCZ patients with the 16p11.2 duplication

      To date no studies have been performed on iNs derived from SCZ patient iPSCs harboring the 16p11.2 duplication. Comparison of phenotypes shared between patient-derived and isogenic lines would allow the causal assignment of such phenotypes to the duplication, versus genetic background. We therefore assembled a 16p11.2 duplication SCZ patient-derived iPSC cohort to assess early neurodevelopmental alterations that may contribute to SCZ risk. iPSCs were derived from cryopreserved lymphocytes (CPL) of three SCZ patients carrying the 16p11.2 duplication alongside age/ethnicity matched controls from a well-characterized SCZ cohort (
      • Shi J.
      • Levinson D.F.
      • Duan J.
      • Sanders A.R.
      • Zheng Y.
      • Pe'er I.
      • et al.
      Common variants on chromosome 6p22.1 are associated with schizophrenia.
      ,
      • Levinson D.F.
      • Duan J.
      • Oh S.
      • Wang K.
      • Sanders A.R.
      • Shi J.
      • et al.
      Copy number variants in schizophrenia: confirmation of five previous findings and new evidence for 3q29 microdeletions and VIPR2 duplications.
      ,
      • Sanders A.R.
      • Levinson D.F.
      • Duan J.
      • Dennis J.M.
      • Li R.
      • Kendler K.S.
      • et al.
      The Internet-based MGS2 control sample: self report of mental illness.
      ) (Fig S9a). iPSCs were characterized based on morphology, alkaline phosphatase uptake, and the presence of pluripotency markers (Nanog, Oct4, Sox2, Fig S9b,c). CNV microarrays confirmed the presence and size of 16p11.2 duplications in each SCZ patient (Fig S9d); 16p11.2 CNVs closely matched the region disrupted in isogenic DUP lines employed (740kbp (
      • Tai D.J.
      • Ragavendran A.
      • Manavalan P.
      • Stortchevoi A.
      • Seabra C.M.
      • Erdin S.
      • et al.
      Engineering microdeletions and microduplications by targeting segmental duplications with CRISPR.
      )).
      To assess the role of 16p11.2 duplication in altered neurodevelopment, iENs were derived from SCZ patient iPSCs harboring 16p11.2 duplications (SCZ), alongside healthy control lines (“Healthy”). MEA (Fig 4a) analyses of SCZ iENs showed progressive deficits in network synchrony throughout development (Fig 4c), however, mean spontaneous firing rate (Fig 4b) was unchanged. At 7WIV (Fig 4d), SCZ iEN showed unaltered firing rate (Fig 4e), and significant reductions in synchronous firing (Fig 4f) and network burst frequency (Fig 4g). As expected, there was high variability between SCZ patients, potentially linked to background/sex (Fig S10, Supplemental Discussion). These data reveal network synchrony as a significantly altered phenotype induced by 16p11.2 duplication.
      Figure thumbnail gr4
      Figure 4Analysis of neuronal network activity and transcriptome in schizophrenia patient-derived iENs. a) Representative well of patient iENs on MEA. b) Developmental time course of healthy and Schizophrenia (SCZ) patient iEN mean firing rate. c) Developmental time course of healthy and SCZ patient iEN synchrony index. d) Representative raster plot of Healthy and SCZ patient activity. Well and plate averages of firing rate (e), synchrony index (f) and network burst frequency (g) at 7WIV (N=6, 3 patients per condition from 2 independent differentiations, n=30 wells per condition). h) Volcano plot of dysregulated gene expression in SCZ patients with top 10 DEGs highlighted (black, 14315 expressed with mean read counts>5). i) Significantly enriched GO terms within SCZ iENs overlapping with isogenic lines. Volcano plot of top overlapping GO terms; “homophilic cell adhesion” (j), “Glutamatergic Synapse” (k) and “Calcium ion binding” (l) and heatmap of aggregated DEGs from these terms (m). n) Venn diagram of overlapping DEGs from isogenic (FDR<0.1) and patient (FDR<0.3) lines showing shared direction. An enrichment of 2.11, p=0.002, was observed. o) Heat plot of significantly upregulated (FDR< 0.1) isogenic DEGs with shared direction in patient lines. p) Heat plot of significantly downregulated (FDR< 0.1) isogenic DEGs with shared direction in patient lines.
      To assess transcriptomic changes, mRNA was harvested from healthy and SCZ iENs at 7WIV and RNA-sequencing was performed. 568 DEGs (FDR<0.1, Fig 4h, Table S5) were observed, with a significant increase in the expression of genes within the 16p11.2 locus (Fig S11a,b) with the exception of MAZ and QPRT, which were unchanged with regard to Healthy iEN. An enrichment in genes affected by de novo risk variants in SCZ and ASD, but no SCZ GWAS loci were identified (Fig S11c-f). Interestingly, a number of GO terms were shared between isogenic and patient lines (Fig 4i); “Homophilic cell adhesion” (Fig 4j), “Glutamatergic synapse” (Fig 4k) and “Calcium ion binding” (Fig 4l), and DEGs arising from these GO terms were well conserved within patient lines (Fig 4m). SynGO analysis revealed a shared dysregulation of “Synaptic vesicle exocytosis” in both isogenic and patient-derived lines (Fig S11g,h). We also observed an enrichment in bipolar disorder variants in both isogenic and patient samples when compared to post-mortem cross-disorder brain transcriptomic data sets (Fig S12, (
      • Gandal M.J.
      • Haney J.R.
      • Parikshak N.N.
      • Leppa V.
      • Ramaswami G.
      • Hartl C.
      • et al.
      Shared molecular neuropathology across major psychiatric disorders parallels polygenic overlap.
      ,
      • Gandal M.J.
      • Zhang P.
      • Hadjimichael E.
      • Walker R.L.
      • Chen C.
      • Liu S.
      • et al.
      Transcriptome-wide isoform-level dysregulation in ASD, schizophrenia, and bipolar disorder.
      )). Overlapping DEGs were observed between isogenic and patient DEGs (Fig 4n, 11 up, 6 down). Assessment of DEGs revealed low consistency in upregulated genes (Fig 4o), but downregulated genes showed high consistency between isogenic and patient lines (Fig 4p). We analyzed recent 16p11.2 duplication transcriptomic data sets from a range of neuronal models/tissues (Fig S13-S16 (
      • Sundberg M.
      • Pinson H.
      • Smith R.S.
      • Winden K.D.
      • Venugopal P.
      • Tai D.J.C.
      • et al.
      16p11.2 deletion is associated with hyperactivation of human iPSC-derived dopaminergic neuron networks and is rescued by RHOA inhibition in vitro.
      ,
      • Tai D.J.C.
      • Razaz P.
      • Erdin S.
      • Gao D.
      • Wang J.
      • Nuttle X.
      • et al.
      Tissue- and cell-type-specific molecular and functional signatures of 16p11.2 reciprocal genomic disorder across mouse brain and human neuronal models.
      ,
      • Urresti J.
      • Zhang P.
      • Moran-Losada P.
      • Yu N.K.
      • Negraes P.D.
      • Trujillo C.A.
      • et al.
      Cortical organoids model early brain development disrupted by 16p11.2 copy number variants in autism.
      ,
      • Rein B.
      • Tan T.
      • Yang F.
      • Wang W.
      • Williams J.
      • Zhang F.
      • et al.
      Reversal of synaptic and behavioral deficits in a 16p11.2 duplication mouse model via restoration of the GABA synapse regulator Npas4.
      )). Notably, GO terms identified in isogenic and SCZ iEN were identified in multiple previous studies (Fig S17). The presence of shared gene ontologies between isogenic, patient, and previous 16p11.2 duplication RNA-seq experiments suggests the presence of conserved molecular markers of 16p11.2 duplication-dependent dysfunction.
      We set out to assess neuroarchitecture of SCZ versus healthy lines. Sholl analysis revealed a deficit in distal dendrites (Fig 5a); however, no alteration in branch point analyses were observed (Fig S18a) suggesting shorter dendritic length, confirmed by a reduction in total and maximum dendrite length (Fig 5b, S18b). SCZ patient iENs showed a reduction in the number of Syn1 puncta (Fig 5d,e), but no change in puncta size (Fig S18c) and morphological/presynaptic deficits were highly consistent between patient lines (Fig S18d-j).
      Figure thumbnail gr5
      Figure 5Deficits in neuroarchitecture and calcium handling in schizophrenia patient-derived 16p11.2 duplication iENs. a) Sholl analysis of control and 16p11.2 duplication schizophrenia (SCZ) lines. b) Total neurite length (n=∼200 cells). c) Representative traces of Healthy (blue) and SCZ (tan) iENs. Scale bar = 50 μM. d) Synapsin1 puncta density (total normalized to cell number per field of view, (n= 20 images per condition). e) Representative image of MAP2 (green) and Syn1 (magenta) stained healthy and SCZ iENs. Scale bar = 100 μM. f) Average calcium peak duration (duration of event normalized to control condition per experiment). g) Time course of a representative spontaneous event (SE). h) Representative trace of Healthy (blue) and SCZ (tan) calcium events (average trace of 5 independent calcium events from 10 cells from 3 coverslips). i) Calcium event frequency (normalized per experiment to control condition). Representative calcium trace of Healthy (j, blue) and SCZ (k, tan) lines. l) Raster plot of representative Healthy (blue) and SCZ (red) coverslips (points indicate the time (x-axis) of calcium events for each cell within the coverslip (y-axis)). m) Synchronous calcium event frequency (normalized to control condition within each independent experiment). n) Representative heatmap of pairwise correlation of each cell to each other cell within a field of view (no correlation between a pair = red, 50% of spontaneous event activity correlated in a pairwise fashion = yellow). o) Pairwise correlation coefficient (normalized to control condition within each independent experiment). N=6; 2 clones, 3 independent differentiations, n=20-24 coverslips.
      “Calcium ion-binding” DEGs were observed in both isogenic and patient transcriptomic profiles. Calcium imaging revealed alterations to calcium event duration in SCZ iENs (Fig 5f-h), as observed in isogenic DUP lines. However, SCZ lines also showed a significant reduction in resting calcium levels (Fig S19a) and an increase in calcium event amplitude (Fig S19b); phenotypes absent in isogenic 16p11.2 duplication lines. These deficits were also evident following KCl-induced depolarization (Fig S20). SCZ patients showed no significant alteration in the mean frequency of calcium events (Fig 5i-k) consistent with MEA activity (Fig 4e), but the strength of these results were likely impacted by patient line variability (Fig S19c-j, see Supplemental Discussion). SCZ patient-derived lines showed a reduction in the number of synchronous events (Fig 5l,m) and in the pairwise correlation of neuronal firing within the network (Fig 5n,o).

      Discussion

      The 16p11.2 duplication is one of the most penetrant risk factors for SCZ, unlike deletions of this region (
      • Niarchou M.
      • Chawner S.
      • Doherty J.L.
      • Maillard A.M.
      • Jacquemont S.
      • Chung W.K.
      • et al.
      Psychiatric disorders in children with 16p11.2 deletion and duplication.
      ). However, identifying the role of 16p11.2 duplication in the etiology of SCZ has been challenging due to the inability to access appropriate neural tissue, the limited availability of 16p11.2 duplication carriers with SCZ and the heterogenetic nature of SCZ. To address these issues, we employed SCZ patient iN to study the 16p11.2 duplication. To complement this, we assessed 16p11.2 duplication using an isogenic line, whereby the 16p11.2 duplication was introduced into iPSCs derived from a healthy subject (
      • Tai D.J.
      • Ragavendran A.
      • Manavalan P.
      • Stortchevoi A.
      • Seabra C.M.
      • Erdin S.
      • et al.
      Engineering microdeletions and microduplications by targeting segmental duplications with CRISPR.
      ). This complementary patient/isogenic approach has been proposed as a powerful manner in which to assess the role of CNVs in genetically defined NDDs by reducing phenotypic noise from diverse patient backgrounds (
      • Brennand K.J.
      • Landek-Salgado M.A.
      • Sawa A.
      Modeling heterogeneous patients with a clinical diagnosis of schizophrenia with induced pluripotent stem cells.
      ). Using this method, we identified novel 16p11.2 duplication-dependent alterations that likely contribute to the molecular and neuronal etiology of SCZ during early neurodevelopment.
      Neuronal activity was dysfunctional in isogenic iEN, with a strong reduction in activity at more mature time points (7WIV, Figure 2, Figure 3). Interestingly, assessment of MEA activity in the presence of AMPAr and NMDAr inhibitors at 4 and 7WIV indicate a strong enhancement of synaptic activity during this period (Fig S3), coinciding with the development of network activity. Moreover, it is at these time points that 16p11.2 activity-dependent iEN phenotypes emerged, supporting reductions in “Glutamatergic synapse” gene expression, observed in both isogenic and patient lines, as key to 16p11.2 duplication activity phenotypes. This is particularly compelling, considering the enrichment in synaptic genes in SCZ GWAS, exome and RNA sequencing studies (
      • Ripke S.
      • O'Dushlaine C.
      • Chambert K.
      • Moran J.L.
      • Kahler A.K.
      • Akterin S.
      • et al.
      Genome-wide association analysis identifies 13 new risk loci for schizophrenia.
      ,
      • Forstner A.J.
      • Hecker J.
      • Hofmann A.
      • Maaser A.
      • Reinbold C.S.
      • Muhleisen T.W.
      • et al.
      Identification of shared risk loci and pathways for bipolar disorder and schizophrenia.
      ,
      • Hall L.S.
      • Medway C.W.
      • Pain O.
      • Pardinas A.F.
      • Rees E.G.
      • Escott-Price V.
      • et al.
      A transcriptome-wide association study implicates specific pre- and post-synaptic abnormalities in schizophrenia.
      ). Thus, alterations in synaptic gene expression are likely to contribute to neuronal activity deficits in isogenic and patient lines, supported by previous studies (Fig S21, Supplemental Discussion).
      In addition, we observed DEGs associated with “Calcium ion binding” and “Neuron projection development”, alongside dysregulation of calcium homeostasis and neuroarchitecture. Deregulated neuronal calcium signaling is an emerging finding among patient and genetic models of SCZ (
      • Ripke S.
      • O'Dushlaine C.
      • Chambert K.
      • Moran J.L.
      • Kahler A.K.
      • Akterin S.
      • et al.
      Genome-wide association analysis identifies 13 new risk loci for schizophrenia.
      ,
      • Vidal-Domenech F.
      • Riquelme G.
      • Pinacho R.
      • Rodriguez-Mias R.
      • Vera A.
      • Monje A.
      • et al.
      Calcium-binding proteins are altered in the cerebellum in schizophrenia.
      ,
      • Khan T.A.
      • Revah O.
      • Gordon A.
      • Yoon S.J.
      • Krawisz A.K.
      • Goold C.
      • et al.
      Neuronal defects in a human cellular model of 22q11.2 deletion syndrome.
      ,
      • Park S.J.
      • Jeong J.
      • Park Y.U.
      • Park K.S.
      • Lee H.
      • Lee N.
      • et al.
      Disrupted-in-schizophrenia-1 (DISC1) Regulates Endoplasmic Reticulum Calcium Dynamics.
      ) and “Calcium ion binding” was found to be the most significantly altered gene set within the SCZ brain (
      • Vidal-Domenech F.
      • Riquelme G.
      • Pinacho R.
      • Rodriguez-Mias R.
      • Vera A.
      • Monje A.
      • et al.
      Calcium-binding proteins are altered in the cerebellum in schizophrenia.
      ). In addition, patient post mortem studies have consistently found impaired dendritic architecture in the SCZ cortex (
      • Forrest M.P.
      • Parnell E.
      • Penzes P.
      Dendritic structural plasticity and neuropsychiatric disease.
      ) suggesting that 16p11.2 duplication may impair dendritic morphology early in neuronal development and this is maintained throughout SCZ progression. These conserved phenotypes may therefore constitute early 16p11.2 duplication-dependent neurodevelopmental alterations contributing to SCZ risk (See Supplemental Discussion).
      Perhaps the most compelling deficit was in iEN network synchrony, observed in isogenic and patients, by both calcium network imaging and MEAs. Neuronal network activity plays key roles during brain development, sensory processing, and cognition (
      • Uhlhaas P.J.
      • Singer W.
      Abnormal neural oscillations and synchrony in schizophrenia.
      ) and neuronal synchrony has been hypothesized to play a major role in the coding and propagation of information, with higher synchrony thought to confer faster responses with greater robustness of the information carried (
      • Panzeri S.
      • Macke J.H.
      • Gross J.
      • Kayser C.
      Neural population coding: combining insights from microscopic and mass signals.
      ). Importantly, neuronal network synchrony alterations occur in SCZ and other neurodevelopmental disorders (
      • Mathalon D.H.
      • Sohal V.S.
      Neural Oscillations and Synchrony in Brain Dysfunction and Neuropsychiatric Disorders: It's About Time.
      ), supporting observed reductions in network activity and synchrony as potentially relevent to SCZ pathophysiology. Of note, the strength of this phenotype in SCZ lines appeared to be influenced by additional genetic risk or resilience factors or patient sex (see Supplemental Discussion).
      Taken together, we observed the presence of early neuronal deficits in isogenic and SCZ patient-derived 16p11.2 duplication neurons representing neurodevelopmental alterations that may contribute to the etiology of SCZ. Moreover, these deficits are dependent on impaired glutamatergic neuronal activity, supporting previous assertions of excitatory dysfunction as a driver of deficits in SCZ (
      • Lewis D.A.
      • Curley A.A.
      • Glausier J.R.
      • Volk D.W.
      Cortical parvalbumin interneurons and cognitive dysfunction in schizophrenia.
      ,
      • Chung D.W.
      • Fish K.N.
      • Lewis D.A.
      Pathological Basis for Deficient Excitatory Drive to Cortical Parvalbumin Interneurons in Schizophrenia.
      ). Finally, these deficits coincide with impaired neuroarchitecture, the glutamatergic synapse and calcium signaling, consistent with GWAS (
      • Fromer M.
      • Pocklington A.J.
      • Kavanagh D.H.
      • Williams H.J.
      • Dwyer S.
      • Gormley P.
      • et al.
      De novo mutations in schizophrenia implicate synaptic networks.
      ), post-mortem studies (
      • Vidal-Domenech F.
      • Riquelme G.
      • Pinacho R.
      • Rodriguez-Mias R.
      • Vera A.
      • Monje A.
      • et al.
      Calcium-binding proteins are altered in the cerebellum in schizophrenia.
      ), and genetic models of SCZ (
      • Naujock M.
      • Speidel A.
      • Fischer S.
      • Kizner V.
      • Dorner-Ciossek C.
      • Gillardon F.
      Neuronal Differentiation of Induced Pluripotent Stem Cells from Schizophrenia Patients in Two-Dimensional and in Three-Dimensional Cultures Reveals Increased Expression of the Kv4.2 Subunit DPP6 That Contributes to Decreased Neuronal Activity.
      ,
      • Khan T.A.
      • Revah O.
      • Gordon A.
      • Yoon S.J.
      • Krawisz A.K.
      • Goold C.
      • et al.
      Neuronal defects in a human cellular model of 22q11.2 deletion syndrome.
      ,
      • Park S.J.
      • Jeong J.
      • Park Y.U.
      • Park K.S.
      • Lee H.
      • Lee N.
      • et al.
      Disrupted-in-schizophrenia-1 (DISC1) Regulates Endoplasmic Reticulum Calcium Dynamics.
      ). These findings strongly implicate deregulated calcium homeostasis, neuronal network properties, and morphological development as 16p11.2 duplication-dependent mechanisms that impair neurodevelopment and contribute to SCZ pathophysiology.

      Acknowledgements

      PP was funded by R01NS114977 awarded by the National Institute of Mental Health. We thank the Northwestern Sequencing core (NU-Seq) and Gene Editing and Transduction and Nanotechnology Core (GET-IN) for their services. Patient samples were sourced from the dbGAP study (phs000021.v2.p1 and phs000167.v1.p1). Schematics were created with BioRender.com.

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