Cortical overgrowth in a preclinical forebrain organoid model of CNTNAP2-associated autism spectrum disorder

Job O De Jong,Frank Provenzano,Huixiang Zhu,Barbara Corneo,Bin Xu,Matthieu Genestine,Ceyda Llapashtica,Christoph Kellendonk,Raju Tomer,Karlla W Brigatti,Jonathan A Javitch,Yan Sun,Kevin Strauss,Sander Markx,Francesco Brundu,Giuseppe P Cortese,Bianca Migliori,Steven A Kushner

doi:10.1038/s41467-021-24358-4

Abstract

We utilized forebrain organoids generated from induced pluripotent stem cells of patients with a syndromic form of Autism Spectrum Disorder (ASD) with a homozygous protein-truncating mutation in CNTNAP2, to study its effects on embryonic cortical development. Patients with this mutation present with clinical characteristics of brain overgrowth. Patient-derived forebrain organoids displayed an increase in volume and total cell number that is driven by increased neural progenitor proliferation. Single-cell RNA sequencing revealed PFC-excitatory neurons to be the key cell types expressing CNTNAP2. Gene ontology analysis of differentially expressed genes (DEgenes) corroborates aberrant cellular proliferation. Moreover, the DEgenes are enriched for ASD-associated genes. The cell-type-specific signature genes of the CNTNAP2-expressing neurons are associated with clinical phenotypes previously described in patients. The organoid overgrowth phenotypes were largely rescued after correction of the mutation using CRISPR-Cas9. This CNTNAP2-organoid model provides opportunity for further mechanistic inquiry and development of new therapeutic strategies for ASD.

Highlights

We utilized forebrain organoids generated from induced pluripotent stem cells of patients with a syndromic form of Autism Spectrum Disorder (ASD) with a homozygous proteintruncating mutation in contactin-associated-protein-like 2 (CNTNAP2), to study its effects on embryonic cortical development
Because the sample size of the initial cohort in which the increased head circumference was described was small[10], we expanded this assessment by measuring the head circumferences of an additional 17 male and 20 female pediatric patients from the Old-Order Amish founder population all carrying the inherited homozygous c.3709DelG mutation in CNTNAP2 and all presenting with the same syndromic ASD
We utilized forebrain organoids generated from human induced pluripotent stem cells (hiPSCs) derived from patients carrying the homozygous c.3709DelG mutation in CNTNAP2 and healthy controls to investigate the effects of this mutation on cortical embryonic development

Summary

Introduction

We utilized forebrain organoids generated from induced pluripotent stem cells of patients with a syndromic form of Autism Spectrum Disorder (ASD) with a homozygous proteintruncating mutation in CNTNAP2, to study its effects on embryonic cortical development. Patients with this mutation present with clinical characteristics of brain overgrowth. Rodent models with modified genes associated with ASD have facilitated this effort, but translating promising preclinical findings to the clinic has remained challenging[2] This is at least partly due to differences in the biology between species[3,4,5] and underscores the need for elucidating human-specific disease mechanisms for psychiatric disorders. Cntnap[2] null mice display several behavioral phenotypes reminiscent of the neurodevelopmental syndrome associated with homozygous LoF mutations, including deficits in social interaction and cognition, an increase in repetitive-stereotyped behaviors, and a lowered seizure threshold[16]

Methods

Results

Conclusion