Modeling Human Primary Microcephaly With hiPSC-Derived Brain Organoids Carrying CPAP-E1235V Disease-Associated Mutant Protein.

Hsiao-Lung An,Tang K Tang,Hung-Chih Kuo

doi:10.3389/fcell.2022.830432

Abstract

The centrosome is composed of a pair of centrioles and serves as the major microtubule-organizing center (MTOC) in cells. Centrosome dysfunction has been linked to autosomal recessive primary microcephaly (MCPH), which is a rare human neurodevelopmental disorder characterized by small brain size with intellectual disability. Recently, several mouse models carrying mutated genes encoding centrosomal proteins have been generated to address the genotype–phenotype relationships in MCPH. However, several human-specific features were not observed in the mouse models during brain development. Herein, we generated isogenic hiPSCs carrying the gene encoding centrosomal CPAP-E1235V mutant protein using the CRISPR-Cas9 genome editing system, and examined the phenotypic features of wild-type and mutant hiPSCs and their derived brain organoids. Our results showed that the CPAP-E1235V mutant perturbed the recruitment of several centriolar proteins involved in centriole elongation, including CEP120, CEP295, CENTROBIN, POC5, and POC1B, onto nascent centrioles, resulting in the production of short centrioles but long cilia. Importantly, our wild-type hiPSC-derived brain organoid recapitulated many cellular events seen in the developing human brain, including neuronal differentiation and cortical spatial lamination. Interestingly, hiPSC-CPAP-E1235V-derived brain organoids induced p53-dependent neuronal cell death, resulting in the production of smaller brain organoids that mimic the microcephaly phenotype. Furthermore, we observed that the CPAP-E1235V mutation altered the spindle orientation of neuronal progenitor cells and induced premature neuronal differentiation. In summary, we have shown that the hiPSC-derived brain organoid coupled with CRISPR/Cas9 gene editing technology can recapitulate the centrosome/centriole-associated MCPH pathological features. Possible mechanisms for MCPH with centriole/centrosome dysfunction are discussed.

Highlights

The centrosome in animal cells is composed of a pair of microtubule-based centrioles surrounded by pericentriolar material (PCM) proteins and serves as the microtubuleorganizing center (MTOC) in cells and a regulator of cellcycle progression (Nigg and Holland, 2018)
Two independent mutant human-induced pluripotent stem cell (hiPSC) clones (CPAP-E1235V#1 and #2) were obtained and room temperature (RT)-PCR analysis of the full-length CPAP cDNA confirmed a homozygous mutation at 3704 A→T in exon 16 of CPAP, leading to an E1235V amino acid substitution (Figures 1A,B); no other variants were present in the CPAP gene of mutant iPSCs
A further analysis by transferasemediated dUTP nick end labeling (TUNEL) staining revealed a significant increase in apoptosis of the pluripotent stem cells (NANOG+ and OCT4+) of these two CPAP-E1235V mutant clones (Figure 2C; Supplementary Figure S1), indicating that the mutant cells are more susceptible to apoptosis

Summary

Introduction

The centrosome in animal cells is composed of a pair of microtubule-based centrioles surrounded by pericentriolar material (PCM) proteins and serves as the microtubuleorganizing center (MTOC) in cells and a regulator of cellcycle progression (Nigg and Holland, 2018). Centrosome abnormalities trigger mitotic errors and genomic instability, resulting in human disorders such as cancer, while gene mutations that affect the cilia structures and/or ciliary function cause a number of genetic diseases, collectively known as ciliopathies (Reiter and Leroux, 2017). The primate cerebral cortex has undergone extensive expansion and complexification, as evidenced by the increases in cortical size, surface area, and network density. Much of this expansion can be attributed to the presence of the outer subventricular zone (OSVZ), which represents a separate progenitor layer that contains a unique stem cell subset known as outer radial glia cells (oRG) in the developing cortex of primates/humans (Fietz et al, 2010; Hansen et al, 2010). Mutations in the STIL, CEP135, CPAP, and RTTN genes were reported to cause MCPH in humans (Thornton and Woods, 2009; Jayaraman et al, 2018; Vandervore et al, 2019)

Methods

Results

Conclusion