Modeling primary microcephaly with human brain organoids reveals fundamental roles of CIT kinase activity
Gianmarco Pallavicini, … , Ferdinando Di Cunto, Stephanie L. Bielas
Gianmarco Pallavicini, … , Ferdinando Di Cunto, Stephanie L. Bielas
Published September 24, 2024
Citation Information: J Clin Invest. 2024;134(21):e175435. https://doi.org/10.1172/JCI175435.
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Research Article Cell biology Neuroscience

Modeling primary microcephaly with human brain organoids reveals fundamental roles of CIT kinase activity

Abstract

Brain size and cellular heterogeneity are tightly regulated by species-specific proliferation and differentiation of multipotent neural progenitor cells (NPCs). Errors in this process are among the mechanisms of primary hereditary microcephaly (MCPH), a group of disorders characterized by reduced brain size and intellectual disability. Biallelic citron rho-interacting serine/threonine kinase (CIT) missense variants that disrupt kinase function (CITKI/KI) and frameshift loss-of-function variants (CITFS/FS) are the genetic basis for MCPH17; however, the function of CIT catalytic activity in brain development and NPC cytokinesis is unknown. Therefore, we created the CitKI/KI mouse model and found that it did not phenocopy human microcephaly, unlike biallelic CitFS/FS animals. Nevertheless, both Cit models exhibited binucleation, DNA damage, and apoptosis. To investigate human-specific mechanisms of CIT microcephaly, we generated CITKI/KI and CITFS/FS human forebrain organoids. We found that CITKI/KI and CITFS/FS organoids lost cytoarchitectural complexity, transitioning from pseudostratified to simple neuroepithelium. This change was associated with defects that disrupted the polarity of NPC cytokinesis, in addition to elevating apoptosis. Together, our results indicate that both CIT catalytic and scaffolding functions in NPC cytokinesis are critical for human corticogenesis. Species differences in corticogenesis and the dynamic 3D features of NPC mitosis underscore the utility of human forebrain organoid models for understanding human microcephaly.

Authors

Gianmarco Pallavicini, Amanda Moccia, Giorgia Iegiani, Roberta Parolisi, Emily R. Peirent, Gaia Elena Berto, Martina Lorenzati, Rami Y. Tshuva, Alessia Ferraro, Fiorella Balzac, Emilia Turco, Shachi U. Salvi, Hedvig F. Myklebust, Sophia Wang, Julia Eisenberg, Maushmi Chitale, Navjit S. Girgla, Enrica Boda, Orly Reiner, Annalisa Buffo, Ferdinando Di Cunto, Stephanie L. Bielas

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Figure 4

CIT-affected organoids demonstrate changes to pseudostratified neuroepithelium.

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CIT-affected organoids demonstrate changes to pseudostratified neuroepi...
(A) Schematic of dorsal forebrain organoid differentiation and microfabricated compartment generation with the corresponding time stamps. Neural differentiation begins with hPSC aggregate generation at 0DD, followed by neural induction at 1.5DD. An illustration of the microfabricated compartment depicts the orientation of compartment components. Neural aggregates are positioned on glass coverslips in a 3 × 3 pattern at 7DD, and the compartment is sealed with a UV adhesive. Neural differentiation medium fills the tissue culture dish to facilitate medium exchange to the compartment containing the developing tissue. Two days later, on 9DD, the compartment is embedded with Matrigel. Inverted confocal microscopy is performed on 21DD, 28DD, and 35DD. Scale bars: 200 μm. (B and C) Representative images of developing organoids and rosettes (white numbers) across time using Lifeact-GFP. Affected CITFS/FS and CITKI/KI rosettes exhibited large lumens and a reduction in neuroepithelial complexity. Scale bars: 250 μm. (D) Illustration of rosette measurements performed across CIT organoids, with diameter measurements and Ri calculations across organoid rosettes. (E and F) Quantification of Ri (dr/dl) in CIT+/KI and in CITKI/KI organoids (E) and CIT+/+ and in CITFS/FS organoids (F). (G and H) Representative insets of developing rosettes across time using Lifeact-GFP and H2B-mCherry. Control rosettes maintained a pseudostratified neuroepithelium at all 3 time points, while many affected CITKI/KI and CITFS/FS rosettes showed transition toward a simple epithelial architecture. Multinucleated cells were apparent in the rosette, and examples are shown (white arrowheads). Scale bars: 25 μm. Quantification was done from a minimum of 2 independent compartment preparations per genotype. Each compartment contained 9 or fewer organoids per preparation. Data indicate the mean ± SEM. *P < 0.05, ***P < 0.001, and ****P < 0.0001, by repeated-measures, 2-way ANOVA.