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 1

CitKI/KI mice show grossly normal CNS morphological structure with an ataxic phenotype.

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CitKI/KI mice show grossly normal CNS morphological structure with an a...
(A) Representative image of Cit+/+, CitKI/KI, and CitFS/FS P10 brains. Scale bar: 5 mm. (B) Kaplan-Meier survival curves for CitKI/KI mice versus CitFS/FS (n = 12) mice. The log-rank (Mantel-Cox) test was used to compare survival between experimental groups (****P < 0.0001). (C) Cresyl Violet staining of sagittal sections of Cit+/+, CitKI/KI, and CitFS/FS P10 brains. Scale bar: 1 mm. (D) Upper panel: Cresyl violet staining of sagittal sections from the midline (vermis) of Cit+/+, CitKI/KI, and CitFS/FS P10 cerebella showing the anterior, central, and posterior sectors; scale bar: 500 μm. Middle panel: High-magnification Nissl stain of lobule V inset from the midline (vermis); scale bar: 50 μm. Lower panel: Immunofluorescence analysis for the Purkinje Cell marker calbindin 1 of sagittal sections of lobule V from the midline (vermis) obtained from P10 mice; scale bar: 25 μm. (E) Quantification of Purkinje cell layer (PCL) perimeter in the vermis of P10 Cit+/+, CitKI/KI, and CitFS/FS mice. (F) Quantification of Purkinje cell density per millimeter in the vermis of Cit+/+, CitKI/KI, and CitFS/FS P10 mice. Purkinje cells were stained for calbindin 1. (G) Representative picture extracted from videos of Cit+/+ and CitKI/KI mice slipping during beam walking test. (H) Quantification of the mean number of slips for 3 consecutive days of the beam-walking test on the same animal. Data indicate the mean ± SEM. **P < 0.01, ***P < 0.001, and ****P < 0.0001, by 1-way ANOVA followed by Bonferroni post hoc analysis (E and F) and unpaired, 2-tailed Student’s t test (H). Each dot represents an independent animal.