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 2

CitKI/KI mice show increased apoptosis, DNA damage, and cytokinesis failure in CNS tissue, but less than CitFS/FS mice.

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CitKI/KI mice show increased apoptosis, DNA damage, and cytokinesis fai...
(A) Immunofluorescence analysis of P4 cerebella from mice of the indicated genotypes for the apoptotic marker cCASP3 (green) and Hoechst (gray). Scale bar: 25 μm. (B) Quantification of cCASP3+ cells in A. (C) Immunofluorescence analysis of P4 cerebella from mice of the indicated genotypes for the DNA damage marker γH2AX (yellow) and Hoechst (gray). Scale bar: 25 μm. (D) Quantification of γH2AX+ cells in C. (E and F) Western blot analysis of total lysate from P4 cerebella from mice of the indicated genotypes. The levels of cCASP3 (E) and γH2AX (F) were analyzed relative to the internal loading control vinculin (Vinc). Each dot represents an independent replicate. (G) Immunofluorescence analysis for the apoptotic marker cCASP3 (green), TUNEL assay (red), and Hoechst (gray) of E14.5 cortex obtained from embryos of the indicated genotypes. Scale bars: 10 μm. (H) Quantification of cCASP3 and TUNEL+ cells in (G). (I) Representative images of E12.5 NPCs from Cit+/+ and Cit KI/KI embryos, immunostained for 53BP1 (magenta) and Hoechst (gray) 18 hours after plating. Scale bar: 10 μm. (J) Quantification of 53BP1 nuclear foci in I; more than 250 cells were counted for each genotype in each replicate (n = 3). (K) High-power fields of Cresyl violet–stained coronal sections of P10 cortex obtained from mice of the indicated genotypes. Arrowheads indicate binucleated cells. Scale bar: 10 μm. (L) Quantification of binucleated cells in K. In microscopy quantifications, every dot represents an independent animal and at least 9 fields per genotype were analyzed. Data indicate the mean ± SEM. *P < 0.05, **P < 0.01,***P < 0.001, and ****P < 0.0001, by 1-way ANOVA followed by Holm-Šidák post hoc analysis.