Modeling primary microcephaly with human brain organoids reveals fundamental roles of CIT kinase activity
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
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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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 5

CIT-affected organoids show aNPC apical endfeet surface area increases and mitotic defects.

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CIT-affected organoids show aNPC apical endfeet surface area increases ...
(A and B) Apical endfeet surface area and representative images of 35DD organoid apical surface labeled with Lifeact-GFP in microfabricated compartments. The apical endfeet surface area was increased in affected CITKI/KI (A) and CITFS/FS (B) organoids compared with CIT+/KI and CIT+/+ control organoids. Scale bars: 10 μm. (C) Representative images from videos of CIT+/KI and CITKI/KI rosettes with aNPC mitotic division (white dashed rectangles) at the apical surface. Panel shows the time details of this division. Scale bar: 50 μm. (D) Quantification of the percentage of division occurring at the central lumen or misplaced from the lumen in C. (E) Quantification of the percentage of correct division, mitotic failure, and cytokinesis failure in C. (F) Measurement of the time spent from anaphase to G1 ascension in CIT+/KI and CITKI/KI aNPC divisions in 35DD organoid rosettes. Each dot indicates a single dividing cell. (G) Representative images from videos of CIT+/+ and CITFS/FS rosettes with aNPC mitotic division (white dashed rectangles) at the apical surface. Panel shows the time details of this division. Scale bar: 50 μm. (H) Quantification of the percentage of division occurring at the central lumen or misplaced from the lumen in G. (I) Quantification of the percentage of correct division, mitotic failure, and cytokinesis failure in G. (J) Measurement of the time spent from anaphase to G1 ascension in CIT+/+ and CITFS/FS aNPC divisions in 35DD organoid rosettes. Each dot indicates a single dividing cell. Quantification was done from a minimum of 2 independent compartment preparations per genotype. The compartments each contained 9 or fewer organoids per preparation. Data indicate the mean ± SEM. *P < 0.05, **P < 0.01, and ***P < 0.001, by unpaired, 2-tailed Student’s t test for the apical endfeet area (A and B), Fisher’s exact probability test for the percentage distribution (D, E, H, and I), and Mann-Whitney U test for the time from anaphase to G1 ascension (F and J).