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 3

Modeling CIT variants using human models of neurodevelopment.

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Modeling CIT variants using human models of neurodevelopment. (A) Scheme...
(A) Scheme of CIT-K and CIT-N protein isoforms. Homozygous and compound heterozygous variants localized in the N-terminus and within the kinase domain of CIT-K. Missense variants are depicted in green, and LOF FS and splice (SpEx) variants are depicted in red. (B) CRISPR/Cas9-mediated targeting of the CIT locus at exon 4. Sequences and chromatograms of the 7 bp deletion are shown, with the corresponding alteration of the protein sequence highlighted in red. (C) Western blot analysis of CIT expression in NPCs of the indicated genotypes. The CIT-K isoform was absent in the CRISPR/Cas9-edited CITFS/FS line. (D) Western blot analysis of CIT expression in NPCs of the indicated genotypes. Variability in CIT-K abundance was detected in CITKI/KI NPCs compared with unaffected controls. (E) Immunofluorescence of Aurora B (green) midbody arms and CIT (red) show the presence of CIT in the midbody central bulge in the indicated genotypes of 35DD dorsal forebrain organoids. DNA stained with Hoechst (blue). Scale bars: 10 μm. (F) Immunofluorescence of Aurora B (green) midbody arms and the midbody central bulge markers CIT (red) and MKLP1 (red) in 35DD dorsal forebrain organoids. DNA stained with Hoechst (blue). CIT was absent in the midbody central bulge in CITFS/FS 35DD organoids. Immunofluorescence with the midbody central bulge marker MKLP1 (red) shows the presence of this structure flanked by Aurora B (green) midbody arms in CITFS/FS 35DD organoids. Scale bars: 10 μm.