Conference Agenda

Neural stem cells (NSCs) and neural progenitors (NPs) are fundamental players in the tremendous complexity of the mammalian central nervous system (CNS); impairments in the mechanisms controlling their specification, expansion, and behavior result in severe neurodevelopmental and neuropsychiatric disorders. New developments in genetic and cell technologies have revolutionized how human NSCs/NPs are obtained and studied, providing new insights into the decryption of the molecular and cellular codes underlying the NSC programs during neural development and into understanding how the same processes are impaired in brain diseases. Here I will present the generation of stem cell-based systems to better dissect the molecular bases of human brain development and diseases.

Pitt-Hopkins syndrome (PTHS) is a rare neurodevelopmental condition characterized by severe intellectual disability, epilepsy, specific facial gestalt and autonomic nervous system dysfunction. PTHS is due to haploinsufficiency of TCF4, a basic helix-loop-helix (bHLH) transcription factor. De novo heterozygous loss-of-function (LoF) variants are associated with mild clinical presentations while missense variants in the bHLH domain are associated with an altered sub-nuclear localization and more severe phenotypes.

Using exome sequencing, we identified three de novo missense variants in TCF4 within or nearby the bHLH domain, in unrelated individuals with a malformative syndrome including craniofacial hallmarks not compatible with PTHS, and without intellectual disability. Functional analysis in NIH-3T3 cells suggests that these new variants are not associated with an altered sub-nuclear localization in support of a possible gain-of-function (GoF) mechanism. To test this hypothesis we overexpressed the de novo variants in zebrafish embryos and we analyzed their craniofacial development identifying specific alterations in ethmoidal cartilages formation (primary palate) due to altered chondrogenesis and ECM deposition. We also performed transcriptome analysis on embryos overexpressing wild-type and one missense variant associated with the atypical presentation to identify new potential TCF4 targets. Finally, we started the generation, by a gene editing approach, of a zebrafish avatar of human patients carrying a de novo TCF4 variant associated with the atypical phenotype and, in comparison, a tcf4^+/- line as a new in vivo tool to better characterize PTHS-associated molecular pathology.

Overall, our in vitro and in vivo results suggested and corroborated the hypothesis that the new TCF4 variants might confer a GoF phenotype.

Joubert syndrome (JS), a recessive ciliopathy characterized by intellectual disabilities, ataxia and other manifestations, is characterized by defects in cerebellar development and by an unusual midbrain–hindbrain malformation called the molar tooth sign (MTS), probably caused by a connectivity failure involving efferent fibers that originate in the cerebellar nuclei. The molecular alterations leading to the MTS and the related morphological and functional effects on the cerebello-thalamo-cortical circuitry are still unknown. Here we propose to address morphology defects leading to the MTS-like malformations of JS patients by analyzing cerebellar nuclei development in a mouse model carrying a mutation of the Zfp423 gene, whose human ortholog is mutated in JS-19. The Zfp423/ZNF423 gene encodes a 30-zinc-finger transcription factor involved in key developmental pathways. We previously demonstrated that ZFP423 is highly expressed in the neurogenic areas of the cerebellum and that Zfp423/ZNF423 participates in the DNA-damage response (DDR), raising questions regarding its role as a regulator of neural progenitor cell cycle progression in cerebellar development. Mouse Zfp423 mutants display an increase of DNA damage in the cerebellar nuclei neurogenic area and a marked alteration of the cerebellar nuclei which: 1) display a delay in the differentiation of glutamatergic neurons; 2) show a perturbed expression of established markers (Tbr1 and Lmx1a); 3) and an alteration of efferent glutamatergic tracts, likely altering cerebello-thalamic circuits. Our in vivo evidence suggests that Zfp423 is a key gene of cerebellar nuclei development, and sheds light on the mechanisms leading to the formation of the MTS in JS patients.

Development of the central nervous system occurs through a series of steps that include rapid expansion, differentiation and migration of neural stem cells. Errors in this process are among the bases of primary hereditary microcephaly (MCPH), a group of disorders characterized by brain size reduction and mild to moderate intellectual disability. Citron Kinase (CITK) loss of function mutation is the genetic basis for MCPH17. The rodent model of MCPH17, characterized by frameshift (FS) mutations of CITK, displays DNA double strand breaks (DSBs) accumulation and CITK-depleted cells show impaired homologous recombination (HR). To assess the role of CITK in the DNA damage response during human neurodevelopment and uncover HR impairment, we generated 3D human forebrain organoids from wild-type and CITK FS stem cells. CITK FS forebrain organoids show accumulation of DNA damage and reduced levels of BRCA1, key player of HR. Moreover, CITK-depleted cells show impairment in BRCA1 loading at DNA DSBs sites, reduced levels of HR downstream effectors BRCA2 and RAD51 protein and reduced mobility of the DSBs foci. These data indicate that HR impairment after CITK loss is due to altered foci dynamics and reduced recruitment of BRCA1 protein at DSBs. Finally, we found that treatment of CITK-depleted cells with microtubule stabilizing agents recovers DNA damage accumulation and BRCA1 levels. Together these data indicate that CITK is required during human neurodevelopment to maintain genomic stability and for efficient BRCA1 loading at DSB through microtubules dynamics regulation.