Mechanisms controlling human brain development, despite their relevance, are still poorly understood, given the challenges of studying human embryos and developmental divergence from animal models. Human cell-based in vitro models can be used to understand mechanisms of human brain development and its possible alterations, thereby bypassing substantial interspecies differences. Since uncovering the molecular and cellular bases of a disorder is critical to developing an evidence-based treatment, research conducted for this Ph.D. project used iPSCs, neural stem cell derivatives, and CRISPR-Cas9 technology to investigate the molecular basis of primary microcephaly (MCPH), a genetic neurodevelopmental disorder in humans. This rare and incurable pathology has a birth incidence that ranges from 1.3 to 150/100,000, depending on the population type and consanguineous marriage rate. Mutations of various genes linked to mitotic spindle assembly or cell cycle can lead to MCPH; the second most commonly affected gene is WDR62 (MCPH2), a scaffold protein localized to the spindle poles during mitosis and involved in symmetric versus asymmetric cell division choices in neural progenitors during cortical development.
Thursday, 06 July 2023 07:14
Human neural stem cell models to dissect WDR62-related microcephaly
WDR62 is a scaffold protein localized to the spindle poles during mitosis and involved in symmetric versus asymmetric cell division choices in neural progenitors during brain development. Pathogenic mutations of WDR62 account for the second most common of genetic primary microcephaly (MCPH2). The research conducted for this Ph.D. project used patient’s iPSCs, neural stem cell derivatives, cerebral organoids, and CRISPR-Cas9 technology to investigate the molecular basis of MCPH2 – enlightening the consequences of a specific WDR62 mutation, and more broadly contributing to a wider comprehension of WDR62-related MCPH pathological mechanisms.
In this Ph.D. project, MCPH2-patient iPSCs carrying the homozygous WDR62 D955AfsX112 de novo truncating mutation (Mut iPSCs) were used. The mutation consists of a 4 bp deletion in exon 23 of WDR62 leading to a premature stop codon, and to a C-terminus truncated protein. An iPSCs line harboring the WDR62 D955AfsX112 heterozygous mutation (from one parent) was characterized and two additional MCPH2 pathogenic mutations were assessed. Furthermore, isogenic corrected lines (Iso) from Mut iPSCs were generated as a gold standard control. Subsequently, Mut and Iso iPSCs neuroepithelial stem (iPS-NES) cells were differentiated and cerebro-cortical neurons used to establish a 2D MCPH2 in vitro model. Further, a 3D neural progenitor in vitro model generating human iPS-derived Iso, Het, and Mut cerebral organoids (hCOs) was also used.
Based on immunofluorescence, in Mut iPSCs and Mut iPS-NES cells, WDR62 failed to localize to the spindle poles during mitosis, with a diffuse pattern around chromatin, whereas localization at spindle poles was restored in Iso iPSCs and Iso iPS-NES cells. Similarly, WDR62 was correctly localized to the spindle poles during mitosis in Het iPSCs. The absence of WDR62 from spindle poles in Mut iPS-NES cells impaired mitotic progression. Conversely, during interphase, there was a novel WDR62 localization pattern, with the protein localized to the Golgi apparatus in both Mut and Iso iPS-NES cells. Several approaches were used to investigate this finding, including WDR62 signal specificity assessment through WDR62 siRNA-mediated knockdown, FLAG-tagged WDR62 mis-expression, and through Golgi apparatus fragmentation, which revealed WDR62 signal localized in the generated Golgi ministacks. Based on these results, we inferred that the mutation, leading to a C-terminus truncated peptide, did not impact WDR62 expression and protein production, and that the D955AfsX112 mutation mainly affected localization during mitosis, although its Golgi apparatus localization was maintained. Additionally, analysis in the FLAG-tagged WDR62 mis-expression system revealed a common pattern among various patient-encountered mutant forms of WDR62.
To extend and validate our findings in a 3D neural progenitor model, Iso, Het, and Mut hCOs at day in vitro 30 (DIV30) were analyzed with confocal imaging. At DIV30, in hCOs were radial glia (RG)-like neural progenitors organized into neural rosettes. In Iso and Het mitotic RG-like cells within the hCOs, WDR62 was present at spindle poles, confirming the WDR62 localization pattern we had detected in iPS-NES cells. Conversely, WDR62 was mis-localized from spindle poles and diffused around chromatin in mitotic RG-like cells in Mut hCOs. Further, WDR62 and Golgi apparatus were localized in the apical domain of the RG-like cells, an observation also validated in human fetal brain sections. RG-like cell mitotic spindle orientation was assessed with the hCO platform, revealing higher asymmetric cell divisions in Mut hCOs and indicating a premature differentiative fate.
Lastly, the mechanism translocating WDR62 from the Golgi apparatus to the spindle poles was explored. Microtubules were pharmacologically disrupted in CTRL iPS-NES cells, with a lack of WDR62 from spindle poles in mitotic cells after treatment. Therefore, we concluded that the D955AfsX112 mutation hampered the microtubule-mediated WDR62 shuttling from the Golgi apparatus to the spindle poles, causing: i) diminished capability of Mut iPS-NES cells to regain the cell cycle after drug-induced arrest; ii) shortened primary cilia in Mut iPS-NES cells; iii) an increase in percentage of asymmetric cell divisions in the population of RG-like cells in Mut hCOs; and iv) impaired neuronal generation timing as a final outcome. Indeed, during directed neocortical differentiation, there was altered neuronal generation timing leading to incorrect cell fate acquisition.
Collectively, these results documented a novel side of Golgi apparatus localization in interphase cells and consequences of the D955AfsX112 (and perhaps others) mutation on protein dynamic localization pattern. Impairment of Golgi apparatus-spindle pole shuttling caused severe neurodevelopmental abnormalities, delineating new mechanisms in MCPH2 etiology.