Modeling neuronal heterotopias using iPSC derived neural stem cells, neurons and cerebral organoids derived from patients with mutations in FAT4 and DCHS1

Klaus, Johannes (2017): Modeling neuronal heterotopias using iPSC derived neural stem cells, neurons and cerebral organoids derived from patients with mutations in FAT4 and DCHS1. Dissertation, LMU München: Medizinische Fakultät

Abstract

Malformations of the brain are the result of disturbances in the regulation of proliferation, differentiation and migration of cells in the developing central nervous system. In the cortex, malformations often become apparent as mislocalized neuronal tissue, so-called heterotopias. In mouse models, neuronal heterotopias could be shown to be the consequence of either disturbed migration of neurons or instability of the scaffold of radial glia processes which neurons use as a guide during migration. Mouse models revealed many aspects of the mechanism underlying the formation of cortical malformations, however, their use is limited due to structural and functional differences between mouse and humans. Induced pluripotent stem cells (iPSC) offer a promising way to derive human cells of any tissue of interest from patients and control individuals to study the phenotype of cells affected by disease causing mutations. These protocols, however, usually yield two-dimensional monolayer cultures, and do not allow insights into the effects of three-dimensional tissue context on cellular processes. Organoids offer a possibility to overcome this problem, since they represent three-dimensional, embryonic structures which reflect the three-dimensional structure of organs. Cerebral organoids in particular have been shown to reflect the three-dimenisonal organization, cell type composition, and transcriptional footprints of the developing brain. This work made use of the availability of fibroblasts derived from patients with van Maldergem Syndrome, a disease which often comprises the development of neuronal heterotopia and which has been shown to be caused by mutations in FAT4 or DCHS1, to investigate possible mechanisms leading to the development of heterotopia seen in patients. To do this, iPSC were generated from the fibroblasts and differentiated to neural progenitor cells (NPCs) and neurons or three-dimensional cerebral organoids to analyze effects of the mutations on progenitor cells or cortical structures in a three-dimensional context. Analysis of organoids revealed differences between control cell derived organoids and mutant cell derived organoids in the organization of cortical zones. The separation of the neuronal layer and the progenitor layer was less clear, and nodules of neurons appeared in the progenitor zone. In DCHS1 mutant derived organoids, neurites of neurons showed a changed morphology. Especially FAT4 mutant derived organoids showed changes in the morphology of radial glia cells, which possess less straight and often truncated processes and often were delaminate from the apical surface. These results could be further supported by knockdown of FAT4 in control organoids, which revealed a similar phenotype. To see whether neurons in isolation also show defects, their movement behavior was analyzed by time lapse imaging, which revealed that indeed neurons derived from mutant iPSC cells showed changes in their migration: they moved more slowly, less straight and in a more saltatory fashion. Finally, single cell RNA sequencing of cells from organoids, which allows for analysis of the transcriptome of single cells, revealed striking changes in cytoskeletal genes in both DCHS1 and FAT4 mutant cells. Specifically the expression of tubulins was changed, demonstrating changes in the cytoskeleton, which is a promising candidate to explain the changes seen in organoids and neurons. This is further underlined by western blot analysis of cell extracts from neural progenitor cells which showed changes in the expression of stabilized microtobules, hinting towards a generalized change in the regulation of the microtubule cytoskeleton. Taken together, this work is one of the first to model neuronal heterotopia in cerebral organoids. It further shows that the phenotype seen in patients is most likely is the result of disturbances in neurons as well as in progenitor cells. Furthermore, it suggests that the mutations analyzed lead to changes in the regulation of the cytoskeleton, which suggests a new function of FAT4 and DCHS1 in regulating processes important for neural development.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Themengebiete: 600 Technik, Medizin, angewandte Wissenschaften ; 600 Technik, Medizin, angewandte Wissenschaften > 610 Medizin und Gesundheit
• Fakultäten: Medizinische Fakultät
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 9. Oktober 2017
• 1. Bericht­erstatter:in: Götz, Magdalena