Investigating pathomechanisms underlying malformations of human cortical development

Abstract

During human development the brain has to form a complex neuronal network requiring a well-orchestrated sequence of neurogenesis, neuronal migration and correct positioning within cortical layers followed by synaptogenesis. Malformations associated with cortical development (MCD) can be the result of interferences with one or several elements of this sequence and subsequently lead to different manifestations of MCD with severe consequences including epilepsy, cognitive impairment and intellectual deficiency. In order to investigate the resulting cortical disorganization various mouse models have been used to approximate pathological human corticogenesis. Numerous aspects of MCD can be recapitulated in mouse models making it possible to draw conclusions regarding underlying pathomechanisms. These models are limited in regard of human specific aspects of brain development though. <br /> The advent of human induced pluripotent stem cells (hiPSCs) and three-dimensional (3D) organoid cell culture enables scientists to circumvent the limitations of mouse models and to address human specific features of MCD in a spatio-temporal context. The capability of self-organization in combination with the patterning into forebrain organoids makes it a suitable in vitro system to study early corticogenesis and linked developmental disorders in a human context. <br /> In addition, using single-cell RNA sequencing (scRNA seq.) technologies it becomes possible to resolve the cytoarchitecture of organoids in order to look at the cell composition on single cell based resolution. Information based on single cell transriptomic data allows us to deconstruct the 3D model into different cell types and identify aspects of pathological mechanisms adding up to findings acquired by the repertoire of molecular biology methods. <br /> The beginning of this compendium describes the protocol we developed for the generation of standardized and reproducible forebrain-type organoids from hiPSC, constituting the prerequisite for the investigation of two distinct forms of MCD found in humans. One of these two studies deals with the characterization and underlying pathomechanism of a form of lissencephaly induced by the heterozygous loss of the genes PAFAH1B1 and YWHAE, called Miller-Dieker syndrome (MDS). Using iPSC- derived organoids from Miller-Dieker-syndrome patients a disturbance of the cortical niche was identified, which leads to alterations in N-cadherin/ b-catenin signaling consequently leading to a non-cell autonomous radial glia cell expansion defect. Moreover, with this study we demonstrate a proof of concept regarding the use of our organoid protocol for disease modeling in vitro.The subsequent part of this compendium is a study in which the role of the Echinoderm Microtubule-Associated Protein-like 1 (Eml1/EML1) gene in subcortical heterotopia formation is investigated in an in vivo context. With the help of a Heterotopic Cortex (HeCo) mutant mouse model and human patient fibroblasts as well as hiPSC derived cortical progenitors, heterotopia formation driver as well as a potentially underlying primary cilia and Golgi apparatus phenotype were identified. <br /> As mentioned before, mouse model can reflect certain aspects of human cortical development but it remains challenging to recapitulate human specific features of neurodevelopmental disorders. The last part of this compendium focuses on the formation of subcortical heterotopia in human patients, characterized by the presence of abnormally positioned neurons, alongside megalencephaly and polymicrogyria induced by mutations in the aforementioned gene EML1. Investigation of iPSC-derived cerebral organoids from EML1-patients and EML1-KO lines showed so far unrecognized abundance of perturbed progenitor cells with increased basal radial glia cell and extracellular matrix (ECM) gene expression profiles in the heterotopic area. This cell population additionally displayed a massive aberrant YAP1 mediated expansion. <br /> Congruencies of major phenotypic findings in hiPSC-derived organoids and the HeCo mouse model reinforces not only the idea that forebrain-type organoids reflect in vivo results, but it also emphasizes the synergistic and complementary potential of our in vitro model by bridging an evolutionary gap. <br /> Taken together, the studies presented in this compendium deliver greater insight into human MCD mechanisms and support the idea that organoid-based systems serve as promising models to study early human cortical development and associated disorders.