Modeling neurodevelopment in 2D and 3D cultures with different DNA glycosylase backgrounds
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The brain is under constant attack from exogenous and endogenous sources of DNA damage, and due to the non-proliferative identity of the neuronal cells, a highly functional repair systems need to be in place. One of the repair systems functioning in the brain is base excision repair. This is a repair pathway that recognizes a diverse array of small base lesions. It is initiated by DNA glycosylases, one of which is alkyladenine DNA glycosylase (AAG). This DNA glycosylase recognizes damage that has been introduced by alkylating agents. Several studies have addressed how this glycosylase impacts the brain of mice and showed that Aag overexpression results in alkylation-triggered neurodegeneration, and that this neurodegeneration is inhibited when Aag is absent. Currently however, little is known about the impact of Aag on neurodevelopment and on human brain functioning in particular. Neurodevelopment has intrigued scientists for decades. The human brain is a complex organ, which differs greatly from models previously used, as for example mice. The inaccessibility of human tissues has thus made it difficult to study human specific traits of neurodevelopment and neurodegeneration. In recent years the ability to produce pluripotent stem cells from normal somatic tissue has expanded the research on human based traits in neurodevelopment. The two main models are 2D models, allowing generation of a homogenous cell population of interest, and 3D models that result in production of heterogenous tissues. The latter has been proven to replicate the human-based traits better than any previously used models. The main aim of this thesis is to investigate the impact of altered AAG expression on human neurodevelopment. This has been accomplished by the production of both cerebral organoids and cortical excitatory neurons. In each of these modeling systems three different hiPSC genotypes were used: AAGWT, AAGE125Q, containing a point mutation making AAG enzymatically inactive, and AAG-/-. We successfully generated both cerebral organoids and cortical excitatory neurons from each of the genotypes. In order to determine the cell diversity in the organoids single-cell RNA sequencing (scRNA-seq) was performed on 1 month old organoids. The scRNA-seq resulted in the identification of several different cell types and brain regions, making this an excellent model to study genetic differences between genotypes. Interestingly, while loss of AAG protein, as well as catalytic activity were not vital for neurodevelopment scRNA-seq experiments suggest that this DNA glycosylase might be important for development of particular cell types, such as cells with forebrain features. Cortical excitatory neurons were produced by modifications of previously published work, and their identity confirmed by IF staining and RT-qPCR . Current experiments suggested that all three tested genotypes are able to form cortical excitatory neurons. In summary, the establishment of conditions for scRNA-seq of cerebral organoids and of differentiation protocols provide valuable tools that will help to explore in depth the role of altered AAG in establishment of human-specific traits of neurodevelopment.