Responsiveness of MGE NPCs to radiation-induced DNA damage in mouse embryonic development: The role of p53

Brain development is a complex process that begins during the early embryonic stages and continues after birth. An important step in brain development is the generation of brain cells, the neurons. This process is called neurogenesis and starts with neural stem cells. These cells are destined to become several types of cells residing in our brain, including neurons. First, these neural stem cells have to undergo several stages to acquire more neuron-like characteristics. As such, neural stem cells differentiate to neural progenitors (NPC), which have both stem cell- and neuron-like characteristics, before becoming mature and functional neurons. They reach their final destination in the brain, the cortex, by migration through the different layers of the cortex. In our brain, two main types of neurons can be found. These are interneurons which are characterized by an inhibitory function, and excitatory neurons, with a stimulatory function. The balance between these neurons is crucial for a properly functioning brain. An imbalance can lead to neurological disorders such as schizophrenia or autism. During embryonic brain development, NPCs are very sensitive to DNA damage. In general, DNA damage can be induced by external and internal factors, such as radiation, nutrition or alcohol. They cause damage to the DNA where either one or both strands of the DNA are altered or broken. If a cell has DNA damage, various repair mechanisms can be activated to repair this damage. These repair mechanisms are essential for keeping the entire set of DNA intact and preventing neurological disorders. In this thesis, we use radiation exposure to induce DNA damage. It is known that radiation induces breaks in both DNA strands, called double strand breaks (DSBs). DSBs are one of the most severe DNA lesions which can be repaired by two specific repair mechanisms. In general, when DNA damage is detected in a cell they initiate a DNA damage response. First, the DNA damage is recognized in the cell, causing different types of molecules to go to the site of damage. An important step is the activation of the p53 gene. p53 is considered a guardian of the DNA and is the main regulator of the DNA damage response. This gene ensures a cell cycle arrest, giving DNA repair mechanisms time to do their work. However, depending on the severity of the damage, p53 can also direct the cell to cell death (apoptosis) or senescence. We investigated the effect of radiation exposure on cell cultures of excitatory and inhibitory NPCs, and embryonic brains. First, we were able to confirm the successful establishment of excitatory and inhibitory NPC cultures as they expressed genes which are specifically found in these cells. When these NPCs were exposed to irradiation, it showed that 2h after irradiation, more DNA damage and more repair mechanisms were present in irradiated cells than in non-irradiated cells. Furthermore, we found a cell cycle arrest in irradiated cells. To investigate whether this cell cycle arrest is mediated by p53, a mouse model with genetic inactivation of Trp53 (p53 gene in mice) was used. The results show that genetic inactivation of Trp53 did not affect the cell cycle arrest observed after irradiation, suggesting that the cell cycle arrest is not p53-dependent. Finally, the migration process of neurons was examined and we found that migration speed of the cells was reduced after irradiation.