Effects of ionizing radiation exposure on human brain development, modeled in dorsal forebrain organoids

Exposure to ionizing radiation (IR) during critical periods of neurogenesis is currently discouraged, as observations in survivors of the atomic bombs in Hiroshima and Nagasaki have shown that it can profoundly affect fetal brain development and possibly lead to a reduction in brain size (microcephaly). In clinical practice, the use of IR is increasing every year, especially for radiotherapy, and a better understanding of the potential adverse effects, such as microcephaly , is To date, i t is uncle ar which molecular mechanisms are at the basis of this defect in humans as the few mechanistic studies conducted so far have been performed in rodents. Although mouse models are excellent for studying the effects of radiation exposure and have provided valuable insights, they are not perfect for mimicking human brain development due to significant differences in neurogenesis between humans and mice. To ad dress this gap and better replicate aspects of human brain developmental physiology we used human embry onic stem cell derived dorsal forebrain organoid s as a model for human corticogenesis. The cortical organoids generated in this study were characterized at the cellular and molecular levels, demonstrating their ability to recapitulate key aspects of human neurogenesis. Following characterization, o rganoids were irradiated with a moderate (0.5 Gy) and a high (2 Gy) dose of X rays at two different time points, representing very early and mid corticogenesis. Organoid growth was monitored until 14 days after ir radiation and immunostainings and transcriptomic analyses were performed at early (2 h, 6 h, and 24 h) and late (14 days) time points after irradiation. Irradiation caused a dose and developmental timing dependent reduction in organoid size, which was more prominent in developmentally younger organoids. This coincided with a dose dependent canonical p53/DREAM dependent DNA damage response ( DDR), consisting of cell cycle arrest, DNA repair and apoptosis. The DDR was delayed and less pronounced in older orga noids compared to young organoids. Besides the DDR, we observed IR induced premature differentiation of neural progenitor cells (NPCs) and changes in metabolism. Importantly, our transcriptomic analysis furthermore demonstrated a coordinated repression of primary microcephaly (MCPH) genes, especially in high dose irradiated organoids. We found that this was a human specific feature, as i t was not observed in mouse embryonic brains or primary mouse NPCs . To validate this finding, qRT PCR experiments confirmed radiation induced downregulation of selected MCPH genes ( ASPM , CIT , KNL1 and WDR62 ) in human, but not mouse NPCs. Knockdown of TP53 and pharmacological inhibition of E2Fs, showed that the repression of MCPH genes was dependent on the p53 E2F/DREAM axis , underscoring a new mechanism underlying IR induced microcephaly that was only possible due to the use of a human model . Overall, our study demonstrated that human dorsal forebrain organoid s are an excellent model for investigating prena tal DNA damage induced microcephaly and uncovering potentially targetable human specific pathways. Moreover, it builds on the currently scarce knowledge regarding the impacts of IR during the early stages of human brain development, indicating that exposure to IR during these periods should continue to be minimized.