Assistant Professor of Pediatrics Stanford University School of Medicine Stanford, California, United States
Background: Human brain development involves fine sculpture of the neuronal network through eliminating redundant synapses by astrocytes; malfunction of this process is associated with ASD. Neonates experienced hypoxic episodes have higher risk of developing ASD. The mechanistic link between hypoxia exposure and ASD development has not been revealed in human species mainly due to lack of available models. We previously developed human cortical organoids (hCOs) from induced pluripotent stem cells (iPSCs) to recapitulate human brain development and residing astrocytes in hCOs also mimic in vivo development features. This hCO model has provided us unprecedent insight into neurodevelopmental diseases. Objective: Objectives of this study include 1) To apply human cortical organoid (hCO) model to study hypoxia effects on astrocytes at neonatal-equivalent stages; 2) To uncover mechanistic link between hypoxia exposure and ASD development; 3) To provide novel therapeutic targets that benefit long term life quality of neonates. Design/Methods: We generated hCOs from 4 iPSC lines and cultured them for 10 months, a timepoint resembling neonatal stages of brain development. We exposed astrocytes isolated from hCOs to hypoxia conditions and performed RNA-sequencing. To evaluate their capability of eliminating synapses in vitro we performed synaptosome engulfment assays. We validated the in vitro findings in a mouse model. Results: We identified hypoxia decreases astrocyte engulfment of synaptosomes. Transcriptome analyses revealed circadian rhythm pathway dysregulation under hypoxia, which we confirmed using PER2::LUCIFERASE assay. Knocking-down a key clock gene up-regulated by hypoxia, REV-ERBα rescued synaptosome elimination defect in hypoxic astrocytes. Increased REV-ERBα activity using agonists was sufficient to decrease synaptosome engulfment by astrocytes, which was replicated in adult mouse hippocampus. Both hypoxia and REV-ERBα agonists increases MEGF10 protein level in human and mouse astrocytes. In addition, MEGF10 is regulated directly by circadian rhythm as demonstrated by its protein level oscillation over day/night cycle, suggesting MEGF10 could mediate the effect of REV-ERBα on astrocyte synapse clearance.
Conclusion(s): Our study identified a novel mechanistic link between hypoxia, circadian rhythm disruptions, and synapse elimination by astrocytes. These findings uncover previously unknown role of circadian rhythm in neuropsychiatric disorders development including ASD.
