Ethanol causes cell death and neuronal differentiation defect during initial neurogenesis of the neural retina by disrupting calcium signaling in human retinal organoids

Abstract

Background: Over 90% of children with fetal alcohol syndrome live with ocular aberration due to the susceptible and intricate human eye development process. Initial neurogenesis of the neural retina around six-week gestation is the critical period of human eye development while sustaining the highest risk of prenatal ethanol exposure because of ignorance of early pregnancy. However, the influence and mechanism of short-term ethanol exposure on this developmental process of the human neural retina remain largely unknown. Methods: To faithfully recapitulate the initial retinal neurogenesis of human neural retina, human embryonic stem cell derived retinal organoids (hROs) were induced and identified by immunostaining. Morphological measurement was performed to primarily assess the influence of short-term ethanol exposure on the growth of neural retina. TUNNEL assay, immunostaining, and flow cytometry were utilized to detect cell death, retinal ganglion cell differentiation, and cell cycle progression in hROs. Bulk RNA-seq analysis and cnet plotting were performed to screen signaling pathway and regulated genes of ethanol treatment. GCaMP5G-expressing human embryonic stem cells were constructed by transduction of pLOV-CMV-GCaMP5G and fluorescence-activated cell sorting. Two-photon microscope live calcium imaging were utilized to reveal altered calcium signaling dynamics after ethanol treatment. Quantitative RT-PCR was performed to verify the expression of screened potential targeted genes of ethanol treatment. Results: The hROs from D24 to D30 well recapitulate the initial neurogenesis of the human neural retina around six-week gestation in vivo at the histological, cellular, and molecular level. 1% (v/v) ethanol slowed the growth of hROs by inducing robust cell death and retinal ganglion cell differentiation defect. Calcium signaling dynamics was proved significantly altered and derived from ethanol-induced down-regulation of RYR1and CACNA1S. Moreover, the calcium-binding protein RET, one of the downstream effector genes of the calcium signaling pathway, synergistically integrates ethanol and calcium signals to abort neuron differentiation and cause cell death. Conclusion: Our study demonstrated that short-term ethanol exposure greatly impaired the initial neurogenesis of hROs by disrupting the RYR1 related calcium signaling. These results may help us elaborate on more detailed principles of ethanol-induced teratogenesis and instruct the rational application of alcohol and ethanol-contained drugs during pregnancy.