A Shared Pathogenic Mechanism for Valproic Acid and SHROOM3 Knockout in a Brain Organoid Model of Neural Tube Defects

Abstract

Neural tube defects (NTDs), including anencephaly and spina bifida, are common major malformations of fetal development resulting from incomplete closure of the neural tube. These conditions lead to either universal death (anencephaly) or severe lifelong complications (spina bifida). Despite hundreds of genetic mouse models of neural tube defect phenotypes, the genetics of human NTDs are poorly understood. Furthermore, pharmaceuticals, such as antiseizure medications, have been found clinically to increase the risk of NTDs when administered during pregnancy. Therefore, a model that recapitulates human neurodevelopment would be of immense benefit to understand the genetics underlying NTDs and identify teratogenic mechanisms. Using our self-organizing single rosette cortical organoid (SOSR-COs) system, we have developed a high-throughput image analysis pipeline for evaluating the SOSR-CO structure for NTD-like phenotypes. Similar to small molecule inhibition of apical constriction, the antiseizure medication valproic acid (VPA), a known cause of NTDs, increases the apical lumen size and apical cell surface area in a dose-responsive manner. GSK3β and HDAC inhibitors caused similar lumen expansion; however, RNA sequencing suggests VPA does not inhibit GSK3β at these concentrations. The knockout of SHROOM3, a well-known NTD-related gene, also caused expansion of the lumen, as well as reduced f-actin polarization. The increased lumen sizes were caused by reduced cell apical constriction, suggesting that impingement of this process is a shared mechanism for VPA treatment and SHROOM3-KO, two well-known causes of NTDs. Our system allows the rapid identification of NTD-like phenotypes for both compounds and genetic variants and should prove useful for understanding specific NTD mechanisms and predicting drug teratogenicity.

Loss of SHROOM3 affects neuroepithelial cell shape through regulating cytoskeleton proteins in cynomolgus monkey organoids

“…Furthermore, SHROOM3 KO led to alterations in the lumen area of the neuroepithelium, indicating a role for SHROOM3 in regulating apical contraction in these cells. The results obtained from our cynomolgus monkey organoids align closely with previous research conducted on human organoids ( Takla et al, 2023 ), suggesting a conserved function of SHROOM3 across species. Our findings indicate that abnormalities associated with NTDs may manifest during the early neuroepithelial stage, prior to neural tube closure.…”

Abnormal cell sorting and altered early neurogenesis in a human cortical organoid model of Protocadherin-19 clustering epilepsy

“…To better understand PCDH19 function during early human corticogenesis, we differentiated WT hESC lines as 2D or 3D neural cultures using Dual-SMAD inhibition ( Shi et al, 2012 ) or a single rosette brain organoid method we developed called self-organizing single rosette cortical organoids (SOSR-COs) ( Takla et al, 2023 ; Tidball et al, 2023 ). PCDH19 mRNA levels and protein localization patterns were examined in the cultures.…”

Brain organoid protocols and limitations

“…The unpredictable number and organization of rosettes within each organoid results in a lack of reproducibility and fidelity ( Table 1 ). Therefore, the self-organizing single rosette (SOSR) organoid was developed and adopted by several laboratories in the past few years (Knight et al, 2018 ; Wang et al, 2022 ; Takla et al, 2023 ). These well-defined SOSR organoids with reproducible size and cytoarchitecture offer improved reproducibility and fidelity (Knight et al, 2018 ; Tidball et al, 2023 ) than the existing organoid protocols (Lancaster et al, 2013 ; Pasca et al, 2015 ) and can be used as a reliable model to recapitulate the neural development and related disorders in the human brain.…”