Neuroligin-3 R451C Induces Gain-of-Function Gene Expression in Astroglia in an Astroglia-Enriched Brain Organoid Model. Neuroligin-3 R451C Induces Gain-of-Function Gene Expression in Astroglia in an Astroglia-Enriched Brain Organoid Model

Astroglia are integral to brain development and the emergence of neurodevelopmental disorders. However, studying the pathophysiology of human astroglia using brain organoid models has been hindered by inefficient astrogliogenesis. In this study, we introduce a robust method for generating astroglia-enriched organoids through BMP4 treatment during the neural differentiation phase of organoid development. Our RNA sequencing analysis reveals that astroglia developed within these organoids exhibit advanced developmental characteristics and enhanced synaptic functions compared to those grown under traditional two-dimensional conditions, particularly highlighted by increased neurexin (NRXN)-neuroligin (NLGN) signaling. Cell adhesion molecules, such as NRXN and NLGN, are essential in regulating interactions between astroglia and neurons. We further discovered that brain organoids derived from human embryonic stem cells (hESCs) harboring the autism-associated NLGN3 R451C mutation exhibit increased astrogliogenesis. Notably, the NLGN3 R451C astroglia demonstrate enhanced branching, indicating a more intricate morphology. Interestingly, our RNA sequencing data suggest that these mutant astroglia significantly upregulate pathways that support neural functions when compared to isogenic wild-type astroglia. Our findings establish a novel astroglia-enriched organoid model, offering a valuable platform for probing the roles of human astroglia in brain development and related disorders. Overall design: To assess the effects of the NLGN3 R451C mutation in human astroglia, we conducted RNA-seq experiments on WT/R451C astroglia cultured in 3D (three replicates per group) and WT astroglia cultured in 2D (three replicates).

星形胶质细胞（Astroglia）是大脑发育以及神经发育障碍发生过程中不可或缺的组成部分。然而，利用脑类器官模型研究人类星形胶质细胞的病理生理机制，却受限于星形胶质细胞生成效率不足的问题。本研究建立了一种稳健的方法，可在类器官发育的神经分化阶段通过骨形态发生蛋白4（BMP4）处理，构建富集星形胶质细胞的脑类器官。我们的RNA测序（RNA-seq）分析显示，相较于传统二维培养条件下的星形胶质细胞，此类类器官内发育的星形胶质细胞展现出更为成熟的发育表型与更强的突触功能，该差异在神经连接蛋白（Neurexin, NRXN）-神经黏附蛋白（Neuroligin, NLGN）信号通路的活化增强上体现得尤为显著。诸如NRXN与NLGN这类细胞黏附分子，对调控星形胶质细胞与神经元间的相互作用至关重要。我们进一步发现，携带自闭症相关NLGN3 R451C突变的人类胚胎干细胞（human embryonic stem cells, hESCs）所构建的脑类器官，其星形胶质细胞生成率显著升高。值得注意的是，携带NLGN3 R451C突变的星形胶质细胞分支能力显著增强，提示其细胞形态更为复杂精细。有趣的是，相较于同基因背景的野生型（WT）星形胶质细胞，此类突变型星形胶质细胞显著上调了一系列支持神经功能的信号通路。本研究构建了一种新型的富集星形胶质细胞的脑类器官模型，为探究人类星形胶质细胞在大脑发育及相关神经疾病中的功能与作用机制提供了极具价值的研究平台。整体实验设计：为评估NLGN3 R451C突变对人类星形胶质细胞的影响，我们对以下样本开展了RNA-seq实验：三维培养的野生型与R451C突变型星形胶质细胞（每组设置3个生物学重复），以及二维培养的野生型星形胶质细胞（3个生物学重复）。