CRISPRi-based screens in iAssembloids to elucidate neuron-glia interactions. CRISPRi-based screens in iAssembloids to elucidate neuron-glia interactions

The sheer complexity of the brain has complicated our ability to understand its cellular mechanisms in health and disease. Genome-wide association studies have uncovered genetic variants associated with specific neurological phenotypes and diseases. In addition, single-cell transcriptomics have provided molecular descriptions of specific brain cell types and the changes they undergo during disease. Although these approaches provide a giant leap forward towards understanding how genetic variation can lead to functional changes in the brain, they do not establish molecular mechanisms. To address this need, we developed a 3D co-culture system termed iAssembloids (induced multi-lineage assembloids) that enables the rapid generation of homogenous neuron-glia spheroids. We characterize these iAssembloids with immunohistochemistry and single-cell transcriptomics and combine them with large-scale CRISPRi-based screens. In our first application, we ask how glial and neuronal cells interact to control neuronal death and survival. Our CRISPRi-based screens identified that GSK3β inhibits the protective NRF2-mediated oxidative stress response in the presence of reactive oxygen species elicited by high neuronal activity, which was not previously found in 2D monoculture neuron screens. We also apply the platform to investigate the role of APOE-𝜀4, a risk variant for Alzheimer’s Disease, in its effect on neuronal survival. We find that APOE-𝜀4 expressing astrocytes may promote more neuronal activity as compared to APOE-𝜀3 expressing astrocytes. This platform expands the toolbox for the unbiased identification of mechanisms of cell-cell interactions in brain health and disease. Overall design: Examination of cellular differention and composition in 3D neuron-astrocyte-microglia co-culture models (iAssembloids)

大脑本身的极端复杂性，极大阻碍了我们对其健康与疾病状态下细胞机制的认知。全基因组关联研究（Genome-wide Association Studies）已发现与特定神经表型及疾病相关的遗传变异。此外，单细胞转录组学（single-cell transcriptomics）已为特定脑细胞类型及其在疾病进程中的变化提供了分子层面的描述。尽管这些研究手段在解析遗传变异如何引发大脑功能改变方面取得了重大突破，但仍无法阐明其分子机制。为解决这一难题，我们开发了一款名为iAssembloids（诱导多谱系组装类器官，induced multi-lineage assembloids）的3D共培养体系，可快速生成均质化的神经元-胶质细胞球体。我们通过免疫组织化学（immunohistochemistry）与单细胞转录组学对该iAssembloids体系进行表征，并将其与大规模基于CRISPR干扰（CRISPRi）的筛选技术相结合。在本研究的首个应用场景中，我们探究胶质细胞与神经元如何相互作用以调控神经元的死亡与存活。我们基于CRISPR干扰的筛选发现，在高神经元活性引发的活性氧（reactive oxygen species, ROS）存在的情况下，糖原合成激酶3β（GSK3β）会抑制具有保护作用的核因子红细胞2相关因子2（NRF2）介导的氧化应激应答——这一发现此前在2D单培养神经元筛选中从未被观测到。我们还将该平台应用于探究阿尔茨海默病（Alzheimer’s Disease）风险变异APOE-ε4对神经元存活的影响机制。我们发现，与表达APOE-ε3的星形胶质细胞相比，表达APOE-ε4的星形胶质细胞可促进更强的神经元活性。该平台为无偏地解析大脑健康与疾病状态下的细胞间相互作用机制提供了新的研究工具。整体实验设计：对3D神经元-星形胶质细胞-小胶质细胞共培养模型（iAssembloids）中的细胞分化与组成进行分析。