Relationship of the Human Astrocyte Matrisome with Synaptic Networks

项目摘要-摘要 星形胶质细胞是神经系统中高度丰富的细胞，它们在 神经元突触网络的协调。他们影响的主要机制之一 神经元突触被认为涉及通过不同的细胞外蛋白环境(即星形胶质细胞)进行信号传递 母体)。然而，目前还不清楚星形胶质细胞基质的哪些成分是必需的，以及 足以形成和加强神经元兴奋性突触，尤其是在人类特有的细胞中 由于目前使用传统单层对人类神经网络进行实验研究的局限性 培养和未成熟的有机物质。为了克服这些技术限制，我们将利用最近优化的 一种产生和分析由特定数字组成的生物工程神经有机体的方法 有丝分裂后星形胶质细胞以及从人类多能干细胞直接转分化的神经元 细胞。具体地说，我们将使用这些生物工程神经有机体来测试成熟的人类 星形胶质细胞产生一种细胞类型受限的、多组分的、依赖活动的母体，它加速了 神经元突触网络的形成和功能。我们的初步数据证实了我们的有机化合物的可行性- 基于一种方法来检验这一假说，并已经确定了潜在的潜在候选蛋白质 星形胶质细胞到神经元对突触的影响。在目标1中，我们将确定人类星形胶质细胞 凝血酶敏感蛋白1是人类神经元突触的充分和必要的诱导物。我们将使用 基因工程和药物治疗相结合得出凝血酶敏感蛋白1是否促进 结构和功能兴奋性突触网络，如果它通过信号传递给神经元α2增量- 1受体。在目标2中，我们将定义细胞外人类星形胶质细胞基质并测试其对 用一种新的间接共培养方法形成神经元突触。Cocultures将通过蜂窝网络实现 海藻酸凝胶胶囊化研究细胞外星形胶质细胞基质组分， 测试它们对神经元器官的影响，并确定相关的受体-配体对。我们将调查是否 星形胶质细胞来源的胞外血栓反应蛋白1和/或硫酸软骨素蛋白多糖影响突触 构成和功能。最后，在目标3中，我们将测试神经元活动如何影响突触促进 人类星形胶质细胞染色体的特征。神经元将被激活，并使用光遗传工具进行起搏， 而对共同培养的星形胶质细胞的最终影响将使用单个细胞的组合来确定 RNA测序、药物治疗、蛋白质分析和钙成像。我们目标的完成 将向科学界提供新的实验细胞系和方案，并可能识别新的 在基于有机物的模型系统中加速神经元突触网络形成的方法。总的来说，我们 期待我们的研究通过识别和定义 人脑星形胶质细胞和神经元突触之间的细胞间信号机制。

PROJECT SUMMARY-ABSTRACT Astrocytes are highly-abundant cells in the nervous system and they play a critical role in the orchestration of neuronal synaptic networks. One of the primary mechanisms through which they influence neuronal synapses is thought to involve signaling via a diverse milieu of extracellular proteins (i.e., the astrocyte matrisome). However, it remains unclear which components of the astrocyte matrisome are necessary and sufficient for the formation and strengthening of neuronal excitatory synapses, especially in human-specific cells due to current limitations in experimentally investigating human neural networks using traditional monolayer cultures and immature organoids. To overcome these technical limitations, we will utilize our recently optimized approach which generates and analyzes bioengineered neural organoids that are composed of specific numbers of post-mitotic astrocytes as well as neurons that are directly transdifferentiated from human pluripotent stem cells. Specifically, we will use these bioengineered neural organoids to test the hypothesis that mature human astrocytes produce a cell type-restricted, multi-component, activity-dependent matrisome that accelerates the formation and function of neuronal synaptic networks. Our preliminary data confirms feasibility of our organoid- based approach to test this hypothesis and has identified top candidate proteins potentially underlying the astrocyte-to-neuron influence on synapses. In Aim 1, we will determine whether human astrocyte Thrombospondin 1 protein is a sufficient and necessary inducer of human neuronal synapses. We will use a combination of genetic engineering and drug treatment to conclude whether Thrombospondin 1 promotes structural and function excitatory synaptic networks and if it acts through signaling to the neuronal alpha2delta- 1 receptor. In Aim 2, we will define the extracellular human astrocyte matrisome and test its influence upon neuronal synapse formation using a novel indirect coculture approach. Cocultures will be enabled by cellular encapsulation within alginate hydrogel capsules to elucidate the extracellular astrocyte matrisome components, test their effect upon neuronal organoids, and identify relevant receptor-ligand pairs. We will investigate whether astrocyte-derived extracellular Thrombospondin 1 and/or chondroitin sulfate proteoglycans influences synapse formation and function. Finally, in Aim 3, we will test how neuronal activity influences the synapse-promoting characteristics of the human astrocyte matrisome. Neurons will be activated and paced using optogenetic tools, and the resultant effect on cocultured astrocytes will be determined using a combination of single cell RNAsequencing, pharmacological treatment, protein assays, and calcium imaging. The completion of our aims will deliver novel experimental cell lines and protocols to the scientific community, and may identify novel approaches to accelerate neuronal synaptic network formation in organoid-based model systems. Broadly, we expect our studies to make significant contributions to the neurobiology field by identifying and defining intercellular signaling mechanisms between human astrocytes and neuronal synapses.