Relationship of the Human Astrocyte Matrisome with Synaptic Networks

项目摘要 星形胶质细胞是神经系统中高度丰富的细胞，它们在神经系统中起关键作用。 神经元突触网络的协调。它们影响的主要机制之一 神经元突触被认为涉及经由细胞外蛋白质的不同环境的信号传导（即，星形胶质细胞 matrisome）。然而，目前还不清楚星形胶质细胞基质体的哪些成分是必需的， 足以形成和加强神经元兴奋性突触，特别是在人类特异性细胞中 由于目前使用传统单层在实验上研究人类神经网络的局限性 培养物和未成熟的类器官。为了克服这些技术限制，我们将利用我们最近优化的 一种生成和分析由特定数量组成的生物工程神经类器官的方法 有丝分裂后星形胶质细胞以及直接从人多能干细胞转分化的神经元 细胞具体来说，我们将使用这些生物工程神经类器官来测试成熟人类的假设， 星形胶质细胞产生细胞类型限制的、多组分的、活性依赖性的基质体， 神经元突触网络的形成和功能。我们的初步数据证实了类器官的可行性- 的方法来测试这一假设，并确定了潜在的潜在潜在候选蛋白质， 星形胶质细胞对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 血小板反应蛋白1是人类神经元突触的充分和必要的诱导剂。我们将使用一个 基因工程和药物治疗的组合，以得出结论是否血小板反应蛋白1促进 结构和功能兴奋性突触网络，如果它通过信号传导到神经元α 2 δ- 1受体。在目标2中，我们将定义细胞外的人星形胶质细胞基质体，并测试其对 神经元突触的形成使用一种新的间接共培养方法。共同培养将通过细胞 包封在藻酸盐水凝胶胶囊内以阐明细胞外星形胶质细胞基质体组分， 测试它们对神经元类器官的作用，并鉴定相关的受体-配体对。我们将调查是否 星形胶质细胞源性细胞外血小板反应蛋白1和/或硫酸软骨素蛋白多糖影响突触 形成和功能。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 人星形胶质细胞基质体的特征。神经元将被激活并使用光遗传学工具起搏， 并将使用单细胞和细胞因子的组合来确定对共培养的星形胶质细胞的最终作用。 RNA测序、药物治疗、蛋白质测定和钙成像。完成我们的目标 将为科学界提供新的实验细胞系和方案，并可能发现新的 在基于类器官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 我希望我们的研究通过识别和定义神经生物学领域做出重大贡献。 人星形胶质细胞和神经元突触之间的细胞间信号传导机制。

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.