Relationship of the Human Astrocyte Matrisome with Synaptic Networks

人星形胶质细胞基质体与突触网络的关系

• 批准号: 10562919
• 负责人: Robert Conrad Krencik
• 金额: $ 45.51万
• 依托单位: METHODIST HOSPITAL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-23 至 2027-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10562919
• 关键词: Adherent Culture; Alginates; Astrocytes; Back; Biological Assay; Biological Models; Biomedical Engineering; Brain; Brain Diseases; Calcium; Cell Line; Cell Lineage; Cells; Chondroitin Sulfate Proteoglycan; Clinical; Coculture Techniques; Communities; Comprehension; Data; Disease; Encapsulated; Environment; Excitatory Synapse; Extracellular Protein; Feedback; Genetic Engineering; Health; Human; Human Characteristics; Hydrogels; Image; Intervention; Knowledge; Lead; Ligands; Mitotic; Modeling; Nervous system structure; Neurobiology; Neurons; Neurosciences Research; Organoids; Pharmacological Treatment; Pharmacotherapy; Play; Proteins; Protocols documentation; Role; Signal Transduction; Synapses; THBS1 gene; Technology; Testing; Thrombospondin 1; base; capsule; cell type; density; excitatory neuron; extracellular; glutamatergic signaling; high throughput screening; human pluripotent stem cell; improved; in vivo; innovation; multi-electrode arrays; neural network; novel; novel strategies; optogenetics; physical separation; pre-clinical; receptor; relating to nervous system; synaptic function; synaptogenesis; therapeutic target; tool

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.

项目概要-摘要 星形胶质细胞是神经系统中含量丰富的细胞，在神经系统中发挥着至关重要的作用。 神经元突触网络的编排。它们影响的主要机制之一 神经元突触被认为涉及通过多种细胞外蛋白环境（即星形胶质细胞）进行信号传导 基质体）。然而，目前尚不清楚星形胶质细胞基质体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，特别是在人类特异性细胞中 由于目前使用传统单层实验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 生成和分析由特定数字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从人多能干细胞直接转分化的神经元 细胞。具体来说，我们将使用这些生物工程神经类器官来检验成熟人类的假设 星形胶质细胞产生细胞类型限制的、多成分的、活性依赖性基质体，可加速 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类器官的可行性 基于方法来检验这一假设，并确定了潜在的最佳候选蛋白质 星形胶质细胞到神经元对突触的影响。在目标 1 中，我们将确定人星形胶质细胞是否 血小板反应蛋白 1 蛋白是人类神经元突触的充分且必要的诱导剂。我们将使用一个 结合基因工程和药物治疗来确定血小板反应蛋白 1 是否促进 兴奋性突触网络的结构和功能以及它是否通过向神经元 alpha2delta 发出信号来起作​​用 1个受体。在目标 2 中，我们将定义细胞外人星形胶质细胞基质体并测试其对 使用新型间接共培养方法形成神经元突触。共培养将通过细胞实现 封装在藻酸盐水凝胶胶囊中以阐明细胞外星形胶质细胞基质体成分， 测试它们对神经元类器官的影响，并识别相关的受体-配体对。我们将调查是否 星形胶质细胞来源的细胞外血小板反应蛋白 1 和/或硫酸软骨素蛋白聚糖影响突触 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。神经元将使用光遗传学工具被激活和调节， 并且将使用单细胞的组合来确定对共培养星形胶质细胞的最终影响 RNA 测序、药物治疗、蛋白质测定和钙成像。完成我们的目标 将向科学界提供新的实验细胞系和方案，并可能鉴定新的 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。广义上来说，我们 期望我们的研究通过识别和定义为神经生物学领域做出重大贡献 人类星形胶质细胞和神经元突触之间的细胞间信号传导机制。