Relationship of the Human Astrocyte Matrisome with Synaptic Networks - 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.
