Stimulus‐dependent modifications in astrocyte‐derived extracellular vesicle cargo regulate neuronal excitability

Abstract Extracellular vesicles have now emerged as key players in cell‐to‐cell communication. This is particularly important in the central nervous system, where glia–neuron cross‐talk helps maintain normal neuronal function. Astrocyte‐derived extracellular vesicles (ADEVs) secreted constitutively promote neurite outgrowth and neuronal survival. However, extracellular stimuli can alter the cargo and downstream functions of ADEVs. For example, ADEVs secreted in response to inflammation contain cargo microRNAs and proteins that reduce neurite outgrowth, neuronal firing, and promote neuronal apoptosis. We performed a comprehensive quantitative proteomic analysis to enumerate the proteomic cargo of ADEVs secreted in response to multiple stimuli. Rat primary astrocytes were stimulated with a trophic stimulus (adenosine triphosphate, ATP), an inflammatory stimulus (IL‐1β) or an anti‐inflammatory stimulus (IL10) and extracellular vesicles secreted within a 2 hr time frame were collected using sequential ultracentrifugation method. ADEVs secreted constitutively without exposure to any stimulus were used a control. A tandem mass tag‐based proteomic platform was used to identify and quantify proteins in the ADEVs. Ingenuity pathway analysis was performed to predict the downstream signaling events regulated by ADEVs. We found that in response to ATP or IL10, ADEVs contain a set of proteins that are involved in increasing neurite outgrowth, dendritic branching, regulation of synaptic transmission, and promoting neuronal survival. In contrast, ADEVs secreted in response to IL‐1β contain proteins that regulate peripheral immune response and immune cell trafficking to the central nervous system.