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Activated human astrocyte-derived extracellular vesicles modulate neuronal uptake, differentiation and firing.

ABSTRACT: Astrocytes in the central nervous system (CNS) provide supportive neural functions and mediate inflammatory responses from microglia. Increasing evidence supports their critical roles in regulating brain homoeostasis in response to pro-inflammatory factors such as cytokines and pathogen/damage-associated molecular pattern molecules in infectious and neurodegenerative diseases. However, the underlying mechanisms of the trans-cellular communication are still unclear. Extracellular vesicles (EVs) can transfer a large diversity of molecules such as lipids, nucleic acids and proteins for cellular communications. The purpose of this study is to characterize the EVs cargo proteins derived from human primary astrocytes (ADEVs) under both physiological and pathophysiological conditions. ADEVs were isolated from human primary astrocytes after vehicle (CTL) or interleukin-1? (IL-1?) pre-treatment. Label-free quantitative proteomic profiling revealed a notable up-regulation of proteins including actin-associated molecules, integrins and major histocompatibility complex in IL-1?-ADEVs compared to CTL-ADEVs, which were involved in cellular metabolism and organization, cellular communication and inflammatory response. When fluorescently labelled ADEVs were added into primary cultured mouse cortical neurons, we found a significantly increased neuronal uptake of IL-1?-ADEVs compared to CTL-ADEVs. We further confirmed it is likely due to the enrichment of surface proteins in IL-1?-ADEVs, as IL-1?-ADEVs uptake by neurons was partially suppressed by a specific integrin inhibitor. Additionally, treatment of neurons with IL-1?-ADEVs also reduced neurite outgrowth, branching and neuronal firing. These findings provide insight for the molecular mechanism of the ADEVs' effects on neural uptake, neural differentiation and maturation, and its alteration in inflammatory conditions.

SUBMITTER: You Y

PROVIDER: S-EPMC6968484 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Activated human astrocyte-derived extracellular vesicles modulate neuronal uptake, differentiation and firing.

You Yang Y Borgmann Kathleen K Edara Venkata Viswanadh VV Stacy Satomi S Ghorpade Anuja A Ikezu Tsuneya T

Journal of extracellular vesicles 20191226 1

Astrocytes in the central nervous system (CNS) provide supportive neural functions and mediate inflammatory responses from microglia. Increasing evidence supports their critical roles in regulating brain homoeostasis in response to pro-inflammatory factors such as cytokines and pathogen/damage-associated molecular pattern molecules in infectious and neurodegenerative diseases. However, the underlying mechanisms of the trans-cellular communication are still unclear. Extracellular vesicles (EVs) c ...[more]

PMID: 32002171

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Project description:Extracellular vesicles (EVs) mediate cell-to-cell communication by horizontally transferring biological materials from host cells to target cells. During exposure to pathogens, pathogen-associated molecular patterns (e.g., lipopolysaccharide, LPS) get in contact with endothelial cells and stimulate the secretion of endothelial cell-derived EVs (E-EVs). The triggered EVs secretion is known to have a modulating influence on the EVs-receiving cells. Macrophages, a major component of innate immunity, are polarized upon receiving external inflammatory stimuli, in which toll-like receptor4 (TLR4)-nuclear factor kappa B (NFκB) pathway plays a key role. However, the functions of LPS-induced E-EVs (ELPS-EVs) in modulating macrophage phenotype and activation remain elusive. We collected the EVs from quiescent endothelial cells (ENor-EVs) and ELPS-EVs to detect their stimulatory role on NR8383 macrophages. Isolated EVs were characterized by transmission electron microscopy (TEM), western blot assay, and nanoparticle tracking analysis (NTA). NR8383 macrophages were stimulated with ELPS-EVs, ENor-EVs, or PBS for 24 h. Hereafter, the uptake of EVs by the macrophages was investigated. Upon EVs stimulation, cellular viability was determined by MTT assay, while macrophage phenotype was analyzed by flow cytometry and immunofluorescence analysis. Furthermore, a western blot assay was conducted to evaluate the potentially involved TLR4-NFκB pathway. Interestingly, upon exposure to LPS, endothelial cells secreted significantly higher amounts of EVs (i.e., ELPS-EVs) when compared to quiescent cells or cells in PBS. The ELPS-EVs were also better internalized by NR8383 macrophages than ENor-EVs. The cellular viability of ELPS-EVs-treated macrophages was 1.2 times higher than those in the ENor-EVs and PBS groups. In addition, ELPS-EVs modulated NR8383 macrophages towards a proinflammatory macrophage M1-like phenotype. This was indicated by the significantly upregulated expressions of proinflammatory macrophage biomarkers CD86 and inducible nitric oxide synthase (iNOS) observed in ELPS-EVs-treated macrophages. The TLR4-NFκB signaling pathway was substantially activated in ELPS-EVs-treated macrophages, indicated by the elevated expressions of makers TLR4 and phosphorylated form of nuclear factor kappa B p65 subunit (p-NFκBp65). Overall, our results indicate that E-EVs play a crucial role in macrophage phenotype modulation under inflammatory conditions.

Project description:BACKGROUND:Stress precipitates mood disorders, characterized by a range of symptoms present in different combinations, suggesting the existence of disease subtypes. Using an animal model, we previously described that repetitive stress via restraint or immobilization induced depressive-like behaviors in rats that were differentially reverted by a serotonin- or noradrenaline-based antidepressant drug, indicating that different neurobiological mechanisms may be involved. The forebrain astrocyte protein aldolase C, contained in small extracellular vesicles, was identified as a potential biomarker in the cerebrospinal fluid; however, its specific origin remains unknown. Here, we propose to investigate whether serum small extracellular vesicles contain a stress-specific protein cargo and whether serum aldolase C has a brain origin. METHODS:We isolated and characterized serum small extracellular vesicles from rats exposed to restraint, immobilization, or no stress, and their proteomes were identified by mass spectrometry. Data available via ProteomeXchange with identifier PXD009085 were validated, in part, by western blot. In utero electroporation was performed to study the direct transfer of recombinant aldolase C-GFP from brain cells to blood small extracellular vesicles. RESULTS:A differential proteome was identified among the experimental groups, including aldolase C, astrocytic glial fibrillary acidic protein, synaptophysin, and reelin. Additionally, we observed that, when expressed in the brain, aldolase C tagged with green fluorescent protein could be recovered in serum small extracellular vesicles. CONCLUSION:The protein cargo of serum small extracellular vesicles constitutes a valuable source of biomarkers of stress-induced diseases, including those characterized by depressive-like behaviors. Brain-to-periphery signaling mediated by a differential molecular cargo of small extracellular vesicles is a novel and challenging mechanism by which the brain might communicate health and disease states to the rest of the body.

Dataset Information

Activated human astrocyte-derived extracellular vesicles modulate neuronal uptake, differentiation and firing.

Publications

Activated human astrocyte-derived extracellular vesicles modulate neuronal uptake, differentiation and firing.

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