Microglial derived extracellular vesicles activate autophagy and mediate multi‐target signaling to maintain cellular homeostasis

Abstract

Microglia, the immunocompetent cells of the central nervous system (CNS), play an important role in maintaining cellular homeostasis in the CNS. These cells secrete immunomodulatory factors including nanovesicles and participate in the removal of cellular debris by phagocytosis or autophagy. Accumulating evidence indicates that specifically the cellular exchange of small extracellular vesicles (EVs), participates in physiology and disease through intercellular communication. However, the contribution of microglial‐derived extracellular vesicles (M‐EVs) to the maintenance of microglia homeostasis and how M‐EVs could influence the phenotype and gene function of other microglia subtypes is unclear. In addition, knowledge of canonical signalling pathways of inflammation and immunity gene expression patterns in human microglia exposed to M‐EVs is limited. Here, we analysed the effects of M‐EVs produced in vitro by either tumour necrosis factor alpha (TNFα) activated or non‐activated microglia BV2 cells. We showed that M‐EVs are internalized by both mouse and human C20 microglia cells and that the uptake of M‐EVs in microglia induced autophagic vesicles at various stages of degradation including autophagosomes and autolysosomes. Consistently, stimulation of microglia with M‐EVs increased the protein expression of the autophagy marker, microtubule‐associated proteins 1A/1B light chain 3B isoform II (LC3B‐II), and promoted autophagic flux in live cells. To elucidate the biological activities occurring at the transcriptional level in C20 microglia stimulated with M‐EVs, the gene expression profiles, potential upstream regulators, and enrichment pathways were characterized using targeted RNA sequencing. Inflammation and immunity transcriptome gene panel sequencing of both activated and normal microglia stimulated with M‐EVs showed involvement of several canonical pathways and reduced expression of key genes involved in neuroinflammation, inflammasome and apoptosis signalling pathways compared to control cells. In this study, we provide the perspective that a beneficial activity of in vitro cell culture produced EVs could be the modulation of autophagy during cellular stress. Therefore, we use a monoculture system to study microglia‐microglia crosstalk which is important in the prevention and propagation of inflammation in the brain. We demonstrate that in vitro produced microglial EVs are able to influence multiple biological pathways and promote activation of autophagy in order to maintain microglia survival and homeostasis.

Oligodendroglia-derived extracellular vesicles activate autophagy via LC3B/BAG3 to protect against oxidative stress with an enhanced effect for HSPB8 enriched vesicles

“…We demonstrate that both EV subsets can be internalized by the C20 recipient microglial cell lines or primary mixed neural culture cells, validating that engineered EVs from oligodendroglia origins can be internalized into different cell types. Additionally, we show that EV uptake is not overtly changed under inflammatory conditions [ 41 ]. Furthermore, elucidating the biological functions of the engineered EVs-mediated cell-to-cell communication is a prerequisite for understanding how signaling processes could influence cellular inflammatory responses [ 42 ].…”

Microglia and Astrocyte Function and Communication: What Do We Know in Humans?

“…Microglia release EVs when their purinergic receptors (P2X7) come into contact with high levels of ATP released from astrocytes ( Bianco et al, 2005 ) ( Figure 5 ). These EVs have been shown to be released from human primary astrocytes under normal conditions and with IL1β treatment ( You et al, 2020 ) and from microglia TNFα or non-activated microglia ( van den Broek et al, 2020 ). When a neuronal injury occurs, astrocytes and microglia communicate to release microglial EVs containing IL1β in order to survey the parenchyma and to start an inflammatory response if needed to respond to pathogens ( Budnik et al, 2016 ).…”

Engineering extracellular vesicles for Alzheimer's disease: An emerging cell‐free approach for earlier diagnosis and treatment

“…Significant research has already been conducted investigating the role of native glial cell-derived EVs in the CNS microenvironment. Recently, part of the multifaceted role of microglia in the brain and specifically in AD pathology was attributed to microglial EVs and their role in multitarget signaling (Van den Broek et al, 2020). Because glial cellderived EVs naturally interact with many of the cell types of interest in the brain, they represent promising candidates for targeting the underlying mechanisms of AD pathology.…”