Differential intracellular trafficking of extracellular vesicles in microglia and astrocytes

Abstract

Extracellular vesicles (EVs) have emerged as key players in cell-to-cell communication in both physiological and pathological processes in the Central Nervous System. Thus far, the intracellular pathways involved in uptake and trafficking of EVs within different cell types of the brain are poorly understood. In our study, the endocytic processes and subcellular sorting of EVs were investigated in primary glial cells, particularly linked with the EV-associated α-synuclein (α-syn) transmission. Mouse microglia and astrocytic primary cultures were incubated with DiI-stained mouse brain-derived EVs. The internalization and trafficking pathways were analyzed in cells treated with pharmacological reagents that block the major endocytic pathways. Brain-derived EVs were internalized by both glial cell types; however, uptake was more efficient in microglia than in astrocytes. Colocalization of EVs with early and late endocytic markers (Rab5, Lamp1) indicated that EVs are sorted to endo-lysosomes for subsequent processing. Blocking actin-dependent phagocytosis and/or macropinocytosis with Cytochalasin D or EIPA inhibited EV entry into glial cells, whereas treatment with inhibitors that strip cholesterol off the plasma membrane, induced uptake, however differentially altered endosomal sorting. EV-associated fibrillar α-Syn was efficiently internalized and detected in Rab5- and Lamp1-positive compartments within microglia. Our study strongly suggests that EVs enter glial cells through phagocytosis and/or macropinocytosis and are sorted to endo-lysosomes for subsequent processing. Further, brain-derived EVs serve as scavengers and mediate cell-to-glia transfer of pathological α-Syn which is also targeted to the endolysosomal pathway, suggesting a beneficial role in microglia-mediated clearance of toxic protein aggregates, present in numerous neurodegenerative diseases.

α-Synuclein oligomers potentiate neuroinflammatory NF-κB activity and induce Cav3.2 calcium signaling in astrocytes

“…Primary astrocytes were treated with 5 ng/ml interleukin 1β (IL-1β) (ΑF-211-1113, Peprotech) with or without 20 μM Bay 11-7085 (14795, Cayman) or 20 ng/ml tumor necrosis factor α (TNFα) (AF-315-01A, Peprotech), or 1 ng/ml interferon γ (IFNγ) (AF-315-05, Peprotech) in ABM without FGF2/EGF for the indicated time points. Prior to treatment, the cells were deprived of growth factors for 6 h. Mixed astroglial cultures were treated with 300 ng/ml PFFs in ABM medium for 3 h. The PFFs were prepared as previously described [ 27 ].…”

Biogenesis and delivery of extracellular vesicles: harnessing the power of EVs for diagnostics and therapeutics

“…The genes identified within the “Packaging” cluster play crucial roles in encapsulating proteins into EVs, specifically facilitating their transport from the ER to the Golgi by COPII machinery ( Bisnett et al, 2020 ). Furthermore ( Figure 2B ), it was identified that the RAS protein family involvement in Golgi to plasma membrane transportation affects EV biogenesis ( Pantazopoulou et al, 2023 ).…”

Kaempferia parviflora Extracellular Vesicle Loaded with Clarithromycin for the Treatment of Helicobacter pylori Infection

“…Labeling EVs with Dil staining did not affect EVs size distribution. 55 Importantly, Dil can be used for studying biological event without being cytotoxic and interfering with their bioactivity. 56,57 The internalization of PDEVs by recipient cells involves three mechanisms: endocytosis, receptor-mediated cell signaling, and phagocytosis.…”