Differential intracellular trafficking of extracellular vesicles in microglia and astrocytes

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. Researchers from the Biomedical Research Foundation Academy of Athens-BRFAA investigated the endocytic processes and subcellular sorting of EVs 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. This 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.

Brain-derived sEVs are internalized in primary microglia and astrocytes

Fig. 2

Primary cells were incubated with Dil-stained brain-derived sEVs (depicted in red) for 6 h and 24 h. Cells were washed, and internalization of sEVs was monitored 6 h and 24 h post-incubation. Cells were fixed and immunostained with an antibody against α-Tubulin (α-Tub) (gray), while cell nuclei were stained with DAPI (blue). Confocal images were deconvolved and analyzed with the Imaris Imaging software. sEVs were defined as cytoplasmic (sEVs in, red) or membranous (sEVs mem, cyan blue) using the ‘’Distance transformation’’ module of the Imaris Imaging software, computing the distance of the sEV puncta from the ‘’α-Tubulin’’ surface. Representative Imaris images depict the internalization of sEVs masked with the α-Tubulin surface (left panel, scale bar 15 μm) and in/mem sEVs (right panel, scale bar 5 μm) in microglia (a) and astrocytes (h), 6 h and 24 h post-addition. ‘’sEVs mem’’ were depicted with arrowheads. Graphs show the total volume of internalized sEVs per cell (bi), the number of puncta per cell (cj), the mean volume of sEVs (dk), the ratio of the volume of sEVs (in/mem) per total volume (el), the ratio of the number of puncta (in/mem) per total number (fm), and the mean volume of sEVs (in/mem) (gn), in microglia and astrocytes, respectively. Data are presented as the mean ± SEM of minimum three independent cell preparations, with at least two replicates per assay; Student’s t test was used for (b), (d), (i), and (k), Mann–Whitney test for (c) and (j), one-way ANOVA with Tukey’s correction for (g) and (n), two-way ANOVA with Tukey’s correction for (e) and (m), and multiple t test for (f) and (m). Statistical significance was set as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Pantazopoulou M, Lamprokostopoulou A, Karampela DS, Alexaki A, Delis A, Coens A, Samiotaki M, Kriebardis AG, Melki R, Pagakis SN, Stefanis L, Vekrellis K. (2023) Differential intracellular trafficking of extracellular vesicles in microglia and astrocytes. Cell Mol Life Sci 80(7):193. [article]

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