Dataset Information

Differential intracellular trafficking of exosomes in microglia and astrocytes

ABSTRACT: Exosomes 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 exosomes within different cell types of the brain are poorly understood. In our study, the endocytic processes and subcellular sorting of exosomes were investigated in primary glial cells, particularly linked with the exosome- associated α-synuclein (α-syn) transmission. Mouse microglia and astrocytic primary cultures were incubated with DiI-stained mouse brain-derived exosomes. The internalization and trafficking pathways were analysed in cells treated with pharmacological reagents that block the major endocytic pathways. Brain-derived exosomes were internalized by both glial cell types; however, uptake was more efficient in microglia than in astrocytes. Colocalization of exosomes with early and late endocytic markers (Rab5, Lamp1) indicated that exosomes are sorted to endolysosomes for subsequent processing. Treatment with Cytochalasin D, that blocks actin-dependent phagocytosis and/or macropinocytosis, inhibited exosome entry into glial cells, whereas treatment with inhibitors that strip off cholesterol from the plasma membrane, induced uptake, however differentially altered endosomal sorting. Exosome-associated fibrillar α-Syn was efficiently internalized and detected in Rab5- and Lamp1- positive compartments within microglia. Our study strongly suggests that exosomes enter glial cells through an actin network-dependent endocytic pathway and are sorted to endolysosomes for subsequent processing. Further, brain-derived exosomes are capable of mediating cell-to-glia transmission of pathological α-Syn that is also targeted to the endosomal pathway, suggesting a possible beneficial role in microglia-mediated clearance of toxic protein aggregates, present in numerous neurodegenerative diseases.

INSTRUMENT(S): Q Exactive HF-X

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Extracellular Exosome

SUBMITTER: Martina Samiotaki

LAB HEAD: Kostas Vekrellis

PROVIDER: PXD038287 | Pride | 2023-07-20

REPOSITORIES: Pride

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Differential intracellular trafficking of extracellular vesicles in microglia and astrocytes.

Pantazopoulou Marina M Lamprokostopoulou Agaristi A Karampela Dimitra Sotiria DS Alexaki Anastasia A Delis Anastasios A Coens Audrey A Samiotaki Martina M Kriebardis Anastasios G AG Melki Ronald R Pagakis Stamatis N SN Stefanis Leonidas L Vekrellis Kostas K

Cellular and molecular life sciences : CMLS 20230630 7

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. Mo ...[more]

PMID: 37391572

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Project description:Background: Cell-to-cell propagation of α-synuclein (α-syn) aggregates is thought to contribute to the pathogenesis of Parkinson’s disease (PD) and underlie the spread of α-syn neuropathology. Increased pro-inflammatory cytokine levels and activated microglia are present in PD and activated microglia can promote α-syn aggregation. However, it is unclear how microglia influence α-syn cell-to-cell transfer. Methods: We developed a clinically relevant mouse model to monitor α-syn prion-like propagation between cells; we transplanted wild-type mouse embryonic midbrain neurons into a mouse striatum overexpressing human α-syn (huα-syn) following adeno-associated viral injection into the substantia nigra. In this system, we depleted or activated microglial cells and determined the effects on the transfer of huα-syn from host nigrostriatal neurons into the implanted dopaminergic neurons, using the presence of huα-syn within the grafted cells as a readout. Results: First, we compared α-syn cell-to-cell transfer between host mice with a normal number of microglia to mice in which we had pharmacologically ablated 80% of the microglia from the grafted striatum. With fewer host microglia, we observed increased accumulation of huα-syn in grafted dopaminergic neurons. Second, we assessed the transfer of α-syn into grafted neurons in the context of microglia activated by one of two stimuli, lipopolysaccharide (LPS) or interleukin-4 (IL-4). LPS exposure led to a strong activation of microglial cells (as determined by microglia morphology and cytokine production) and significantly higher amounts of huα-syn in grafted neurons. In contrast, injection of IL-4 did not change the proportion of grafted dopamine neurons that contained huα-syn relative to controls. RNA sequencing analysis revealed differential gene expression between LPS and IL-4 injected mice; many genes were upregulated in LPS including those involved with the inflammatory response. Conclusions: The absence or the hyperstimulation of microglia affected α-syn transfer in the brain. Our results suggest that under resting, non-inflammatory conditions, microglia modulate the transfer of α-syn. Pharmacological regulation of neuroinflammation could represent a future avenue for limiting the spread of PD neuropathology.

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