Abstract
Astrocytes are the most abundant glial cell type in the central nervous system (CNS) and are known to fulfill critical homeostatic functions. Dysfunction of activated astrocytes is also known to participate in the development of several neurological diseases. Astrocytes can be uniquely identified by expression of the intermediate filament protein glial acidic fibrillary protein (GFAP). Herein, we report on the development of a rigorous and sensitive methodology to identify GFAP+ exosomes in primary culture using flow cytometry. We then demonstrate that activated astrocytes release increased amounts of exosomes in response to treatment with interleukin-1β. Using this methodology, we report the identification of GFAP+ exosomes in blood and then use a mouse model of inflammatory demyelination, experimental autoimmune encephalomyelitis (EAE), to examine whether the abundance of GFAP+ exosomes in blood circulation changes during clinical illness. We find a detectable increase in the presence of GFAP+ exosomes in EAE mice when compared with non-EAE, control mice. Our data provide a novel perspective on the presence of GFAP in blood as it identifies exosomes as potential astrocyte-derived signals within blood. These data are complementary to previous clinical studies that reported elevated GFAP protein in blood samples from multiple sclerosis (MS) patients during a clinical relapse. These data also reveal the existence of a potential systemic role for astrocyte-derived exosomes in CNS conditions involving inflammation such as multiple sclerosis.
Highlights
Exosomes are small, extracellular, membrane-bound vesicles (50–150 nm in diameter) secreted by cells as a means of selective transfer of biologically active materials from one cell to another that function as a mechanism of intercellular communication (Guescini et al, 2010; Mause and Weber, 2010; Ostrowski et al, 2010; Gyorgy et al, 2011)
We sought to determine whether flow cytometry could be adopted as a means to reliably identify exosomes in media samples conditioned by primary astrocytes in culture
Using this step-wise approach we identified exosomes bound to Abs specific for the astrocyte marker glial acidic fibrillary protein (GFAP) as well as canonical exosome markers TSG101 and the tetraspanin protein, CD63 (Figure 1F)
Summary
Extracellular, membrane-bound vesicles (50–150 nm in diameter) secreted by cells as a means of selective transfer of biologically active materials from one cell to another that function as a mechanism of intercellular communication (Guescini et al, 2010; Mause and Weber, 2010; Ostrowski et al, 2010; Gyorgy et al, 2011). Exosomes develop from multivesicular bodies (MVBs), known as endosomes. These MVBs have a single outer limiting membrane (LM) that surrounds multiple luminal vesicles (LVs). LVs are formed from the LM by inward budding This process enables the incorporation of selected membrane proteins onto the surface of the MVBs (Bobrie et al, 2011). Formation and shedding of exosomes can be regulated by intracellular calcium levels (Wiedmer et al, 1990; Pasquet et al, 1996) or released constitutively, but release can be stimulated by inflammatory stimuli
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