A Refined Bead-Free Method to Identify Astrocytic Exosomes in Primary Glial Cultures and Blood Plasma

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.

Astrocyte Support for Oligodendrocyte Differentiation can be Conveyed via Extracellular Vesicles but Diminishes with Age

“…Emerging evidence now points to a contributing role for EVs as a major component of the SASP 47 . We have previously shown that young cultured astrocytes release EVs, which are readily identifiable by the astrocyte marker GFAP 48 . To test whether EVs were released from aged astrocytes in culture, we isolated EVs from the conditioned media of young and aged astrocytes and assessed the presence of EVs by negative staining and transmission electron microscopy (EM).…”

“…All procedures involving animals in this study were only conducted with prior approval from the Institutional Animal Care and Use Committee (IACUC) at the University of Connecticut School of Medicine and in accordance with guidelines set forth by the National Research Council of the National Academies Guide for the Care and Use of Laboratory Animals. Cultures were generated from cerebral cortices of neonatal C57BL/6 mouse pups (P0-P3) using a neural tissue dissociation kit (Miltenyi Biotec, Cambridge, MA, USA), as previously described 48,86,87 and consistent with previous reports of 90-97% GFAP + cells 88 . Culture flasks were subjected to weekly media changes (Dulbecco's modified eagle medium, 1% Pen-strep, and 10% heat-inactivated fetal bovine serum).…”

“…Western blot. Western blot analysis was performed as previously described 48 . Briefly, astrocytes were lysed in RIPA buffer (with protease inhibitor cocktail) and separated by SDS-PAGE.…”

The roles of extracellular vesicle microRNAs in the central nervous system

“…This study also demonstrated a correlation between the quantity of EVs in the CSF and the number of active lesions. Increased astrocyte‐derived GFAP positive EVs within the circulation of EAE mice has also been reported (Willis et al, ) and suggests that increases in EV‐mediated intercellular signaling may correlated with disease severity in both mice and humans.…”

“…Biologically active components of EVs (termed “cargo”) can include proteins, lipids, and various RNA species (e.g., mRNAs, miRNAs, tRNAs; reviewed by Yáñez‐Mó et al, ). Virtually all cells are capable of releasing EVs, which can contain cargo that is either unique to the cell‐of‐origin (e.g., glial fibrillary acidic protein [GFAP] is present within EVs derived from astrocytes) or non‐specific (i.e., found within all or most EVs independent of the cell of origin; Keerthikumar et al, ; Kowal et al, ; Willis et al, ). EV cargo may not always be representative of the cellular contents of the secreting cell, suggesting that its cargo may be selectively sorted and packaged for deliberate secretion; however, the potential contribution of culture additive contaminations make this a matter of current debate (Colombo et al, ; Jovičić & Gitler, ; Nolte‐'t Hoen et al, ; Tosar et al, ; Valadi et al, ; Wei, Batagov, Carter, & Krichevsky, ).…”