Time‐dependent uptake and trafficking of vesicles capturing extracellular S100B in cultured rat astrocytes

Abstract

Astrocytes, the most heterogeneous glial cells in the central nervous system, contribute to brain homeostasis, by regulating a myriad of functions, including the clearance of extracellular debris. When cells are damaged, cytoplasmic proteins may exit into the extracellular space. One such protein is S100B, which may exert toxic effects on neighboring cells unless it is removed from the extracellular space, but the mechanisms of this clearance are poorly understood. By using time-lapse confocal microscopy and fluorescently labeled S100B (S100B-Alexa ) and fluorescent dextran (Dextran ), a fluid phase uptake marker, we examined the uptake of fluorescently labeled S100B-Alexa from extracellular space and monitored trafficking of vesicles that internalized S100B-Alexa . Initially, S100B-Alexa and Dextran internalized with distinct rates into different endocytotic vesicles; S100B-Alexa internalized into smaller vesicles than Dextran . At a later stage, S100B-Alexa -positive vesicles substantially co-localized with Dextran -positive endolysosomes and with acidic LysoTracker-positive vesicles. Cell treatment with anti-receptor for advanced glycation end products (RAGE) antibody, which binds to RAGE, a 'scavenger receptor', partially inhibited uptake of S100B-Alexa , but not of Dextran . The dynamin inhibitor dynole 34-2 inhibited internalization of both fluorescent probes. Directional mobility of S100B-Alexa -positive vesicles increased over time and was inhibited by ATP stimulation, an agent that increases cytosolic free calcium concentration ([Ca ] ). We conclude that astrocytes exhibit RAGE- and dynamin-dependent vesicular mechanism to efficiently remove S100B from the extracellular space. If a similar process occurs in vivo, astroglia may mitigate the toxic effects of extracellular S100B by this process under pathophysiologic conditions. This study reveals the vesicular clearance mechanism of extracellular S100B in astrocytes. Initially, fluorescent S100B internalizes into smaller endocytotic vesicles than dextran molecules. At a later stage, both probes co-localize within endolysosomes. S100B internalization is both dynamin- and RAGE-dependent, whereas dextran internalization is dependent on dynamin. Vesicle internalization likely mitigates the toxic effects of extracellular S100B and other waste products.

Human induced pluripotent stem cell–derived extracellular vesicles reduce hepatic stellate cell activation and liver fibrosis

“…Before studying the iPSC-EV-dependent gene regulation of HSCs, we investigated the mechanism of action (MOA) of iPSC-EVs. Our group and others have demonstrated that cell-derived EVs are effectively engulfed by target cells and modulate a variety of cell responses (23)(24)(25)(26). Based on this evidence, we assessed whether this is true for iPSC-EVs as well.…”

The S100B story: from biomarker to active factor in neural injury

“…Data have also been reported indicating that extracellular S100B is captured by vesicles and re‐uptaken by astrocytes in a RAGE‐dependent manner (Lasic et al . ). Interestingly, the activity of S100B has been associated with multimerization, so that tetrameric S100B has been shown to exhibit a higher binding affinity than dimeric S100B to RAGE (Ostendorp et al .…”

“…RAGE is a ubiquitous, transmembrane immunoglobulin-like receptor that binds to a diverse range of extracellular ligands and intracellular effectors, initiating a complex intracellular signaling cascade, which may also be associated with a series of pathological conditions, including neuroinflammatory reaction to neural injury, and concomitantly resulting in an up-regulation of RAGE itself (for review, Bongarzone et al 2017). Data have also been reported indicating that extracellular S100B is captured by vesicles and re-uptaken by astrocytes in a RAGE-dependent manner (Lasic et al 2016). Interestingly, the activity of S100B has been associated with multimerization, so that tetrameric S100B has been shown to exhibit a higher binding affinity than dimeric S100B to RAGE (Ostendorp et al 2005).…”

Astrocytic modulation of information processing by layer 5 pyramidal neurons of the mouse visual cortex

“…In Schwann cells and glioblastoma, endogenous S100 is localized in vesicle-like structures (Davey et al 2001;Perrone et al 2008) that are translocated across the cell in response to increasing intracellular Ca 2+ or decreasing intracellular Zn 2+ concentration through an endoplasmic reticulum-Golgi-independent pathway (Davey et al 2001) suggesting that it could be released from vesicles. Additional support is provided by the observation that extracellular S100 is recaptured in a time-dependent manner by vesicle endocytosis (Lasič et al 2016). The exact concentration of the protein at the site of release is difficult to estimate, but on the basis of our measurements of S100β-evoked decrease in [Ca 2+ ]e (Fig.…”