Lasič Eva, Galland Fabiana, Vardjan Nina, Šribar Jernej, Križaj Igor, Leite Marina Concli, Zorec Robert, Stenovec Matjaž
Laboratory of Neuroendocrinology - Molecular Cell Physiology, Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia.
Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil.
J Neurochem. 2016 Oct;139(2):309-323. doi: 10.1111/jnc.13754. Epub 2016 Aug 19.
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.
星形胶质细胞是中枢神经系统中最具异质性的神经胶质细胞，通过调节包括清除细胞外碎片在内的无数功能，对大脑内环境稳态发挥作用。当细胞受损时，细胞质蛋白可能会进入细胞外空间。其中一种蛋白是S100B，除非将其从细胞外空间清除，否则它可能会对邻近细胞产生毒性作用，但这种清除机制尚不清楚。通过使用延时共聚焦显微镜以及荧光标记的S100B（S100B- Alexa）和荧光葡聚糖（葡聚糖）（一种液相摄取标记物），我们检测了细胞外空间中荧光标记的S100B- Alexa的摄取情况，并监测了内化S100B- Alexa的囊泡的运输过程。最初，S100B- Alexa和葡聚糖以不同的速率内化到不同的内吞囊泡中；S100B- Alexa内化到比葡聚糖更小的囊泡中。在后期，S100B- Alexa阳性囊泡与葡聚糖阳性的内溶酶体以及酸性的溶酶体追踪染料阳性囊泡大量共定位。用抗晚期糖基化终产物受体（RAGE）抗体处理细胞，该抗体与“清道夫受体”RAGE结合，部分抑制了S100B- Alexa的摄取，但不影响葡聚糖的摄取。发动蛋白抑制剂dynole 34-2抑制了两种荧光探针的内化。S100B- Alexa阳性囊泡的定向移动随时间增加，并受到ATP刺激的抑制，ATP刺激是一种增加胞质游离钙浓度（[Ca]）的试剂。我们得出结论，星形胶质细胞表现出依赖RAGE和发动蛋白的囊泡机制，以有效地从细胞外空间清除S100B。如果体内发生类似过程，在病理生理条件下，星形胶质细胞可能通过该过程减轻细胞外S100B的毒性作用。这项研究揭示了星形胶质细胞中细胞外S100B的囊泡清除机制。最初，荧光S100B内化到比葡聚糖分子更小的内吞囊泡中。在后期，两种探针在内溶酶体内共定位。S100B的内化既依赖发动蛋白也依赖RAGE，而葡聚糖的内化仅依赖发动蛋白。囊泡内化可能减轻细胞外S100B和其他废物的毒性作用。
