Release of astroglial vimentin by extracellular vesicles: Modulation of binding and internalization of C3 transferase in astrocytes and neurons
Release of astroglial vimentin by extracellular vesicles: Modulation of binding and internalization of C3 transferase in astrocytes and neurons
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DOI：
10.1002/glia.23566
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发表时间：
2019-04-01
期刊：
影响因子：
6.2
通讯作者：
Hoeltje, Markus
中科院分区：
文献类型：
--
作者：
Adolf, Andrej;Rohrbeck, Astrid;Hoeltje, Markus
肉毒梭菌 C3 转移酶 (C3bot) ADP 核糖基化 rho 蛋白，可改变多种细胞类型（包括星形胶质细胞和神经元）的细胞功能。中间丝蛋白波形蛋白以及跨膜整合素参与 C3bot 进入细胞的内化。然而，这些蛋白质对 C3bot 与细胞表面的结合以及随后的细胞摄取的确切贡献仍有待阐明。通过比较野生型和 Vim(-/-) 小鼠的原代星形胶质细胞培养物，我们证明缺乏波形蛋白的星形胶质细胞表现出 rhoA 的 ADP 核糖基化延迟，同时形态反应减弱。这种功能损伤可以通过细胞外过量的重组波形蛋白来挽救。使用带有突变的整合素结合 RGD 基序 (C3bot-G89I) 的 C3bot 进行的结合测定揭示了整合素参与 C3bot 星形胶质细胞的结合。 C3bot 的轴突效应依赖于波形蛋白，并假定星形胶质细胞和神经元之间存在分子串扰的潜在机制。我们使用体外划伤模型提供了外泌体星形胶质细胞释放波形蛋白的功能证据。野生型星形胶质细胞释放的外泌体波形蛋白+颗粒促进 C3bot 与神经元膜的相互作用。当培养 Vim(-/-) 星形胶质细胞时，这种效应消失。这些发现的特异性通过重组波形蛋白传播增强的 C3bot 与来自大鼠脊髓和小鼠大脑的突触体的结合来证实。我们假设反应性星形胶质细胞释放的波形蛋白+外泌体提供了一种新的分子机制，构成脊髓损伤后 C3bot 的轴突（神经保护）和可塑性增强作用。
Clostridium botulinum C3 transferase (C3bot) ADP-ribosylates rho proteins to change cellular functions in a variety of cell types including astrocytes and neurons. The intermediate filament protein vimentin as well as transmembrane integrins are involved in internalization of C3bot into cells. The exact contribution, however, of these proteins to binding of C3bot to the cell surface and subsequent cellular uptake remains to be unraveled. By comparing primary astrocyte cultures derived from wild-type with Vim(-/-) mice, we demonstrate that astrocytes lacking vimentin exhibited a delayed ADP-ribosylation of rhoA concurrent with a blunted morphological response. This functional impairment was rescued by the extracellular excess of recombinant vimentin. Binding assays using C3bot harboring a mutated integrin-binding RGD motif (C3bot-G89I) revealed the involvement of integrins in astrocyte binding of C3bot. Axonotrophic effects of C3bot are vimentin dependent and postulate an underlying mechanism entertaining a molecular cross-talk between astrocytes and neurons. We present functional evidence for astrocytic release of vimentin by exosomes using an in vitro scratch wound model. Exosomal vimentin+ particles released from wild-type astrocytes promote the interaction of C3bot with neuronal membranes. This effect vanished when culturing Vim(-/-) astrocytes. Specificity of these findings was confirmed by recombinant vimentin propagating enhanced binding of C3bot to synaptosomes from rat spinal cord and mouse brain. We hypothesize that vimentin+ exosomes released by reactive astrocytes provide a novel molecular mechanism constituting axonotrophic (neuroprotective) and plasticity augmenting effects of C3bot after spinal cord injury.