Program for Neurotrauma, Neuroproteomics & Biomarkers Research, University of Florida, Gainesville, FL, 32611, USA.
The Departments of Psychiatry, University of Florida, Gainesville, FL, 32611, USA.
Mol Neurobiol. 2018 Mar;55(3):2112-2124. doi: 10.1007/s12035-017-0474-x. Epub 2017 Mar 10.
A number of neuronal and glial proteins were previously found to be released in free-standing soluble form from cultured brain cells into cell-conditioned media. Here, we sought to examine if similar proteins are also contained in neural and astroglial cell-released extracellular microvesicles/exosomes (MV/E). In this study, MV/E were isolated from cell-conditioned media from control and cytotoxin-challenged rat cerebrocortical mixed culture (CTX) and mouse neuroblastoma N2a cells. Cytotoxin challenges included pro-necrosis calcium ionophore A23187, pro-apoptosis staurosporine (STS), and excitotoxin N-methyl-D-aspartate. Based on established nanoparticle characterization method (dynamic light scattering, NanoTracker, and transmission electron microscopy), we confirmed that these released vesicles are in fact characteristic representation of MV/E by morphology (lipid bilayered vesicles) and by particle size (132-142 nm for CTX and 49-77 nm for N2a cells). We indeed identified neural cell body protein UCH-L1, axonal injury marker αII-spectrin and its breakdown products (SBDPs), astroglial markers GFAP and its breakdown products (GFAP-BDP), dendritic protein BIII-tubulin, synaptic protein synaptophysin, and exosome marker Alix in microvesicles from CTX and/or N2a cells. Furthermore, SBDPs, GFAP-BDP, UCH-L1, and synaptophysin are especially dominant in MV/E isolated from cytotoxin-treated CTX cells. Similarly, SBDPs, βIII-tubulin, and UCH-L1 are more prominently observed in cytotoxin-challenged N2a cells. Lastly, when isolated MV/E from A23187- or STS-challenged N2a cells were introduced to healthy N2a culture, they are capable of evoking cytotoxicity in the latter. Taken together, our study identified that microvesicles/exosomes isolated form healthy and injured brain cells contain certain neural and astroglial proteins, as well as possibly other cytotoxic factors that are capable of propagating cytotoxic effects.
一些神经元和神经胶质蛋白先前被发现以游离的可溶性形式从培养的脑细胞释放到细胞条件培养基中。在这里，我们试图研究类似的蛋白质是否也存在于神经和星形胶质细胞释放的细胞外微泡/外泌体（MV/E）中。在这项研究中，MV/E 是从对照和细胞毒素挑战的大鼠大脑皮质混合培养物（CTX）和小鼠神经母细胞瘤 N2a 细胞的细胞条件培养基中分离出来的。细胞毒素挑战包括促坏死钙离子载体 A23187、促凋亡星形孢菌素（STS）和兴奋毒性 N-甲基-D-天冬氨酸。基于已建立的纳米颗粒表征方法（动态光散射、NanoTracker 和透射电子显微镜），我们通过形态（双层脂质囊泡）和粒径（CTX 为 132-142nm，N2a 细胞为 49-77nm）证实了这些释放的囊泡实际上是 MV/E 的特征表现。我们确实在 CTX 和/或 N2a 细胞的微泡中鉴定到神经元胞体蛋白 UCH-L1、轴突损伤标志物αII- spectrin 及其降解产物（SBDPs）、星形胶质细胞标志物 GFAP 及其降解产物（GFAP-BDP）、树突蛋白 BIII-tubulin、突触蛋白 synaptophysin 和外泌体标志物 Alix。此外，SBDPs、GFAP-BDP、UCH-L1 和 synaptophysin 在来自细胞毒素处理的 CTX 细胞的 MV/E 中特别占主导地位。同样，SBDPs、βIII-tubulin 和 UCH-L1 在受到细胞毒素挑战的 N2a 细胞中更明显。最后，当从 A23187 或 STS 挑战的 N2a 细胞中分离的 MV/E 引入健康的 N2a 培养物时，它们能够在后者中引起细胞毒性。总之，我们的研究表明，从健康和受损脑细胞中分离的微泡/外泌体含有某些神经和星形胶质细胞蛋白，以及可能具有传播细胞毒性作用的其他细胞毒性因子。
