Bijelić Dunja, Adžić Marija, Perić Mina, Reiss Gebhard, Milošević Milena, Andjus Pavle R, Jakovčevski Igor
Centre for Laser Microscopy, Institute of Physiology and Biochemistry "Ivan Djaja", Faculty of Biology, University of Belgrade, Belgrade, Serbia.
Laboratory for Human Molecular Genetics, Institute of Molecular Genetics and Genetic Engineering, University of Belgrade, Belgrade, Serbia.
Front Cell Dev Biol. 2022 Aug 26;10:952208. doi: 10.3389/fcell.2022.952208. eCollection 2022.
Understanding processes that occur after injuries to the central nervous system is essential in order to gain insight into how the restoration of function can be improved. Extracellular glycoprotein tenascin-C (TnC) has numerous functions in wound healing process depending on the expression time, location, isoform and binding partners which makes it interesting to study in this context. We used an injury model, the mixed culture of cortical astrocytes and microglia, and observed that without TnC microglial cells tend to populate gap area in greater numbers and proliferate more, whereas astrocytes build up in the border region to promote faster gap closure. Alternatively spliced domain of TnC, fibronectin type III-like repeat D (FnD) strongly affected physiological properties and morphology of both astrocytes and microglia in this injury model. The rate of microglial proliferation in the injury region decreased significantly with the addition of FnD. Additionally, density of microglia also decreased, in part due to reduced proliferation, and possibly due to reduced migration and increased contact inhibition between enlarged FnD-treated cells. Overall morphology of FnD-treated microglia resembled the activated pro-inflammatory cells, and elevated expression of iNOS was in accordance with this phenotype. The effect of FnD on astrocytes was different, as it did not affect their proliferation, but stimulated migration of reactivated astrocytes into the scratched area 48 h after the lesion. Elevated expression and secretion of TNF-α and IL-1β upon FnD treatment indicated the onset of inflammation. Furthermore, on Western blots we observed increased intensity of precursor bands of β1 integrin and appearance of monomeric bands of P2Y12R after FnD treatment which substantiates and clarifies its role in cellular shape and motility changes. Our results show versatile functions of TnC and in particular FnD after injury, mostly contributing to ongoing inflammation in the injury region. Based on our findings, FnD might be instrumental in limiting immune cell infiltration, and promoting astrocyte migration within the injury region, thus influencing spaciotemporal organization of the wound and surrounding area.
了解中枢神经系统损伤后发生的过程对于深入了解如何改善功能恢复至关重要。细胞外糖蛋白腱生蛋白-C（TnC）在伤口愈合过程中具有多种功能，这取决于其表达时间、位置、异构体和结合伙伴，因此在这种情况下研究它很有意思。我们使用了一种损伤模型，即皮质星形胶质细胞和小胶质细胞的混合培养，并观察到在没有TnC的情况下，小胶质细胞倾向于大量聚集在间隙区域并增殖更多，而星形胶质细胞则在边界区域聚集以促进更快的间隙闭合。TnC的可变剪接结构域，纤连蛋白III型样重复序列D（FnD）在该损伤模型中强烈影响星形胶质细胞和小胶质细胞的生理特性和形态。随着FnD的添加，损伤区域中小胶质细胞的增殖速率显著降低。此外，小胶质细胞的密度也降低了，部分原因是增殖减少，也可能是由于迁移减少以及经FnD处理的细胞增大后接触抑制增加。经FnD处理的小胶质细胞的整体形态类似于活化的促炎细胞，诱导型一氧化氮合酶（iNOS）的表达升高与此表型一致。FnD对星形胶质细胞的作用不同，因为它不影响其增殖，但在损伤后48小时刺激重新活化的星形胶质细胞迁移到划痕区域。FnD处理后肿瘤坏死因子-α（TNF-α）和白细胞介素-1β（IL-1β）的表达和分泌升高表明炎症开始。此外，在蛋白质免疫印迹分析中，我们观察到FnD处理后β1整合素前体条带的强度增加以及P2Y12R单体条带的出现，这证实并阐明了其在细胞形状和运动变化中的作用。我们的结果显示了TnC特别是FnD在损伤后的多种功能，主要促成损伤区域持续的炎症反应。基于我们的发现，FnD可能有助于限制免疫细胞浸润，并促进星形胶质细胞在损伤区域内的迁移，从而影响伤口和周围区域的时空组织。
