Gangoso Ester, Talaverón Rocío, Jaraíz-Rodríguez Myriam, Domínguez-Prieto Marta, Ezan Pascal, Koulakoff Annette, Medina José M, Giaume Christian, Tabernero Arantxa
MEMOLIFE Laboratory of Excellence and Paris Science Lettre Research University, Center for Interdisciplinary Research in Biology (CIRB)/Centre National de la Recherche Scientifique, Unité Mixte de Recherche 7241/Institut National de la Santé et de la Recherche Médicale U1050, Collège de France, Université Pierre et Marie Curie, Paris, France.
Departamento de Bioquímica y Biología Molecular, Instituto de Neurociencias de Castilla y León (INCYL), Universidad de Salamanca, Salamanca, Spain.
Front Mol Neurosci. 2017 Dec 15;10:418. doi: 10.3389/fnmol.2017.00418. eCollection 2017.
The non-receptor tyrosine kinase c-Src is an important mediator in several signaling pathways related to neuroinflammation. Our previous study showed that cortical injection of kainic acid (KA) promoted a transient increase in c-Src activity in reactive astrocytes surrounding the neuronal lesion. As a cell-penetrating peptide based on connexin43 (Cx43), specifically TAT-Cx43, inhibits Src activity, we investigated the effect of TAT-Cx43 on neuronal death promoted by cortical KA injections in adult mice. As expected, KA promoted neuronal death, estimated by the reduction in NeuN-positive cells and reactive gliosis, characterized by the increase in glial fibrillary acidic protein (GFAP) expression. Interestingly, TAT-Cx43 injected with KA diminished neuronal death and reactive gliosis compared to KA or KA+TAT injections. In order to gain insight into the neuroprotective mechanism, we used models. In primary cultured neurons, TAT-Cx43 did not prevent neuronal death promoted by KA, but when neurons were grown on top of astrocytes, TAT-Cx43 prevented neuronal death promoted by KA. These observations demonstrate the participation of astrocytes in the neuroprotective effect of TAT-Cx43. Furthermore, the neuroprotective effect was also present in non-contact co-cultures, suggesting the contribution of soluble factors released by astrocytes. As glial hemichannel activity is associated with the release of several factors, such as ATP and glutamate, that cause neuronal death, we explored the participation of these channels on the neuroprotective effect of TAT-Cx43 Our results confirmed that inhibitors of ATP and NMDA receptors prevented neuronal death in co-cultures treated with KA, suggesting the participation of astrocyte hemichannels in neurotoxicity. Furthermore, TAT-Cx43 reduced hemichannel activity promoted by KA in neuron-astrocyte co-cultures as assessed by ethidium bromide (EtBr) uptake assay. In fact, TAT-Cx43 and dasatinib, a potent c-Src inhibitor, strongly reduced the activation of astrocyte hemichannels. In conclusion, our results suggest that TAT-Cx43 exerts a neuroprotective effect through the reduction of hemichannel activity likely mediated by c-Src in astrocytes. These data unveil a new role of c-Src in the regulation of Cx43-hemichannel activity that could be part of the mechanism by which astroglial c-Src participates in neuroinflammation.
非受体酪氨酸激酶c-Src是几种与神经炎症相关的信号通路中的重要介质。我们之前的研究表明，向皮质注射海藻酸（KA）可促使神经元损伤周围反应性星形胶质细胞中的c-Src活性短暂增加。由于基于连接蛋白43（Cx43）的细胞穿透肽，特别是TAT-Cx43，可抑制Src活性，我们研究了TAT-Cx43对成年小鼠皮质KA注射所促进的神经元死亡的影响。正如预期的那样，KA通过NeuN阳性细胞减少和反应性胶质增生促进神经元死亡，反应性胶质增生的特征是胶质纤维酸性蛋白（GFAP）表达增加。有趣的是，与KA或KA+TAT注射相比，与KA一起注射的TAT-Cx43减少了神经元死亡和反应性胶质增生。为了深入了解神经保护机制，我们使用了模型。在原代培养的神经元中，TAT-Cx43不能预防KA所促进的神经元死亡，但当神经元生长在星形胶质细胞上时，TAT-Cx43可预防KA所促进的神经元死亡。这些观察结果证明星形胶质细胞参与了TAT-Cx43的神经保护作用。此外，在非接触共培养中也存在神经保护作用，这表明星形胶质细胞释放的可溶性因子起了作用。由于胶质半通道活性与几种导致神经元死亡的因子如ATP和谷氨酸的释放有关，我们探讨了这些通道在TAT-Cx43神经保护作用中的参与情况。我们的结果证实，ATP和NMDA受体抑制剂可预防KA处理的共培养中的神经元死亡，这表明星形胶质细胞半通道参与了神经毒性。此外，通过溴化乙锭（EtBr）摄取试验评估，TAT-Cx43降低了神经元-星形胶质细胞共培养中KA所促进的半通道活性。事实上，TAT-Cx43和强效c-Src抑制剂达沙替尼强烈降低了星形胶质细胞半通道的激活。总之，我们的结果表明，TAT-Cx43可能通过降低星形胶质细胞中可能由c-Src介导的半通道活性发挥神经保护作用。这些数据揭示了c-Src在调节Cx43半通道活性中的新作用，这可能是星形胶质细胞c-Src参与神经炎症机制的一部分。
