DataSheet_3_The circadian rhythms regulated by Cx43-signaling in the pathogenesis of Neuromyelitis Optica.zip

IntroductionNeuromyelitis Optica (NMO) is an inflammatory demyelinating disease of the central nervous system (CNS). NMO manifests as selective and severe attacks on axons and myelin of the optic nerve and spinal cord, resulting in necrotic cavities. The circadian rhythms are well demonstrated to profoundly impact cellular function, behavior, and disease. This study is aimed to explore the role and molecular basis of circadian rhythms in NMO.MethodsWe used an Aquaporin 4(AQP4) IgG-induced NMO cell model in isolated astrocytes. The expression of Cx43 and Bmal1 were detected by real-time PCR and Western Blot. TAT-Gap19 and DQP-1105 were used to inhibit Cx43 and glutamate receptor respectively. The knockdown of Bmal1 were performed with the shRNA containing adenovirus. The levels of glutamate, anterior visual pathway (AVP), and vasoactive intestinal peptide (VIP) were quantified by ELISA kits.ResultsWe found that Bmal1 and Clock, two essential components of the circadian clock, were significantly decreased in NMO astrocytes, which were reversed by Cx43 activation (linoleic acid) or glutamate. Moreover, the expression levels of Bmal1 and Clock were also decreased by Cx43 blockade (TAT-Gap19) or glutamate receptor inhibition (DQP-1105). Furthermore, adenovirus-mediated Bmal1 knockdown by shRNA (Ad-sh-Bmal1) dramatically decreased the levels of glutamate, AVP, and VIP from neurons, and significantly down-regulated the protein level of Cx43 in NMO astrocytes with Cx43 activation (linoleic acid) or glutamate treatment. However, Bmal1 knockdown did not alter these levels in normal astrocytes with Cx43 blockade (TAT-Gap19) or glutamate receptor inhibition (DQP-1105).DiscussionCollectively, these results suggest that Cx43-glutamate signaling would be a critical upstream regulator that contributes to the NMO-induced rhythmic damage in SCN astrocytes.

引言神经节苷脂性多发性硬化症（NMO）是一种影响中枢神经系统（CNS）的炎症性脱髓鞘疾病。NMO表现为对视神经和脊髓轴突及髓鞘的特异性严重攻击，导致坏死性空洞。昼夜节律已被充分证明对细胞功能、行为和疾病产生深远影响。本研究旨在探讨昼夜节律在NMO中的作用及其分子基础。方法我们利用了由水通道蛋白4（AQP4）IgG诱导的NMO细胞模型，在分离的星形胶质细胞中检测Cx43和Bmal1的表达，通过实时PCR和Western Blot进行。使用TAT-Gap19和DQP-1105抑制Cx43和谷氨酸受体。通过含腺病毒的shRNA敲低Bmal1。通过ELISA试剂盒定量谷氨酸、前视觉通路（AVP）和血管活性肠肽（VIP）的水平。结果我们发现，昼夜节律的两个关键组分Bmal1和Clock在NMO星形胶质细胞中显著降低，这种降低可通过Cx43激活（亚油酸）或谷氨酸逆转。此外，Cx43阻断（TAT-Gap19）或谷氨酸受体抑制（DQP-1105）也降低了Bmal1和Clock的表达水平。进一步地，通过腺病毒介导的shRNA敲低Bmal1（Ad-sh-Bmal1）显著降低了神经元中谷氨酸、AVP和VIP的水平，并显著下调了NMO星形胶质细胞中Cx43激活（亚油酸）或谷氨酸处理后的Cx43蛋白水平。然而，Bmal1敲低并未改变正常星形胶质细胞中Cx43阻断（TAT-Gap19）或谷氨酸受体抑制（DQP-1105）后的这些水平。讨论总体而言，这些结果表明Cx43-谷氨酸信号通路可能是NMO诱导的SCN星形胶质细胞节律性损伤的关键上游调节因子。