Department of Neuropsychiatry, Division of Neuroscience, Graduate School of Medicine, Mie University, Tsu 514-8507, Japan.
Biomolecules. 2024 Feb 8;14(2):204. doi: 10.3390/biom14020204.
To explore the developmental processes of epileptogenesis/ictogenesis, this study determined age-dependent functional abnormalities associated with purinergic transmission in a genetic rat model (S286L-TG) of autosomal-dominant sleep-related hypermotor epilepsy (ADSHE). The age-dependent fluctuations in the release of ATP and L-glutamate in the orbitofrontal cortex (OFC) were determined using microdialysis and ultra-high-performance liquid chromatography with mass spectrometry (UHPLC-MS). ATP release from cultured astrocytes was also determined using UHPLC-MS. The expressions of P2X7 receptor (P2X7R), connexin 43, phosphorylated-Akt and phosphorylated-Erk were determined using capillary immunoblotting. No functional abnormalities associated with purinergic transmission could be detected in the OFC of 4-week-old S286L-TG and cultured S286L-TG astrocytes. However, P2X7R expression, as well as basal and P2X7R agonist-induced ATP releases, was enhanced in S286L-TG OFC in the critical ADSHE seizure onset period (7-week-old). Long-term exposure to a modest level of P2X7R agonist, which could not increase astroglial ATP release, for 14 d increased the expressions of P2X7R and connexin 43 and the signaling of Akt and Erk in astrocytes, and it enhanced the sensitivity of P2X7R to its agonists. Akt but not Erk increased P2X7R expression, whereas both Akt and Erk increased connexin 43 expression. Functional abnormalities, enhanced ATP release and P2X7R expression were already seen before the onset of ADSHE seizure in S286L-TG. Additionally, long-term exposure to the P2X7R agonist mimicked the functional abnormalities associated with purinergic transmission in astrocytes, similar to those in S286L-TG OFC. Therefore, these results suggest that long-term modestly enhanced purinergic transmission and/or activated P2X7R are, at least partially, involved in the development of the epileptogenesis of ADSHE, rather than that of ictogenesis.
为了探索癫痫发生/发作的发展过程，本研究在常染色体显性遗传性睡眠相关运动过度癫痫（ADSHE）的基因大鼠模型（S286L-TG）中确定了与嘌呤能传递相关的年龄依赖性功能异常。使用微透析和超高效液相色谱与质谱联用（UHPLC-MS）测定眶额皮质（OFC）中 ATP 和 L-谷氨酸释放的年龄依赖性波动。还使用 UHPLC-MS 测定培养的星形胶质细胞中 ATP 的释放。使用毛细管免疫印迹测定 P2X7 受体（P2X7R）、连接蛋白 43、磷酸化-Akt 和磷酸化-Erk 的表达。在 4 周龄 S286L-TG 和培养的 S286L-TG 星形胶质细胞的 OFC 中未检测到与嘌呤能传递相关的功能异常。然而，在关键的 ADSHE 发作起始期（7 周龄），S286L-TG OFC 中 P2X7R 表达以及基础和 P2X7R 激动剂诱导的 ATP 释放增强。长期暴露于适度水平的 P2X7R 激动剂（不能增加星形胶质细胞 ATP 释放）14 天可增加星形胶质细胞中 P2X7R 和连接蛋白 43 的表达以及 Akt 和 Erk 的信号传导，并增强 P2X7R 对其激动剂的敏感性。Akt 而非 Erk 增加 P2X7R 的表达，而 Akt 和 Erk 均增加连接蛋白 43 的表达。在 S286L-TG 出现 ADSHE 发作之前，已经观察到功能异常、增强的 ATP 释放和 P2X7R 表达。此外，长期暴露于 P2X7R 激动剂模拟了星形胶质细胞中与嘌呤能传递相关的功能异常，类似于 S286L-TG OFC。因此，这些结果表明，长期适度增强的嘌呤能传递和/或激活的 P2X7R 至少部分参与了 ADSHE 癫痫发生的发展，而不是发作的发展。
