Department of Neurology, The Affiliated Hospital of Zunyi Medical University, Zunyi, China.
The Collaborative Innovation Center of Tissue Damage Repair and Regeneration, Zunyi Medical University, Zunyi, China.
Epilepsia Open. 2024 Aug;9(4):1252-1264. doi: 10.1002/epi4.12945. Epub 2024 May 3.
The objective of this study is to determine whether inhibition of mitophagy affects seizures through Clathrin-mediated endocytosis (CME).
Pentylenetetrazol (PTZ) was intraperitoneally injected daily to establish a chronic PTZ-kindled seizure. The Western blot (WB) was used to compare the differences in Parkin protein expression between the epilepsy group and the control group. Immunofluorescence was used to detect the expression of MitoTracker and LysoTracker. Transferrin-Alexa488 (Tf-A488) was injected into the hippocampus of mice. We evaluated the effect of 3-methyladenine (3-MA) on epilepsy behavior through observation in PTZ-kindled models.
The methylated derivative of adenine, known as 3-MA, has been extensively utilized in the field of autophagy research. The transferrin protein is internalized from the extracellular environment into the intracellular space via the CME pathway. Tf-A488 uses a fluorescent marker to track CME. Western blot showed that the expression of Parkin was significantly increased in the PTZ-kindled model (p < 0.05), while 3-MA could reduce the expression (p < 0.05). The fluorescence uptake of MitoTracker and LysoTracker was increased in the primary cultured neurons induced by magnesium-free extracellular fluid (p < 0.05); the fluorescence uptake of Tf-A488 was significantly decreased in the 3-MA group compared with the control group (p < 0.05). Following hippocampal injection of Tf-A488, both the epilepsy group and the 3-MA group exhibited decreased fluorescence uptake, with a more pronounced effect observed in the 3-MA group. Inhibition of mitophagy by 3-MA from day 3 to day 9 progressively exacerbated seizure severity and shortened latency.
It is speculated that the aggravation of seizures by 3-MA may be related to the failure to remove damaged mitochondria in time and effectively after inhibiting mitochondrial autophagy, affecting the vesicle endocytosis function of CME and increasing the susceptibility to epilepsy.
Abnormal mitophagy was observed in a chronic pentylenetetrazol-induced seizure model and a Mg-free-induced spontaneous recurrent epileptiform discharge model. A fluorescent transferrin marker was utilized to track clathrin-mediated endocytosis. Using an autophagy inhibitor (3-methyladenine) on primary cultured neurons, we discovered that inhibition of autophagy led to a reduction in fluorescent transferrin uptake, while impairing clathrin-mediated endocytosis function mediated by mitophagy. Finally, we examined the effects of 3-methyladenine in an animal model of seizures showing that it exacerbated seizure severity. Ultimately, this study provides insights into potential mechanisms through which mitophagy regulates clathrin-mediated endocytosis in epilepsy.
本研究旨在探讨通过网格蛋白介导的内吞作用（CME）抑制线粒体自噬是否会影响癫痫发作。
通过腹腔注射戊四氮（PTZ）每日建立慢性 PTZ 点燃癫痫模型。采用 Western blot 比较癫痫组和对照组之间 Parkin 蛋白表达的差异。免疫荧光检测 MitoTracker 和 LysoTracker 的表达。将转铁蛋白-Alexa488（Tf-A488）注入小鼠海马。通过观察 PTZ 点燃模型中 3-甲基腺嘌呤（3-MA）对癫痫行为的影响来评估其作用。
腺嘌呤的甲基衍生物，即 3-MA，已广泛应用于自噬研究领域。转铁蛋白从细胞外环境通过 CME 途径内化到细胞内空间。Tf-A488 利用荧光标记物跟踪 CME。Western blot 显示，PTZ 点燃模型中 Parkin 的表达显著增加（p<0.05），而 3-MA 可降低其表达（p<0.05）。无镁细胞外液诱导的原代培养神经元中 MitoTracker 和 LysoTracker 的荧光摄取增加（p<0.05）；与对照组相比，3-MA 组 Tf-A488 的荧光摄取明显减少（p<0.05）。海马内注射 Tf-A488 后，癫痫组和 3-MA 组的荧光摄取均减少，3-MA 组的效果更为明显。3-MA 从第 3 天到第 9 天抑制线粒体自噬，逐渐加重癫痫发作的严重程度并缩短潜伏期。
推测 3-MA 加重癫痫发作可能与抑制线粒体自噬后不能及时有效清除受损线粒体有关，影响 CME 的囊泡内吞作用，增加癫痫易感性。
在慢性戊四氮诱导的癫痫发作模型和无镁诱导的自发性反复癫痫样放电模型中观察到异常的线粒体自噬。利用荧光转铁蛋白标记物跟踪网格蛋白介导的内吞作用。在原代培养神经元中使用自噬抑制剂（3-甲基腺嘌呤），我们发现抑制自噬会导致荧光转铁蛋白摄取减少，同时损害由线粒体自噬介导的网格蛋白介导的内吞作用功能。最后，我们在癫痫动物模型中检查了 3-甲基腺嘌呤的作用，发现其加重了癫痫发作的严重程度。最终，该研究提供了有关线粒体自噬调节癫痫发作中网格蛋白介导的内吞作用的潜在机制的见解。
