背景:涉及PTEN诱导的推定激酶1(PINK1)和PARKIN的通路在线粒体自噬中起关键作用,由青蒿琥酯(ART)激活的过程。我们建议患有抗N-甲基-D-天冬氨酸受体(NMDAR)脑炎的患者表现出不足的线粒体自噬,ART通过PINK1/PARKIN途径增强线粒体自噬,从而提供神经保护。
方法:选择8-10周龄的成年雌性小鼠,建立抗NMDAR脑炎的被动转移模型。我们在设定的时间范围内对这些小鼠进行了行为测试。免疫组织化学等技术,免疫荧光,和蛋白质印迹用于评估标志物,包括PINK1,PARKIN,LC3B,p62、caspase3和切割的caspase3。TUNEL测定法用于检测神经元凋亡,而透射电子显微镜(TEM)用于检测线粒体自噬体。原代培养海马神经元,治疗,然后通过免疫荧光分析mtDNA,mtROS,TMRM.
结果:与对照组相比,实验组的线粒体自噬水平没有显著改变,然而,凋亡神经元显着增加。此外,与对照组相比,实验组发现线粒体渗漏和损伤的标志物升高,但是这些标志物在ART治疗后显示出改善。ART有效激活PINK1/PARKIN通路,增强线粒体自噬,减少神经元凋亡。行为评估显示,ART改善了被动转移模型(PTM)中抗NMDAR脑炎小鼠的症状。PINK1的敲除导致线粒体自噬水平降低,随后的ART干预并没有缓解抗NMDAR脑炎PTM小鼠的症状,表明ART的疗效是通过激活PINK1/PARKIN途径介导的。
结论:在抗NMDAR脑炎发作时,观察到线粒体损伤;然而,通过PINK1/PARKIN途径激活线粒体自噬可以减轻这种损伤。这种调节反馈机制有助于去除受损的线粒体,防止神经元凋亡,从而保护神经组织。ART激活PINK1/PARKIN通路以增强线粒体自噬,从而发挥神经保护作用,并可能达到治疗抗NMDAR脑炎的治疗目标。
BACKGROUND: The pathway involving PTEN-induced putative kinase 1 (PINK1) and PARKIN plays a crucial role in mitophagy, a process activated by artesunate (ART). We propose that patients with anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis exhibit insufficient mitophagy, and ART enhances mitophagy via the PINK1/PARKIN pathway, thereby providing neuroprotection.
METHODS: Adult female mice aged 8-10 weeks were selected to create a passive transfer model of anti-NMDAR encephalitis. We conducted behavioral tests on these mice within a set timeframe. Techniques such as immunohistochemistry, immunofluorescence, and western blotting were employed to assess markers including PINK1, PARKIN, LC3B, p62, caspase3, and cleaved caspase3. The TUNEL assay was utilized to detect neuronal apoptosis, while transmission electron microscopy (TEM) was used to examine mitochondrial autophagosomes. Primary hippocampal neurons were cultured, treated, and then analyzed through immunofluorescence for mtDNA, mtROS, TMRM.
RESULTS: In comparison to the control group, mitophagy levels in the experimental group were not significantly altered, yet there was a notable increase in apoptotic neurons. Furthermore, markers indicative of mitochondrial leakage and damage were found to be elevated in the experimental group compared to the control group, but these markers showed improvement following ART treatment. ART was effective in activating the PINK1/PARKIN pathway, enhancing mitophagy, and diminishing neuronal apoptosis. Behavioral assessments revealed that ART ameliorated symptoms in mice with anti-NMDAR encephalitis in the passive transfer model (PTM). The knockdown of PINK1 led to a reduction in mitophagy levels, and subsequent ART intervention did not alleviate symptoms in the anti-NMDAR encephalitis PTM mice, indicating that ART\'s therapeutic efficacy is mediated through the activation of the PINK1/PARKIN pathway.
CONCLUSIONS: At the onset of anti-NMDAR encephalitis, mitochondrial damage is observed; however, this damage is mitigated by the activation of mitophagy via the PINK1/PARKIN pathway. This regulatory feedback mechanism facilitates the removal of damaged mitochondria, prevents neuronal apoptosis, and consequently safeguards neural tissue. ART activates the PINK1/PARKIN pathway to enhance mitophagy, thereby exerting neuroprotective effects and may achieve therapeutic goals in treating anti-NMDAR encephalitis.