Qiu Miaomiao, Zhao Xiaoming, Guo Tao, He Hongyun, Deng Yihao
School of Basic Medical Sciences, Kunming University of Science and Technology, Kunming, 650500, China.
Anning First People's Hospital Affiliated to Kunming University of Science and Technology, Kunming, 650500, China.
Cell Death Discov. 2024 Aug 18;10(1):368. doi: 10.1038/s41420-024-02144-7.
Autophagosome-lysosome fusion defects play a critical role in driving autolysosomal dysfunction, leading to autophagic/lysosomal impairment in neurons following ischemic stroke. However, the mechanisms hindering autophagosome-lysosome fusion remain unclear. Soluble N-ethylmaleimide-sensitive factor (NSF) is an essential ATPase to reactivate STX17 and VAMP8, which are the paired molecules to mediate fusion between autophagosomes and lysosomes. However, NSF is frequently inactivated to inhibit the reactivation of STX17 and VAMP8 in ischemic neurons. Herein, we investigated whether autophagosome-lysosome fusion could be facilitated to alleviate autophagic/lysosomal impairment in ischemic neurons by over-expressing NSF. Rat model of middle cerebral artery occlusion (MCAO) and HT22 neuron ischemia model of oxygen-glucose deprivation (OGD) were prepared, respectively. The results demonstrated that NSF activity was significantly suppressed, accompanied by reduced expressions of STX17 and VAMP8 in penumbral neurons 48 h post-MCAO and in HT22 neurons 2 h post-OGD. Moreover, the attenuated autolysosome formation accompanied by autophagic/lysosomal dysfunction was observed. Thereafter, NSF activity in HT22 neurons was altered by over-expression and siRNA knockdown, respectively. After transfection with recombinant NSF-overexpressing lentiviruses, both STX17 and VAMP8 expressions were concurrently elevated to boost autophagosome-lysosome fusion, as shown by enhanced immunofluorescence intensity co-staining with LC3 and LAMP-1. Consequently, the OGD-created autophagic/lysosomal dysfunction was prominently ameliorated, as reflected by augmented autolysosomal functions and decreased autophagic substrates. By contrast, NSF knockdown conversely aggravated the autophagic/lysosomal impairment, and thereby exacerbated neurological damage. Our study indicates that NSF over-expression induces neuroprotection against ischemic neuronal injury by restoring autophagic/lysosomal dysfunction via the facilitation of autophagosome-lysosome fusion. Over-expression of NSF promotes fusion by reactivating STX17 and VAMP8. Black arrows represent the pathological process after cerebral ischemia, green arrows represent the mechanism of remission after NSF over-expression, and red arrows represent the effect on the pathological process after NSF knockdown.
自噬体-溶酶体融合缺陷在驱动自噬溶酶体功能障碍中起关键作用，导致缺血性中风后神经元出现自噬/溶酶体损伤。然而，阻碍自噬体-溶酶体融合的机制仍不清楚。可溶性N-乙基马来酰亚胺敏感因子（NSF）是一种必需的ATP酶，可重新激活STX17和VAMP8，它们是介导自噬体与溶酶体融合的配对分子。然而，NSF在缺血神经元中经常失活，以抑制STX17和VAMP8的重新激活。在此，我们研究了通过过表达NSF是否可以促进自噬体-溶酶体融合，以减轻缺血神经元中的自噬/溶酶体损伤。分别制备了大脑中动脉闭塞（MCAO）大鼠模型和氧糖剥夺（OGD）的HT22神经元缺血模型。结果表明，在MCAO后48小时的半暗带神经元和OGD后2小时的HT22神经元中，NSF活性显著受到抑制，同时STX17和VAMP8的表达降低。此外，观察到自噬溶酶体形成减弱，并伴有自噬/溶酶体功能障碍。此后，分别通过过表达和siRNA敲低改变HT22神经元中的NSF活性。用重组NSF过表达慢病毒转染后，STX17和VAMP8的表达同时升高，以促进自噬体-溶酶体融合，这通过与LC3和LAMP-1共染色增强的免疫荧光强度得以体现。因此，OGD造成的自噬/溶酶体功能障碍得到显著改善，表现为自噬溶酶体功能增强和自噬底物减少。相比之下，NSF敲低则相反地加重了自噬/溶酶体损伤，从而加剧了神经损伤。我们的研究表明，NSF过表达通过促进自噬体-溶酶体融合恢复自噬/溶酶体功能障碍，从而诱导对缺血性神经元损伤的神经保护作用。NSF的过表达通过重新激活STX17和VAMP8促进融合。黑色箭头表示脑缺血后的病理过程，绿色箭头表示NSF过表达后的缓解机制，红色箭头表示NSF敲低后对病理过程的影响。
