Progressive photoreceptor death is the main cause of retinal degeneration diseases. Determining the underlying mechanism of this process is essential for therapy improvement. Autophagy has long been considered to be involved in neuronal degeneration diseases, and the regulation of autophagy is thought to have potential implications for neurodegenerative disease therapies. However, whether autophagy is protective or destructive varies among diseases and is controversial. In the present study, we established an N-methyl-N-nitrosourea (MNU)-induced photoreceptor cell damage model in vitro that faithfully replicated photoreceptor cell death in retinal degeneration diseases. Cell viability was tested by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assays. Reactive oxygen species (ROS) levels were assessed through 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescence. Autophagy was confirmed by observing autophagosomes using transmission electron microscopy (TEM). A lysosome tracker was used to identify acidic lysosomes in cells. We also measured the expression of some proteins related to autophagy, apoptosis and lysosomal degradation by western blot and immunofluorescence assays. We found that MNU could decrease photoreceptor cell viability in a time- and dose-dependent manner, and this change was accompanied by concomitant increases in ROS and the expression of the apoptosis-inducing protein cleaved caspase-3. Moreover, autophagy was activated by MNU treatment during this process. Inhibition of autophagy with 3-methyladenine accelerated cell damage. Lysosome dysfunction was confirmed by autophagosome enlargement and increased cathepsin expression, which was accompanied by mTOR dephosphorylation. In conclusion, autophagy was activated through inhibition of the PI3K/mTOR pathway in the context of MNU-induced photoreceptor cell death. Prolonged mTOR dephosphorylation and autophagy activation resulted in autophagic vacuole accumulation, as indicated by inefficient degradation in lysosomes, and further led to apoptosis.

中文翻译：

---

自噬，溶酶体功能障碍和mTOR抑制MNU诱导的感光细胞损伤。

进行性光感受器死亡是视网膜变性疾病的主要原因。确定该过程的潜在机制对于改善治疗至关重要。自噬长期以来一直被认为与神经元变性疾病有关，自噬的调节被认为对神经退行性疾病疗法具有潜在的影响。但是，自噬是保护性的还是破坏性的，在各种疾病之间存在差异，并存在争议。在本研究中，我们建立了由N-甲基-N-亚硝基脲（MNU）诱导的光感受器细胞损伤模型，该模型忠实地复制了视网膜变性疾病中的光感受器细胞死亡。通过3-（4，5-二甲基噻唑-2-基）-5-（3-羧基-甲氧基苯基）-2-（4-磺基苯基）-2H-四唑鎓（MTS）测定法测试细胞活力。通过2,7-二氯二氢荧光素二乙酸酯（DCFH-DA）荧光评估活性氧（ROS）水平。通过使用透射电子显微镜（TEM）观察自噬体来确认自噬。溶酶体追踪剂用于鉴定细胞中的酸性溶酶体。我们还通过蛋白质印迹和免疫荧光测定法测量了一些与自噬，凋亡和溶酶体降解有关的蛋白质的表达。我们发现，MNU可以以时间和剂量依赖的方式降低感光细胞的活力，并且这种变化伴随着ROS的增加和凋亡诱导蛋白裂解的caspase-3的表达。此外，在此过程中，通过MNU处理激活了自噬。用3-甲基腺嘌呤抑制自噬可加速细胞损伤。自噬体增大和组织蛋白酶表达增加证实了溶酶体功能障碍，并伴有mTOR去磷酸化。总之，在MNU诱导的感光细胞死亡的背景下，通过抑制PI3K / mTOR途径激活了自噬。延长的mTOR去磷酸化和自噬激活导致自噬液泡积累，这在溶酶体中降解效率低下，进一步导致细胞凋亡。