Autophagy, lysosome dysfunction and mTOR inhibition in MNU-induced photoreceptor cell damage.
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.
['Li|Ying|Y|', 'Wang|Chenguang|C|', 'Liu|Yang|Y|', 'You|Jiaxin|J|', 'Su|Guanfang|G|']
D000818:Animals D017209:Apoptosis D001343:Autophagy D002460:Cell Line D002470:Cell Survival D008247:Lysosomes D008770:Methylnitrosourea D051379:Mice D019869:Phosphatidylinositol 3-Kinases D010786:Photoreceptor Cells D051057:Proto-Oncogene Proteins c-akt D017382:Reactive Oxygen Species D015398:Signal Transduction D058570:TOR Serine-Threonine Kinases
Autophagy Lysosome dysfunction Photoreceptor cells Neurodegeneration Retinitis pigmentosa