Suppressing Autophagy Protects Retinal Photoreceptor Cells From Light-induced Injury

Autophagy, a conserved cellular self-degradation process, not only. serves to protect cells at critical times during development and nutrient stress, but also contributes to cell death. Photoreceptor cells are unique neurons which when directly exposed to the light, transduces light stimuli into visual signal. However, intense light exposure can be cytotoxic to the retina. So far, the precise mechanism underlying retina light injury remains unknown, and the effective therapy is still unavailable.1.Light exposure leads to autophagy in661W cellsAim:though different autophagy tests investigate whether2600Lux light exposure induces autophagy in661W cells. Method:(1) use661W cell line exposed by2600Lux visual light to build photoreepter cells light-injured model.(2) The level of the autophagic marker LC3II and LC3I in cell lysates was examined by western blot.the morphological change of the autophalysosomes was observed by electron microscope.(3) After using3-MA and LY294002two different autophagy inhibitors to preprocess661W cells,Quantitative assessment of cell death percentage (the number of PI positive cells/the number of total cells%) was performed through PI staining to observe wether suppressing autophagy protect661W cells from light damage.Results:(1) after the661W cells were exposed to2600Lux light for1or2d, the cellular LC3II level was remarkably increased.Further quantitative analysis revealed that the ratio of LC3II/LC3I from the light-treated cell lysate was significantly higher than that from the untreated cell lysate(P<0.001).After2-d light exposure, the661W cells exhibited features typical of autophagy, such as sequestration of some cytosolic material,including organelles, within double-membrane bound vesicles termed autophagosomes.(2) the pretreatment of cells with10-301M LY29400230min before light exposure significantly protected661W cells from light-induced damage and decreased the cell death percentage from63%to about20%based on PI staining assay results.The optimum concentration of LY294002was around201M.Pre-incubation of661W cells with2.5-7.5mM3MA led to a significant decrease in the light-induced cell death, reducing the death percentage from63%to about30%.3MA at5mM showed the best protection.(P<0.001) These results indicate that autophagy is a key stage in the light-induced cell death and that blocking autophagy effectively protects photoreceptor cells from light damage.2. MAPK signaling is activated and contributes to the light-induced cell deathAim:we examined the effect of MAPK signal pathways on light-injured661W cells under the condition of visual light exposure.Methods:(1) use661W cell line exposed by2600Lux visual light to build photoreepter cells light-injured model.(2) use western blot to test expression levels of phosphorylated ERK, ERK, phosphorylated JNK,JNK,phosphorylated p38and p38. Quantitative assessment of cell death percentage (the number of PI positive cells/the number of total cells%) was performed through PI staining after661W cells were pretreated with PD980519and SP600125and SB203580.(3) The cells lysates werecollected at the indicated time periods and subjected to western blot analysis of phosphorylated ERK、ERK and LC3II、 LC3I.The cells were stained with LysoTracker and then examined under confocal laser microscope. Results:(1) Cell lysates were collected at the indicated time periods and subjected to western blot analysis of phosphorylated ERK, ERK, phosphorylated JNK, JNK, phosphorylated p38and p38. The phosphorylation of ERK1/2, c-Jun N-terminal kinase (JNK) and p38MAPK was significantly increased after the exposure of cells to2600Lux light, while the levels of non-phosphorylated forms of ERK1/2and p38were only slightly increased(P＜.001).pretreatment of cells with40-801M PD98059remarkably suppressed the light-induced cell death,reducing the death percentage from61%to about30%, whereas pre-incubation with SP600125or SB203580failed to protect cells from light damage.(P＜0.001).(2)401M PD98059at30min prior to light exposure markedly reversed the light-induced upregulation of both p-ERK and ERK as determined by western blot. Further quantitative analysis demonstrated that the levels of non-phosphorylated and phosphorylated forms of ERK were significantly reduced in the PD98059-pretreated group.Next, we examined the change in the level of autophagic marker LC3II after treatment with PD98059. After exposure to light for2d remarkably caused the up-regulation of LC3II/LC3I ratio, but the addition of401M PD98059significantly attenuated the lightinduced increase in the LC3II/LC3I ratio.Consistent with this, we also observed numerous cellular lysosomes detected by Lyso Tracker staining. Pretreatment with401M PD98059markedly suppressed the formation of cellular lysosomes even with lightexposure. These observations suggest that blocking the ERK pathway effectively suppressed the light-induced autophagy in661W cells(P<0.001).Here, we found that visible light exposure activated the mitogen-activated protein kinases (MAPK) pathway and led to remarkable autophagy in photoreceptor cells (661W cells).Among the activated downstream factors of MAPK pathway, ERK, not JNK or p-38, played a critical role in light-induced death mechanism. Inhibiting the activation of ERK with its specific inhibitor PD98059significantly suppressed light-induced autophagy and protected661W cells from light injury.These results indicate that autophagy is an essential event in light-induced photoreceptor death and that directly blocking autophagy or suppressing autophagy by inhibiting the ERK pathway could effectively attenuates light-induced damage. These observations may have a potential application in the treatment of retinal light injury.