MTOR Interact With PARP-1 To Regulate Visible Light-induced Parthanatos In Photoreceptors

BackgroundRetinal degenerative diseases?RDD?such as age-related macular degeneration?AMD?,retinitis pigmentosa?RP?and Stargardt's disease may seriously damage the visual function and eventually cause irreversible blindness.The pathogenesis of retinal degenerative diseases includes multiple mutations of genes related to retinal function and risk environmental factors.Photoreceptor cell death is the basic pathological feature of retinal degenerative diseases.Retinal light damage is considered to be one of the important pathogenic factors.Excessive light,as one of the harmful environmental factors,plays an important role in the pathogenesis of retinal degenerative diseases.Photoreceptor cell death is an important pathological feature of retinal degenerative diseases.However,the specific mechanism of photoreceptor cell death induced by light is still not clear.Excessive light may cause photothermal damage,photomechanical damage and photochemical damage to the retina.Among these damage,photochemical damage plays a leading role in retinal light damage.After the retina is exposed to intense light stimulation,rhodopsin can absorb photons and activate ionization effect to form oxidation free radicals.Under the excessive light stimulation,the synthesis and degradation of free radicals are unbalanced,which leads to the increase of the level of oxidative stress.At this time,the unbalanced antioxidant system could not effectively eliminate the generated reactive oxygen species,then the internal antioxidant system is damaged,resulting in the overload of oxidative stress in the mammalian retina.Under certain-situations,the process of apoptosis is regulated by a series of signal cascades.Caspase dependent pathway is a classic apoptosis pathway,which regulates apoptosis and inflammation by activating a series of caspase,and maintains homeostasis.Generally,there are two pathways through which the caspase family proteases can be activated:one is the death signal-induced,death receptor-mediated pathway;the other is the stress-induced,mitochondrion-mediated pathway.In previous study,we found that in the glucose deprivation model of photoreceptor cell 661W,the protein levels of apoptosis related proteins caspase-3 and caspase-9 increased significantly.The treatment of caspase inhibitor z-VAD-fmk can inhibit the expression of Caspase-3 and caspase-9,and inhibit the apoptosis of mitochondria and cells.After using caspase inhibitor z-VAD-fmk,caspase-3 and other proteins decreased,mitochondrial apoptosis pathway was inhibited,and apoptosis was also inhibited.However,z-VAD-fmk,a caspase inhibitor,could not block the apoptosis induced by light injury,indicating that there are other possible death mechanisms in light-induced injury.Parthanatos is a unique form of cell death which depends on the overactivation of PARP-1.This pathway involves rapid activation of PARP-1,synthesis and accumulation of a large number of par polymers,followed by mitochondrial depolarization,transfer of apoptosis inducing factor?AIF?to the nucleus,degradation of large segments of chromosomes,ultimately leading to cell death.In the model of light-induced damage,excessive light leads to oxidative stress injury,which leads to excessive ROS penetrating the nuclear membrane and DNA strand breakage.DNA damage fragment specifically overactivates PARP-1 in the nucleus,causes the exhaustion of NAD⁺pool,slows down the rate of glycolysis,impedes the transmission of electronic respiratory chain,and depletion of adenine nucleoside triphosphate?ATP?pool to initiate programmed cell death,leading to cell death cascade.Therefore,we speculate that parthanatos death may be involved in the death mechanism of retinal photoreceptor cells in the light-induced injury.Mammalian rapamycin target protein?mTOR?is a serine threonine protein kinase which exists in almost all eukaryotic cells,regulating cell growth,protein synthesis,gene expression and metabolic balance.The dysfunction of mTOR pathway is related to many neurodegenerative diseases,and the activation of mTOR signal is widely involved in various kinds of retinopathy,including diabetic retinopathy and age-related macular degeneration.Therefore,mTOR is also the main target for the treatment of many retinal degenerative diseases.Rapamycin,an inhibitor of mTOR,may reduce ROS production and oxidative stress,improve mitochondrial dysfunction,and protect photoreceptor cells from degenerative death.661W cell line was obtained by immortalization of mouse photoreceptors,and its expression of SV40-T antigen was controlled by the promoter encoding human retinal receptor binding protein gene.The cell line has both structural and biochemical characteristics of cone-shaped photoreceptor cells,so is an effective model for the study of photoreceptor cell biology in vitro.In this study,661W cells were used to simulate the degeneration of photoreceptor cells in light-induced retina to elucidate the mechanism of photoreceptor cell death induced by light and whether the neuroprotective effect of mTOR and PARP-1 signaling pathway on photoreceptor cell death was mediated by apoptosis inducible factor?AIF?.In order to investigate the mechanism of retina protection by inhibiting mTOR and PARP-1 signaling pathway,a mouse model of light injury was also established in vivo.Methods1.To explore the protective effect of mTOR and PARP-1 signal pathway inhibition on photoreceptor cells in the light-induced damage.1.1 661W cells with mTOR/PARP-1 knockdown were constructed with lentivirus-mediated siRNA technique.1.2 The cells were divided into 661W group,Scramble group?negative control group?,Lt 661W group,Lt Scramble group,Lt mTOR KD group and Lt PARP-1 KD group.Hoechst/PI method and Western Blot method were used to detect cell death in different experimental groups and to explore the protection mechanism of inhibiting mTOR and PARP-1 signal pathway.2.To explore the protective effect of AIF inhibition on photoreceptor cells in the light-induced damage.?1?661W cells with AIF knockdown were constructed with lentivirus-mediated siRNA technique.?2?The cells were divided into 661W group,Scramble group?negative control group?,Lt 661W group,Lt Scramble group,Lt AIF KD group.Hoechst/PI method and Western Blot method were used to detect apoptosis in different experimental groups and to explore the protection mechanism of inhibiting AIF signal pathway.3.Detection of AIF nuclear translocation under light exposed condition.?1?661W cells were transiently transfected with AIF^GFP plasmid to construct the cell line expressed with AIF^GFP.?2?AIF^GFPFP cells were divided into dark group and light damaged group.After co-staining with Hoechst,the translocation of AIF to the nucleus was observed under confocal microscope.?3?The cells were divided into 661W group,Scramble group?negative control group?,Lt 661W group,Lt Scramble group,Lt mTOR KD group and Lt PARP-1 KD group.The nuclear/cytoplasm fraction were separated and the expression of 57kDa AIF protein in the nuclear fraction was detected with Western Blot.4.To explore the role of SIRT1 signaling pathway between mTOR and PARP-1signaling pathway.The cells were divided into 661W group,Scramble group?negative control group?,Lt 661W group,Lt Scramble group,Lt mTOR KD group and Lt PARP-1 KD group.Western Blot and quantitative real-time PCR were used to detect SIRT1 protein and mRNA expression in different experimental groups.The effects of SIRT1inhibitor EX527 on apoptosis of 661W,Lt mTOR KD and Lt PARP-1 KD were observed by Hoechst/PI staining.The influence of EX527 on mTOR signaling pathway in Lt PARP-1 KD cells/PARP-1 signaling pathway in Lt mTOR KD cells were detected by Western Blot.5.To explore the effect of mTOR and PARP-1 inhibition on mouse retina under light injury.The mice were divided into four groups:normal?Control?group,light exposed group?Lt+vehicle?,rapamycin treated light-exposed group?Lt+RAPA?and 3AB treated light-exposed group?Lt+3AB?.The neuroptotection of RAPA/3AB against light injury was assessed with ERG functionally based on the amplitude of a wave b wave.Retina slice was stained with HE and the thickness and the number of nuclei of ONL were quantitatively determined to evaluate the protection of RAPA/3AB against retina light injury.The influence of RAPA/3AB treatment on mTOR/PARP-1signaling pathway?mTOR,p-mTOR,p-4EBP1,p-s6k1,PARP-1,PAR,AIF?in light-damaged retina were also determined with Western Blot.Results1.The death percentage of 661W cells significantly increased under the excessive light exposed condition,while the inhibition of mTOR and PARP-1 was able to reduce the death percentage.Interestingly,intensive light exposure may activate mTOR and PARP-1 pathway,resulting in the increase of 57kDa AIF protein.However,the activation of PARP-1 pathway was significantly inhibited by mTOR Knock Down.Similarly,the phosphorylation of mTOR pathway was also suppressed by PARP-1 Knock Down.Compared with the Lt Scramble group,there was a decrease of 57kDa AIF protein in both Lt mTOR KD group and Lt PARP-1 KD group.2.The inhibition of AIF signaling pathway can reduce the death percentage of photoreceptor cells under light injury.However,AIF Knock Down can not inhibit the activation of PARP-1 or mTOR pathway under the light exposed condition.3.The active form of 57 kDa AIF was detected in light-exposed cells.In addition,the AIF^GFP was primarily located in the cytoplasm under dark condition,while AIF clearly translocated into nuclei after light exposure,as indicated by light-blue fluorescence in Hoechst-stained nuclei.AIF activation was also suppressed by mTOR/PARP-1 knockdown,as indicated by a reduced 57 kDa AIF form in nuclear fraction of mTOR/PARP-1 KD cells,even under light exposure conditions,indicating that excessive light exposure may induce AIF translocation;However,AIF knockdown failed to influence the levels of PARP-1 and mTOR signals,indicating that both PARP-1 and mTOR are upstream factors of AIF.4.Treatment with EX527 remarkably mitigated the PARP-1 knockdown-induced suppression of the mTOR signal,with increased levels again in p-mTOR,p-S6K1,p-4EBP1 after light exposure compared to untreated PARP-1 KD cells.Similarly,treatment with EX527 also attenuated mTOR knockdown-induced inhibition of the PARP-1 signal,as indicated by re-upregulation of PARP-1 and PAR levels compared to untreated mTOR KD cells.Moreover,treatment with EX527 significantly mitigated the protection caused by mTOR/PARP-1 knockdown following light exposure.These results suggest that SIRT1 is an important intermediate factor involved in crosstalk between the PARP-1 and mTOR signals.5.Treatment with 3AB or rapamycin significantly mitigated light-induced damage in retina structurally,as evindenced by increased ONL thicknesses and abundances of photoreceptor nuclei compared to the vehicle light-treated group.Intraperitoneal administration of 3AB or rapamycin significantly mitigated light-induced damage to the function of the retina,as indicated by attenuated a-and b-wave amplitude decreases related to the vehicle light-treated group.The PARP-1and mTOR pathways are activated and interact in light-injured retina.Light exposure induces parthanatos-like injury in the retina.Conlusion1.PARP-1 and mTOR signals are both activated by light exposure,and there is crosstalk between the signals.A parthanatos-like death mechanism was evaluated in light-injured 661W cells,with over-activation of PARP-1 and translocation of 57kDa AIF.2.AIF inhibition may provided significant neuroprotection against light-induced injury.3.Both PARP-1 and mTOR are upstream factors of AIF.Either PARP-1 or mTOR inhibition may suppress the translocation of 57kDa AIF and provided significant neuroprotection against light-induced injury.4.SIRT1 is an important intermediate factor involved in crosstalk between the PARP-1 and mTOR signals in light-injured 661W cells.5.PARP-1/mTOR inhibition mitigated light-induced damage to both function and structure of the retina,provides remarkable protection on retina against light injury by suppressing AIF pathway.