The Protection Of PARP Inhibition On Light-injured Photoreceptor Cilia

BackgroundCilia are sensory organelles that protrude from the cell surface.With a small size and complex structure,cilia play an important sensory role,sensing changes of extracellular mechanical and chemical signals and assisting intracellualr signal transduction to trigger cascade responses.Recent studies have demonstrated that various human diseases are closely related to the disorders of ciliary structure,length and function,and these diseases are collectively referred as "ciliopathy".Photoreceptor cells are structurally composed of an inner segment(IS)and an outer segment(OS).The outer segment(OS),also known as photosensitive cilia,does not show the usual hair-like shape and is the most highly modified and the most special sensory cilia in the body.Therefore,defects in cilia usually lead to retinal phenotypes of diseases,such as retinitis pigmentosa(RP),Leber's congenital amaurosis(LCA),etc.The function of photoreceptor cells is to convert light stimuli into bioelectrical signals,then form vision.However,excessive light exposure(over intensity or over exposing duration)may leads to the damage of photoreceptor cells,causing compromised visual function and even blindness.The retina light injury is closely related to the progress of retinal degenerative diseases(RDD),such as retinitis pigmentosa(RP)and age-related macular degeneration(AMD).Poly(ADP-ribose)polymerase(PARP)is a nuclear enzyme involved in the cellular response to DNA damage.Itis activated when DNA damaged and broken,and plays an important role in DNA repair and transcription.However,excessive activation of PARP can lead to cell death.Up to now,the studies on retinal ciliopathy are limited,and it lacts effective clinical treatment for retina degenerative diseases,which are classified as "ciliopathy",yetpeople's daily exposure to light is inevitable.Therefore,the need to protect vision from light-induced damage is even more urgent.In this study,we plan to explore the protection of PARP inhibition on retinal cilia during light injury,and attempt to decipher the molecular mechanism.Objective1.We are going to investigate the changes of the expression level of PARP protein with a light-injured photoreceptor cell(661W)model and verify the protection of PARP inhibition with PARP knockdown 661 W cells.2.We are going to investigate the structure and quantity of cilia on the cell surface,detect the expression level of cilia related proteins,verify the damage caused by light,and determine the protection of PARP inhibition on light-injured cilia.3.We are going to investigate the structure of retinal cilia,detect the expression level of retinal cilia related protein,verify the retinal injury caused by light in vivo,and determine the protection of PARP inhibition on light-injured retinal cilia.4.We are going to explore the molecular mechanism of the protection of PARP inhibition on cilia.Methods1.The protection of PARP inhibition on light-injured photoreceptorcells.Photoreceptor cells(661W)were exposed to 1500 lux visible light and Western blot was used to detect the change of PARP protein expression level.A PARP knockdown cell line was established with lentivirus-mediated sh RNA technique.The death percentage of cells in different groups were assessed by PI/ Hoechst staining under the condition of light injury.2.The protective effect of PARP inhibition on light-injured photoreceptorcell cilia.The changes of cilia on photoreceptor cell surface in light injury were observed with transmission electron microscopy(TEM).The primary cilia were stained with immunofluorescence and the percentage of cells with cilium was calculated.The percentage of cells with cilium in control and PARP knockdown group were compared under light injury to evaluate the protection of PARP inhibition on cilia.In addition,the expression levels of cilia-related proteins(CEP164,Arl13b)were detected with western blot.3.The protection of PARP inhibition on light-injured retinal cilia.An animal model of retinal light injury was established with C57BL/6J mice,and the mice were treated with PARP inhibitor(ABT-888).The changes of retinal structure and thickness were observed with HE staining under the microscrope,and the morphological changes of photoreceptor outer segments were observed with transmission electron microscope.The location of cilia-related proteins(CEP164,Arl13b)in mice retina was detected with immunofluorescence staining.The expression level of cilia-related proteins(CEP164,Arl13b)in mice retina was determined to evaluate the protection of PARP inhibitor on retinal cilia in light injury with western blot.4.The potential molecular mechanism of PARP inhibition on cilia in light injury.The localization of p Ser473-Akt and primary cilia in photoreceptor cells and in mouse retina were observed by immunofluorescence.In vitro,the activation of PI3K/Akt pathway was detected with western blot.The relationship between PARP inhibition and activation of PI3K/Akt pathway was further confirmed.The photoreceptor cells with PARP knockdown were treated with PI3 K inhibitor(LY294002),and the percentage of cells with cilium was calculated with immunofluorescence staining,which was used to evaluate the change of the protection.In addition,661 W cells were treated with Akt activator(SC79)and the percentage of cells with cilium was also calculated with immunofluorescence staining to evaluate the protection.Results1.661 W cells were exposed to light,and the expression level of PARP protein increased in a time-dependent manner,which reached the peak on day 2.The photoreceptor cells with PARP knockdown showed remarkable resistance against light damage and the death percentage was significantly less than that in control group cells,and there was statistical difference between the two groups.2.On the day 1 of light exposure,the number of cilia on the photoreceptor cell surface significantly decreased,and it was difficult to find the normal cilia structure on day 2 detected with transmission electron microscopy.However,in the PARP knockdown group,the percentage of cells with cilium was maintained at the normal level on the day 1 of light exposure,and the expression of cilia-related proteins(CEP164,Arl13b)were maintained at a higher level compared with the control group,and there was statistical difference between the two groups.3.Light exposure led to a significant reduction in the number of cells in the outer nuclear layer,thinned the outer nuclear layer and the photoreceptor layer,caused the swollen of OSs structure,vacuolar degeneration and disordered arrangement of the disc structure.In the mice treated with PARPinhibitor(ABT-888),the retinal structure was markedly improved.CEP164 is significantly expressed in IS layer,while Arl13 b is expressed in IS and OS.In the lightexposed mice treated with PARP inhibitor,the expression level of cilia-related proteins(CEP164,Arl13b)were maintained in the retina with statistical difference compared with that in control group.4.The p Ser473-Akt is located at the basal body of cilium or between two adjacent cilia in cells,and is expressed in the IS layer in mice retina.The treatment with PI3 K inhibitor(LY294002)was able to significantly attenuate the protection of PARP knockdown on cilia under the light condition.However,the treatment with Akt activator(SC79)was able to significantly maintained the the percentage of cells with cilium as compared with control group.Conclusions1.Light exposure led to the activation of PARP,and overactivation of PARP is involved in cell death caused by light injury.2.PARP inhibition can protect the structure of retinal cilia against light injury,and reduce the death percentage of photoreceptor cells.3.The protection of PARP inhibition on retinal cilia was possible due to its function on maintaining the expression of cilia-related proteins(CEP164,Arl13b).4.The protection of PARP inhibition on retinal cilia was possible due to its function on the activation of PI3K/Akt pathway,which promotes the translocation of p Ser473-Akt in the cilia basal body or between two adjacent cilia.