The Mechanism Of Pitx2-PARP1 Signaling Axis Inducing Photoreceptor Cell Death In PDE6b Mutation

Objective(s): Retinitis pigmentosa(RP)is a genetic retinal neurodegenerative disease,characterized by the pathogenic gene mutation and progressive apoptosis of retina photoreceptor cells.Inhibition of poly-ADP-ribose polymerase 1(PARP1)could relieve phosphodiesterase 6 mutation-induced retinitis pigmentosa(RP).Previous studies found that Pitx2 was highly expressed in photoreceptor cells in RP mice retina,which could induce the reduction of photoreceptor cell layers,and increased with the expression of PARP1.This research is to explore the relationship between the changes of Pitx2 and PARP1 and photoreceptor cell death,find potential target molecules related to RP,and provide new targets and ideas for the diagnosis,treatment and prevention of RP.Methods: ATAC-seq and RNA-seq were performed for retina tissues of C3 H and rd1 mice.The differentially expressed genes(DEGs)were identifed,followed by the construction of PARP1-DEG co-expression and protein–protein interaction(PPI)networks.Gene ontology-biological process and pathway enrichment of DEGs were performed by cluster Profler software.Meanwhile,the data of RNA-seq and ATAC-seq are comprehensively analyzed.Construction of AAV9 vector overexpressing and interfering with pitx2 gene.The retina was transfected with AAV9 vector,overexpressed and knocked down Pitx2,and divided into four groups: m-Pitx2(Pitx2 overexpression group),si-pitx2(Pitx2 interference group),Ctrl-Pitx2(empty body sham treatment control group),and blank(blank control group).The rate of LC3 and PARP1 positive cells in the outer nuclear layer of the retina was detected by immunostaining,and the TUNEL positive cell rate,the number of photoreceptor cells and the thickness of the outer nuclear layer were detected and counted by TUNEL.The expression level of LC3 and PARP1 in mouse retina was detected by Western blot.To verify the relationship between pitx2 and PARP1 and photoreceptor cell death.Construction of LC3 lentivirus vector with double fluorescent markers.The retina was transfected with LC3 lentivirus vector labeled with double fluorescence.The expression of LC3 and P62 in the outer nuclear layer of retina of rd1 and WT mice was detected by Western blot.To determine the difference of autophagy level between WT mice and rd1 mice.To construct an in vitro culture model of retinal explant,use AAV9 vector to transfect the retinal explant,knock down and over-express pitx2 gene,and combine with various drug interventions,conduct immunostaining on the cultured retinal grafts to detect the rate of LC3,PARP1,pitx2 positive cells in the outer nuclear layer of the retina,TUNEL detection and statistics of the TUNEL positive cell rate of the outer nuclear layer of the retina,the number of photoreceptor cell layers and the thickness of the outer nuclear layer.Western blot test was used to detect the expression level of LC3,P62 and PARP1 in the retina of mice.Western blot test was used to determine the regulatory relationship of pitx2,PARP1 and LC3 in the retina of rd1 mice.Result(s): At the late stage of photoreceptor cell death,1061 DEGs were identified between rd1 and WT mice.340 up-regulated genes and 721 down-regulated genes.The up-regulated genes are closely related to epithelial tube morphogenesis,neural tube development and T cell differentiation.The down-regulated genes are closely related to visual perception,light stimulation sensory perception and monovalent inorganic cation transport.The enrichment analysis of KEGG pathway showed that the up-regulated genes were mainly concentrated in PPAR signal pathway and B cell receptor signal pathway,while the down-regulated genes were mainly concentrated in light transduction and synaptic vesicle cycle.313 DEGs were co-expressed with PARP1,of which 102 were positively correlated and 211 were negatively correlated.ATAC-seq analysis obtained 1394 up-regulated signal peaks and 891 down-regulated signal peaks.There is overlap between genes with different accessible peaks and DEGs in the co-expression network.The up-regulated overlapping genes are Asxl3 and Nyap2,and the down-regulated overlapping genes are Tmem136 and Sus.In vivo experiment: After the retina was transfected with LC3 lentivirus vector,the rate of LC3 positive cells in the outer nuclear layer of the retina of rd1 mice was higher than that of WT mice.Western blot detection at the corresponding time point: compared with WT mice,the expression of LC3 in rd1 mice increased and the expression of P62 decreased,with a statistically significant difference.In vitro experiment: After the treatment of retinal explant with autophagy inducer Rapamycin,the rate of LC3 positive cells in the outer nuclear layer of retina increased,the rate of PARP1 positive cells decreased,the rate of TUNEL positive cells decreased,and the number and thickness of photoreceptor cells increased.Western blot showed that the expression of LC3 increased,while the expression of P62 and PARP1 decreased.The AAV9 virus vector transfected the retina in vivo and overexpressed Pitx2 gene,resulting in a decrease in the rate of LC3 positive cells in the outer nuclear layer of the retina,an increase in the rate of PARP1 positive cells,an increase in the rate of TUNEL positive cells,a decrease in the thickness of photoreceptor cells,and a decrease in the number of outer nuclear layers.At the same time,the level of LC3 protein decreased and the level of PARP1 protein increased.Knock down the Pitx2 gene,resulting in an increase in the rate of LC3 positive cells in the outer nuclear layer of the retina,a decrease in the rate of PARP1 positive cells,a decrease in the rate of TUNEL positive cells,an increase in the thickness of photoreceptors,and an increase in the number of outer nuclear layers.At the same time,the level of LC3 protein increased and the level of PARP1 protein decreased.The rate of PARP1 and TUNEL positive cells in the outer nuclear layer and the number and thickness of photoreceptor cells in the outer nuclear layer did not change significantly compared with that in the rd1 blank control group;When 3-MA was used to inhibit autophagy and Olaparib was used to intervene in rd1 retinal explants,the rate of PARP1 and TUNEL positive cells in the outer nuclear layer and the number and thickness of photoreceptor cells had no significant change compared with the rd1 blank control group.Conclusion(s): In rd1 mice,up-regulation of pitx2 level leads to decreased LC3 expression and increased PARP1 expression,resulting in photoreceptor cell death.pitx2 targets PARP1 to promote photoreceptor cell death through Pitx2-LC3-PARP1 signaling axis.