P21cip1 Delay The Retinal Degeneration Of Rd1 Mice By Suppressing Cdk Activity

Purpose:Retinitis pigmentosa（RP）is a common hereditary retinal degenerative disease characterized by the death of retinal photoreceptors.At present,there is no effective method to prevent or reverse the disease process.The rd1 mouse is the most commonly used animal model in RP research.Previously our research team and collaborators made three important findings regarding the mechanism of rd1 rod death.First we found that rd1 rod death is mediated by Bmi1-Cdk-Rb-E2f pathway;Second,we found epigenetic modifier Ezh2 can regulate Akt activity,thus affect rd1 rod death;Finally,we also identified that c AMP induced Neogenin expression is important for rd1rod death.Ezh2 and Neogenin are both downstream target genes of E2f,thus knocking out Bmi1 or E2f,inhibiting Ezh2,and neutralizing Neogenin with peptides can both delay the rd1 rod death in varied degrees.These results indicated that rod cell death in rd1 mice is closely related to E2f upregulation.E2f transcription factor is an important factor that regulates the cell cycle,and its activity is mainly inhibited by the Rbprotein.The function（phosphorylation state）of the Rbprotein is regulated by Cyclin and Cdk;the activity of Cdk is controlled by its inhibitor（CKI）.In this way,CKI,Cdk/Cyclin,and Rb/E2f together form the Rb-E2f pathway that sequentially transmits signals inside and outside the cell to control the cell cycle.We found previously that knocking out the Rbgene（RbKO）in the retina can also cause a large number of ectopic division and apoptosis of rod cells,and this phenotype is medicated by E2f1 up-regulation,which can be rescued by E2f1KO.Since the death of rod cells in rd1 and RbKO mice are both related to the up-regulation of E2f,we hypothesized that further knockout Rbgene in the rd1 retina may accelerate the death of rod cells.However,we found that RbKO significantly delayed rd1 rod death,indicating that there are other mechanisms to control the rd1 rod death.The purpose of this study is to identify this mechanism in order to deepen our understanding of the pathogenesis of RP and provide prospective ideas and translational medical evidence for the treatment of RP.Methods:Using the alpha-cre which specifically expressed in the periphery of the retina from E10,the Rbgene was knocked out in the retina and crossed with rd1/rd1,p21^-/-,Cdk1^floxed,and Cdk2^floxed mice to obtain different lines of mouse.The retinas of these mutant mice at postnatal day 14（P14）and P21were collected and subjected to immunofluorescence staining in frozen sections to observe changes in cell types,cell proliferation,cell death,DNA damage,and the number and distribution of microglia.The expression of p21Cip1 was also analyzed.The m RNA and protein expression were analyzed by PCR and Western blots.Cdk inhibitor CR8 was injected into subretinal space to directly observe the effect of the drug on retinal degeneration of rd1mice.Results:1.During our observation period（21 days after birth）,rd1 mice mainly lost rod cells in the retina,and the outer nuclear layer（ONL）became significantly thinner;but other retinal cell types were basically normal,and retinal lamination developed normally.There were no obvious Ki67 positive dividing cells,but there was an increase of activated microglial cells in the ONL,and retinal angiogenesis was partially delayed.2.During our observation period（21 days after birth）,the retina of RbKO mice had ectopic division of all retinal cells,and there were many Ki67 positive cells;in addition to a large number of rod cell apoptosis,Pkca⁺ bipolar cells and Brn3⁺ganglion cells also died by apoptosis,but the number and distribution of cone cells,horizontal cells,and amacrine cells were basically normal.Müller cells increased and partially ectopically located to the ONL.The number of microglial cells increased and distributed to the whole retina.The development of inner retinal angiogenesis and retinal lamination delayed.3.The ONL of rd1;RbKO double mutant mice is thicker than that of rd1 and RbKO mice,and the difference is more obvious in P21.Other cell types, microglia,vascular development,and retinal lamination were similar to RbKO mice.However,the cell cycle was significantly activated,and Ki67- positive cells doubled compared to RbKO mice,indicating that cell cycle activation did exist in the retina of rd1 mice;meanwhile,the expression of CKI proteins P21^cip1 increased,many P21^cip1-positive cells were located in the outer nuclear layer.4.The ONL of rd1;RbKO;p21KO triple mutant mice is similar to that of rd1 and RbKO mice,and was significantly thinner than that of rd1;RbKO double mutant mice.These results indicated that RbKO delays the apoptosis of rod cells in rd1 mice by up-regulating P21cip1.5.rd1;RbKO;p21KO;Cdk1^+/-;Cdk2^+/-quintuple mutant mice have a thicker ONL than rd1;RbKO double-mutant mice,and rescued the thinner ONL in rd1;RbKO;p21KO triple-mutant mice.This indicates that RbKO up- regulates P21cip1,which can suppress the activity of Cdk1 and Cdk2,thus delays the apoptosis of rod cells in rd1 mice.6.The rd1;Cdk1^+/-;Cdk2^+/-triple mutant mice have a thicker ONL than rd1 mice.Except for some microglia invasion,other retinal cell types and lamination were basically normal.7.ChIP experiment indicated that Bmi1 can bind to the promoter of p21cip1 gene in rd1 retina.8.Vitreal injection of CR8,a small molecular Cdk inhibitor,could delay rod cell death and increase the amplitudes of dark-adpated ERG a/b waves in rd1 mice at P14 but not P21.Conclusions:Through the observation of a series of gene knockout mice in this study,it was found that increased Cdk activity is an important mechanism for the rod death in rd1 mice.The rd1 mutation can increase the ectopic division cells in the RbKO retina,but RbKO significantly delays the death of rod cells in rd1 mice.The mechanism is mainly through the up-regulation of P21cip1,which inhibits the activity of Cdk（especially Cdk1 and Cdk2）.This also indicates that abnormal cell division is not the mechanism of rod cell death in rd1 mice.Inhibition of Cdk activity by gene knockout or small molecular drug can delay the apoptosis of rod cells in rd1 mice.As Bmi1 can bind to the promoter of p21cip1 gene in rd1 retina,p21cip1 may also mediate the protection effects of Bmi1KO on rd1 rods.