The Impact Of P21 Dysfunction In The Aging Processof Werner Syndrome Mice And Its Molecualr Mechanism

As of 2016,the population of 60 years old or older has increased to 220 million in China,the elder people’s proportion is more than 16%,the trend of population is serious.To elongate the lifespan,have a healthy aging process and prevent aging-related diseaseshave becomemost importanttopics.The first important topic is the prevention of aging-related diseases,such as osteoporosis,cataracts,type Ⅱ diabetes,atherosclerosis and so on.The high incidence of these aging diseases seriously affected the quality of life of the elderly.Along with the rapid development of science and technology is developing in high speed,more and more people are trying to prolong lifespan and improve the quality of the elder people’s life.Werner Syndrome(WS)isa rare autosomal recessive disease,caused by mutations in the WRN gene.The patients will go ageing rapidly after puberty,with high risk of osteoporosis,cataracts,type II diabetes,atherosclerosis and so on.These characteristics of Werner Syndrome make it an excellent model for study of ageing.Werner Syndrome is manifested when WRN protein is dysfunction and telomere DNAisdamaged.According to this,Terc and WRN double knockout mice has been established and faithfully manifests WS.With the passage of WS mice generation by generation,telomeres are shortened due to the homozygous loss of Terc knock out.Along with the generation passaging,the occurrence of ageing phenotypesinWS mice become earlier and earlier.The first generation of mTer-/-mice generated from mTer+/-parents are named G1 WS mice(Generationl of Werner Syndrome),and the generation generated by G1 WS parents are G2 WS mice.Then we get G3,G4,G5 WS mice by the same mating strategy.When the telomeres have shortened too certain extend,such as the generation after G3,the lifespan and the half-life period become much shorter then G1 WS mice or WT mice.The incidence of osteoporosis,cataracts,type II diabetes,atherosclerosis may become earlier.These phenotypes provided us good read outs for monitoring the ageing process of WS mice.p21(coded by Cdkn1a)is an inhibitor of cell cycle,it may inhabit the process of cell cycle by bonding to the compound of CDKs.Besides,it also may compete with DNA polymerase and bond to PCNA(Proliferating cell nuclear antigen),then the activity of DNA polymerase will be suppressed,the process of replication and restoration will be suppressed.The dysfunction of p21 will release cell cycle partly and rescue the ageing without tumor,while p53 is wild type.Therefore,we knocked out p21 in WS mice and observed the influence of p21 on WS mice’s ageing phenotypes.We crossed p21-/-mice with WS mice and obtained mTer-/-Wrn-/-p21-/-TKO mice(triple knockout mice).The mice were then bred generation by generation and obtained G2,G3,G4 TKO mice.Surprisingly,we found that p21 knock out have accelerated the ageing of WS mice,rather than rescued it.Comparing the survival curve of WT,WS and TKO mice,we found that the lifespan of G4 WS mice was 430 days while the lifespan of G4 TKO mice decreased to 196 days.The half-life of WS mice was 184 days while the half-life of G4 TKO mice decreased to 133 days.However,the survival curve of G2 TKO mice was similar to that of G2 WS mice.These results indicated the impact of the dysfunction of p21 in accelerating the ageing of WS was stronger along with the shortening of telomeres.By X-ray of femur,we found that the 4 month old G4 TKO mice had the lower bone density than the G4 WS or WT mice of same age,indicating that osteoporosis had occurred in 4 month old G4 TKO mice.Furthermore,we detected the cellular senescence of mouse testis and small intestine tissue by SA-β-gal staining.It showed that the testis and small intestine tissue of G4 TKO mice had higher rate of SA-β-gal positive cells comparing with G4 WS mice and WT mice.We also detected the level of cellular proliferation in testis and small intestine by BrdU incorporation and immunohistochemistry.The results showed that the testis and small intestine of G4 TKO mice showed the highest rate of positive cell for BrdU incorporation and Ki67 staining,the second was in G4 WS mice,and the least positive rate was in WT mice.These results indicated that the cells in G4 TKO mice had higher level of proliferation than that in G4 WS mice and WT mice.We also detected the level of apoptosis in testis and small intestine by TUNEL assay and immunohistochemistry.The G4 TKO mice’s testis and small intestine showed higher rate of positive cell for TUNEL assay and Bax than that of G4 WS mice and WT mice.These results showed that the cells in G4 TKO mice had higher level of apoptosis than that in G4 WS mice and WT mice.Then we detected the level of y-H2AX in MEFs by immunofluorescence.It showed that more G3 TKO MEFs were positive for y-H2AX,suggesting more damages in G3 TKO MEFs than that in G3 WS MEFs and WT MEFs.At last,we detected cell cycle of MEFs by Flow Cytometry.We found that G4 TKO MEFs had enchanced G2/M arrest,suggesting that the dysfunction of p21 disabled the G1/S checkpoint and the cell with damages were blocked in G2/M.In summary,our study in mice and in MEFs showed that the dysfunction of p21 accelerated the ageing process in WS mice.Since the p21 is an important component of the G1/S checkpoint,we suspect that the function of the G1/S checkpoint for checking DNA integrity is weakened due to the absence of p21,which lead to the absence of G1/S arrest for those cells bearing DNA damges,and the accumulation of cells with DNA damages.The unrepaired DNA damages activate p53-mediated DNA damage response pathway,cause aging and apoptosis.In the other hand,the dysfunction of p21 accerlates the cellular proliferation and causes cellular over-proliferation of organs,which exhausts the stem cell reservoir and induces the failure of testis and small intestine.Our results suggest that it may not rescue ageing by release the inhibition of cell cycle only,such as knockout of p21.Especially in the cells with telomere DNA damages,the release of cell cycle inhibition may induce higher DNA damage response and accelerate the process of ageing.We may try to rescue ageing process by help repairing the DNA damages or eliminating the ageing cells.