The Changes In Expression Of P35, P25 And Cdk5 Kinase Activity In Hippocampal Neuron Irradiated With X-ray

【Background】The malignant tumor incidence rate roars up globally these years with the speeding up of life tempo and global aging.Now it has become 'the second killer'of human following the cardiovascular diseases which damage people's health seriously as all know.And it endangers human health seriously.Radiotherapy is one of the major therapeutic tools on malignant tumor and the radical cure tools on partial of them.According to WHO statistics in 1998,malignant tumor control rate of all over the body is 45%,operation occupies 22%and radiotherapy 18%.Over 70%malignant tumor patients had radiotherapeutic experience.However,X-ray can result in normal tissues injury at the same time of killing tumor cells.Radiation injury of brain is a radioactive syndrome caused by various kinds of reason and one of the most serious long-term complications after pate malignant tumors radiotherapy.Following oncotherapy level improvement and life span unceasing prolongation,the incidence of radiation brain injury goes up year by year.Radiation brain injury impacts living quality,life span and prognosis of patients seriously.But the mechanism of radiation brain injury is not clear,so the effective treatment is still lack up to now.Therefore,it is very important to study the radiation brain injury and explore new therapeutic targets.Cyclin-dependent kinase 5(CDK5) is a proline-directed protein kinase that phosphorylates serine and threonine residues.It is a special member of the cyclin-dependent kinase family(CDK).CDK5 phosphorylates a diverse list of substrates including KSP protein motif on MAP1b,KSPXK on tau or KSPXX on neurofilament protein.However,in contrast to other members of the cyclin-dependent kinase family,CDK5 is not directly involved in mediating progression through the cell cycle.Although CDK5 is expressed in most tissues,its activity is found predominantly in post-mitotic neurons.The reason for the localized activity of CDK5 is that its activators,p35 and p39,are expressed almost exclusively in the central nervous system(CNS).CDK5 is critical to CNS development.Furthermore,CDK5 likely plays a role in synaptic plasticity,cellular motility and adhesion,drug addiction and neurodegeneration.As monomeric CDK5 displays on enzymatic activity,an interaction with either p35 or p39 anchored to membrane is necessary for CDK5 activation.The proteolysis of p35 to p25,is likely important in regulating CDK5 activity.The truncated activators,p25,appear to have greater stability than their precursors and therefore,this may result in prolonged CDK5 activity resulting in hyperphosphorylation of available substrates.There is evidence to suggest that overexpression of CDK5 and p25 in neurons results in apoptosis.In addition,the expression and activity of CDK5 have been shown to increase during neuronal apoptosis.CDK5 inhibitors protected against both necrotic and apoptotic neuronal cell death in several different models.Focusing on the links between CDK5 activity and components of the cytoskeletal membrane and adhesion systems has allowed us to postulate a role for CDK5 in directing intracellular traffic in neurons.Microtubule dynamics and axonal transport might be targets for CDK5 through its phosphorylation of the microtubule-associated protein(MAP).Aberrant phosphorylation of the neuonal cytoskeleton by CDK5 could alter celluar trafficking and transport and contribute to neuronal dysfunction and death.So it is hypothesized that p25(a truncated form of p35) cause aberrant activation of CDK5,leading to the breakdown of cytoskeletal proteins and intracellular trafficking,which,in turn,contributes to neurodegeneration.Cleavage of p35 to p25,may be a critical step in this pathological cascade,including neurite retraction,microtubule collaose and apoptosis.But the exact mechanisms by which CDK5 can facilitate cell death processes have not been fully elucidated.There is no doubt that CDK5 is critical for the development and functioning of the CNS.In addition,there is increasing evidence that overactivation of CDK5 may contribute to neurodegenerative processes.So the question is whether CDK5 is involved in the pathogenesis of RBI.Thus,the aim of this study is to investigate the relationship between aberrant activation of CDK5 and RBI.On this ground,we exposed the hippocampal neurons at 12days to single x-ray doses of 30Gy,and then we observed the changes in the expression of p35/p25 by western-blot,and finally we observed the effect of inhibition of CDK5 so as to investigate if dysfunction of CDK5 is involved in the pathogenesis of RBI.【Objective】To study the changes in expression of p35,p25 and CDK5 kinase activity in cultured rats hippocampal neurons irradiated with x-ray and provide theoretic basis for prevention and treatment of radiation brain injury.【Methods】(1) The cultured rat hippocampal neurons at 12days were exposed to single x-irradiation with doses of 30Gy.(2) Western-blot was used to detect the levels of protein p35/p25,which correlates with the activity of CDK5.The levels of protein p35/p25 were assayed separately at 3.5h,4h,5h,6h after irradiation.(3) To observe the effect of roscovitine treatment on neuronal apoptosis induced by x-irradiation,the cells were stained with 4',6-diamidino-2-phenylindole(DAPI) at 24 h after irradiation,the apoptotic changes were measured quantitatively by nuclear pyknosis.(4) All values were expressed as mean±SD.Statistical significance was defined as P≤0.05. 【Results】(1) Western-blot showed that p35(35KD) significantly increased(F(4,15) = 19.338,P=0.000,N=4).Compared with the control group the 3.5 hours group significantly increased to 1.52(P＜0.001),the 4 hours group significantly increased to 1.44(P＜0.001).The result also show that p25(25KD) significantly increased (F(4,15) =6.617,P=0.003,N=4).To compared withthe control group,the 6 hours group significantly increased after x-irradiation(P＜0.001 ).The changes of p35,p25 expression levels suggested that CDK5 may participate in the neuronal death of the hippocampus after x-irradiation.(2) Apoptosis experiment showed that administration roscovitine(10μM) at 15 minutes before x-irradiation can protect the hippocampal neuronal death induced by irradiation(F(2,13.121)= 146.226,P= 0.000,N=9).The percentage of nuclear pyknosis in the group of 30Gy(24.8%±3.97%) was significantly increased over that in the group of 0Gy at 24 h after irradiation(1.82%±1.08%,P＜0.001),while the percentage of nuclear pyknosis in the group of 30Gy & roscovitine(7.74%±2.27%) was significantly different from that in the group of 30Gy at 24 h after irradiation(P＜0.001).There was significant difference between the group of 30Gy & roscovitine and the group of 0Gy(P＜0.001).【Conclusion】(1) x-irradiation may activates CDK5 by enhancing the levels of p35,p25 in hippocampal neurons.(2) Inhibiting CDK5 activity by the CDK5 inhibitor roscovitine can significantly protect hippocampal neuronal cell from death,which suggest that dysfunction of CDK5 may be involved in the pathogenesis of radiation hippocampal neuron injury.