The Study Of ATM Inhibitor Ku-55933 In The Damage Of Irradiated Neural Stem Cells

Background and objective:Radiotherapy is an effective treatment modality for head and neck tumors.Due to the special anatomical location of the head and neck tumor,it is inevitable to damage the normal brain tissue during the implementation of radiotherapy.When the brain area holding the cognitive function is damaged by irradiation,it may lead to the occurrence of radiation-induced cognitive impairment.Current studies have shown that the mechanisms of cognitive impairment induced by ionizing radiation are mainly believed to be related to neurogenesis,intracerebral inflammation and oxidative stress,and cerebrovascular injury,among which neurogenesis disorder is considered as a key factor.Neurogenesis is a process in which neural stem cells self-renew and eventually differentiate into neurons,which establish synaptic connections between neurons and exert neural functions.When this process isdisturbed and inhibited,neurogenesis is impaired.Neurogenesis mainly occurs in the dentate gyrus of the hippocampus,the subependymal area of the lateral ventricle,and the olfactory bulb.At present,researches on the molecular mechanism of neurogenesis disorder caused by ionizing radiation have largely focused more on the changes in the microenvironment of neural stem cells caused by ionizing radiation,while the repair capability of neural stem cells in response to the damage caused by ionizing radiation has not received enough attention.The previous experimental results of my research group showed that radiation-induced cognitive impairment was related to the ability of DNA damage repair in the hippocampus.Therefore,we speculate that DNA damage repair in hippocampal stem/progenitor cells may be associated with neurogenesis.So in this study primary cultured neural stem cells(NSCs)were used,we investigated the effects of low dose ionizing radiation on various activities of NSCs such as proliferation,apoptosis and differentiation in detail.We also used ku-55933,a specific ATM inhibitor,to inhibit the DNA damage response(DDR)of NSCs,and then assess how the impaired DDR affected the activities of irradiated NSCs.The results may provide a theoretical reference for the study of the molecular mechanism of radiation-induced cognitive impairment.Methods:In the first part of this study,we investigated the effect of ionizing radiation on apoptosis,proliferation and differentiation of neural stem cells.The NSCs were isolated from the cerebral cortex of embryo in C57BL/6 pregnant mice on E12 day of gestation.And the cultured cells were identified as neural stem cells by Nestin staining using immunofluorescence microscopy.Then,neural stem cells were irradiated with 0,0.1,0.2,0.5,1Gy X-rays,and the changes in various biological endpoints were detected at different time points after irradiation.1.Annexin v-PI apoptosis kit was used to determine the apoptosis of irradiated NSCs by flow cytometry.2.Cell counter was used to evaluate the changes in proliferation of irradiated NSCs.3.Immunofluorescence microscopy was used for Tuj1 or GFAP staining,and the positive rate of Tuj1 or GFAP was calculated to investigate the effect of ionizing radiation on the differentiation ability of neural stem cells into neurons and glial cells.In the second part of this study,we investigated the effect of ATM inhibitor ku-55933 on apoptosis,proliferation and differentiation of irradiated neural stem cells.the proliferation of neural stem cells under different drug concentrations of ATM inhibitor ku-55933 was first examined by cell counting experiment to determine the optimal working concentration of ku-55933.After that,the neural stem cells were pretreated with the ATM inhibitor ku-55933 with the optimal drug concentration,that is,ku-55933 was added to the medium 1 hour before the cells were irradiated,and the medium was changed to normal drug-free medium before irradiation.Then,the changes of DNA damage response of irradiated neural stem cells pretreated with ku-55933 were explored.The changes of several biological endpoints of irradiated neural stem cells pretreated by ku-55933 were determined.The apoptosis rate of neural stem cells was measured by flow cytometry.The proliferation of neural stem cells was detected by a disposable automatic cell counter.The ability of neural stem cells to differentiate into neurons was determined by immunofluorescence microscopy.Results:1.①The primary neural stem cells from the cerebral cortex of C57BL/6 mouse embryo were cultured for 3-5 days to form the suspended neurospheres,which manifest as strong positive staining for Nestin.②Neural stem cells were exposed to 0 Gy,0.1 Gy,0.2Gy,0.5Gy and 1 Gy,and we detectd the apoptosis rate of NSCs at different times(24,48,72 hours)after radiation.We found that 0.1 and 0.2 Gy of X-raya did not induce obvious apoptosis,but 0.5 Gy of X-rays could increased apopotosis rate of NSCs at 72 hours after radiation.Furthermore,the results showed that obvious apoptosis could be induced 24 h,48h,72h after 1 Gy X-exposure.And we found that at 72h after irradiation,the apoptosis rate of NSCs induced by 1Gy X-ray was 2 times higher than that of unexposed control cells.However,the previous data of our research group showed that the apoptosis rate of primary neurons only increased by 1.9 times at 48h after receiving 50Gy X radiation,indicating that neural stem cells are very sensitive to ionizing radiation compared with mature neurons.③Neural stem cells were irradiated with 0,0.1,0.2,0.5 and 1 Gy of X-rays,and the number of cells was counted at different times(24,48,72,96 hours after irradiation).The results showed that 0.1 Gy of X-rays did not significantly affect neural stem cell proliferation.As the dose increased to 0.2-1 Gy,ionizing radiation inhibited the proliferation of neural stem cells in a dose-dependent manner.We concluded that even a low-dose of radiation(0.2Gy)can inhibit NSC proliferation.This suggests that neural stem cells are highly sensitive to ionizing radiation.In addition,it was found that 0.1,0.2 Gy X-irradiation did not affect their capability to differentiate into neurons,but their capability to differentiate into neurons upon receiving 0.5Gy and 1Gy X-rays was significantly decreased.However,the capability of neural stem cells to differentiate into glial cells was not significantly affected by X-ray exposure at the used doses.2.Neural stem cells were irradiated with 0.5Gy of X-rays,and western blot results showed that the phosphorylation of ATM was significantly increased.After pretreatment with ku-55933,and western blot results showed that the ATM phosphorylation was significantly inhibited,which confirmed the inhibitory effect of ku-55933 on DNA damage response.Meanwhile,our results showed that ku-55933 pretreatment further inhibited the proliferation and differentiation of irradiated neural stem cells into neurons,which was statistically significant compared with the untreated irradiated cells.Conclusion:1.Neural stem cells are very sensitive to ionizing radiation.Lower dose ionizing radiation can lead to increased apoptosis of neural stem cells,proliferation inhibition and decreased capability to differentiate into neurons,but has no significant effect on the differentiation into glial cells.2.ATM is involved in the regulation of proliferation and neuronal differentiation of irradiated neural stem cells,which can further inhibit proliferation and neuronal differentiation of irradiated neural stem cells.This study suggests that the proliferation and differentiation of irradiated neural stem cells may be regulated by DNA damage repair response.