The Role Of Atg7-Mediated Autophagy In Ionizing Radiation-Induced Neural Stem Cell Damage

Objective:Nowadays,radiotherapy has become an essential treatment modality for patients with brain,head and neck cancer.With the development of radiotherapy technology,the survival of patients has significantly prolonged.However,during the implementation of radiotherapy,the normal brain tissue is inevitably damaged which leads to irreversible cognitive decline in patients during long-term survival after treatment.It has been reported that the impairment of neurogenesis is one of the important mechanisms of radiation-induced cognitive decline.Neurogenesis refers to a process by which neural stem cells differentiate into neurons and newborn neurons eventually integrate into neural networks.Autophagy plays an important role in neurogenesis,by which aging and dysfunctional proteins was hydrolysised and recycled.However,the role of autophagy in radiation-induced cognitive decline remains undetermined.Our previous studies have found that minocycline,a broad-spectrum antibacterial tetracycline antibiotic that can cross the blood-brain barrier,protects newborn neurons from radiation-induced apoptosis through inducing autophagy,thus inhibiting the development of rat cognitive decline after whole-brain irradiation.However,it is unclear whether autophagy would play any role in radiation-induced neural stem cell(NSC)damage.In the present study,mouse embryonic neural stem cells were used to investigate the role of autophagy in radiation-induced NSCs damage and how autophagy regulated the self-renewal and differentiation of NSCs in vitro.Methods:The aim of the first part of the study was to confirm whether autophagy was induced in irradiated NSCs and the role of autophagy in radiation-induced NSC apoptosis.First,FACS analysis was performed to detect the percentage of autophagic cells(AO staining).The expression levels of autophagy-related proteins such as p62,Atg7,Atg4B,LC3-? and phosphorylated mTOR were evaluated using Western Blot.To determine the role of autophagy in irradiated NSCs,Atg7,a critical autophagy-related gene,was knocked down using Atg7-targeting shRNA constructs.Autophagy level of irradiated Atg7-deficient NSCs was detected by FACS analysis and Western Blot.Subsequently,the percentage of apoptotic cells was detected in Atg7-deficient NSCs after exposed to different doses of X-rays to confirm the protective role of autophagy.In the second part of this study,we further investigated the protective role of autophagy on self-renewal and differentiation of irradiated NSCs.The self-renewal of Atg7-deficient NSCs after exposed to different doses of X-rays was analyzed by the number and the size of neurospheres using microscopy.The percentage of nestin positive cells in Atg7-deficient NSCs after irradiated with 0.1 Gy X-rays was detected by immunofluorescence microscopy.The percentage of nestin and cleaved-caspase3 double positive cells was detected to confirm the effect of apoptosis on Atg7-deficient NSCs after irradiation.Then z-VAD-FMK,a pan caspase3 inhibitor was applied prior to irradiation,and the neurospheres formed by Atg7-deficient NSCs after irradiation were measured to further confirm the effect of apoptosis.Finally the differentiation of Atg7-deficient NSCs was detected by immunofluorescent microscopy for Tuj 1(neuronal marker)and and GFAP(glial marker)and the length of the longest neurites of neurons were measured.Results:The percentage of positive cells with acidic vacuoles(AO staining)was obviously increased after irradiation in a dose-dependent manner.The accumulation of acidic vacuoles at 1 Gy was increased by 8.08 times compared with control.In agreement with that,the changes in the expression levels of essential autophagy markers such as Atg7,LC3-?,P62,p-mTOR and Atg4B further confirmed radiation-induced autophagy in NSCs in a dose-dependent manner.After Atg7 knocked down using Atg7-targeting shRNA constructs,the expression level of Atg7 was inhibited by 50%compared with that of the Atg7-NC NSCs.Besides,radiation-induced autophagy was inhibited in Atg7-deficient cells manifested as the reduction of autophagic vacuoles and the decrease in the induction of LC3-?.Meanwhile,radiation-induced NSC apoptosis was detected by FACS analysis,it was found that apoptosis increased more significantly in Atg7-deficient NSCs after exposed to 1 Gy X-rays.Moreover,even X-irradiation at lower doses such as 0.1 and 0.2 Gy,which did not cause obvious apoptosis in Atg7-NC NSCs,induced significant apoptosis in Atg7-deficient NSCs.These results indicated that autophagy may protect NSCs from radiation-induced apoptosis.Then,we further investigated the protective role of autophagy in radiation-induced neurosphere formation and NSC differentiation.It was found that the number and the size of the neurospheres formed by irradiated Atg7-deficient NSCs were significantly reduced in contrast to the Atg7-NC NSCs.Meanwhile,The percentage of nestin positive cells significantly reduced in Atg7-deficient NSCs after exposed to 0.1 Gy X-rays in contrast to the NC cells,which further confirmed the protective role of autophagy in NSCs self-renewal.Interestingly,the percentage of nestin and cleaved-caspase3 double positive cells was significantly increased in irradiated Atg7-deficient NSCs,indicating that Atg7 deficiency intensified radiation-induced NSC apoptosis.Additionally,when a pan caspase-3 inhibitor,z-VAD-FMK was used prior to irradiation,the size of the neurospheres formed by Atg7-deficient NSCs returned back to the level of unirradiated NSCs,suggesting that it was apoptosis that inhibited the neurosphere formation in irradiated Atg7-deficient NSCs.Furthermore,we investigated whether autophagy protect differentiation of Atg7-deficient NSCs after irradiation.The percentage of Tuj1 positive cell in Atg7-deficient NSCs significantly decreased after 0.1 Gy of irradiation when cultured in neuronal differentiation medium in contrast to Atg7-NC NSCs.Interestingly,the length of the longest neutrites of neurons increased slightly after irradiation in Atg7-NC NSCs,however the length of the longest neutrites of neurons differentiated from Atg7-deficient NSCs decreased after exposure to irradiation.Meanwhile,The percentage of GFAP positive cell in Atg7-deficient NSCs significantly decreased after exposed to 0.1 Gy X-rays when cultured in glial differentiation medium in contrast to Atg7-NC NSCs.These results suggested that autophagy not only affected the neuronal and glial differentiation but also regulated the neurite growth of irradiated NSCs.Conclusions:Radiation induced autophagy in mouse embryonic NSCs in a dose-dependent manner.Inhibition of autophagy by Atg7 knockdown significantly enhanced cell death and reduced the potential of self-renewal and differentiation of irradiated NSCs,indicating an important protective role of autophagy in maintaining neurogenesis after irradiation.Along with our previous study on neurons,the present study on NSCs not only shed the light of the involvement of autophagy in the development of radiation-induced cognitive decline,but also provided a potential target for preventing cognitive impairment after radiotherapy.