The Effect Of Atg7 Knockout On A Mouse Model Of Tuberous Sclerosis Complex

Hyperactivation of the mammalian target of rapamycin complex 1（mTORC1）caused by TSC1 and TSC2 genes mutation is the major factor of tuberous sclerosis complex（TSC）.The intracranial tumors generated by excessive proliferation of astrocytes mainly resulted in the onset of clinical symptoms and the death of patients.It has been found that mTOR inhibitors can reduce the size of TSC related tumor,and autophagy plays a pivotal role in mTOR inhibitors exerting their antineoplastic effects.However,the effect of autophagy on TSC related intracranial tumors has not been reported so far.Our previous studies found knockout TSC1 in primary mouse astrocytes inhibited the initiation of autophagy,which suggest that autophagy may play an important role in the proliferation of astrocytes in TSC.In order to further clarify the effect and the specific regulatory mechanisms of autophagy on astrocyte proliferation,our study intends to use TSC1 and autophagy related gene 7（Atg7）of astrocyte selective knockout mice,which may provide an important theoretical and experimental basis for TSC targeted therapy based on autophagy.Objective:Explore the role of autophagy initiation-related gene Atg7 in tuberous sclerosis model mice,and observe the effects of Atg7 knockout on survival rate,astrocyte proliferation,mTOR signal pathway activation of TSC model mice,the impact of autophagy-related indicators and the frightened behavior of mice before and after rapamycin administration.Methods:First,we used the Cre-LoxP system to conditionally knock out Atg7(Atg7^GFAPCKO),TSC1(TSC1^GFAPCKO)or dual knockout of Atg7 and TSC1(Atg7/TSC1^GFAPCKO).Wild type mice were used as the control group,Atg7^GFAPCKOmice,TSC1^GFAPCKO mice,Atg7/TSC1^GFAPCKO mice are the experimental groups.Life span of each group with or without administration of rapamycin（1 mg/kg,every other day,ip）were recorded.The proliferation of astrocytes in the hippocampal CA1 and DG area was determined by immunofluorescence staining with GFAP.Western blot was performed to assay the activation of mTOR signaling pathway and expression of autophagy-related proteins.Approach-test and touch-test were used to detect the frightened behavior in mice.Results:First,mice that specifically knock out TSC1 and Atg7 in astrocytes were verified by Western blot and PCR.Then,the results shows that the life span was 20.3±4.1 days in TSC1^GFAPCKO mice and 14.1±2.8 days in Atg7/TSC1^GFAPCKO mice,whereas no obvious change in Atg7^GFAPCKO mice as compared to control mice.The life span of TSC1^GFAPCKO was significantly longer than that of Atg7/TSC1^GFAPCKO mice（P<0.001）.In the immunofluorescence staining assay,TSC1^GFAPCKO and Atg7/TSC1^GFAPCKO mice develop progressive increases in astrocyte number whereas no obvious change in Atg7^GFAPCKO mice as compared to control mice.The expression of p-mTOR,p-P70S6K and p-S6 increased in TSC1^GFAPCKO mice and Atg7/TSC1^GFAPCKO mice while there was no significant difference between Atg7^GFAPCKO mice and control mice.In addition,no statistical discrepancy in astrocyte proliferation and activation of mTOR signaling pathway were observed between TSC1^GFAPCKO mice and Atg7/TSC1^GFAPCKO mice.The up-regμlation of SQSTM1together with the down-regμlation of LC3-II/LC3-Ⅰcould be observed in Atg7^GFAPCKO mice and Atg7/TSC1^GFAPCKO mice,whereas no obvious change in TSC1^GFAPCKO mice as compared to control mice.Behaviorally,TSC1^GFAPCKO and Atg7/TSC1^GFAPCKO mice showed more frightened than the Control group and Atg7^GFAPCKO group（P<0.001）,but there was no significant difference between the two groups.After administration,the life span was 49.4±7.6 days in TSC1^GFAPCKO mice and 23.5±5.4days in Atg7/TSC1^GFAPCKO mice,astrocyte proliferation and the up-regμlation of p-mTOR,p-P70S6K,p-S6 were restored to normal levels by rapamycin in TSC1^GFAPCKO mice and Atg7/TSC1^GFAPCKO mice,that is,no statistical discrepancy in astrocyte proliferation and activation of mTOR signaling pathway were observed among four groups.After that,there was no change in autophagy-related indicators such as LC3-II and SQSTM1/P62 comparing with that before treatment.Noticeably,while the lifetime of the above two groups was prolonged after administration,the survival time of TSC1^GFAPCKO mice the was still distinctly longer than that of Atg7/TSC1^GFAPCKO mice（P<0.01）;TSC1^GFAPCKO and Atg7/TSC1^GFAPCKO mice have no significant difference with Control group and Atg7^GFAPCKO group in the degree of fright.Conclusion:Therefore,we draw a conclusion that Atg7 gene has a protective effect on mice with tuberous sclerosis.Knockout of the Atg7 gene will worsen the survival of tuberous sclerosis model mice,suggesting that activating autophagy may be effective in treating the tuberous sclerosis complex.