A Critical Role Of Tob In The Pathogenesis Of Alzheimer's Disease

Objective: Recent studies suggest that there are abnormal expressions of cell cycle protein and dysfunction of cell cycle in the brain of Alzheimer's disease.Tob protein,as one negative regulator of cell cycle,may play a critical role in the pathogenesis of AD.In our experiment,we used senescence accelerated mouse SAMP8 as the animal model of Alzheimer's disease,and SAMR1 as control.Firstly,we detect the expression of Tob protein in the brain of SAMP8/R1 mice at different ages by Western blot.If there is a relationship between Tob protein and the pathologies of AD,we will interfere with the expression of Tob protein using gene technology,then explore the role of Tob in the development of AD-like behavior and the underlying signal pathway.Methods:(1)By Western Blotting,we detect the age-related changes of the levels of Tob protein family in the brain of SAMP8 and SAMR1 mice.(2)After the microinjection of AAV-Tob1 vector in CA1 subregion,we detect the levels of Tob1 and Tob2 protein in different brain regions of mice by Western Blotting.(3)After the microinjection of AAV-Tob1 vector in CA1 subregion,the mice were used to assess the changes in learning and memory behavior.(4)After the microinjection of AAV-Tob2-shRNA vector in CA1 subregion,we detect the levels of Tob1 and Tob2 protein in different brain regions of mice by Western Blotting.(5)After the microinjection of AAV-Tob2-shRNA vector in CA1 subregion,the mice were used to assess the changes in learning and memory behavior.Results:(1)Compared with SAMR1 at the same age,there were no significant agerelated changes of the levels of Tob1 and Tob2 protein in both the prefrontal cortex(PFC)and hippocampus(HIP)of SAMP8 mice(p>0.05).(2)In the CA1 and DG subregions of the hippocampus,both the levels of Tob1 and Tob2 protein in 9 month-old SAMP8 increased greatly(p<0.05),compared with all other groups;in addition,we observed an increase of the expression of Tob2 protein in the DG subregion of SAMR1,but still lower than that of SAMP8.However,there were no significant alterations of the levels of both Tob1 and Tob2 protein in the CA3 subregion(p>0.05).(3)Among the 3 month-old SAMP8 mices,the expression of Tob1 protein was interfered by the microinjection of AAV-GFP and AAV-Tob1-3*flag-GFP vectors in CA1 subregion.We observed a significant increase of the level of Tob2 protein in hippocampus(p<0.05),but no alterations of the levels of Tob2 protein in hippocampus and both Tob1 and Tob2 protein in the prefrontal cortex(p>0.05).(4)After the microinjection of AAV-GFP and AAV-Tob1-3*flag-GFP vectors in CA1 subregion of the 3 month-old SAMP8 mice,compared with the GFP-controls,Tob1-overexpression group showed impairments of object recognition capability in the test 1h after Novel Object Recognition(NOR)training(p<0.05),and the situation became much more serious in the 24 h test.(5)Among the 7 month-old mices,the expression of Tob1 protein was interfered by the microinjection of AAV-GFP and/or AAV-Tob1-3*flag-GFP vectors in CA1 subregion.Compared SAMR1-GFP group with SAMR1-7month ones,there were no alterations of the levels of Tob1 and Tob2 protein in both hippocampus and the prefrontal cortex(p>0.05).Among the SAMP8 mices,we observed a significant effect of the down-regulation of Tob1 protein in hippocampus;there was also a downward trend in the expression of Tob2 protein of the hippocampus(p=0.079>0.05),but no statistical significance;in addition,there were no significant changes of the levels of both Tob1 and Tob2 protein in the prefrontal cortex(p>0.05).(6)After the microinjection of AAV-GFP and AAV-Tob1-shRNA-GFP vectors in CA1 subregion of the 7 month-old mice,compared with the SAMR1-GFP controls,both SAMP8-Tob1-shRNA and SAMP8-GFP groups showed impairments of object recognition capability in the test both 1h and 24 h after NOR training(p<0.05),and the situation became much more serious in the 24 h test.In addition,there was a significant difference between SAMP8-Tob1-shRNA group and SAMP8-GFP ones in the NOR-24 h test(p<0.05).(7)Among the 3 month-old SAMP8 mices,the expression of Tob2 protein was interfered by the microinjection of AAV-GFP and AAV-Tob2-shRNA-GFP vectors in CA1 subregion.We observed a significant decrease of the level of Tob2 protein in hippocampus(p<0.05),but no significant alterations of the levels of Tob2 protein in hippocampus and both Tob1 and Tob2 protein in the prefrontal cortex(p>0.05).(8)After the microinjection of AAV-GFP andAAV-Tob2-shRNA-GFP vectors in CA1 subregion of the 3 month-old mices,we observed no significant changes in the behavioral tests both 1h and 24 h after the NOR training(p>0.05),and the similar situation happened in the MWM task.Conclusion: 1)There were no significant age-related changes of the levels of Tob1 and Tob2 protein in both the prefrontal cortex(PFC)and hippocampus(HIP)of mices.However,we observed a significant age-related increase of the levels of both Tob1 and Tob2 in the CA1 subregion,and the similar situation happened in the DG subregion.2)The intervention of the expression of Tob1 protein in CA1 subregion by gene technology did not affect the expressions of Tob2 in hippocampus and both Tob1 and Tob2 in the prefrontal cortex.3)There is a relation between Tob1 protein and the pathogenesis of Alzheimer's disease,and Tob1 protein maybe play a negative regulation of the learning memory abilities of SAMP8 mice.4)After intervening the expression of Tob2 protein in CA1 subregion by gene technology,we observed a significant decrease of the level of Tob2 protein in hippocampus,but no significant alterations of the levels of Tob2 protein in hippocampus and both Tob1 and Tob2 protein in the prefrontal cortex.5)After intervening the expression of Tob2 protein in CA1 subregion by gene technology,there were no alterations of the AD-associated behaviors.The current data indicated that the change of the level of Tob1 didn't affect the learning memory ability of SAMP8 mices.