| Object Neurofibrillary tangles,one of the pathological hallmarks of Alzheimer’s disease(AD), is consist of hyperphosphorylated tau protein. And it is generally accepted thatε4 allele of apolipoprotien E(APOE) is the main genetic risk factor for AD. And it participated in various pathological process of AD. So far, the relationship between apoE4 and Aβhad been fully explored. But, the effect of apoE4 on tau phosphorylation had not been fully understood. Now, we are trying to study it in vivo and in vitro. In vivo, we use EFAD mouse model to study the effect of different apoE isforms on tau phosphorylation, totol tau, tau kinases and calpain under a genetic background of AD. In vitro, we incubated N2a/APP695 cells with different recombinant apoE peptides, and explored the same observation target. Finally, we are trying to provide a certain theoretical basis for the treatment of AD.Methods 1. In vivo, the SPF 3- and 7-month old male E3 FAD and E4 FAD mice were used in this experiment( 9-12 per group). The effects of different apoE isforms on the learning and memory of 5xFAD mice were tested by Morris water maze behavior experiment. Modified Marsland’s silver staining was used to observe the morphology of nerve fibers. Through westernblot and immunohistochemical technique, we tested the the total tau level and the level of phosphorylated tau in T205, T231, S396 and S404 sites. And we also examined the expression levels of tau kinases GSK3β, CDK5, P35 and P25. Also, we observed the expression of calpain which is involved in the calpain-CDK5 pathway by western blot.2. In vitro, N2a/APP695 cells, a clone stable expression of mutant APP695 in mouse neuroblastoma cell line, was used as a AD model. And 200 nM recombinant human apoE peptides were added to it, and then cells were divided into apoE3 treated group, apoE4 treated group and control group. Through western blot methods, the expression levels of phosphorylated tau, tau kinases and calpain were examined, and finally to verify the phenomenon and mechanism observed in vivo.Results 1. In vivo results showed, a. The results of Morris water maze suggested that there was no significant difference of learning and memory ability between E3 FAD mice and E4 FAD mice at 3 months. But the ability of learning and memory in E4 FAD mice were significantly decreased at 7 months.b. The results of modified Marsland’s silver staining suggested that the nerve fibers of two groups were integrated and regularly shaped at age of 3 months. And the nerve fibers had a heavy staining in E4 FAD mice. But, at age of 7 months, compared to E3 FAD mice, the nerve fibers were disrupted in E4 FAD mice.c. Western blot suggested that compared with E3 FAD mice, the expression of phosphorylated tau at T205, T231, S396 and S404 sites, tau kinases CDK5, P35 and P25 were increased in E4 FAD mice, but the level of tau kinase GSK3β had no difference between two groups. Also, calpain was increased in E4 FAD mice.2. In vivo results showed, a. After incubated with 200 nm recombinated human apoE peptides for 24 hours, the expression of phosphorylated tau at T205, T231, S396 and S404 sites, tau kinases CDK5, P35, P25 and calpain were increased in N2a/APP695 cells.b. After co-incubated with the inhibitor of CDK5, the phosphorylation of tau at T205, T231, S396 and S404 sites were decreased.Conclusion 1. Tau phosphorylation emerged earlier than cognitive impairment.2. The increased levels of tau phosphorylation were induced by CDK5, but not GSK3β in 5xFAD mice.3. ApoE4 induced tau hyperphosphorylation was through calpain-CDK5 pathway, led to an instability of microtubule, followed by a disruption of the cytoskeleton, and finally impaired the function of cognition. |
PDF Full Text Request