| Objective:In order to investigate the alteration of PTEN at different stages of mimic Alzheimer's disease(AD), and to further speculate its role in AD, We made aluminum toxic mice model, judged the damage degree of the model mice by impairment of learning and memory in behavioral tests and pathological staining with SPs and NFTs, and detect PTEN protein level, phospho-PTEN protein level, PTEN mRNA level, and PTEN immunoreactivity and distribution in the hippocampus and brain cortex. These will be beneficial to understand the etio-pathological mechanism of AD and to find new strategies and targets for AD drug therapy.Methods:①Infusion of 0.5% AlCl3 solution, 2μl/d,consecutively for 5 days, into the lateral cerebro-ventricles of the mice was used to produce the model, infusion of ACSF (artificial cerebrospinal fluid) for the control.②Passive avoidance response tests 2 and 7 days, and Morris water maze tasks 23～30 days after the last injection were used to identify the degrees of learning and memory impairment in the mice.③Immediately after the behavioral tests at the above three time points, MTST (Modified thioflavine S techniques) was used to detect the formation of SPs and NFTs in hippocampus and brain cortex, RT-PCR to detect PTEN mRNA expression and immunoblotting to detect protein levels of PTEN and pPTEN-Ser380 in hippocampus.④The same procedure was used to treat another crop of mice, and immunostaining to detect the distribution and intensity of PTEN in the hippocampus and brain cortex 7, 14, 21, 28, 35 days respectively after the last injection.Results: 1.Aluminum toxic AD-like model:①The avoidance latencies and number errors had no significant difference between the model and the control 2 days post i.c.v..②The latencies of the control significantly prolonged at the 7th day post i.c.v. than that at the 2nd day post i.c.v. (P<0.001), and the error number decreased; the latencies of the model had no significant change but the error number increased; reflecting that the ability of passive avoidance of the model mice is deteriorating(interaction of treatment and time, P<0.05).③The mean performance record of the model was significantly worse than that of the control in place navigation test of Morris water maze task 23～30 days post i.c.v. (P<0.001); in spatial probe test, the lingering time of the model within the target quadrat was significantly shorter than that of the control(P<0.05), reflecting a severe impairment on learning and memory ability of the model mice.④2～3 mice of each group were decapitated immediately after the behavioral tests in the above three time points, and the brain sections of the model at the third time points (30 days post i.c.v.) stained by MTST showed SPs and NFT-like structures in regiones temporalis cortex.2.PTEN mRNA levels of the hippocampus: 4～5 mice of each group were decapitated immediately after the behavioral tests in the above three time points, the complete hippocampuses were stripped out, and the total RNA was extracted; the mRNAs were reversely transcripted and the cDNAs were amplificated by PCR; the PTEN mRNA level of the model at the third time point (30 days post i.c.v.) was found significantly lower than that of the control(P<0.05, 1-tailed), but no significant difference was found between the model and the control at the first two time points.3.Protein levels of PTEN and p-PTEN in the hippocampus: 4～5 mice of each group were decapitated immediately after the behavioral tests in the above three time points, the complete hippocampuses were stripped out and the protein levels of PTEN and pPTEN-Ser380 were detected by Western blotting.①No significant difference of PTEN protein level between the model and the control was found, but a slight increase of the model at the above three time points.②The protein level of pPTEN-Ser380 of the model at the third time point (30 days post i.c.v.) was found significantly lower than that of the control(P<0.05), but no significant difference between the model and the control at the first two time points.4.Immuno-histochemistry staining: The same procedure is used to treat another crop of mice, which were then divided into the model and the control. 5 mice of each group respectively at the 7th, 14th, 21st, 28th, 35th day post i.c.v. were perfusion-fixed with 4% formaldehyde. Coronal sections of the brain were made by Microtome Cryostat and immunostained with anti-PTEN as the primary antibody, and streptomycin-anti-biotin (SP) kite and 3'3'diaminobenzidine (DAB) was used to present the staining. The analysis results of immunoreactive intensities showed that①PTEN is abundantly expressed in the pyramidal neurons of the CA1, CA2, CA3 regions of the hippocampus and in the granule cells of the dentate gyrus; the pyramidal neurons of the CA1 region in the hippocampuses of the model showed significantly higher immunoreactive intensity than that of the control at the 7th day post i.c.v. (P<0.05); it was still higher than that of the control at the 14th day post i.c.v.(P<0.05); no significant difference was found at the 21st and 28th day post i.c.v.; but the immunoreactive intensity of the model was significantly lower than that of the control at the 35th day post i.c.v.(P<0.05);②PTEN is abundantly expressed in pyramidal neurons of brain cortex, and weak immunoreactivity was also seen in other brain cellular stratums; the immunoreactive intensity of the lamina pyramidalis in regiones temporalis of the model mice displayed a dynamic alteration; no significant differnence between the model and control at the 7th day post i.c.v., but a significantly higher immunoreactive intensity was seen of the model than that of matched control at the 14th day post i.c.v.(P<0.01); significantly lower intensities of the model than that of matched control were found at the 28, 35 days post i.c.v.(P<0.05).Conclusion:1. The experimental AD-like mice model with AlCl3 i.c.v. displayed a progressive decline in the ability of learning and memory, with SPs and NFTs-like structure appearing in the regiones temporalis cortex at the end stage. So the model successfully simulated the learning and memory impairment and pathological changes in AD, figuring out the initiative, development and formation of an AD-like process.2. In the anaphase of the aluminum toxic mice, the PTEN mRNA level and phospho-PTEN protein level significantly decreased.3. In the progression of the model induced, PTEN might have a dynamic alteration in stratum pyramidale neurons of the hippocampal CA1 regions and in layer of medium-sized pyramidal cells in regiones temporalis cortex, where it arised at the initiative stage and then descended in the developmental and formative stages until to a degree significantly lower than that of matched control at the end stage.
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