Experimental Study On The Expression Of Synapse-associated Proteins And Ultrastructural Changes In Cerebellum Of Transgenic Mice

Objective: In the present study,APPswe/PS1 d E9(APP/PS1)and APPswe/PS1M146V/tau P301L(3xTg-AD)transgenic mice were used as AD models.In order to investigate the underlying mechanisms of synaptic plasticity in AD cerebellum,immunohistochemistry,Western-blotting and electron microscopy were utilized to observe the changes in the expression of synapse-associated proteins and ultrastructure in the cerebellum of transgenic mice.Methods: The nine-month-old APP/PS1 male mice,3xTg-AD transgenic mice and wild-type(WT)mice were assigned to three groups: APP/PS1,3xTg-AD and WT.The mice were purchased from Chinese Academy of Medical Sciences Institute of Experimental Medicine and The Jackson Laboratory Company.They were raised in a specific pathogen-free facility under a 12:12 h light/dark cycle with ad libitum access to food and water.In each group,mice were further divided randomly into three groups,5 for immunohistochemistry,6 for Western-blotting,3 for transmission electron microscopy.(1)Immunohistochemistry experiment Each group of 4 mice were perfused,and brain tissue was harvested and frozen in-80 ℃,and then sliced at a thickness of 30 μm,block with donkey serum,and incubation with the primary antibodies at 4 ℃ overnight,Sections were incubated with secondary antibodies for 2 h at room temperature,washed with PBS interval were rinsed 3 times every 5 minutes,and visualised with DAB,Amyloid beta,Synaptophysin,BDNF and Trk-B positive cells were assessed under microscope.(2)Western-blotting experiment The cerebellums of mice were dissected and homogenized in tissue protein extraction reagent,and protein concentration was measured using BCA kit.Sample proteins were separated on 12% SDS-polyacrylamide gels.After electrophoresis,the proteins were transferred onto PVDF membranes and blocked with 5% BSA.The membranes were incubated with a primary antibody overnight at 4 ℃,followed by a secondary antibody for 2 h.Analysis of the optical density of the target strip(Synaptophysin、BDNF and Trk-B)using the gel image processing system.(3)Electron microscopy The cerebellums were sliced into 1mm×1mm×1mm sections.The sections were fixed in the 4% paraformaldehyde and washed with PBS,then post-fixed in 1% osmium tetroxide.Tissues were washed in the buffer,dehydrated and infiltrated with propylene oxide.Tissues were embedded in Epon and polymerized.Ultrathin sections were cut with LKB,counterstained with 2% uranyl acetate and lead citrate,examined with electron microscope.Images of individual synapses were analyzed using NIH Image.Results:(1)Aβ plaques mainly located in both Ⅵ and Ⅶ foliole of cerebellar cortex were found in APP/PS1 and 3xTg-AD transgenic mice.(2)The Western-blotting protein levels in the cerebellum decreased in the APP/PS1 group(P < 0.01)and the 3xTg-AD group(P < 0.01)when compared to the WT group.For Immunochemistry experiment,we observed a punctuate pattern of staining for Synaptophysin on the cerebellar cortex.So,we analyzed the integrated optical density in the GL,PCL and ML.Synaptophysin decreased in all layers in the APP/PS1 group(GL: P < 0.01;PCL: P < 0.01;ML: P < 0.01)when compared to the WT group,and the 3xTg-AD group also experienced a substantial decrease(GL: P < 0.01;PCL: P < 0.01;ML: P < 0.01).The protein levels were insignificantly different between the APP/PS1 group and the 3xTg-AD group.Besides,there were no obvious morphological changes in purkinje cells,when compared with that of WT group.(3)The Western-blotting protein levels for both BDNF and Trk-B reduced significantly in the APP/PS1 group(P < 0.01)and the 3xTg-AD group(P < 0.01)respectively as compared to the WT group.For immunohistochemistry experiment,BDNF substantially decreased only in PCL and GL in the APP/PS1 group(GL: P < 0.01;PCL: P < 0.01)and the 3xTg-AD group(GL: P < 0.01;PCL: P < 0.01).Trk-B decreased in all layers in the APP/PS1 group(GL: P < 0.001;PCL: P < 0.001;ML: P < 0.001)and in the 3xTg-AD group(GL:P < 0.001;PCL:P < 0.001;ML:P < 0.001).Furthermore,the Purkinje cell dendrites became shorter and less branched when compared with that of WT group.(4)Compared with the WT group,both the APP/PS1 group and the 3xTg-AD group had the signs of apoptosis,including dim cells,karyopyknosis,high electronic density of nucleus,and chromatin margination,.The mitochondria also showed swelling,vacuolization,mitochondrial cristae broken,and incomplete structure.(5)We observed that the APP/PS1 group and the 3xTg-AD group had the early signs of tangle formation,PHFs-like structures under TEM forming small clumps or strands were found in neurons and nerve fibers.Moreover,the 3xTg-AD group even produced the microtubule fracture.We also found the abnormal accumulation of synaptic vesicles in the nerve terminals in both APP/PS1 group and 3xTg-AD group.(6)In the APP/PS1 group and the 3xTg-AD group,the synaptic morphology also changed significantly in the cortex of cerebellum: increased width of the synaptic cleft(APP/PS1: P < 0.001;3xTg: P < 0.001),decreased thickness of postsynaptic density(PSD)(APP/PS1: P < 0.001;3xTg: P < 0.001),less length of the active zone(P < 0.05),and fewer synaptic curvature(P < 0.001).Conclusions:(1)The cerebellum of transgenic mice had the typical pathological features of AD.(2)These findings suggested a potential association between the expressions of BDNF/Trk B and the changes of synapse-associated proteins and ultrastructure in AD cerebellum of AD transgenic mice(3)There were no significant differences between the APP/PS1 group and the 3xTg-AD group.