| Objective:Alzheimer's disease?AD?is an intractable neurodegenerative disorder that is more common in the elderly population,being characterized clinically by progressive cognitive decline.Currently,there exist no effective drugs for AD treatment.Amyloid-??A??deposition,a key neuropathological characteristic in AD brain,is the primary causative factor in the pathogenesis of AD.Accumulating evidence indicates that the imbalance between A?production and clearance is the main cause of A?abnormal accumulation and aggregation.Hence,it is an effective strategy for the AD prevention and treatment to promote A?clearance in the brain and peripheral of AD patients.There was preliminary evidence that Trichostatin A?TSA?,a histone deacetylase inhibitor,improved conditioned fear memory and long-term potentiation?LTP?impairment in APP/PS1 mice,indicating the neuroprotective properties of TSA.However,there is a lack of systematic studies on cognitive behavior and pathology,particularly about the molecular mechanism underlying the protective function of TSA.In this study,we first evaluated the ameliorative effects of chronic intraperitoneal injection of TSA on short-term recognition memory and long-term spatial memory in APP/PS1 mice by using open field,novel object recognition and Morris water maze.Then,immunofluorescence staining,Western blot and Simoa techniques were also employed to examine the effects of TSA on A?deposition and A?clearance-associated proteins in the hippocampus of APP/PS1 mice.Furthermore,the molecular mechanism underlying the promotion of TSA in A?clearance was explored by a combination of co-immunoprecipitation,mass spectrometry and cell culture.Methods:1.Recognition memory,spatial learning and reference memory tests in miceAPP/PS1 mice and their wild-type?WT?littermates were used at 8-month age in this study,which were randomly divided into the four groups:WT+Vehicle,WT+TSA,APP/PS1+Vehicle and APP/PS1+TSA.Mice were intraperitoneally injected with TSA?2mg/kg?or equivalent vehicle for 30 days before the behavioral tests.Moreover,the injection continued until the end of behavioral tests.On the 31st day of administration,the following three behavioral tests were performed:1.1 Open field test?OFT?:mice were placed into the center of the open field and allowed to move freely for 10 min.Total distance moved and the percentage of time spent in the center area were recorded as a measure for locomotion activity and exploratory behavior.1.2 Novel object recognition test?NORT?:NORT consisted of two phases,familiarization phase and test phase.During the familiarization,mice were allowed to explore two identical objects for 10 min in the open-field.The test phase began 6 h after familiarization,in which the mice were returned to the same open-field,but with one novel object with different in color and shape.Mice were allowed to freely explore the objects for 10 min and the exploration time to each object were recorded.Novel object recognition memory was calculated as the“novel object recognition index”?NOI?=?exploration time of novel object?/?exploration time of familiar and novel objects?×100%.NOI was used for assessing the short-term recognition memory in mice.1.3 Morris water maze?MWM?test:MWM test was divided into the place navigation test and probe test.The escape latency,the time spent in the target quadrant and the number of crossings over the target platform were respectively measured in the place navigation test and the probe test to evaluate the spatial learning and reference memory ability of mice.Furthermore,the visible task was performed to detect visual ability of mice.The swimming speed was recorded during MWM test to exclude mice with motor deficits.2.The examinations of AD-like pathological characteristics and A?production/clearanceTo avoid the irritating effects of behavioral experiments on mice,the brain tissues of mice were collected two weeks after recovery from behavioral tests.Then,the following experiments were performed:2.1 Western blot:the hippocampal protein extracts were electrophoresed using SDS-PAGE gels and then transferred to PVDF membranes and blocked in blocking buffer.The membranes were incubated with primary antibody,followed by secondary antibody.The immunoreactive bands?A?,APP,CTF,BACE1,IDE,NEP,LRP1,etc.?were visualized using the ECL system,and densitometric analyses were performed with Alpha View SA software.2.2 Immunofluorescence:the fixed brains were cut into 30-?m-thick sections using a cryomicrotome.For detecting the number and distribution of A?plaques and microglia in the hippocampus,the sections were incubated with primary antibody and then with secondary antibody.2.3 Simoa assay:the levels of A?400 and A?422 in the plasma and hippocampus were quantified by the Simoa HD-1 Analyzer.3.Analysis of proteins interacted with A?in hippocampus of APP/PS1 mice3.1 Co-immunoprecipitation?Co-IP?:the hippocampal protein extract were pre-incubated with normal IgG?homology with the primary antibody?and protein A/G agarose to remove nonspecifically bound proteins and then centrifuged.The supernatant was collected and incubated with 6E10 antibody and protein A/G agarose.After centrifugation,the resulting immunoprecipitate were washed with PBS buffer and resuspended with 1×SDS-PAGE loading buffer,and then detected by SDS-PAGE electrophoresis.3.2 LC-MS/MS assay:after SDS-PAGE electrophoresis?3.1 experiment?,the gel was stained with Coomassie brilliant blue for 30 min.Thereafter,the gels containing the target protein were collected for analysis by LC-MS/MS analysis.4.Cytology experiment4.1 Cell culture:murine microglia cell line?BV2?and murine microvascular endothelial cell line?bEnd.3?were the common in vitro cell models in the study of AD.Cells were cultured in DMEM with high glucose supplemented with 10%FBS and 1%penicillin/streptomycin at 37°C in a cell incubator under 95%/5%?v/v?mixture of air and CO2.The cells or supernatant were collected under the indicated experimental conditions to detect the relevant indicators.4.2 Effects of TSA on the albumin expression in BV2 cells and the phagocytosis of A?oligomers by BV2 cells4.2.1 BV2 cells were seeded in 6-well plates and were treated with different concentrations of TSA for different time.After treatment for 24 h,cells were harvested and extracted protein,and then analyzed the effects of TSA on the expression of albumin in BV2 cells by Western blot.4.2.2 In order to observe the effects of mouse albumin on A?aggregation,mouse albumin and human A?1-42-42 were incubated overnight at 37°C and then used for Western blot and transmission electron microscope?TEM?assay.Furthermore,the effects of human A?1-42-42 and mouse albumin on BV2 cells migration were investigated by the transwell system.4.2.3 Effects of TSA on the phagocytosis of A?oligomers by BV2 cells:BV2 cells were seeded in 6-well plates and divided into four groups:Vehicle,A?42,A?42+TSA,A?42+TSA+ITSA-1?a specific inhibitor of TSA?.After 24 h of treatment,changes in the morphology of BV2 cells were observed.Cells were then collected and washed with PBS to remove extracellular soluble A?42.The cellular levels of albumin,Ac-H4 and A?were detected by Western blot.4.3 bEnd.3 cells were seeded in 6-well plates and then grouped and treated as 4.2.3.The effects of TSA on the expression levels of albumin and A?in bEnd.3 cells were detected by Western blot.Results:1.TSA significantly reduced the hyperactivity of APP/PS1 mice in the open field.The results of OFT showed that,compared with vehicle-treated WT mice,the total distance was significantly increased in vehicle-treated APP/PS1 mice?P<0.05?and the percentage of time spend in the center area was slightly increased,whereas TSA-treated APP/PS1 mice significantly decreased the the total distance?P<0.001?and slightly decreased the percentage of time spend in the center area compared with vehicle-treated APP/PS1 mice.2.TSA improved the short-term recognition memory of mice.In NORT,the exploration time of the two identical objects in familiarization phase was no different among the four groups?P>0.05?.In test phase,the percentage of novel object recognition index of vehicle-treated APP/PS1 mice was lower than vehicle-treated WT mice?P<0.01?,while the value was higher in TSA-treated APP/PS1 mice?P<0.05?.In addition,TSA treatment also obviously elevated NOI in WT mice?P<0.001?.3.TSA enhanced the spatial learning and reference memory of APP/PS1 mice.In the place navigation test,vehicle-treated APP/PS1 mice exhibited markedly longer escape latency than vehicle-treated WT mice on day 4?P<0.001?and day 5?P<0.05?,while TSA-treated APP/PS1 mice showed apparent improvement on training day 5?P<0.05?.In the probe test,compared with vehicle-treated WT mice,the percentage of swimming time in the target quadrant?P<0.05?and the number of platform crossings?P<0.01?were significantly decreased in vehicle-treated APP/PS1 mice,while that were clearly increased in APP/PS1 mice with TSA treatment?P<0.05?.During the MWM test,the swimming speed was not significant among all test groups?P>0.05?.4.TSA evidently inhibited the elevation of A?level and microgliosis in the hippocampus of APP/PS1 mice.In the detection of pathology in hippocampus of mice:?1?the results of 6E10immunofluororesence and Thioflavin S staining displayed that the number and area of A?plaques were significantly decreased in TSA-treated APP/PS1 mice?P<0.05?;?2?Western blot with 6E10 antibody and D54D2 antibody exhibited that,compared to vehicle-treated WT mice,A?oligomers in hippocampus were markedly increased in vehicle-treated APP/PS1 mice?6E10:P<0.01;D54D2:P<0.001?,while that was significantly decreased in TSA-treated APP/PS1 mice?6E10:P<0.05;D54D2:P<0.001?;?3?the results of Simoa analysis showed that there was a decrement level of soluble A?40?P<0.01?and A?42?P<0.05?in the hippocampus of TSA-treated APP/PS1 mice;?4?results from Iba1immunofluorescence staining presented that microglia accumulated around A?plaques in hippocampus of APP/PS1 mice.Meanwhile,vehicle-treated APP/PS1 mice displayed more higher in the number and area of microglia in the hippocampus than vehicle-treated WT mice?P<0.001?,whereas that was decreased in APP/PS1 mice with TSA treatment?P<0.01?.5.TSA enhanced the clearance of A?via the ubiquitin-proteasome pathway and peripheral pathway without affecting A?production,extracellular proteolytic degradation and LRP1-mediated transport of A?.In the detection of A?production and metabolism:?1?a noticeable upregulation of flAPP was observed in hippocampus of APP/PS1 mice compared to vehicle-treated WT mice?P<0.001?,although no difference in flAPP levels were observed in TSA-treated APP/PS1 mice compared to vehicle-treated APP/PS1 mice?P>0.05?,indicating that the construction of APP/PS1 mice were successful and TSA had no influence on the expression of APP;?2?there was no significant differences in the levels of CTF and BACE1 between four groups?P>0.05?.However,the ratio of A?/flAPP was higher in the hippocampus of vehicle-treated APP/PS1 mice than that in vehicle-treated WT mice?P<0.01?,while TSA treatment attenuated this ratio?P<0.05?,indicating that the decrease of A?by TSA was not via the inhibition of A?production.These results suggested that the promotion of A?clearance by TSA is a potential way to reduce A?level in the brain;?3?the results of intracellular and extracellular clearance of A?by Western blot showed that the levels of extracellular A?-degrading enzyme,IDE and NEP were comparable among four groups?P>0.05?.And no significant changes of intracellular autophagy-related proteins levels such as p62,Beclin 1 and the ratio of LC3B-?/LC3B-I were noticed in the hippocampus of four groups?P>0.05?.In addition,the monomeric ubiqutin was significantly upregulated in vehicle-treated APP/PS1 mice in comparison to vehicle-treated WT mice?P<0.01?,indicating that the damages of ubiquitin-proteasome activity in APP/PS1 mice resulted in the accumulation of monomeric ubiquitin in the hippocampus.Nevertheless,the monomeric ubiqutin level was significantly downregulated in TSA-treated APP/PS1 mice?P<0.05?,implying that the promotion of ubiquitin-proteasome activity by TSA might lead to the decrease of monomeric ubiquitin as A?degraded.These results demonstrated that TSA might clear A?through the ubiquitin-proteasome pathway,rather than autophagy-lysosome pathway;?4?data of Simoa analysis showed that TSA treatment caused a decrease of A?40?P<0.01?and A?42?P<0.05?levels in the plasma of APP/PS1 mice.Nevertheless,the level of LRP1,a transporter responsible for clearance of A?from the brain,was no obviously different among four groups?P>0.05?.These results revealed that the peripheral clearance of A?by TSA was also a probable way to lower the brain A?level,but might not be related to LRP1transport.6.The interaction between albumin and A?mediated the clearance of A?by TSA.The data in the experiment of protein-protein interaction exhibited that:?1?results from Co-IP,LC-MS/MS and immunofluoresence staning demonstrated that there was an interaction between albumin and A?in the hippocampus of APP/PS1,and albumin levels decreased with decreasing A?levels after TSA treatment;?2?the co-accumulation of albumin and A?in the blood vessel around flax cerebri was found by immunofluoresence staning;?3?the levels of albumin and A?oligomers in bEnd.3 cells were higher in A?42+TSA group than A?422 group,suggesting that TSA treatment up-regulated albumin level and promoted the phagocytosis of A?oligomers in bEnd.3 cells.These results indicated that TSA promoted albumin-A?complexes appeared in the blood vessel of flax cerebri,and the promotion of A?transport and clearance by TSA might via the bEnd.3 cells.7.TSA inhibited A?accumulation and enhanced A?clearance via elevating the expression of albumin in BV2 cells.Results of cell culture experiments showed that:?1?in the experiment to detect the effects of TSA on albumin expression and the effects of albumin on A?accumulation,Western blot analysis revealed that albumin was mainly expressed in BV2 cells compared to N2a cells and HT22 cells,and treatment with TSA increased albumin level in BV2 cells?dose:60 nM,P<0.05;125 nM,P<0.01;250 nM,P<0.01;time:24 h,P<0.05;36 h,P<0.01?,while the action of TSA inducing albumin expression was inhibited with ITSA-1?a specific inhibitor of TSA??250 nM,P<0.05;36 h,P<0.01?.?2?Mouse albumin treatment caused an obvious decrement of A?oligomers?P<0.01?,and evidently induced BV2 cells migration in transwell assay?P<0.01?.?3?A?42-treated BV2 cells were converted to round shape and its pseudopodia were less and shorter?P<0.05?.In A?42+TSA group,the pseudopodia of BV2 cells were more and longer?P<0.01?,while ITSA-1treatment caused a decrease of its pseudopodia?P<0.01?.Similarly,TSA treatment promoted microglia to adopt an ramified shape and increased its pseudopodia in the hippocampus of APP/PS1 mice.?4?Compared to A?42-treated BV2 cells,the levels of albumin?P<0.05?,Ac-H4?P<0.001?and A?oligomers?P<0.01?in BV2 cells were upregulated by TSA,while ITSA-1 markedly suppressed the action of TSA,implying that TSA treatment promoted the phagocytosis of A?oligomers in BV2 cells.Conclusion:In summary,the present study further confirmed the improve effects of TSA on the learning and memory and A?pathological characteristics in the brain of APP/PS1 mice.Notably,the most meaningful discovery in this study was that TSA can enhance the expression of albumin in microglia and cerebral microvascular endothelial cell,and albumin then binds and inhibits A?accumulation.Meanwhile,TSA can promote the phagocytosis and transport of A?by microglia and cerebral microvascular endothelial cell to clear A?. |
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