Proteomic Analysis Of Hippocampus Synaptosomes Of Alzheimer's Disease Rats

ObjectiveThis study is to explore the proteins related with Alzheimer's disease (AD) fromthe view of proteomics in both hippocampus synaptosomes and serum of AD rats.The results may help to understand of AD pathogenesis.Methods1.Materials and animal model preparationAdult male SD rats(n=24;age:3 months;weight:250-300g) were used in thisstudy.The rats were assigned to 2 groups randomly:8 for control group and 16 formodel group.AD rats were made by injecting 5μl Aβ_1-40(1μg/μl) into CA1 of the leftlateral hippocampus in stereotaxic coordinates.2.Morris water maze of AD model ratsAll of the 16 AD rats were assessed using the Morris water maze method(including place navigation trail and spatial probe trial) to evaluate spatial learningand memory and 8 rats consistent with denitia standard were chosen as AD model.3.Preparation of serum and hippocampus synaptosomes samples anddetermination of protein concentrationRat serums in both groups were collected and hippocampus synaptosomes wereisolated by sucrose density gradient centrifugation procedures.Both groups of ratserums and hippocampus synaptosomes were mixed together respectively.Albumin,IgG and sodium chloride were removed from serum before concentration.Serumconcentration was measured by Bradford assay.Sodium chloride was removed fromthe synatosomes before concentration.Synatosome protein concentration wascalculated.4.2-DE and MALDI-TOF-MS of serum and hippocampus synaptosomesTwo-dimensional electrophoresis(2-DE) was performed on serum protein of bothgroups,synaptosome protein of control group and model group respectively.Alteredproteins were analyzed by matrix assisted laser desorption/ionization time of flightmass spectrometry (MALDI-TOF-MS). 5.Database query and identification of differential proteinsThe five apparently down-regulated proteins of serum and hippocampussynaptosomes are identified based on peptide mass fingerprint with the MASCOTPeptide Mass Fingerprint software in SWISS-PROT database.Results1.2-DE ofhippocampus synaptosomes and identification of altered proteins12 down-regulated hippocampus synaptosome proteins were found bycomparing 2-DE images of AD rats with normal control rats.3 of the altered 12proteins were chosen and identified by MALDI-TOF-MS.The 3 identified proteinsare:alpha-2-globin chain,peptidyl-prolycis-trans isomerase A(PPIaseA) andcofilin-1 protein.2.2-DE of serum and identification of differential proteins10 down-regulated serum proteins were found by comparing 2-DE images ofAD rats with normal control rats.2 of the altered 10 proteins were chosen andidentified by MALDI-TOF-MS.The 2 identified proteins are:plasma retinol-bindingprotein(RBP) precursor;complement component 4,gene 2(C4b).Conclusions1.In this study,the 3 hippocampus synaptosomes altered proteins chosen from2-DE images and identified by MALDI-TOF-MS of AD rats are different from the 2serum altered proteins chosen from 2-DE images and identified by MALDI-TOF-MSof AD rats.2.The 3 identified altered proteins in AD hippocampus synaptosomes are:alpha-2-globin chain,PPIaseA,cofilin-1 protein.Correlation of the proteins with ADis as follows:(1) The down-regulated Alpha-2-globin chain may decrease cell signaling abilityand leads to cognitive impairment.(2) PPIase A participates in neural cells' caspases cascade reactions thus cancause cell apoptosis,and it also participates in cell signal transmission.The possiblemechanism for down-regulated PPIase A to cause AD is that decreased cell signaling ability may decrease synaptic function and synaptic plasticity,which leads tocognitive impairment.(3) Cofilin-1 participates formation of cerebromedullary tube,migration ofnervous crest as well as apoptosis.The down-regulation that cause apoptosis of nervecells and function damages of axon and dendrite may lead to decrease of synaptictransmission and cognitive impairment in AD.3.The 2 identified altered proteins in AD serum are:RBP precursor,C4b.Correlation of the proteins with AD is as follows:(1) C4b participates in Aβcleaning.Its down-regulation influences cleaning ofAβ,while deposition of Aβleads to decrease of synaptic function and synapticplasticity,thus leads to cognitive impairment.(2) Down-regulated expression of RBP precursor probably causes cell apoptosisand leads to cognitive impairment in AD.In this study,we screened altered proteins related with AD in both serum andhippocampus synaptosomes by proteomics,but mechanisms of these proteins in ADneed to be further studied.