| Alzheimer's disease(AD) is a neurodegeneration disease of central nervous system characterized by senile plaques(SP) which is formed by extracellularβ-amyloid(Aβ) deposits and neurofibrillary tangles(NFT) which is formed by tau hyper-phosphoralation.Presenilin(PS) is an essential component ofγ-secretase, which cleaves the C-terminal ofβ-amyloid precursor protein(APP) to produce Aβthat accumulates into SP.In addition to the critical role of PS in Aβformation in the brain,PS also has many other functions such as embryo development,neuroblast differentiation,neuron apoptosis,tau hyper-phosphoralation and neural cell adhesion.The forebrain-specific(cerebral cortex and hippocampus) and conditional PSI and PS2 double knock-out(PS cDKO) mice appear inactivation of PS at 3 weeks of age postnatally.They exhibit age-dependent AD-like neurodegenerative phenotypes although the brain Aβwas decreased,such as synaptic loss,neuronal cell death, astrogliosis,cerebral cortex,hippocampus and corpus callosum atrophy,lateral ventricle and third ventricle enlargement and tau hyperphosphorylation.Memory loss and synaptic plasticity impairments start from 2 months of age and become more serious progressively.Thus,derived from genetic manipulation of AD genes that reproduce the central features of AD,namely neurodegeneration and dementia,PS cDKO mice is an ideal model for the study of PS functions in adult mice and AD pathology.In PS cDKO mice,Aβproduction is decreased and the oxidative stress induced by Aβis not found.It is not coincidence with the conventional "Aβhypothesis" of AD pathogenesis.Several important studies have indicated that memory impairment and neurodegeneration in PS cDKO mice are not caused by Aβaccumulation but related to the loss of PS function with the evidence of differential up-regulation of inflammatory markers in the cerebral cortex.As a function executor, protein,especially protein expression and post-translational modification(PTM),is more important to the AD pathogenesis.Unfortunately,up to now,the proteomics of PS cDKO mice is still unknown.In this study,a two-dimensional liquid chromatography-based(2-DL) proteomic approach and other molecular biology methods were used to observe the differentially expressed proteins between PS cDKO and wild-type(WT) mice,and the identification of oxidative modified proteins and oxidative stress status in the brain of PS cDKO mice were also performed.The molecular mechanism of AD-like pathology of PS cDKO mice on the protein level was further investigated.On the other hand,the NR2B transgenic(Tg) mice are the model mice with enhanced learning and memory.However,information about the proteomics of NR2B Tg mice is also scant.It would be thus more helpful to the analysis of protein functions in AD when we compare the expression patterns of brain proteins between these two transgenic models via the proteomics approach.This thesis is composed of four chapters.Chapter 1 will mainly describe the establishment of the methods for purification of brain tissue proteins and the reproducibility and reliability of the 2-DL system PF2D.Chapter 2 describes the proteomic investigation of proteins in brain tissues and the identification of the differentially expressed proteins between PS cDKO and the age-,gender-matched WT mice over several age points,and those between NR2B and age-,gender-matched WT mice,respectively.The expression levels of some proteins were further verified by using the Western blot method.The relationship of these differentially expressed proteins with AD pathogenesis will further be discussed.In chapter 3,we present the results from the studies of oxidative stress of PS cDKO mice at different age point by detecting oxidative productions and anti-oxidative enzymes.Finally,we will give a comprehensive literature review in terms of the most recent advances in physiological and pathological relevance of PS and NR2B in neurodegeneration as well as learning and memory functions,and in proteomic studies of AD and the oxidative stress as a major function in AD pathogenesis.Firstly we optimized the method of brain tissue protein purification to make sure that protein could be well separated by PF2D.The brain tissue is aboundant of lipid as much as about 63%of the dry matter of the brain.High contents of lipid and lipid-associated protein in the brain sample may cause the charge loss on the surface of protein and dramatically reduce the binding efficiency of proteins to the stationary phase of the CF column during the first dimensional fractionation.We initially tried several approaches for removing lipids from the rude protein extracts,such as ultra-centrifugation,acetone precipitation,isopropanol/hexane,Freon and Cleanascite. Freon and Cleanascite methods were proved to be superior over the others in terms of its simplicity and a higher protein recovery rate.Thus,it was used to purify the brain proteins in our protein profiling experiments.The good reproducibility of 1st dimension and 2nd dimension is demonstrated after we loaded two repeat samples. Peak area is in direct ratio with protein concentration.Both of them assure the reliability of the system for analysis the differentially expressed protein.Secondly,we separated proteins of brain tissue by using the PF2D system and searched differentially expressed peaks by the Delt vue software.By comparing with the age- and gender-matched WT mice,26 peaks(6 up-regulation and 20 down-regulation) in 2-month-old PS cDKO mice,51 peaks(18 up-regulation and 33 down-regulation) in 4-month-old PS cDKO mice and 108 peaks(64 up-regulation and 44 down-regulation) in 6-month-old PS cDKO mice were observed.Differentially expressed peaks of 6-month-old PS cDKO mice from the second dimensional chromatography were selected for protein identification using the PMF and MS/MS data from matrix assisted laser desorption/ionization time-of-flight(MALDI-TOF) mass spectrometric analysis.Dihydropyrimidinase-like 2(DRP-2),isocitrate dehydrogenase,pyruvate dehydrogenase,creatine kinase,glial fibrillary acidic protein (GFAP),tubulin were identified and they have relations with neuron development and plasticity,neuron injury and ATP synthesis.To further corroborate the expression profiling data from the present proteomic experiment,GFAP was selected for Western blotting analysis by using specific antibody.The result showed that the mean level of the protein in the 6-month-old PS cDKO mice were elevated compared to WT mice's, similar to that observed in the proteomic data.Totally,72 differentially expressed proteins were mapped with 34 proteins up-regulation and 38 down-regulation in 6-month-old NR2B Tg mice.Twenty six cortical proteins with altered expression level were identified by MALDI-TOF/TOF in the NR2B Tg mice.Among them,14 proteins were up-regulated,while 12 proteins were down-regulated,including glutamine synthetase(GS),guanine nucleotide-releasing factor 1,carbonic anhydrase,clathrin light chain B(Lcb), enolase 1,ATP synthase,cytochrome c,THO complex 4,and M-phase phosphoprotein 1.They play important roles in cell cycle control,neuronal development,RNA transcription as well as energy and metabolic homeostasis.These findings were further corroborated in an independent group of and WT mice by Western blot analysis of two selected proteins:Lcb and GS. From analysis of functions of these identified protein in PS cDKO and NR2B Tg mice,we found many proteins have relations with energy metabolic homeostasis, suggesting an important role of proteins associated energy metabolism in the neuron normal functions,and ATP synthesis has close relations with neuron plasticity and learning and memory.These results provide important information in understanding the AD pathological mechanism of PS cDKO mice.Thirdly,previous investigations into oxidative imbalance implicated in underlying AD pathogenic mechanism have been largely based on the AD-like amyloidosis in transgenic animal models.However,data derived from PS cDKO that presents the loss-of-function of pathogenic mechanism of AD with reduced Aβproduction are currently scant.The present study aimed primarily to investigate oxidative stress status in the PS cDKO mice with different ages by measuring levels of peroxidation products including malondialdehyde(MDA),carbonylated proteins and F2-isoprostanes-Ⅵ(iPF2α-Ⅵ) and anti-oxidative enzymes such as superoxide dismutase(SOD),catalase(CAT) and glutathione peroxidase(GSH-px) in response to the presenilin inactivation in the brain.Our results show that levels of lipid peroxidation product MDA were significantly elevated in the cortex of PS cDKO mice at the age as early as 2-month, and the oxidative stress were even severe and maintained at a higher level during the ages from 4- to 7-month,as compared with those of age- and gender-matched. Accordantly,protein oxidative modification levels,as measured as protein carbonyl content by DNPH assay,in the cerebral cortex samples were also significantly higher by 51%,61%and 41%in 2-,4- and 7-month age groups of PS cDKO mice, respectively,as compared with those of WT controls.The finding was further confirmed in the mice,especially in those at ages of 4 and 7-month,using Oxyblot immunolabeling of carbonylated proteins.Levels of anti-oxidative enzymes also have changes.Cortical CAT activities were significantly decreased by 58%and 46%in the 4- and 7-month mice,respectively,compared to that of matched controls. Contrastingly,cortical SOD activities were markedly higher in 2-month and 7-month PS cDKO mice by 12%and 45%,respectively,compared to WT controls.There was no significant difference in cortical GSH-px activities between WT and PS cDKO mice over all age points.However,in 2-month male PS cDKO mice the activities of this enzyme appeared to be increased significantly compared to age- and gender-matched WT.GFAP expression was increased in the cortices of PS cDKO mice in an age-related manner,similar to the changes shown in the MDA and protein oxidation results above.It revealed that the interaction relationship between oxidative stress and inflammatory may be closely associated with the underlying loss-of-function pathogenesis of AD.We also measured the levels of iPF2α-Ⅵby gas chromatograph-mass spectrometer(GC-MS).iPF2α-Ⅵis one of active isoforms of isoprostanes which were produced by lipid peroxidation of cell memberane.Results show that cerebral cortex iPF2α-Ⅵcontents increased in an age-dependent manner,and have significantly difference in 4-month and 7-month PS cDKO mice compared with WT's.Contents of plasma iPF2α-Ⅵsignificantly increased in 7-month PS cDKO mice,and increased in 4- and 7-month male PS cDKO mice.In the present study,we also conducted a preliminary investigation in identification of oxidative modified proteins in the brain of the AD Tg model by using biocytin hydrazide-streptavidin affinity methodology coupled to microTOFQ proteomics approach.2-oxoglutarate dehydrogenase E1 component,alpha-actin-2, hemoglobin subunit beta-land mitotic checkpoint serine/threonine-protein kinase BUB1 were identified only in PS cDKO mice cortex.In conclusion,we identified many proteins by 2-DL coupled to MALDI-TOF/TOF approach and found changes of protein levels to be mainly associated with energy metabolism and neuron plasticity in both PS cDKO and NR2B Tg mice compared to WT mice.Enhanced oxidative stress in the cortex of PS cDKO mice is an early event responsible for the loss-of-function of PS towards the AD-like pathologies unrelated with the brain amyloidosis.The cause-effect relationship between oxidative stress and inflammatory response in this PS-deficient AD-like model needs to be further elucidated.
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