阿尔茨海默病实验免疫治疗与致病机理的研究
|
摘要
β-淀粉样蛋白(β-amyloid peptide,Aβ)和淀粉样前体蛋白(amyloid precursor protein,APP)与阿尔茨海默病(Alzheimer's disease,AD)的发生、发展密切相关。本论文围绕这两种蛋白开展了相关研究。
为了克服从前免疫治疗AD引起的严重副作用,本论文开展了应用不同长度的Aβ的实验免疫研究。本论文表达纯化了的三种重组蛋白Aβ28H8、Aβ35H8和Aβ42H8,与Aβ42(纯品)一起,作为免疫原免疫小鼠,获得血清抗体。四种免疫原诱导产生抗体的滴度分别为1:6400、1:3200、1:6400、1:6400。研究发现,获得的四种血清抗体均能够识别包括Aβ42单体、寡聚体、原纤维和成熟纤维在内的各种聚集形式的Aβ42,具有抑制Aβ42的聚集、诱导Aβ42聚集体解聚的功效,具有抑制和中和Aβ42细胞毒性的作用。与其他三种免疫原相比,Aβ28H8主要诱导了体液免疫应答,因此可以避免由细胞免疫应答引起的炎症反应。由此证明:Aβ28是一种有效且较Aβ42更为安全的免疫原。
为了进一步探讨AD的致病机理,探索APP分子间相互作用的关键区域,本论文以COS-7为靶细胞,通过免疫共沉淀、细胞伤口愈合、transwell、免疫荧光、细胞粘附等实验证实,APP对靶细胞的迁移有阻滞作用,APP分子间可以发生相互作用,并介导靶细胞间的相互聚集,或介导靶细胞在基质Aβ4-8,Aβ11,Aβ28,Aβ35,Aβ42,C99上的粘附,而Aβ42或由Aβ28诱导产生的抗体能够抑制上述所有的作用。因此可以推断:Aβ42的胞外区,特别是Aβ4-8区域,有可能是导致包含该区域的相关分子间发生相互作用的最关键区域。
上述结果为免疫预防与治疗AD的疫苗设计提供了理论指导,为进一步探索AD的致病机理奠定了理论基础。
Alzheimer’s disease (AD), commonly known as senile dementia, is the fourth-ranking cause of death in the modern world.β-amyloid peptide (Aβ) and amyloid precursor protein (APP) are closely related to the occurrence and development of AD. In vivo, APP is proteolytically cleaved byβ-secretase andγ-secretase sequentially and releases Aβ, the latter can aggregate to form senile plaques, with neurotoxicity, is a major pathogenic factor in AD.
In order to overcome the serious side effects caused by previous immunotherapy of AD, experimental immunotherapy study with different length of Aβwas designed and carried out in this thesis. Effects of serum antibodies induced by different length of Aβon Aβ42 aggregation and cytotoxicity were analyzed. Three recombinant expression vectors pET-41b(+)-Aβ28, pET-41b(+)-Aβ35, pET-41b(+)-Aβ42 were first constructed, and then three recombinant proteins Aβ28H8, Aβ35H8, Aβ42H8 were expressed and purified. Aβ28H8, Aβ35H8, Aβ42H8 and Aβ42 were used as immunogens to immune BALB/c mice respectively. The antisera were collected from the mice that were received a total of seven injections. The titers of serum antibodies induced by Aβ28H8, Aβ42H8 and Aβ42 against Aβ42 oligomers were 1:6400, whereas the one by Aβ35H8 was 1:3200. The levels of serum antibody concentration were consistent with the levels of the corresponding antibody titers. The analysis of isotypes of serum antibodies showed that Aβ28 mainly induced humoral immune response, while Aβ42 and Aβ35 mainly induced cellular immune response. It was indicated that immunotherapy for AD with Aβ28 as immunogen could reduce the inflammation caused by cellular immune response. Reactive specificities of serum antibodies with different aggregation forms of Aβ42 were determined by indirect ELISA. The results showed that all the serum antibodies could identify and react with various forms of Aβ42 including Aβ42 monomers, Aβ42 oligomers, Aβ42 protofibrils and Aβ42 mature fibers. This indicated that these antibodies had wide reactogenicities with all existing forms of Aβ42. The inhibitory or induction effects of serum antibodies on Aβ42 aggregation or on the disassembly of Aβ42 fibrils in vitro were observed by electron microscopy. The results showed that serum antibodies could not only prevent Aβ42 aggregation, but also disassemble the preformed Aβ42 oligomers, Aβ42 protofibrils or Aβ42 fibrils and ultimately induced Aβ42 aggregates into much smaller globular units in vitro. There were significant abilities of serum antibodies to inhibit and neutralize Aβ42 cytotoxicity in PC12 cells. These results indicated that Aβ28 was an effective, more importantly, a safer immunogen than Aβ42.
In order to explore the pathogenic mechanism of AD and to reveal the effects of the interactions between APP on occurrence and development of AD, the key sites in APP for the interactions APP were investigated. COS-7 cells were first transfected with different tagged APP, and then we confirmed that APP can interact with each other by using immunofluorescence, co-IP, wound healing assay, transwell assay, and cell aggregation assay etc. These interactions between APPs could inhibit the migration of target cells, promote the adhesion of target cell, and mediate the adhesion between target cells or target cells and different Aβmatrix including Aβ4-8, Aβ11, Aβ28, Aβ35, Aβ42, C99. These effects mediated by interactions between APP could be inhibited by Aβ42 or the serum antibodies induced by Aβ28. These results suggested that the extracellular region of Aβ42, especially Aβ4-8, might play an important role in the interactions between APPs. It was supposed that the adhesion between neurons and extracellular Aβ42 aggregates was mediated by the interactions between molecules including Aβ4-8 region, leading to neurons dysfunction, and eventually AD.
These conclusions obtained in this thesis are significant for providing the theoretical guidance for vaccine design involved in the immunization of prevention and treatment of AD, and for laying the theoretical foundation to reveal the pathogenic mechanism of AD.
为了克服从前免疫治疗AD引起的严重副作用,本论文开展了应用不同长度的Aβ的实验免疫研究。本论文表达纯化了的三种重组蛋白Aβ28H8、Aβ35H8和Aβ42H8,与Aβ42(纯品)一起,作为免疫原免疫小鼠,获得血清抗体。四种免疫原诱导产生抗体的滴度分别为1:6400、1:3200、1:6400、1:6400。研究发现,获得的四种血清抗体均能够识别包括Aβ42单体、寡聚体、原纤维和成熟纤维在内的各种聚集形式的Aβ42,具有抑制Aβ42的聚集、诱导Aβ42聚集体解聚的功效,具有抑制和中和Aβ42细胞毒性的作用。与其他三种免疫原相比,Aβ28H8主要诱导了体液免疫应答,因此可以避免由细胞免疫应答引起的炎症反应。由此证明:Aβ28是一种有效且较Aβ42更为安全的免疫原。
为了进一步探讨AD的致病机理,探索APP分子间相互作用的关键区域,本论文以COS-7为靶细胞,通过免疫共沉淀、细胞伤口愈合、transwell、免疫荧光、细胞粘附等实验证实,APP对靶细胞的迁移有阻滞作用,APP分子间可以发生相互作用,并介导靶细胞间的相互聚集,或介导靶细胞在基质Aβ4-8,Aβ11,Aβ28,Aβ35,Aβ42,C99上的粘附,而Aβ42或由Aβ28诱导产生的抗体能够抑制上述所有的作用。因此可以推断:Aβ42的胞外区,特别是Aβ4-8区域,有可能是导致包含该区域的相关分子间发生相互作用的最关键区域。
上述结果为免疫预防与治疗AD的疫苗设计提供了理论指导,为进一步探索AD的致病机理奠定了理论基础。
Alzheimer’s disease (AD), commonly known as senile dementia, is the fourth-ranking cause of death in the modern world.β-amyloid peptide (Aβ) and amyloid precursor protein (APP) are closely related to the occurrence and development of AD. In vivo, APP is proteolytically cleaved byβ-secretase andγ-secretase sequentially and releases Aβ, the latter can aggregate to form senile plaques, with neurotoxicity, is a major pathogenic factor in AD.
In order to overcome the serious side effects caused by previous immunotherapy of AD, experimental immunotherapy study with different length of Aβwas designed and carried out in this thesis. Effects of serum antibodies induced by different length of Aβon Aβ42 aggregation and cytotoxicity were analyzed. Three recombinant expression vectors pET-41b(+)-Aβ28, pET-41b(+)-Aβ35, pET-41b(+)-Aβ42 were first constructed, and then three recombinant proteins Aβ28H8, Aβ35H8, Aβ42H8 were expressed and purified. Aβ28H8, Aβ35H8, Aβ42H8 and Aβ42 were used as immunogens to immune BALB/c mice respectively. The antisera were collected from the mice that were received a total of seven injections. The titers of serum antibodies induced by Aβ28H8, Aβ42H8 and Aβ42 against Aβ42 oligomers were 1:6400, whereas the one by Aβ35H8 was 1:3200. The levels of serum antibody concentration were consistent with the levels of the corresponding antibody titers. The analysis of isotypes of serum antibodies showed that Aβ28 mainly induced humoral immune response, while Aβ42 and Aβ35 mainly induced cellular immune response. It was indicated that immunotherapy for AD with Aβ28 as immunogen could reduce the inflammation caused by cellular immune response. Reactive specificities of serum antibodies with different aggregation forms of Aβ42 were determined by indirect ELISA. The results showed that all the serum antibodies could identify and react with various forms of Aβ42 including Aβ42 monomers, Aβ42 oligomers, Aβ42 protofibrils and Aβ42 mature fibers. This indicated that these antibodies had wide reactogenicities with all existing forms of Aβ42. The inhibitory or induction effects of serum antibodies on Aβ42 aggregation or on the disassembly of Aβ42 fibrils in vitro were observed by electron microscopy. The results showed that serum antibodies could not only prevent Aβ42 aggregation, but also disassemble the preformed Aβ42 oligomers, Aβ42 protofibrils or Aβ42 fibrils and ultimately induced Aβ42 aggregates into much smaller globular units in vitro. There were significant abilities of serum antibodies to inhibit and neutralize Aβ42 cytotoxicity in PC12 cells. These results indicated that Aβ28 was an effective, more importantly, a safer immunogen than Aβ42.
In order to explore the pathogenic mechanism of AD and to reveal the effects of the interactions between APP on occurrence and development of AD, the key sites in APP for the interactions APP were investigated. COS-7 cells were first transfected with different tagged APP, and then we confirmed that APP can interact with each other by using immunofluorescence, co-IP, wound healing assay, transwell assay, and cell aggregation assay etc. These interactions between APPs could inhibit the migration of target cells, promote the adhesion of target cell, and mediate the adhesion between target cells or target cells and different Aβmatrix including Aβ4-8, Aβ11, Aβ28, Aβ35, Aβ42, C99. These effects mediated by interactions between APP could be inhibited by Aβ42 or the serum antibodies induced by Aβ28. These results suggested that the extracellular region of Aβ42, especially Aβ4-8, might play an important role in the interactions between APPs. It was supposed that the adhesion between neurons and extracellular Aβ42 aggregates was mediated by the interactions between molecules including Aβ4-8 region, leading to neurons dysfunction, and eventually AD.
These conclusions obtained in this thesis are significant for providing the theoretical guidance for vaccine design involved in the immunization of prevention and treatment of AD, and for laying the theoretical foundation to reveal the pathogenic mechanism of AD.
引文
[1] Alzheimer A. Ueber eine eigenartige Erkrankung derHimrinde. Cen Nervenheil Psychiat, 1907, 30: 177.
[2] Hardy J, Selkoe DJ. The amyloid hypothesis of Alzheimer’s disease: progress and problems on the road to therapeutics. Science, 2002, 297: 353–356.
[3] RibéEM, Pérez M, Puig B, et al. Accelerated amyloid deposition, neurofibrillary degeneration and neuronal loss in double mutant APP/tau transgenic mice. Neurobiol Dis, 2005, 20(3): 814-822.
[4] Octave JN. Alzheimer disease: cellular and molecular aspects. Bull Mem Acad R Med Belg. 2005. 160(10-12): 445-449.
[5] Aguzzi A, Haass C. Games played by rogue proteins in prion disorders and Alzheimer’s disease. Science, 2003, 302(5646): 814–818.
[6] Ren Z, Schenk D, Basi GS, et al. Amyloid beta-protein precursor juxtamembrane domain regulates specificity of gamma-secretase-dependent cleavages. J Biol Chem, 2007, 282(48): 35350-35360.
[7] Findeis MA. The role of amyloid beta peptide 42 in Alzheimer's disease. Pharmacology & Therapeutics. 2007, 116(2): 266-286.
[8] Multhaup G. Amyloid precursor protein and BACE function as oligomers. Neurodegener Dis, 2006, 3(4-5): 270-274.
[9] Li M, Chen L, Lee DH, et al. The role of intracellular amyloid beta in Alzheimer's disease. Progress in Neurobiology, 2007, 83(3): 131-139.
[10] Lambert MP, Viola KL, Chromy BA. Vaccination with soluble Abeta oligomers generates toxicity-neutralizing antibodies. J. Neurochem, 2001, 79: 595–605.
[11] Walsh DM, lyubin I, Fadeeva JV, et al. Secreted oligomers of amyloid beta protein potently inhibit hippocampal long-term potentiation in vivo. Nature, 2002, 416: 535–539.
[12] Levine H. Alzheimer's beta-peptide oligomer formation at physiologic concentrations. Anal Biochem, 2004, 335(1): 81-90.
[13] Haass C, Selkoe DJ. Soluble protein oligomers in neurodegeneration: lessons from the Alzheimer's amyloid beta-peptide. Nat Rev Mol Cell Biol, 2007, 8(2): 101-112.
[14] William L., Grant A., Caleb E. Targeting small Aβoligomer’s: The solution to an Alzheimer’s conundrum? Neurosciences, 2001, 24(4): 219-224.
[15] Cappai R, Barnham KJ. Molecular determinants of Alzheimer's disease Aβpeptide neurotoxicity. Future Neurol, 2007, 2: 397-409.
[16] Walsh DM, Klyubin I, Fadeeva JV, et al. Amyloid beta oligomers: their production, toxicity and therapeutic inhibition. Biochem. Soc.Trans, 2002, 30(4): 552-557.
[17] Pereira C, Ferreiro E, Cardoso SM, et al. Cell degeneration induced by amyloid-beta peptides:implications for Alzheimer’s disease. J Mol Neurosci, 2004, 23(1-2): 97-104.
[18] Schliebs R. Basal forebrain cholinergic dysfunction in Alzheimer's disease-interrelationship with beta-amyloid, inflammation and neurotrophin signaling. Neurochem Res, 2005, 30(6-7): 895-908.
[19] Le WD, Xie WJ, Kong R, et al.β-amyloid-induced neurotoxicity of a hybrid septal cell line associated with increased tau phosphorylation and expression ofβ-amyloid precursor protein. J. Neurochem.1997, (69): 978–985.
[20] Engelborghs S, Vreese K, Casteele T, et al. Diagnostic performance of a CSF-biomarker panel in autopsy-confirmed dementia. Neurobiol Aging, 2008, 29: 1143-1159.
[21] Schoonenboom NS, Flier W M, Blankenstein MA, et al. CSF and MRI markers independently contribute to the diagnosis of Alzheimer’s disease. Neurobiol Aging, 2008, 29: 669-675.
[22] Schenk D, Hagen M, Seubert P. Current progress in beta-amyloid immuno therapy. Curr Opin Immunol, 2004(5): 599-606.
[23] Schenk D, Barbour R, Dunn W, et al. Immunization with amyloid-βattenuates Alzheimer -disease-like pathology in the PDAPP mouse. Nature, 1999(400): 173-177.
[24] Hauben E, Nevo U, Yoles E, et al. AutoimmuneT cells as potential neuroprotecitve therapy for spinal cord injury. Lancet, 2000, 355(9200): 286-287.
[25] Becker KJ, McCarron RM, Ruetzler C, et al. Immunologic tolerance to myelin basic protein decreases stroke size after transient focal cerebral ischemia. Proc NatA cad Sci USA, 1997, 94(20): 10873-10878.
[26] Janus C, Pearson J, McLaurin J, et al. Aβpeptide immunization reduces behavioural impairment and plaques in a model of Alzheimer’s disease. Nature, 2000(408): 979-982.
[27] Hock C, Konietzko U, Streffer JR, et al. Antibodies against beta-amyloid slowcognitive decline in Alzheimer’s disease. Neuron, 2003(38): 547-554.
[28] Morgan D, Diamond DM, Gottschall PE, et al. Aβpeptide vaccination prevents memory loss in an animal model of Alzheimer’s disease. Nature, 2000(408): 982-985.
[29] Hock C, Konietzko U, Streffer JR, et al. Antibodies against beta-amyloid slow cognitive decline in Alzheimer’s disease. Neuron, 2003, 38(4): 547-554.
[30] Nicoll JA, Wilkinson D, Holmes C, et al. Neuropathology of human Alzheimer disease after immunization with amyloid-beta peptide: a case report. Nat Med, 2003, 9(4): 448-452.
[31] Weiner H, Selkoe DJ. Inflammation and therapeutic vaccination in CNS diseases. Nature, 2002, 4(20): 879-884.
[32] McLaurin J, Cecal R, Kierstead ME, et al. Therapeutically effective antibodies against amyloid-beta peptide target amyloid-beta residues 4-10 and inhibit cytotoxicity and fibrillogenesis. Nature Medicine, 2002, 8(11): 1263-1269.
[33] Monsonego A, Maron R, Zota V, et al. Immune hyporesponsiveness to amyloid beta-peptide in amyloid precursor protein transgenic mice: implications for the pathogenesis and treatment of Alzheimer's disease. Proc Natl Acad Sci U S A, 2001, 98(18): 10273-10278.
[34] Agadjanyan MG, Ghochikyan A, Petrushina I, et al. Prototype Alzheimer's disease vaccine using the immunodominant B cell epitope from beta-amyloid and promiscuous T cell epitope pan HLA DR-binding peptide. J Immunol, 2005, 174(3): 1580-1586.
[35] Ghochikyan A, Mkrtichyan M, Petrushina I, et al. Prototype Alzheimer's disease epitope vaccine induced strong Th2-type anti-Abeta antibody response with Alum to Quil A adjuvant switch. Vaccine, 2006, 24(13): 2275-2282.
[36] Movsesyan N, Ghochikyan A, Mkrtichyan M, et al. Reducing AD-like pathology in 3xTg-AD mouse model by DNA epitope vaccine - a novel immunotherapeutic strategy. PLoS ONE, 2008, 3(5): 21-24.
[37] Arbel M, Solomon B. Immunotherapy for Alzheimer’s disease: attacking amyloid-beta from the inside. TRENDS in Immunology, 2007, 28(12): 511-513.
[38] Bard F, Cannon C, Barbour R, et al. Peripherally administered antibodies against amyloid beta-peptide enter the central nervous system and reduce pathology in a mouse model of Alzheimer disease. Nat Med, 2000, 6(8): 916-919.
[39] Bard F, barbour R, Cannon C, et al. Epitope and isotype specification ofantibodies toβ-amyloid people for protection against Alzheimer’s disease-like neuropathology. PANS, 2003, 100(4): 2023-2028.
[40] Bard F, Barbour R, Cannon C, et al. Epitope and isotype specificities ofantibodies to beta-amyloid peptide for protection against Alzheimer’s disease like neuropathology. Proc Natl Acad Sci USA, 2003, 100(4): 2023-2028.
[41] DeMattos RB, Bales KR, Cummins DJ, et al. Peripheral anti-A beta anti-body alters CNS and plasma A beta clearance and decreases brain A betaburden in a mouse model of Alzheimer’s disease. Proc Natl Acad Sci USA, 2001, 98(15): 8850-8855.
[42] Wilhelmina H, van den Hurk, Bloemen M, et al. Expression of the gene encoding theβ-amyloid precursor protein APP in Xenopus laevis. Molecular Brain Research, 2001, 97(1): 13-20.
[43] Annaert W, Strooper BA. Cell biological perspective on Alzheimer’s disease. Annu Rev Cell Dev Biol, 2002, 18: 25–51.
[44] Mattson MP. Cellular actions of beta-amyloid precursor protein and its soluble and fibrillogenic derivatives. Physiol Rev, 1997, 77(4): 1081–1132.
[45] Jin LW, Ninomiya H, Roch JM, et al. Peptides containing the RERMS sequence of amyloid beta/A4 protein precursor bind cell surface and promote neurite extension. J Neurosci, 1994, 14(9): 5461–5470.
[46] Reinhard C, Hébert SS, De Strooper B. The amyloid-βprecursor protein: integrating structure with biological function. The EMBO J, 2005, 24(23): 3996–4006.
[47] Multhaup G., Mechler H., Masters CL. Characterization of the high affinity heparin binding site of the Alzheimer’s disease beta A4 amyloid precursor protein (APP) and its enhancement by zinc(II). J Mol Recognit, 1995, 8(4): 247–257.
[48] Scheuermann S, Hambsch B, Hesse L, et al. Homodimerization of amyloid precursor protein and its implication in the amyloidogenic pathway of Alzheimer’s disease. J Biol Chem, 2001, 276(36): 33923–33929.
[49] Wang Y, Ha Y. The X-Ray Structure of an Antiparallel Dimer of the Human Amyloid Precursor Protein E2 Domain. Molecular Cell, 2004, 15(3): 343-353.
[50] Soba P, Eggert S, Wagner K, et al. Homo- and heterodimerization of APP family members promotes intercellular adhesion. The EMBO Journal, 2005, 24(20): 3624-3634.
[51] Kuan YH, Gruebl T, Soba P, et al. PAT1a modulates intracellular transport andprocessing of amyloid precursor protein (APP), APLP1, and APLP2. J Biol Chem, 2006, 281(52): 40114-40123.
[52] Neumann S, Sch?bel S, J?ger S, et al. Amyloid precursor-like protein 1 influences endocytosis and proteolytic processing of the amyloid precursor protein. J Biol Chem, 2006, 281(11): 7583-7594.
[53] Heber S, Herms J, Gajic V, et al. Mice with combined gene knock-outs reveal essential and partially redundant functions of amyloid precursor protein family members. J Neurosci, 2000, 20(21): 7951–7963.
[54] Islam R, Kristiansen LV, Romani S, et al. Activation of EGF Receptor Kinase by L1-mediated Homophilic Cell Interactions. Mol Biol Cell, 2004, 15(4): 2003–2012.
[55] Kaden D, Munter LM, Voigt P, et al. Dissection of the individual domains of APP family members involved in homo- and heterotypic interactions. Alzheimer's and Dementia, 2008, 4(4): T242-243.
[56] Kaden D, Munter LM, Voigt P, et al. Homo-and heterophilic interactions of APP-family-proteins. Alzheimer's and Dementia, 2006, 2(3): S248-249.
[57] Nelson TJ, Alkon DL. Protection against beta-amyloid-induced apoptosis by peptides interacting with beta-amyloid. J Biol Chem, 2007, 282(10): 31238-31249.
[58] Shaked GM, Kummer MP, Lu DC, et al. Aβinduces cell death by direct interaction with its cognate extracellular domain on APP (APP 597–624). The FASEB Journal, 2006, 20(8): 1254-1256.
[59] D'Errico G, Vitiello G, Ortona O, et al. Interaction between Alzheimer's Aβ(25–35) peptide and phospholipid bilayers: The role of cholesterol. Biochimica et Biophysica Acta, 2008, 1778(12): 2710-2716.
[60] Klein WL, Stine WB Jr, Teplow DB. Small assemblies of unmodified amyloid beta-protein are the proximate neurotoxin in Alzheimer’s disease. Neurobiol Aging, 2004, 25(5): 569–580.
[61] Hung LW, Ciccotosto GD, Giannakis E, et al. Amyloid-beta peptide (Abeta) neurotoxicity is modulated by the rate of peptide aggregation: Abeta dimers and trimers correlate with neurotoxicity. J Neurosci, 2008, 28(46): 11950-11958.
[62] Poling A, Morgan-Paisley K, Panos JJ, et al. Oligomers of the amyloid-beta protein disrupt working memory: confirmation with two behavioral procedures. Behav Brain Res, 2008, 193(2): 230-234.
[63] Van Nostrand WE, Melchor JP, Keane DM, et al. Localization of a fibrillar amyloid beta-protein binding domain on its precursor. J Biol Chem, 2002, 277(39): 36392–36398.
[64] Lu DC, Rabizadeh S, Chandra S, et al. A second cytotoxic proteolytic peptide derived from amyloid beta-protein precursor. Nature Med, 2000, 6(4): 397–404.
[65] Yankner BA, Duffy LK, Kirschner DA. Neurotrophic and neurotoxic effects of amyloid beta protein: reversal by tachykinin neuropeptides. Science, 1990, 250(4978): 279-282.
[66] da Cruz e Silva EF, da Cruz e Silva OA. Protein phosphorylation and APP metabolism, Neurochemical Research, 2003, 28(10): 1553-1561.
[67] Nakaya T and Suzuki T. Role of APP phosphrylation in FE65-dependent gene transactivation mediated by AICD. Genes to Cells, 2006(11): 633-645.
[68] Iijima K, Ando K, Takeda S, et al. Neuron-specific phosphorylation of Alzheimer’sβ-amyloid precursor protein by cyclin-dependent kinase 5. J. Neurochem, 2000, 75: 1085–1091.
[69] Standen CL, Brownless J, Grierson AJ, et al. Phosphorylation of thr(668) in the cytoplasmic domain of the Alzheimer’s disease amyloid precursor protein by stress-activated protein kinase 1b (Jun N-terminal kinase-3). J Neurochem, 2001, 76(1): 316–320.
[70] Inomata H, Nakamura Y, Hayakawa A, et al. Scaffold protein JIP-1b enhances amyloid precursor protein phosphorylation by JNK and its association with kinesin light chain 1. J Biol Chem, 2003, 278 (25): 22946–22955.
[71] Scheinfeld MH, Ghersi E, Davies P et al. Amyloid beta protein precursor is phosphorylated by JNK-1 independent of, yet facilitated by, JNK-interacting protein (JIP)-1. J Biol Chem, 2003, 278(43): 42058-42063.
[72] Taru H, Suzuki T. Facilitation of stress-induced phosphorylation of beta-amyloid precursor protein family members by X11-like/Mint2 protein. J Biol Chem, 2004, 279(20): 21628–21636.
[73] Ando K, Iijima K, Elliott JI, et al. Phosphorylation-dependent regulation of the interaction of amyloid precursor protein with Fe65 affects the production ofβ-amyloid. J Biol Chem, 2001 276: 40353–40361.
[74] Lee MS, Kao SC, Lemere CA, er al. APP processing is regulated by cytoplasmic phosphorylation. J Cell Biol, 2003, 163(1): 83-95.
[75] Ando K, Oishi M, Takeda S, et al. Role of Phosphorylation of Alzheimer'sAmyloid Precursor Protein during Neuronal Differentiation. The Journal of Neuroscience, 1999, 19(11): 4421-4427.
[76] Liu F, Su Y, Li B, et al. Regulation of amyloid precursor protein (APP) phosphorylation and processing by p35/Cdk5 and p25/Cdk5. FEBS Lett, 2003, 547 (1-3): 193–196.
[77] Lee MS, Kao SC, Lemere CA, et al. APP processing is regulated by cytoplasmic phosphorylation. J Cell Biol, 2003, 163: 83–95.
[78] Plant LD, Boyle JP, Smith IF, et al. The production of amyloid beta peptide is a critical requirement for the viability of central neurons. J Neurosci, 2003, 23(13): 5531-5535.
[79] von Koch CS, Zheng H, Chen H, et al. Generation of APLP2 KO mice and early postnatal lethality in APLP2/APP double KO mice. Neurobiol Aging, 1997, 18(6): 661-669.
[80] Dawson GR, Seabrook GR, Zheng H, et al. Age-related cognitive deficits, impaired long-term potentiation and reduction in synaptic marker density in mice lacking the beta-amyloid precursor protein. Neuroscience, 1999, 90(1): 1-13.
[81] Pierschbacher MD, Ruoslahti E. Cell attachment activity of fibronectin can be duplicated by small synthetic fragments of the molecule. Nature, 1984, 309(5963): 30-33.
[82] Yamada KM, Kennedy DW. Dualistic nature of adhesive protein function: fibronectin and its biologically active peptide fragments can autoinhibit fibronectin function. J Cell Biol, 1984, 99:29-36.
[83] Hayman EG, Pierschbacher MD, Ruoslahti E. Detachment of cells from culture substrate by soluble fibronectin peptides. J Cell Biol, 1985, 100(6): 1948-1954.
[2] Hardy J, Selkoe DJ. The amyloid hypothesis of Alzheimer’s disease: progress and problems on the road to therapeutics. Science, 2002, 297: 353–356.
[3] RibéEM, Pérez M, Puig B, et al. Accelerated amyloid deposition, neurofibrillary degeneration and neuronal loss in double mutant APP/tau transgenic mice. Neurobiol Dis, 2005, 20(3): 814-822.
[4] Octave JN. Alzheimer disease: cellular and molecular aspects. Bull Mem Acad R Med Belg. 2005. 160(10-12): 445-449.
[5] Aguzzi A, Haass C. Games played by rogue proteins in prion disorders and Alzheimer’s disease. Science, 2003, 302(5646): 814–818.
[6] Ren Z, Schenk D, Basi GS, et al. Amyloid beta-protein precursor juxtamembrane domain regulates specificity of gamma-secretase-dependent cleavages. J Biol Chem, 2007, 282(48): 35350-35360.
[7] Findeis MA. The role of amyloid beta peptide 42 in Alzheimer's disease. Pharmacology & Therapeutics. 2007, 116(2): 266-286.
[8] Multhaup G. Amyloid precursor protein and BACE function as oligomers. Neurodegener Dis, 2006, 3(4-5): 270-274.
[9] Li M, Chen L, Lee DH, et al. The role of intracellular amyloid beta in Alzheimer's disease. Progress in Neurobiology, 2007, 83(3): 131-139.
[10] Lambert MP, Viola KL, Chromy BA. Vaccination with soluble Abeta oligomers generates toxicity-neutralizing antibodies. J. Neurochem, 2001, 79: 595–605.
[11] Walsh DM, lyubin I, Fadeeva JV, et al. Secreted oligomers of amyloid beta protein potently inhibit hippocampal long-term potentiation in vivo. Nature, 2002, 416: 535–539.
[12] Levine H. Alzheimer's beta-peptide oligomer formation at physiologic concentrations. Anal Biochem, 2004, 335(1): 81-90.
[13] Haass C, Selkoe DJ. Soluble protein oligomers in neurodegeneration: lessons from the Alzheimer's amyloid beta-peptide. Nat Rev Mol Cell Biol, 2007, 8(2): 101-112.
[14] William L., Grant A., Caleb E. Targeting small Aβoligomer’s: The solution to an Alzheimer’s conundrum? Neurosciences, 2001, 24(4): 219-224.
[15] Cappai R, Barnham KJ. Molecular determinants of Alzheimer's disease Aβpeptide neurotoxicity. Future Neurol, 2007, 2: 397-409.
[16] Walsh DM, Klyubin I, Fadeeva JV, et al. Amyloid beta oligomers: their production, toxicity and therapeutic inhibition. Biochem. Soc.Trans, 2002, 30(4): 552-557.
[17] Pereira C, Ferreiro E, Cardoso SM, et al. Cell degeneration induced by amyloid-beta peptides:implications for Alzheimer’s disease. J Mol Neurosci, 2004, 23(1-2): 97-104.
[18] Schliebs R. Basal forebrain cholinergic dysfunction in Alzheimer's disease-interrelationship with beta-amyloid, inflammation and neurotrophin signaling. Neurochem Res, 2005, 30(6-7): 895-908.
[19] Le WD, Xie WJ, Kong R, et al.β-amyloid-induced neurotoxicity of a hybrid septal cell line associated with increased tau phosphorylation and expression ofβ-amyloid precursor protein. J. Neurochem.1997, (69): 978–985.
[20] Engelborghs S, Vreese K, Casteele T, et al. Diagnostic performance of a CSF-biomarker panel in autopsy-confirmed dementia. Neurobiol Aging, 2008, 29: 1143-1159.
[21] Schoonenboom NS, Flier W M, Blankenstein MA, et al. CSF and MRI markers independently contribute to the diagnosis of Alzheimer’s disease. Neurobiol Aging, 2008, 29: 669-675.
[22] Schenk D, Hagen M, Seubert P. Current progress in beta-amyloid immuno therapy. Curr Opin Immunol, 2004(5): 599-606.
[23] Schenk D, Barbour R, Dunn W, et al. Immunization with amyloid-βattenuates Alzheimer -disease-like pathology in the PDAPP mouse. Nature, 1999(400): 173-177.
[24] Hauben E, Nevo U, Yoles E, et al. AutoimmuneT cells as potential neuroprotecitve therapy for spinal cord injury. Lancet, 2000, 355(9200): 286-287.
[25] Becker KJ, McCarron RM, Ruetzler C, et al. Immunologic tolerance to myelin basic protein decreases stroke size after transient focal cerebral ischemia. Proc NatA cad Sci USA, 1997, 94(20): 10873-10878.
[26] Janus C, Pearson J, McLaurin J, et al. Aβpeptide immunization reduces behavioural impairment and plaques in a model of Alzheimer’s disease. Nature, 2000(408): 979-982.
[27] Hock C, Konietzko U, Streffer JR, et al. Antibodies against beta-amyloid slowcognitive decline in Alzheimer’s disease. Neuron, 2003(38): 547-554.
[28] Morgan D, Diamond DM, Gottschall PE, et al. Aβpeptide vaccination prevents memory loss in an animal model of Alzheimer’s disease. Nature, 2000(408): 982-985.
[29] Hock C, Konietzko U, Streffer JR, et al. Antibodies against beta-amyloid slow cognitive decline in Alzheimer’s disease. Neuron, 2003, 38(4): 547-554.
[30] Nicoll JA, Wilkinson D, Holmes C, et al. Neuropathology of human Alzheimer disease after immunization with amyloid-beta peptide: a case report. Nat Med, 2003, 9(4): 448-452.
[31] Weiner H, Selkoe DJ. Inflammation and therapeutic vaccination in CNS diseases. Nature, 2002, 4(20): 879-884.
[32] McLaurin J, Cecal R, Kierstead ME, et al. Therapeutically effective antibodies against amyloid-beta peptide target amyloid-beta residues 4-10 and inhibit cytotoxicity and fibrillogenesis. Nature Medicine, 2002, 8(11): 1263-1269.
[33] Monsonego A, Maron R, Zota V, et al. Immune hyporesponsiveness to amyloid beta-peptide in amyloid precursor protein transgenic mice: implications for the pathogenesis and treatment of Alzheimer's disease. Proc Natl Acad Sci U S A, 2001, 98(18): 10273-10278.
[34] Agadjanyan MG, Ghochikyan A, Petrushina I, et al. Prototype Alzheimer's disease vaccine using the immunodominant B cell epitope from beta-amyloid and promiscuous T cell epitope pan HLA DR-binding peptide. J Immunol, 2005, 174(3): 1580-1586.
[35] Ghochikyan A, Mkrtichyan M, Petrushina I, et al. Prototype Alzheimer's disease epitope vaccine induced strong Th2-type anti-Abeta antibody response with Alum to Quil A adjuvant switch. Vaccine, 2006, 24(13): 2275-2282.
[36] Movsesyan N, Ghochikyan A, Mkrtichyan M, et al. Reducing AD-like pathology in 3xTg-AD mouse model by DNA epitope vaccine - a novel immunotherapeutic strategy. PLoS ONE, 2008, 3(5): 21-24.
[37] Arbel M, Solomon B. Immunotherapy for Alzheimer’s disease: attacking amyloid-beta from the inside. TRENDS in Immunology, 2007, 28(12): 511-513.
[38] Bard F, Cannon C, Barbour R, et al. Peripherally administered antibodies against amyloid beta-peptide enter the central nervous system and reduce pathology in a mouse model of Alzheimer disease. Nat Med, 2000, 6(8): 916-919.
[39] Bard F, barbour R, Cannon C, et al. Epitope and isotype specification ofantibodies toβ-amyloid people for protection against Alzheimer’s disease-like neuropathology. PANS, 2003, 100(4): 2023-2028.
[40] Bard F, Barbour R, Cannon C, et al. Epitope and isotype specificities ofantibodies to beta-amyloid peptide for protection against Alzheimer’s disease like neuropathology. Proc Natl Acad Sci USA, 2003, 100(4): 2023-2028.
[41] DeMattos RB, Bales KR, Cummins DJ, et al. Peripheral anti-A beta anti-body alters CNS and plasma A beta clearance and decreases brain A betaburden in a mouse model of Alzheimer’s disease. Proc Natl Acad Sci USA, 2001, 98(15): 8850-8855.
[42] Wilhelmina H, van den Hurk, Bloemen M, et al. Expression of the gene encoding theβ-amyloid precursor protein APP in Xenopus laevis. Molecular Brain Research, 2001, 97(1): 13-20.
[43] Annaert W, Strooper BA. Cell biological perspective on Alzheimer’s disease. Annu Rev Cell Dev Biol, 2002, 18: 25–51.
[44] Mattson MP. Cellular actions of beta-amyloid precursor protein and its soluble and fibrillogenic derivatives. Physiol Rev, 1997, 77(4): 1081–1132.
[45] Jin LW, Ninomiya H, Roch JM, et al. Peptides containing the RERMS sequence of amyloid beta/A4 protein precursor bind cell surface and promote neurite extension. J Neurosci, 1994, 14(9): 5461–5470.
[46] Reinhard C, Hébert SS, De Strooper B. The amyloid-βprecursor protein: integrating structure with biological function. The EMBO J, 2005, 24(23): 3996–4006.
[47] Multhaup G., Mechler H., Masters CL. Characterization of the high affinity heparin binding site of the Alzheimer’s disease beta A4 amyloid precursor protein (APP) and its enhancement by zinc(II). J Mol Recognit, 1995, 8(4): 247–257.
[48] Scheuermann S, Hambsch B, Hesse L, et al. Homodimerization of amyloid precursor protein and its implication in the amyloidogenic pathway of Alzheimer’s disease. J Biol Chem, 2001, 276(36): 33923–33929.
[49] Wang Y, Ha Y. The X-Ray Structure of an Antiparallel Dimer of the Human Amyloid Precursor Protein E2 Domain. Molecular Cell, 2004, 15(3): 343-353.
[50] Soba P, Eggert S, Wagner K, et al. Homo- and heterodimerization of APP family members promotes intercellular adhesion. The EMBO Journal, 2005, 24(20): 3624-3634.
[51] Kuan YH, Gruebl T, Soba P, et al. PAT1a modulates intracellular transport andprocessing of amyloid precursor protein (APP), APLP1, and APLP2. J Biol Chem, 2006, 281(52): 40114-40123.
[52] Neumann S, Sch?bel S, J?ger S, et al. Amyloid precursor-like protein 1 influences endocytosis and proteolytic processing of the amyloid precursor protein. J Biol Chem, 2006, 281(11): 7583-7594.
[53] Heber S, Herms J, Gajic V, et al. Mice with combined gene knock-outs reveal essential and partially redundant functions of amyloid precursor protein family members. J Neurosci, 2000, 20(21): 7951–7963.
[54] Islam R, Kristiansen LV, Romani S, et al. Activation of EGF Receptor Kinase by L1-mediated Homophilic Cell Interactions. Mol Biol Cell, 2004, 15(4): 2003–2012.
[55] Kaden D, Munter LM, Voigt P, et al. Dissection of the individual domains of APP family members involved in homo- and heterotypic interactions. Alzheimer's and Dementia, 2008, 4(4): T242-243.
[56] Kaden D, Munter LM, Voigt P, et al. Homo-and heterophilic interactions of APP-family-proteins. Alzheimer's and Dementia, 2006, 2(3): S248-249.
[57] Nelson TJ, Alkon DL. Protection against beta-amyloid-induced apoptosis by peptides interacting with beta-amyloid. J Biol Chem, 2007, 282(10): 31238-31249.
[58] Shaked GM, Kummer MP, Lu DC, et al. Aβinduces cell death by direct interaction with its cognate extracellular domain on APP (APP 597–624). The FASEB Journal, 2006, 20(8): 1254-1256.
[59] D'Errico G, Vitiello G, Ortona O, et al. Interaction between Alzheimer's Aβ(25–35) peptide and phospholipid bilayers: The role of cholesterol. Biochimica et Biophysica Acta, 2008, 1778(12): 2710-2716.
[60] Klein WL, Stine WB Jr, Teplow DB. Small assemblies of unmodified amyloid beta-protein are the proximate neurotoxin in Alzheimer’s disease. Neurobiol Aging, 2004, 25(5): 569–580.
[61] Hung LW, Ciccotosto GD, Giannakis E, et al. Amyloid-beta peptide (Abeta) neurotoxicity is modulated by the rate of peptide aggregation: Abeta dimers and trimers correlate with neurotoxicity. J Neurosci, 2008, 28(46): 11950-11958.
[62] Poling A, Morgan-Paisley K, Panos JJ, et al. Oligomers of the amyloid-beta protein disrupt working memory: confirmation with two behavioral procedures. Behav Brain Res, 2008, 193(2): 230-234.
[63] Van Nostrand WE, Melchor JP, Keane DM, et al. Localization of a fibrillar amyloid beta-protein binding domain on its precursor. J Biol Chem, 2002, 277(39): 36392–36398.
[64] Lu DC, Rabizadeh S, Chandra S, et al. A second cytotoxic proteolytic peptide derived from amyloid beta-protein precursor. Nature Med, 2000, 6(4): 397–404.
[65] Yankner BA, Duffy LK, Kirschner DA. Neurotrophic and neurotoxic effects of amyloid beta protein: reversal by tachykinin neuropeptides. Science, 1990, 250(4978): 279-282.
[66] da Cruz e Silva EF, da Cruz e Silva OA. Protein phosphorylation and APP metabolism, Neurochemical Research, 2003, 28(10): 1553-1561.
[67] Nakaya T and Suzuki T. Role of APP phosphrylation in FE65-dependent gene transactivation mediated by AICD. Genes to Cells, 2006(11): 633-645.
[68] Iijima K, Ando K, Takeda S, et al. Neuron-specific phosphorylation of Alzheimer’sβ-amyloid precursor protein by cyclin-dependent kinase 5. J. Neurochem, 2000, 75: 1085–1091.
[69] Standen CL, Brownless J, Grierson AJ, et al. Phosphorylation of thr(668) in the cytoplasmic domain of the Alzheimer’s disease amyloid precursor protein by stress-activated protein kinase 1b (Jun N-terminal kinase-3). J Neurochem, 2001, 76(1): 316–320.
[70] Inomata H, Nakamura Y, Hayakawa A, et al. Scaffold protein JIP-1b enhances amyloid precursor protein phosphorylation by JNK and its association with kinesin light chain 1. J Biol Chem, 2003, 278 (25): 22946–22955.
[71] Scheinfeld MH, Ghersi E, Davies P et al. Amyloid beta protein precursor is phosphorylated by JNK-1 independent of, yet facilitated by, JNK-interacting protein (JIP)-1. J Biol Chem, 2003, 278(43): 42058-42063.
[72] Taru H, Suzuki T. Facilitation of stress-induced phosphorylation of beta-amyloid precursor protein family members by X11-like/Mint2 protein. J Biol Chem, 2004, 279(20): 21628–21636.
[73] Ando K, Iijima K, Elliott JI, et al. Phosphorylation-dependent regulation of the interaction of amyloid precursor protein with Fe65 affects the production ofβ-amyloid. J Biol Chem, 2001 276: 40353–40361.
[74] Lee MS, Kao SC, Lemere CA, er al. APP processing is regulated by cytoplasmic phosphorylation. J Cell Biol, 2003, 163(1): 83-95.
[75] Ando K, Oishi M, Takeda S, et al. Role of Phosphorylation of Alzheimer'sAmyloid Precursor Protein during Neuronal Differentiation. The Journal of Neuroscience, 1999, 19(11): 4421-4427.
[76] Liu F, Su Y, Li B, et al. Regulation of amyloid precursor protein (APP) phosphorylation and processing by p35/Cdk5 and p25/Cdk5. FEBS Lett, 2003, 547 (1-3): 193–196.
[77] Lee MS, Kao SC, Lemere CA, et al. APP processing is regulated by cytoplasmic phosphorylation. J Cell Biol, 2003, 163: 83–95.
[78] Plant LD, Boyle JP, Smith IF, et al. The production of amyloid beta peptide is a critical requirement for the viability of central neurons. J Neurosci, 2003, 23(13): 5531-5535.
[79] von Koch CS, Zheng H, Chen H, et al. Generation of APLP2 KO mice and early postnatal lethality in APLP2/APP double KO mice. Neurobiol Aging, 1997, 18(6): 661-669.
[80] Dawson GR, Seabrook GR, Zheng H, et al. Age-related cognitive deficits, impaired long-term potentiation and reduction in synaptic marker density in mice lacking the beta-amyloid precursor protein. Neuroscience, 1999, 90(1): 1-13.
[81] Pierschbacher MD, Ruoslahti E. Cell attachment activity of fibronectin can be duplicated by small synthetic fragments of the molecule. Nature, 1984, 309(5963): 30-33.
[82] Yamada KM, Kennedy DW. Dualistic nature of adhesive protein function: fibronectin and its biologically active peptide fragments can autoinhibit fibronectin function. J Cell Biol, 1984, 99:29-36.
[83] Hayman EG, Pierschbacher MD, Ruoslahti E. Detachment of cells from culture substrate by soluble fibronectin peptides. J Cell Biol, 1985, 100(6): 1948-1954.