A Cyclic Undecamer Peptide Mimics a Turn in Folded Alzheimer Amyloid β and Elicits Antibodies against Oligomeric and Fibrillar Amyloid and Plaques

Abstract

The 39- to 42-residue amyloid β (Aβ) peptide is deposited in extracellular fibrillar plaques in the brain of patients suffering from Alzheimer's Disease (AD). Vaccination with these peptides seems to be a promising approach to reduce the plaque load but results in a dominant antibody response directed against the N-terminus. Antibodies against the N-terminus will capture Aβ immediately after normal physiological processing of the amyloid precursor protein and therefore will also reduce the levels of non-misfolded Aβ, which might have a physiologically relevant function. Therefore, we have targeted an immune response on a conformational neo-epitope in misfolded amyloid that is formed in advance of Aβ-aggregation. A tetanus toxoid-conjugate of the 11-meric cyclic peptide Aβ(22–28)-YNGK′ elicited specific antibodies in Balb/c mice. These antibodies bound strongly to the homologous cyclic peptide-bovine serum albumin conjugate, but not to the homologous linear peptide-conjugate, as detected in vitro by enzyme-linked immunosorbent assay. The antibodies also bound—although more weakly—to Aβ(1–42) oligomers as well as fibrils in this assay. Finally, the antibodies recognized Aβ deposits in AD mouse and human brain tissue as established by immunohistological staining. We propose that the cyclic peptide conjugate might provide a lead towards a vaccine that could be administered before the onset of AD symptoms. Further investigation of this hypothesis requires immunization of transgenic AD model mice.

About the Authors:

Peter Hoogerhout

* E-mail: [email protected]

Affiliation: Department of Vaccinology, Centre for Infectious Disease Control, National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands

Willem Kamphuis

Affiliation: Netherlands Institute for Neuroscience (NIN), Astrocyte Biology & Neurodegeneration, Royal Netherlands Academy of Arts and Sciences, Amsterdam, The Netherlands

Humphrey F. Brugghe

Affiliation: Department of Vaccinology, Centre for Infectious Disease Control, National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands

Jacqueline A. Sluijs

Affiliation: Netherlands Institute for Neuroscience (NIN), Astrocyte Biology & Neurodegeneration, Royal Netherlands Academy of Arts and Sciences, Amsterdam, The Netherlands

Hans A. M. Timmermans

Affiliation: Department of Vaccinology, Centre for Infectious Disease Control, National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands

Janny Westdijk

Affiliation: Department of Vaccinology, Centre for Infectious Disease Control, National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands

Gijsbert Zomer

Affiliation: Department of Vaccinology, Centre for Infectious Disease Control, National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands

Claire J. P. Boog

Affiliation: Department of Vaccinology, Centre for Infectious Disease Control, National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands

Elly M. Hol

Affiliation: Netherlands Institute for Neuroscience (NIN), Astrocyte Biology & Neurodegeneration, Royal Netherlands Academy of Arts and Sciences, Amsterdam, The Netherlands

Germie P. J. M. van den Dobbelsteen

Affiliation: Department of Vaccinology, Centre for Infectious Disease Control, National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands

Introduction

Alzheimer's disease (AD) is a neurodegenerative disorder and the most common cause of dementia in elderly [1], [2]. A characteristic of the disease is formation of plaques in the brain or in brain blood vessels. These plaques originate from a membrane-bound protein, amyloid precursor protein (APP). An α-helical fragment of 39–42 amino acid residues is cleaved by β- and γ-secretases from APP thus forming a soluble amyloid β (Aβ) peptide. Soluble Aβ initially adopts an extended conformation but at high concentrations, soluble Aβ will undergo conformational changes and form oligomers, protofibrils, and fibrils. In AD, fibrillar Aβ is deposited in the brain as amyloid plaques, which is one of the main neuropathological hallmarks of the disease. However, accumulating studies suggest that the soluble oligomeric Aβ instead of insoluble Aβ in amyloid plaques is the culprit in AD [3]–[8] and therapeutic approaches aimed at preventing the formation of these oligomeric isoforms may be able to reduce the progression of the disease. In line with this concept, immunization of transgenic mice [9] with a suspension of “pre-aggregated” Aβ(1–42) and the adjuvant quillaja saponin 21 appeared to be beneficial. Based on these results, a phase I clinical trial was started. Antibodies present in human sera recognized plaques and Aβ deposits in brain blood vessels [10]. The antibodies did not recognize APP or soluble Aβ. In the following phase II clinical trial, 20% of the vaccine recipients generated anti-Aβ antibody titers. Unfortunately, this trial had to be terminated since 6% of the patients developed meningoencephalitis as a vaccine-related side effect. This side effect was caused by a cellular inflammatory reaction, attributed to a T helper cell type 1 response to epitopes located in the central and C-terminal part of Aβ(1–42) [11], [12].

Multiple ongoing studies aim at improving the Aβ vaccination strategy [13]–[18]. The use of T helper cell type 2 directing adjuvants [19], [20] or the use of formulations without any adjuvant [21] are under investigation. In addition, it has been proposed to use C-terminally truncated Aβ peptides [22], [23] or peptide mimics (affitopes) of the N-terminus [24]. Antibodies induced by Aβ(1–42) are dominantly directed against the linear N-terminal epitope [25], [26], although generation of conformation-specific antibodies against other regions within aggregated Aβ has been reported [27]. A disadvantage of a vaccine against the N-terminus of Aβ is that it will interfere with the normal physiological processing of APP. It may not be without risk to administer such a vaccine before onset of symptoms of AD.

By targeting an immune response exclusively on misfolded Aβ the undesired response against the N-terminus of Aβ may be avoided all together. A structural model of fibrillar Aβ(1–42) predicts folding of monomeric Aβ(1–42) into a cross-β unit. Two antiparallel extended β-strands, residues 11–25 and 28–42, are connected via a sharp bend around amino acid residues S26 and N27 [28]. Fig. 1 shows a simplification of the original model. A recent study of a particular oligomer of N-Met-Aβ(1–42) suggests a bend around the sequence V24-G-S-N27 [29].

[Figure omitted. See PDF.]

Figure 1. Early folding of human amyloid β (Aβ), around S26 and N27.

The figure is a simplification of the model designed by Olofsson et al. [28]. Residues in red are solvent accessible; residues in blue (and to a lesser extent in gray) are shielded from the solvent. The peptide cyclo[Aβ(22–28)-YNGK′] is a mimic for misfolded Aβ. YNGK′ is a turn-stabilizing sequence and K′ is a side-chain-modified lysyl residue for selective conjugation to a protein carrier. Other YNGK′-containing cyclic peptides prepared (23–28, 24–29, 25–30, 21–27, 22–28, 23–29, 24–30, and 25–31) did not mimic misfolded Aβ.

https://doi.org/10.1371/journal.pone.0019110.g001

The models of folded Aβ show some resemblance with the β-turn structure in surface loops of meningococcal outer membrane protein PorA. Previously, we have stabilized the β-turn conformation of small meningococcal peptides by adding an artificial sequence YNGK′, in which K′ is a modified lysine residue for selective conjugation to a carrier protein, followed by main chain (“head to tail”) amide cyclization [30], [31]. Likewise, it appeared possible to prepare small cyclic peptides mimicking the turn in folded Aβ. A panel of cyclic decameric and undecameric peptides spanning six or seven residues from the region 21–31 of Aβ and YNGK′ was prepared and conjugated to tetanus toxoid (TTd). The conjugates were used for immunization of mice. A tetanus toxoid-conjugate of one of the peptides, cyclo[Aβ(22–28)-YNGK′], elicited antibodies that specifically recognized misfolded Aβ.

Results and Discussion

Antigen preparation

Oligomeric (≈35 kD) and fibrillar Aβ(1–42) were prepared according to published procedures [32]. A well-established method [33] was used for synthesis of a panel of cyclic decameric and undecameric peptides spanning six or seven residues from the region 21–31 (AEDVG25SNKGA30I) of Aβ (i.e. 23–28, 24–29, 25–30, 21–27, 22–28, 23–29, 24–30, and 25–31) and the artificial stabilizing sequence YNGK′. Linear Ac-K′-Aβ(21–31)-NH2 was prepared to serve as control. The peptides obtained were conjugated to tetanus toxoid (TTd) to give experimental vaccines or to bovine serum albumin (BSA) to give coating antigens for enzyme-linked immunosorbent assay (ELISA ) [34].

Immunization of mice

Balb/c mice were immunized twice with oligomeric or fibrillar Aβ(1–42) without adjuvant or with the different amyloid peptide/TTd-conjugates using aluminum phosphate as adjuvant. The pooled sera from each group immunized with peptide conjugate were analyzed by ELISA using plain TTd, homologous peptide/BSA-conjugate and oligomeric or fibrillar Aβ(1–42) as coating antigens. The sera showed a good antibody titer against TTd and the homologous cyclopeptide (titers of 104–105 were obtained, data not shown).

Six out of eight pooled anti-cyclo-Aβ peptide antisera were not cross-reactive with oligomeric or fibrillar Aβ(1–42) in ELISA or a dot blot assay. Antibodies against cyclo[Aβ(23–29)-YNGK′] showed a weak cross-reactivity. However, anti-cyclo[Aβ(22–28)-YNGK′] antibodies were strongly cross-reactive with oligomeric or fibrillar Aβ(1–42) in ELISA (Fig. 2) and the antibodies recognized Aβ deposits in AD mouse and human brain tissue (Fig. 3). The anti-peptide antibodies did not recognize full length APP in Western blot assay (Fig. 4).

[Figure omitted. See PDF.]

Figure 2. Binding of anti-cyclopeptide antibodies to oligomeric amyloid β (Aβ) as determined by ELISA.

Threefold serial dilutions (starting from 1/150) of pooled antisera obtained were tested. Antisera were raised by immunization with oligomeric (black ▴) or fibrillar (black ▪) Aβ(1–42) and TTd-conjugates of cyclo[Aβ(22–28)-YNGK′] (red ▴) or linear K′-Aβ(22–28)-YNG (red ▪). Monoclonal antibody 6E10 (green •) served as control. Antibody binding to fibrillar Aβ(1–42) gave comparable results.

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[Figure omitted. See PDF.]

Figure 3. Immunohistochemical staining of brain tissue.

A, a brain section (hippocampus) of an 85-year-old female AD patient (NBB 1999-030) was stained with mouse anti-cyclo[Aβ(22–28)-YNGK′] serum and (B) co-stained with an antibody against APP (6E10). The neuropathological diagnosis of the is patient is Braak stage for neurofibrillary tangles 6 and for amyloid B [43]. C, immunostaining of a plaque-loaded cortex of a 9 month old APPswe/PS1dE9 transgenic mouse.

https://doi.org/10.1371/journal.pone.0019110.g003

[Figure omitted. See PDF.]

Figure 4. Western blot of a lysate of SW13 adrenal cells transfected with amyloid precursor protein (APP).

The cells were transfected with either human APP695wt (wild type) or with APP695swe (Swedish mutation). Monoclonal antibody 6E10 is specific for the largely unstructured N-terminus of amyloid β (Aβ) and recognized both APPs (amino acid residues 2–8 in Aβ correspond to residues 598–604 in APP695). Anti-cyclo[Aβ (22–28)-YNGK′] antibodies stained neither of the APPs under the denaturing conditions of the assay and thus seem to recognize a conformational epitope in Aβ.

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The cyclization of the peptide was essential to produce a functional antigen. Conjugates of linear control peptides, i.e. Ac-K′-Aβ(21–31)- NH2 or the fully homologous Ac-K′-Aβ(22–28)-YNG-NH2, induced good anti-peptide antibody titers but the antibodies failed to recognize oligomeric or fibrillar Aβ(1–42) (Fig. 2). The incorporation of the artificial sequence YNGK′ in the cyclopeptide was essential to mimic the turn in folded Aβ(1–42). Incorporation of the sequence GAIK′, i.e. Aβ(29–31)-K′, instead of YNGK′ did not provide a turn-mimicking peptide, since antibodies against cyclo[Aβ(22–31)-K′] were not functional.

The anti-cyclo[Aβ(22–28)-YNGK′] antibodies were very specific for the common Aβ sequence. Amyloid β with the Dutch, Arctic, or Italian mutations (E22Q, E22G, or E22K, respectively) was not recognized. Unrelated but partially homologous proteins like the islet amyloid polypeptide (amylin)or the human Wiskott-Aldrich Syndrome protein (WASP) were also not recognized (data not shown).

Finally, antibodies elicited with oligomeric or fibrillar Aβ(1–42) were non-reactive with cyclo[Aβ(22–28)-YNGK′]-BSA. This was expected since antibodies induced by Aβ(1–42) are directed against the N-terminus [25], [26].

The cross-reactivity of anti-cyclo[Aβ(22–28)-YNGK′] antibodies with oligomeric Aβ(1–42) was confirmed after a second immunization experiment. Individual sera of mice immunized with cyclo[Aβ(22–28)-YNGK′]-TTd were analyzed this time. All sera (8/8) gave a good response against TTd and the homologous peptide (titers 104–105, data not shown), but not all individual sera were clearly cross-reactive with oligomeric or fibrillar Aβ(1–42). Five sera showed titers ≥2000 (Fig. 5), whereas one serum showed no response (titer<100). Two immunizations with oligomeric Aβ(1–42) gave two sera with titers as high as 40×103 and 50×103 on the homologous coating. Three titers were considered to be intermediate (2000–8000) and three titers were low (100–2000).

[Figure omitted. See PDF.]

Figure 5. IgG response of individual Balb/c mice against Aβ (1–42) as determined by ELISA.

As indicated on the x-axis, groups of eight mice were immunized with cyclo[Aβ (22–28)-YNGK′]-TTd, oligomeric Aβ(1–42), or fibrillar Aβ (1–42). A, coating antigen: oligomeric Aβ (1–42); B, coating antigen: fibrillar Aβ (1–42). A mouse with a serum titer ≤100 is considered to be a non-responder. The two titers out of range in the right panel of A have values of 40×103 and 50×103. The figure demonstrates that anti- cyclo[Aβ (22–28)-YNGK′] antibodies recognize oligomeric and fibrillar Aβ (1–42).

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Two immunizations with fibrillar Aβ(1–42) gave only two sera with titers ≥2000 on the homologous coating, two very low responders and four non-responders (titer<100). Poor responsiveness to aggregated Aβ(1–42) was found earlier in clinical and animal studies [35], [36].

In conclusion, cyclo[Aβ(22–28)-YNGK′] mimics a conformational epitope in folded Aβ(1–42) that normally does not induce an antibody response. Cyclo[Aβ(22–28)-YNGK′]-conjugate may be an interesting vaccine candidate against AD. Since the response is directed specifically to misfolded Aβ, it is unlikely that vaccination will interfere with normal physiological processing of the amyloid precursor protein. Thus, vaccination might be possible at an early stage of AD symptoms or before. This seems to be indicated since plaque deposits have already reached a near maximum level at a mild stage of AD [37].

Further studies will aim at improvement of the immune response to cyclo[Aβ(22–28)-YNGK′]-TTd by variation of the dose of cyclopeptide-conjugate, the adjuvant, and the number of administrations, preferably in transgenic AD model mice.

Materials and Methods

Ethics statement

Tissue from AD patients was obtained from the Netherlands Brain Bank (NBB; Amsterdam, The Netherlands). The NBB performs brain autopsies with short post-mortem intervals, and the brain donors have given written informed consent for using the tissue and for accessing the extensive neuropathological and clinical information for scientific research, in compliance with ethical and legal guidelines [38]. The study was approved by the independent Review Board (“Medisch Ethische Toetsingscommissie, METc”) of the VU University Medical Center, Amsterdam, The Netherlands.

Morphological studies of mouse brain tissue were approved by the Ethical Committee on Animal Experiments of the Royal Netherlands Academy of Arts and Sciences (approval ID: NIN 06.51).

Immunization of mice was approved by the Ethical Committee on Animal Experiments of the National Institute of Public Health and the Environment (approval ID: 2007 00331 and 2008 00179), as required under Dutch Law on the use of laboratory animals. All efforts were made to minimize suffering of the animals.

Materials

Human Aβ(1–42) (Fig. 1), hAβ(1–40/42) analogues, and hIAPP were purchased from AnaSpec (Freemont, CA, USA). AlPO4 suspension (1.5 mg/ml), pH 7.0, and tetanus toxoid (TTd) were produced by the RIVM. Monoclonal antibody 6E10 against Aβ(1–17), was purchased from Abcam (Cambridge, MA, USA) or Covance Research Products, Dedham, MA, USA).

Disaggregation of Aβ(1–42)

Lyophilized Aβ(1–42) was dissolved in trifluoroacetic acid at a concentration of 1.0 mM, left to stand at room temperature for 1 h and dried under a stream of nitrogen and, thereafter, in a vacuum (1 mm Hg) for 15 min. The peptide was then redissolved in hexafluoroisopropanol at a concentration of 1.0 mM and, after 1 h of incubation at room temperature, dried as described above [39]. The peptide was stored at −20°C for 18–20 h.

Preparation of oligomeric or fibrillar Aβ(1–42)

Disaggregated of Aβ(1–42) was dissolved in dimethyl sulfoxide at a concentration of 5.0 mM, diluted 50-fold with either phosphate buffered saline (PBS), pH 7.2, or 10 mM hydrochloric acid. The solution in PBS was incubated at 4°C for 24 h (to give oligomers), whereas the solution in 10 mM HCl was incubated at 37°C for 24 h (to give fibrils) [32].

Peptide synthesis, purification, and conjugation

The α-(2,4-dimethoxybenzyl) ester of Nα-fluorenylmethoxycarbonyl-L-aspartic acid (Fmoc-Asp-ODmb) was coupled through its side-chain to a polymer for the synthesis of peptide amides (for later conversion of the starting Asp into Asn). Synthesis of cyclic peptides, like cyclo[GK′EDVGSNKYN]≡cyclo[EDVGSNKYNGK′] in which K′ is N ε-(S-acetylmercaptoacetyl)lysyl, was then performed as described earlier [33]. The following Aβ-derived and YNGK′-extended cyclopeptides were prepared: 23–28, 24–29, 25–30, 21–27, 22–28, 23–29, 24–30, and 25–31. Linear Ac-K′-Aβ(22–31)-NH2 and linear Ac-K′-Aβ(22–28)-YNG-NH2 were also prepared. The peptides were purified by reverse-phase high performance liquid chromatography and coupled to either bromoacetylated tetanus toxoid or maleimidyl-modified bovine serum albumin (BSA) (modifying reagent: NHS-PEO2-Maleimide, Pierce) [34].

Immunization of mice

Groups of eight female Balb/c mice of 6–8 weeks of age were immunized with 25 µg oligomeric or fibrillar Aβ(1–42) in 0.3 ml phosphate buffered saline (PBS), pH 7.2, without adjuvant or with 50 µg of peptide/TTd-conjugate and 75 µg of AlPO4 in 0.3 ml PBS on days 0 and 28. The mice were bled on day 42 under oxygen/nitrous oxide/isoflurane anesthesia.

Western blot

Human carcinoma SW13 cells [40] were transfected with APP695wt (wild type) and APP695swe (Swedish mutation) and proteins were isolated by homogenization in 0.1 M NaCl, 0.01 M Tris HCl, 0.001 M EDTA, pH 7.6, with proteinase inhibitors. The samples were supplemented with 2× loading buffer (100 mM Tris pH 6.8, 4% SDS, 20% glycerol, 0.2 M DTT, 0.006% bromophenol blue) and run on a 7.5% SDS-PAGE gel. The gel was semi-dry blotted on nitrocellulose and probed overnight with the 6E10 antibody (1∶1000) or the mouse anti-cyclo[Aβ(22–28)-YNGK′] serum (1∶500). After washing, the blots were incubated with anti-mouse Cy5 (1∶300), bands were visualized with the Odyssey Infrared Imaging System.

Dot blot

Dilutions of cell lysates (WASPpos HL60 cells and WASPneg Vero cells) in PBS were spotted (1 µl/spot) on polyvinylidene difluoride membrane (Immobilon-P, pore size 0.45 µM, Millipore). The blots were incubated with mouse anti-cyclo[Aβ(22–28)-YNGK′] serum, control monoclonal antibody 6E10, or polyclonal anti-WASP antibodies. After washing, the blots were incubated with horseradish peroxidase-conjugated 2nd antibody, and visualized with GZ11 signal reagent [41].

ELISA

Microtiter plates (Greiner 655092) were coated with Aβ(1–42) or peptide-BSA conjugates. Freshly prepared Aβ(1–42) oligomers or fibrils were diluted to a final concentration of 2.5 µM (11.3 µg/ml) in 0.04 M sodium carbonate/bicarbonate buffer, pH 9.7. Peptide-BSA conjugates in phosphate buffered saline, pH 7.2 (PBS), had a total protein concentration of 0.5 µg/ml. Aliquots (100 µl) of these solutions were transferred into wells of the plates. The plates were incubated for 90 min at 37°C. The plates were further processed as described earlier [42]. Titers were calculated as the reciprocal serum dilution at 50% of the maximum optical density (OD50).

Immunohistochemical staining

Human brain sections of the hippocampus of several donors with Alzheimer disease, Braak 5 or 6 [43], were used (Netherlands Brain Bank). In addition, brain sections of 9 month old APPswe/PS1dE9 [42] mice with a significant plaque deposition were used. Cryosections (10 µm) were cut from unfixed, directly frozen tissue, thaw-mounted, dried for 1 hour and stored in a sealed box at −20 C. For immunostaining, sections were fixed in 4% PFA-PBS solution for 10 min, washed in 0.05 M phosphate buffer (PB) for 10 min with 2 exchanges and blocked with 10% normal donkey serum (NDS) +0.4% Triton X-100 in 0.05 M PB for 1 hour at RT. The blocking solution was discarded and diluted mouse sera (1∶300; first antibody) in 3% NDS +0.4% Triton X-100 in 0.05 M PB was added and incubated O/N at RT. Sections were washed with 0.05 M PB; at least 30 min with one or more exchanges. Then sections were incubated with Donkey-anti-Mouse ∼Cy3 1∶1400 in 0.05 M PB for 2 hours. Sections were washed with 0.05 M PB; at least 30 min with one or more exchanges. Sections were sealed in Vectashield with Dapi (Vector). Mouse Monoclonal 6E10 to beta amyloid 3–8 (Signet-Covance) was used as positive control (1∶15,000).

Acknowledgments

The human brain tissue was obtained from The Netherlands Brain Bank, Netherlands Institute for Neuroscience, Amsterdam.

Author Contributions

Conceived and designed the experiments: PH WK JW GZ EMH GPJMvdD. Performed the experiments: PH WK HFB JAS HAMT JW GZ. Analyzed the data: PH WK JW GZ CJPB EMH GPJMvdD. Contributed reagents/materials/analysis tools: PH WK HFB JAS HAMT JW GZ. Wrote the paper: PH WK EMH.

Citation: Hoogerhout P, Kamphuis W, Brugghe HF, Sluijs JA, Timmermans HAM, Westdijk J, et al. (2011) A Cyclic Undecamer Peptide Mimics a Turn in Folded Alzheimer Amyloid β and Elicits Antibodies against Oligomeric and Fibrillar Amyloid and Plaques. PLoS ONE6(4): e19110. https://doi.org/10.1371/journal.pone.0019110

References

1. Blennow K, de Leon MJ, Zetterberg H (2006) Alzheimer's disease. Lancet 368: 387–403.K. BlennowMJ de LeonH. Zetterberg2006Alzheimer's disease.Lancet368387403

2. Goedert M, Spillantini MG (2006) A century of Alzheimer's disease 1. Science 314: 777–781.M. GoedertMG Spillantini2006A century of Alzheimer's disease 1.Science314777781

3. Glabe CG (2008) Structural classification of toxic amyloid oligomers. J Biol Chem 283: 29639–29643.CG Glabe2008Structural classification of toxic amyloid oligomers.J Biol Chem2832963929643

4. Haass C, Selkoe DJ (2007) Soluble protein oligomers in neurodegeneration: lessons from the Alzheimer's amyloid beta-peptide. Nat Rev Mol Cell Biol 8: 101–112.C. HaassDJ Selkoe2007Soluble protein oligomers in neurodegeneration: lessons from the Alzheimer's amyloid beta-peptide.Nat Rev Mol Cell Biol8101112

5. Hillen H, Barghorn S, Striebinger A, Labkovsky B, Muller R, et al. (2010) Generation and therapeutic efficacy of highly oligomer-specific beta-amyloid antibodies. J Neurosci 30: 10369–10379.H. HillenS. BarghornA. StriebingerB. LabkovskyR. Muller2010Generation and therapeutic efficacy of highly oligomer-specific beta-amyloid antibodies.J Neurosci301036910379

6. Klein WL, Stine WB Jr, Teplow DB (2004) Small assemblies of unmodified amyloid beta-protein are the proximate neurotoxin in Alzheimer's disease. Neurobiol Aging 25: 569–580.WL KleinWB Stine JrDB Teplow2004Small assemblies of unmodified amyloid beta-protein are the proximate neurotoxin in Alzheimer's disease.Neurobiol Aging25569580

7. Lambert MP, Velasco PT, Chang L, Viola KL, Fernandez S, et al. (2007) Monoclonal antibodies that target pathological assemblies of Abeta. J Neurochem 100: 23–35.MP LambertPT VelascoL. ChangKL ViolaS. Fernandez2007Monoclonal antibodies that target pathological assemblies of Abeta.J Neurochem1002335

8. Walsh DM, Selkoe DJ (2007) A beta oligomers - a decade of discovery. J Neurochem 101: 1172–1184.DM WalshDJ Selkoe2007A beta oligomers - a decade of discovery.J Neurochem10111721184

9. Schenk D, Barbour R, Dunn W, Gordon G, Grajeda H, et al. (1999) Immunization with amyloid-beta attenuates Alzheimer-disease-like pathology in the PDAPP mouse. Nature 400: 173–177.D. SchenkR. BarbourW. DunnG. GordonH. Grajeda1999Immunization with amyloid-beta attenuates Alzheimer-disease-like pathology in the PDAPP mouse.Nature400173177

10. Hock C, Konietzko U, Papassotiropoulos A, Wollmer A, Streffer J, et al. (2002) Generation of antibodies specific for beta-amyloid by vaccination of patients with Alzheimer disease. Nat Med 8: 1270–1275.C. HockU. KonietzkoA. PapassotiropoulosA. WollmerJ. Streffer2002Generation of antibodies specific for beta-amyloid by vaccination of patients with Alzheimer disease.Nat Med812701275

11. Gilman S, Koller M, Black RS, Jenkins L, Griffith SG, et al. (2005) Clinical effects of Abeta immunization (AN1792) in patients with AD in an interrupted trial. Neurology 64: 1553–1562.S. GilmanM. KollerRS BlackL. JenkinsSG Griffith2005Clinical effects of Abeta immunization (AN1792) in patients with AD in an interrupted trial.Neurology6415531562

12. Pride M, Seubert P, Grundman M, Hagen M, Eldridge J, et al. (2008) Progress in the active immunotherapeutic approach to Alzheimer's disease: clinical investigations into AN1792-associated meningoencephalitis. Neurodegener Dis 5: 194–196.M. PrideP. SeubertM. GrundmanM. HagenJ. Eldridge2008Progress in the active immunotherapeutic approach to Alzheimer's disease: clinical investigations into AN1792-associated meningoencephalitis.Neurodegener Dis5194196

13. Brody DL, Holtzman DM (2008) Active and passive immunotherapy for neurodegenerative disorders 2. Annu Rev Neurosci 31: 175–193.DL BrodyDM Holtzman2008Active and passive immunotherapy for neurodegenerative disorders 2.Annu Rev Neurosci31175193

14. Foster JK, Verdile G, Bates KA, Martins RN (2009) Immunization in Alzheimer's disease: naive hope or realistic clinical potential? 1. Mol Psychiatry 14: 239–251.JK FosterG. VerdileKA BatesRN Martins2009Immunization in Alzheimer's disease: naive hope or realistic clinical potential? 1.Mol Psychiatry14239251

15. Hawkes CA, McLaurin J (2007) Immunotherapy as treatment for Alzheimer's disease. Expert Rev Neurother 7: 1535–1548.CA HawkesJ. McLaurin2007Immunotherapy as treatment for Alzheimer's disease.Expert Rev Neurother715351548

16. Solomon B (2008) Immunological approaches for amyloid-beta clearance toward treatment for Alzheimer's disease. Rejuvenation Res 11: 349–357.B. Solomon2008Immunological approaches for amyloid-beta clearance toward treatment for Alzheimer's disease.Rejuvenation Res11349357

17. Wisniewski T (2009) AD vaccines: conclusions and future directions 3. CNS Neurol Disord Drug Targets 8: 160–166.T. Wisniewski2009AD vaccines: conclusions and future directions 3.CNS Neurol Disord Drug Targets8160166

18. Woodhouse A, Dickson TC, Vickers JC (2007) Vaccination strategies for Alzheimer's disease: A new hope? Drugs Aging 24: 107–119.A. WoodhouseTC DicksonJC Vickers2007Vaccination strategies for Alzheimer's disease: A new hope?Drugs Aging24107119

19. Cribbs DH, Ghochikyan A, Vasilevko V, Tran M, Petrushina I, et al. (2003) Adjuvant-dependent modulation of Th1 and Th2 responses to immunization with beta-amyloid. Int Immunol 15: 505–514.DH CribbsA. GhochikyanV. VasilevkoM. TranI. Petrushina2003Adjuvant-dependent modulation of Th1 and Th2 responses to immunization with beta-amyloid.Int Immunol15505514

20. Ghochikyan A, Mkrtichyan M, Petrushina I, Movsesyan N, Karapetyan A, et al. (2006) Prototype Alzheimer's disease epitope vaccine induced strong Th2-type anti-Abeta antibody response with Alum to Quil A adjuvant switch. Vaccine 24: 2275–2282.A. GhochikyanM. MkrtichyanI. PetrushinaN. MovsesyanA. Karapetyan2006Prototype Alzheimer's disease epitope vaccine induced strong Th2-type anti-Abeta antibody response with Alum to Quil A adjuvant switch.Vaccine2422752282

21. Cao C, Lin X, Wahi MM, Jackson EA, Potter H Jr (2008) Successful adjuvant-free vaccination of BALB/c mice with mutated amyloid beta peptides. BMC Neurosci 9: 25.C. CaoX. LinMM WahiEA JacksonH. Potter Jr2008Successful adjuvant-free vaccination of BALB/c mice with mutated amyloid beta peptides.BMC Neurosci925

22. Lemere CA (2009) Developing novel immunogens for a safe and effective Alzheimer's disease vaccine 2. Prog Brain Res 175: 83–93.CA Lemere2009Developing novel immunogens for a safe and effective Alzheimer's disease vaccine 2.Prog Brain Res1758393

23. Lemere CA, Masliah E (2010) Can Alzheimer disease be prevented by amyloid-beta immunotherapy? Nat Rev Neurol 6: 108–119.CA LemereE. Masliah2010Can Alzheimer disease be prevented by amyloid-beta immunotherapy?Nat Rev Neurol6108119

24. Schneeberger A, Mandler M, Otawa O, Zauner W, Mattner F, et al. (2009) Development of Affitope Vaccines for Alzheimer's Disease (AD) - From Concept to Clinical Testing 1. J Nutr Health Aging 13: 264–267.A. SchneebergerM. MandlerO. OtawaW. ZaunerF. Mattner2009Development of Affitope Vaccines for Alzheimer's Disease (AD) - From Concept to Clinical Testing 1.J Nutr Health Aging13264267

25. Lee M, Bard F, Johnson-Wood K, Lee C, Hu K, et al. (2005) Abeta42 immunization in Alzheimer's disease generates Abeta N-terminal antibodies. Ann Neurol 58: 430–435.M. LeeF. BardK. Johnson-WoodC. LeeK. Hu2005Abeta42 immunization in Alzheimer's disease generates Abeta N-terminal antibodies.Ann Neurol58430435

26. Lemere CA, Beierschmitt A, Iglesias M, Spooner ET, Bloom JK, et al. (2004) Alzheimer's disease abeta vaccine reduces central nervous system abeta levels in a non-human primate, the Caribbean vervet. Am J Pathol 165: 283–297.CA LemereA. BeierschmittM. IglesiasET SpoonerJK Bloom2004Alzheimer's disease abeta vaccine reduces central nervous system abeta levels in a non-human primate, the Caribbean vervet.Am J Pathol165283297

27. Vasilevko V, Pop V, Kim HJ, Saing T, Glabe CC, et al. (2010) Linear and conformation specific antibodies in aged beagles after prolonged vaccination with aggregated Abeta. Neurobiol Dis 39: 301–310.V. VasilevkoV. PopHJ KimT. SaingCC Glabe2010Linear and conformation specific antibodies in aged beagles after prolonged vaccination with aggregated Abeta.Neurobiol Dis39301310

28. Olofsson A, Sauer-Eriksson AE, Ohman A (2006) The solvent protection of alzheimer amyloid-beta-(1–42) fibrils as determined by solution NMR spectroscopy. J Biol Chem 281: 477–483.A. OlofssonAE Sauer-ErikssonA. Ohman2006The solvent protection of alzheimer amyloid-beta-(1–42) fibrils as determined by solution NMR spectroscopy.J Biol Chem281477483

29. Yu L, Edalji R, Harlan JE, Holzman TF, Lopez AP, et al. (2009) Structural characterization of a soluble amyloid beta-peptide oligomer. Biochemistry 48: 1870–1877.L. YuR. EdaljiJE HarlanTF HolzmanAP Lopez2009Structural characterization of a soluble amyloid beta-peptide oligomer.Biochemistry4818701877

30. Oomen CJ, Hoogerhout P, Bonvin AM, Kuipers B, Brugghe H, et al. (2003) Immunogenicity of peptide-vaccine candidates predicted by molecular dynamics simulations. J Mol Biol 328: 1083–1089.CJ OomenP. HoogerhoutAM BonvinB. KuipersH. Brugghe2003Immunogenicity of peptide-vaccine candidates predicted by molecular dynamics simulations.J Mol Biol32810831089

31. Oomen CJ, Hoogerhout P, Kuipers B, Vidarsson G, van AL, et al. (2005) Crystal structure of an Anti-meningococcal subtype P1.4 PorA antibody provides basis for peptide-vaccine design. J Mol Biol 351: 1070–1080.CJ OomenP. HoogerhoutB. KuipersG. VidarssonAL van2005Crystal structure of an Anti-meningococcal subtype P1.4 PorA antibody provides basis for peptide-vaccine design.J Mol Biol35110701080

32. Stine WB Jr, Dahlgren KN, Krafft GA, LaDu MJ (2003) In vitro characterization of conditions for amyloid-beta peptide oligomerization and fibrillogenesis. J Biol Chem 278: 11612–11622.WB Stine JrKN DahlgrenGA KrafftMJ LaDu2003In vitro characterization of conditions for amyloid-beta peptide oligomerization and fibrillogenesis.J Biol Chem2781161211622

33. Brugghe HF, Timmermans HA, Van Unen LM, Ten Hove GJ, Van de Werken G, et al. (1994) Simultaneous multiple synthesis and selective conjugation of cyclized peptides derived from a surface loop of a meningococcal class 1 outer membrane protein. Int J Pept Protein Res 43: 166–172.HF BruggheHA TimmermansLM Van UnenGJ Ten HoveG. Van de Werken1994Simultaneous multiple synthesis and selective conjugation of cyclized peptides derived from a surface loop of a meningococcal class 1 outer membrane protein.Int J Pept Protein Res43166172

34. Drijfhout JW, Hoogerhout P (2000) Methods of preparing peptide-carrier conjugates. In: Chan WC, White PD, editors. Fmoc Solid Phase Peptide Synthesis: A Practical Approach:. Oxford University Press. pp. 229–241.JW DrijfhoutP. Hoogerhout2000Methods of preparing peptide-carrier conjugates.WC ChanPD WhiteFmoc Solid Phase Peptide Synthesis: A Practical Approach:Oxford University Press229241

35. Holmes C, Boche D, Wilkinson D, Yadegarfar G, Hopkins V, et al. (2008) Long-term effects of Abeta42 immunisation in Alzheimer's disease: follow-up of a randomised, placebo-controlled phase I trial 1. Lancet 372: 216–223.C. HolmesD. BocheD. WilkinsonG. YadegarfarV. Hopkins2008Long-term effects of Abeta42 immunisation in Alzheimer's disease: follow-up of a randomised, placebo-controlled phase I trial 1.Lancet372216223

36. Lemere CA, Maier M, Jiang L, Peng Y, Seabrook TJ (2006) Amyloid-beta immunotherapy for the prevention and treatment of Alzheimer disease: lessons from mice, monkeys, and humans. Rejuvenation Res 9: 77–84.CA LemereM. MaierL. JiangY. PengTJ Seabrook2006Amyloid-beta immunotherapy for the prevention and treatment of Alzheimer disease: lessons from mice, monkeys, and humans.Rejuvenation Res97784

37. Perrin RJ, Fagan AM, Holtzman DM (2009) Multimodal techniques for diagnosis and prognosis of Alzheimer's disease. Nature 461: 916–922.RJ PerrinAM FaganDM Holtzman2009Multimodal techniques for diagnosis and prognosis of Alzheimer's disease.Nature461916922

38. Huitinga I, Rademaker M, Klioueva N (2008) The art of brain banking in Europe: ethical, legal and practical guidelines for donor recruitment, tissue handling and tissue distribution. J Neural Transm 115: 1715.I. HuitingaM. RademakerN. Klioueva2008The art of brain banking in Europe: ethical, legal and practical guidelines for donor recruitment, tissue handling and tissue distribution.J Neural Transm1151715

39. Zeng H, Zhang Y, Peng L, Shao H, Menon NK, et al. (2001) Nicotine and amyloid formation. Biol Psychiatry 49: 248–257.H. ZengY. ZhangL. PengH. ShaoNK Menon2001Nicotine and amyloid formation.Biol Psychiatry49248257

40. Sarria AJ, Lieber JG, Nordeen SK, Evans RM (1994) The presence or absence of a vimentin-type intermediate filament network affects the shape of the nucleus in human SW-13 cells. J Cell Sci 107(Pt 6): 1593–1607.AJ SarriaJG LieberSK NordeenRM Evans1994The presence or absence of a vimentin-type intermediate filament network affects the shape of the nucleus in human SW-13 cells.J Cell Sci107Pt 615931607

41. Zomer G, Hamzink M, de Haan A, Kersten G, Reubsaet K (2009) Development and optimization of a quantitative Western blot and dot blot procedure for the determination of residual host cell proteins present in inactivated polio vaccine using GZ11 based signal reagent. In: Shen X, Yang XL, Zhang XR, Cui ZJ, Kricka LJ, editors. Bioluminescence and Chemiluminescence Light Emission:. Biology and Scientific Applications: World Scientific. pp. 287–290.G. ZomerM. HamzinkA. de HaanG. KerstenK. Reubsaet2009Development and optimization of a quantitative Western blot and dot blot procedure for the determination of residual host cell proteins present in inactivated polio vaccine using GZ11 based signal reagent.X. ShenXL YangXR ZhangZJ CuiLJ KrickaBioluminescence and Chemiluminescence Light Emission:Biology and Scientific Applications: World Scientific287290

42. Westdijk J, Van den IJssel J, Thalen M, Beuvery C, Jiskoot W (1997) Quantification of cell-associated and free antigens in Bordetella pertussis suspensions by antigen binding ELISA. J Immunoassay 18: 267–284.J. WestdijkJ. Van den IJsselM. ThalenC. BeuveryW. Jiskoot1997Quantification of cell-associated and free antigens in Bordetella pertussis suspensions by antigen binding ELISA.J Immunoassay18267284

43. Braak H, Braak E (1991) Neuropathological stageing of Alzheimer-related changes. Acta Neuropathol 82: 239–259.H. BraakE. Braak1991Neuropathological stageing of Alzheimer-related changes.Acta Neuropathol82239259