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Preparation and characterization of toxic Aβ aggregates for structural and functional studies in Alzheimer's disease research

Nature Protocols volume 5, pages 1186–1209 (2010)Cite this article

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Abstract

The amyloid cascade hypothesis, supported by strong evidence from genetics, pathology and studies using animal models, implicates amyloid-β (Aβ) oligomerization and fibrillogenesis as central causative events in the pathogenesis of Alzheimer's disease (AD). Today, significant efforts in academia, biotechnology and the pharmaceutical industry are devoted to identifying the mechanisms by which the process of Aβ aggregation contributes to neurodegeneration in AD and to the identity of the toxic Aβ species. In this paper, we describe methods and detailed protocols for reproducibly preparing Aβ aggregates of defined size distribution and morphology, including monomers, protofibrils and fibrils, using size exclusion chromatography. In addition, we describe detailed biophysical procedures for elucidating the structural features, aggregation kinetics and toxic properties of the different Aβ aggregation states, with special emphasis on protofibrillar intermediates. The information provided by this approach allows for consistent correlation between the properties of the aggregates and their toxicity toward primary neurons and/or cell lines. A better understanding of the molecular and structural basis of Aβ aggregation and toxicity is crucial for the development of effective strategies aimed at prevention and/or treatment of AD. Furthermore, the identification of specific aggregation states, which correlate with neurodegeneration in AD, could lead to the development of diagnostic tools to detect and monitor disease progression. The procedures described can be performed in as little as 1 day, or may take longer, depending on the exact toxicity assays used.

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Figure 1: Aβ fibril formation pathway.
Figure 2
Figure 3: SEC fractionation and biophysical characterization of Aβ42 monomers and protofibrils.
Figure 4: Application to fAD-associated Aβ mutant peptides.
Figure 5: Application to synthetic and recombinant Aβ peptides.
Figure 6: Neurotoxicity of SEC-purified Aβ monomers and protofibrils.

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Acknowledgements

This work was supported by the Swiss Federal Institute of Technology Lausanne (EPFL) and by grants from the Swiss National Foundation (Grant # 310000-110027), the National Institute on Aging, USA (Grant # AG19970) (D.M.H.) and from the Alzheimer's Association (D.M.H.). We thank AC Immune (S.A.), Lausanne, Switzerland for financially supporting Asad Jan. We also thank Professor Andrea Pfeifer, Dr. Andreas Muhs and Dr. Oskar Adolfsson from AC Immune (S.A.), Lausanne, Switzerland for thoughtful discussions. We also thank Professor Carl Frieden, Washington University, St. Louis, USA and Professor Rudolf Glockshuber, ETH, Zurich, Switzerland for kindly providing recombinant Aβ peptides. We gratefully acknowledge Dr. Graham Knott at Bio-Electron Microscopy Facility (CIME), EPFL, Lausanne for technical support with TEM; Dr. Harald Wutzel, Laboratory of Polymers, EPFL, Lausanne for help with dynamic light-scattering measurements; and Dr. Michel Prudent, LMNN, EPFL, Lausanne for help with mass spectrometry measurements.

Author information

Authors and Affiliations

  1. Laboratory of Molecular Neurobiology and Neuroproteomics, Brain Mind Institute, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland

    Asad Jan & Hilal A Lashuel

  2. Department of Neurological Sciences, Rush University Medical Center, Chicago, Illinois, USA

    Dean M Hartley

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  1. Asad Jan
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Contributions

A.J. and H.A.L. contributed to the fractionation data and biophysical characterization of SEC fractions. D.M.H. provided the Aβ toxicity data and protocols for preparing and treating neuronal cultures in modified neurobasal media for toxicity studies. A.J., D.M.H. and H.A.L. contributed to writing the paper.

Corresponding authors

Correspondence to Dean M Hartley or Hilal A Lashuel.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Figure 1

(a) Circular dichroism (CD) spectroscopy analysis of SEC fractions from Superdex 75 HR 10/30 (A1340 and A1342) in a Jasco-810 CD Spectrometer using a 10 mm pathiength cell (black line = A1342 protofibrils; red line = A1342 monomer obtained using DMSO method; blue line = A1342 monomer obtained using the 6 M guanidine-HC1 method; green line = A1340 monomer obtained using the 6 M guanidine-HC1 method). (b) A1340 (monomeric = M) and A1342 (monomeric = M and protofibriliar = PF) SEC fractions were obtained from a Superdex 75 HR 10/30 column and were electrophoresed on NuPAGE 4-12% Bis Tris gels (invitrogen, cat. No. NPO336BOX). The proteins were detected by (left) silver staining (SiiverXpress, invitrogen Cat. No. LC61 00) and also, after having been transferred on a Nitroceilulose membrane (Whatman, Cat. No. 10401396), immunobiotting with anti-A13 antibody 6E10 (Signet, Cat. No. 9320). (C) Thiofiavin-T (ThT) binding over time by protofibriliar and monomeric A1342 fractions (10 pMAI342; 96 h, 37°C without agitation). (d-e) Fibril formation byA1342 crude (stock solution obtained after DMSO solubilization method): sAI342 was solubilized using DMSO (1 mg mi-1) and incubated at 37°C with gentle agitation (4,000 rpm) (d) ThT binding and (e) representative TEM image of resultant fibrils after 24 h of incubation. (t) SEC fractionation of synthetic A1340 (Keck facility), solubilized using DMSO method (1 mg mi-1), on a Superdex 75 HR 10/30 SEC column into protofibrils and monomeric A13 fractions. A1340 was solubilized and fractionated, without incubation (red line), or fractionated after incubation at room temperature, overnight (black line). (g) SEC fractionation of recombinant rAI340 from independent research groups, solubilized using DMSO method (0.5-1 mg mi-1), on a Superdex 75 HR 10/30 SEC. rAI340 1 (black line; 0.5 mg mi-1) was a generous gift from Prof. Rudolf Giockshuber, ETH, Zurich, Switzerland and rAI340 2 (green line; 1 mg mi-1) was generously provided by Prof. Carl Frieden, Washington University, St. Louis, USA. (Abbreviations: ON = Overnight; RT= Room temperature; PF=A13 Protofibrils; M= Monomeric A13; CR= Crude A1342; a.u. = arbitrary units; the error bars in (c and d) represent STD in duplicate samples; scale bar= 200 nm). (TIFF 1575 kb)

Supplementary Figure 2

SEC sub-fractionation of A42 protofibrils on a Superose 6 HR 10130 SEC column. (a) A1342 was solubilized using DMSO method (2mg mi-i) and fractionated into protofibrillar (Fi-F4) and monomeric (F5-F6) fractions on a Superose 6 HR 10/30 SEC coiumn. (b) Anaiyticai SEC of Fi-F4 on Superose 6 pc 3.2/30 SEC coiumn. (c) CD spectroscopy anaiysis of A13 42 protofibriiiar fractions from Superose6 HR 10/30 as in Suppiementary Figure ia. (d-f) Representative TEM images of (d) Fl, (e) F3 and (f) F5. (Abbreviations: F= Fraction; scaie bar= 200 nm). (TIFF 1005 kb)

Supplementary Figure 3

Stability of crude A42 solution and SEC isolated protofibrillar and monomeric A42 fractions. (a) A1342 was solubilized (DM50 method, 1 mg mV1) and split into three aliquots (— 300 p 1/aliquot). The first aliquot was injected immediately into a Superdex 75 HR 10/30 column. The second and third aliquots were incubated at 4°C for 4 and 24 h respectively, and then injected. At each time point, the solution was centrifuged (16,000 g, 4°C, 10 mm) and 250 p1 of the supernatant were injected. (b) A42 protofibrils were obtained by SEC and the fraction (50 pM, 1 ml) was split in two aliquots (500 pI/aliquot). The first aliquot was re-injected immediately into a Superdex 75 HR 10/30 column. The second aliquot was incubated at 4°C for 24 h then re-injected as above. At each time point, the solution was centrifuged (16,000 g, 4°C, 10 mm) and 400 p1 of supernatant were injected. (c) ThT binding by crude, protofibrillar and monomeric A1342 after incubation at 4°C. (d-i) Representative TEM images: (d and g) A1342 crude soon after solubilization and after 4 h of incubation at 4°C, (e and h) A1342 protofibrils soon after fractionation and after 24 h of incubation at 4°C and (f and i) monomeric A13 42 soon after fractionation and after 24 h of incubation at 4°C. (Abbreviations: PF= A1342 Protofibrils; M= Monomeric A1342; CR= Crude A1342; a.u. = arbitrary units; the error bars in (c) represent STD in duplicate samples; scale bar= 200 nm). (TIFF 1813 kb)

Supplementary Figure 4

Comparative analysis of A concentration determination by amino acid analysis (AAA), BCA method and UV A280 nm method. (a) The protein concentration of four A13 samples, from different fractionation experiments, was determined by AAA, BCA kit (Pierce Cat. No. 23227) and UV A280 nm methods. AAA was carried out in FGCZ, ETH, Zurich, Switzerland. A3 standard curve, obtained from serial dilutions of A340 in H20, was used for BOA. UV A280 nm for different fractions was determined in a 10 mm path-length cuvette using a Cary spectrophotometer. (b) Protein concentration of eight A13 samples, from different fractionation experiments, was determined by BOA kit and UVA280 nm methods. A13 concentration by BOA method was determined using a standard curve generated from both A1340 and bovine serum albumin (BSA) serial dilutions. UV A280 nm was carried out as in (a) above. BSA was provided as part of the PIEROE BOA kit. (c) Generation of standard curve of A1340 for BOA assay. A1340 was freshly solubilized in double distilled H20 (1 mg mi-1 200 pM by net weight; 80% peptide content) and serial dilutions (5 - 200 pM) were prepared (in HO) for generating a standard curve for BOA assay. The stock (200 pM) A1340 solution was aliquoted into sterile tubes (Fisherbrand, Oat. No. 05-669-27; 200 p1/tube) and frozen at -20°O. After 24 h of freezing, one of the aliquots was thawn on ice, and serial dilutions (5-200 pM) were carried out in H20. Protein concentration for both series of dilutions was determined by the BOA kit. Synthetic A 3 (Keck facility) was used for all measurements outlined in (a-c). (TIFF 554 kb)

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Jan, A., Hartley, D. & Lashuel, H. Preparation and characterization of toxic Aβ aggregates for structural and functional studies in Alzheimer's disease research. Nat Protoc 5, 1186–1209 (2010). https://doi.org/10.1038/nprot.2010.72

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