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Amyloid-β(1–42) Protofibrils Formed in Modified Artificial Cerebrospinal Fluid Bind and Activate Microglia

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Abstract

Soluble aggregated forms of amyloid-β protein (Aβ) have garnered significant attention recently for their role in Alzheimer’s disease (AD). Protofibrils are a subset of these soluble species and are considered intermediates in the aggregation pathway to mature Aβ fibrils. Biological studies have demonstrated that protofibrils exhibit both toxic and inflammatory activities. It is important in these in vitro studies to prepare protofibrils using solution conditions that are appropriate for cellular studies as well as conducive to biophysical characterization of protofibrils. Here we describe the preparation and characterization of Aβ(1–42) protofibrils in modified artificial cerebrospinal fluid (aCSF) and demonstrate their prominent binding and activation of microglial cells. A simple phosphate/bicarbonate buffer system was prepared that maintained the ionic strength and cell compatibility of F-12 medium but did not contain numerous supplements that interfere with spectroscopic analyses of Aβ protofibrils. Reconstitution of Aβ(1–42) in aCSF and isolation with size exclusion chromatography (SEC) revealed curvilinear β-sheet protofibrils <100 nm in length and hydrodynamic radii of 21 nm. Protofibril concentration determination by BCA assay, which was not possible in F-12 medium, was more accurately measured in aCSF. Protofibrils formed and isolated in aCSF, but not monomers, markedly stimulated TNFα production in BV-2 and primary microglia and bound in significant amounts to microglial membranes. This report demonstrates the suitability of a modified aCSF system for preparing SEC-isolated Aβ(1–42) protofibrils and underscores the unique ability of protofibrils to functionally interact with microglia.

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Abbreviations

AD:

Alzheimer’s disease

Aβ:

Amyloid-β protein

aCSF:

Artificial cerebrospinal fluid

BCA:

Bicinchoninic acid

DAPI:

4′,6-diamidino-2-phenylindole dihydrochloride

HFIP:

Hexafluoroisopropanol

SEC:

Size exclusion chromatography

ThT:

Thioflavin T

TNFα:

Tumor necrosis factor α

References

  • Bamberger ME, Harris ME, McDonald DR, Husemann J, Landreth GE (2003) A cell surface receptor complex for fibrillar β-amyloid mediates microglial activation. J Neurosci 23:2665–2674

    PubMed  CAS  Google Scholar 

  • Bitan G, Kirkitadze MD, Lomakin A, Vollers SS, Benedek GB, Teplow DB (2003) Amyloid β-protein (Aβ) assembly: Aβ40 and Aβ42 oligomerize through distinct pathways. Proc Natl Acad Sci USA 100:330–335

    Article  PubMed  CAS  Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  PubMed  CAS  Google Scholar 

  • Cleary JP, Walsh DM, Hofmeister JJ, Shankar GM, Kuskowski MA, Selkoe DJ, Ashe KH (2005) Natural oligomers of the amyloid-β protein specifically disrupt cognitive function. Nat Neurosci 8:79–84

    Article  PubMed  CAS  Google Scholar 

  • Combs CK, Karlo JC, Kao SC, Landreth GE (2001) β-amyloid stimulation of microglia and monocytes results in TNFα-dependent expression of inducible nitric oxide synthase and neuronal apoptosis. J Neurosci 21:1179–1188

    PubMed  CAS  Google Scholar 

  • Dahlgren KN, Manelli AM, Stine WB Jr, Baker LK, Krafft GA, LaDu MJ (2002) Oligomeric and fibrillar species of amyloid-β peptides differentially affect neuronal viability. J Biol Chem 277:32046–32053

    Article  PubMed  CAS  Google Scholar 

  • Dickson DW (2004) Apoptotic mechanisms in Alzheimer neurofibrillary degeneration: cause or effect? J Clin Invest 114:23–27

    PubMed  CAS  Google Scholar 

  • Dickson DW, Lee SC, Mattiace LA, Yen SHC, Brosnan C (1993) Microglia and cytokines in neurological disease, with special reference to AIDS and Alzheimer disease. Glia 7:75–83

    Article  PubMed  CAS  Google Scholar 

  • Fassbender K, Walter S, Kuhl S, Landmann R, Ishii K, Bertsch T, Stalder AK, Muehlhauser F, Liu Y, Ulmer AJ, Rivest S, Lentschat A, Gulbins E, Jucker M, Staufenbiel M, Brechtel K, Walter J, Multhaup G, Penke B, Adachi Y, Hartmann T, Beyreuther K (2004) The LPS receptor (CD14) links innate immunity with Alzheimer’s disease. FASEB J 18:203–205

    PubMed  CAS  Google Scholar 

  • Georganopoulou DG, Chang L, Nam JM, Thaxton CS, Mufson EJ, Klein WL, Mirkin CA (2005) Nanoparticle-based detection in cerebral spinal fluid of a soluble pathogenic biomarker for Alzheimer’s disease. Proc Natl Acad Sci USA 102:2273–2276

    Article  PubMed  CAS  Google Scholar 

  • Gravina SA, Ho L, Eckman CB, Long KE, Otvos L Jr, Younkin LH, Suzuki N, Younkin SG (1995) Amyloid β protein (Aβ) in Alzheimer’s disease brain. Biochemical and immunocytochemical analysis with antibodies specific for forms ending at Aβ40 or Aβ42(43). J Biol Chem 270:7013–7016

    Article  PubMed  CAS  Google Scholar 

  • Haass C, Selkoe DJ (2007) Soluble protein oligomers in neurodegeneration: lessons from the Alzheimer’s amyloid β-peptide. Nat Rev Mol Cell Biol 8:101–112

    Article  PubMed  CAS  Google Scholar 

  • Harper JD, Wong SS, Lieber CM, Lansbury PT Jr (1997) Observation of metastable Aβ amyloid protofibrils by atomic force microscopy. Chem Biol 4:119–125

    Article  PubMed  CAS  Google Scholar 

  • Harper JD, Wong SS, Lieber CM, Lansbury PT Jr (1999) Assembly of Aβ amyloid peptides: an in vitro model for a possible early event in Alzheimer’s disease. Biochemistry 38:8972–8980

    Article  PubMed  CAS  Google Scholar 

  • Hartley DM, Walsh DM, Ye CP, Diehl T, Vasquez S, Vassilev PM, Teplow DB, Selkoe DJ (1999) Protofibrillar intermediates of amyloid β-protein induce acute electrophysiological changes and progressive neurotoxicity in cortical neurons. J Neurosci 19:8876–8884

    PubMed  CAS  Google Scholar 

  • Jan A, Hartley DM, Lashuel HA (2010) Preparation and characterization of toxic Aβ aggregates for structural and functional studies in Alzheimer’s disease research. Nat Protoc 5:1186–1209

    Article  PubMed  CAS  Google Scholar 

  • Jarrett JT, Berger EP, Lansbury PT Jr (1993) The carboxy terminus of the β amyloid protein is critical for the seeding of amyloid formation: Implications for the pathogenesis of Alzheimer’s disease. Biochemistry 32:4693–4697

    Article  PubMed  CAS  Google Scholar 

  • Jin M, Shepardson N, Yang T, Chen G, Walsh D, Selkoe DJ (2011) Soluble amyloid β-protein dimers isolated from Alzheimer cortex directly induce Tau hyperphosphorylation and neuritic degeneration. Proc Natl Acad Sci USA 108:5819–5824

    Article  PubMed  CAS  Google Scholar 

  • Kayed R, Head E, Thompson JL, McIntire TM, Milton SC, Cotman CW, Glabe CG (2003) Common structure of soluble amyloid oligomers implies common mechanism of pathogenesis. Science 300:486–489

    Article  PubMed  CAS  Google Scholar 

  • Kheterpal I, Lashuel HA, Hartley DM, Walz T, Lansbury PT Jr, Wetzel R (2003) Aβ protofibrils possess a stable core structure resistant to hydrogen exchange. Biochemistry 42:14092–14098

    Article  PubMed  CAS  Google Scholar 

  • Lee EB, Leng LZ, Zhang B, Kwong L, Trojanowski JQ, Abel T, Lee VM (2006) Targeting amyloid-β peptide (Aβ) oligomers by passive immunization with a conformation-selective monoclonal antibody improves learning and memory in Aβ precursor protein (APP) transgenic mice. J Biol Chem 281:4292–4299

    Article  PubMed  CAS  Google Scholar 

  • Liu S, Liu Y, Hao W, Wolf L, Kiliaan AJ, Penke B, Rube CE, Walter J, Heneka MT, Hartmann T, Menger MD, Fassbender K (2012) TLR2 Is a primary receptor for Alzheimer’s amyloid β peptide to trigger neuroinflammatory activation. J Immunol 188:1098–1107

    Article  PubMed  CAS  Google Scholar 

  • McGeer EG, McGeer PL (1998) The importance of inflammatory mechanisms in Alzheimer disease. Exp Gerontol 33:371–378

    Article  PubMed  CAS  Google Scholar 

  • McGeer PL, Itagaki S, Tago H, McGeer EG (1987) Reactive microglia in patients with senile dementia of the Alzheimer type are positive for the histocompatibility glycoprotein HLA-DR. Neurosci Lett 79:195–200

    Article  PubMed  CAS  Google Scholar 

  • McNay EC, Gold PE (1999) Extracellular glucose concentrations in the rat hippocampus measured by zero-net-flux: effects of microdialysis flow rate, strain, and age. J Neurochem 72:785–790

    Article  PubMed  CAS  Google Scholar 

  • Meda L, Cassatella MA, Szendrei GI, Otvos L Jr, Baron P, Villalba M, Ferrari D, Rossi F (1995) Activation of microglial cells by β-amyloid protein and interferon-γ. Nature 374:647–650

    Article  PubMed  CAS  Google Scholar 

  • Nichols MR, Moss MA, Reed DK, Lin WL, Mukhopadhyay R, Hoh JH, Rosenberry TL (2002) Growth of β-amyloid(1–40) protofibrils by monomer elongation and lateral association. Characterization of distinct products by light scattering and atomic force microscopy. Biochemistry 41:6115–6127

    Article  PubMed  CAS  Google Scholar 

  • O’Nuallain B, Freir DB, Nicoll AJ, Risse E, Ferguson N, Herron CE, Collinge J, Walsh DM (2010) Amyloid β-protein dimers rapidly form stable synaptotoxic protofibrils. J Neurosci 30:14411–14419

    Article  PubMed  Google Scholar 

  • Paranjape GS, Gouwens LK, Osborn DC, Nichols MR (2012) Isolated amyloid-β(1–42) protofibrils, but not isolated fibrils, are robust stimulators of microglia. ACS Chem Neurosci 3:302–311

    Article  PubMed  CAS  Google Scholar 

  • Reed-Geaghan EG, Savage JC, Hise AG, Landreth GE (2009) CD14 and Toll-like receptors 2 and 4 are required for fibrillar Aβ-stimulated microglial activation. J Neurosci 29:11982–11992

    Article  PubMed  CAS  Google Scholar 

  • Selkoe DJ (2004) Cell biology of protein misfolding: The examples of Alzheimer’s and Parkinson’s diseases. Nat Cell Biol 6:1054–1061

    Article  PubMed  CAS  Google Scholar 

  • Stewart CR, Stuart LM, Wilkinson K, van Gils JM, Deng J, Halle A, Rayner KJ, Boyer L, Zhong R, Frazier WA, Lacy-Hulbert A, Khoury JE, Golenbock DT, Moore KJ (2010) CD36 ligands promote sterile inflammation through assembly of a Toll-like receptor 4 and 6 heterodimer. Nat Immunol 11:155–161

    Article  PubMed  CAS  Google Scholar 

  • Tzounopoulos T, Kim Y, Oertel D, Trussell LO (2004) Cell-specific, spike timing-dependent plasticities in the dorsal cochlear nucleus. Nat Neurosci 7:719–725

    Article  PubMed  CAS  Google Scholar 

  • Udan ML, Ajit D, Crouse NR, Nichols MR (2008) Toll-like receptors 2 and 4 mediate Aβ(1–42) activation of the innate immune response in a human monocytic cell line. J Neurochem 104:524–533

    PubMed  CAS  Google Scholar 

  • Walsh DM, Selkoe DJ (2007) Aβ oligomers - a decade of discovery. J Neurochem 101:1172–1184

    Article  PubMed  CAS  Google Scholar 

  • Walsh DM, Lomakin A, Benedek GB, Condron MM, Teplow DB (1997) Amyloid β-protein fibrillogenesis: detection of a protofibrillar intermediate. J Biol Chem 272:22364–22372

    Article  PubMed  CAS  Google Scholar 

  • Walsh DM, Hartley DM, Kusumoto Y, Fezoui Y, Condron MM, Lomakin A, Benedek GB, Selkoe DJ, Teplow DB (1999) Amyloid β-protein fibrillogenesis: structure and biological activity of protofibrillar intermediates. J Biol Chem 274:25945–25952

    Article  PubMed  CAS  Google Scholar 

  • Ye CP, Selkoe DJ, Hartley DM (2003) Protofibrils of amyloid β-protein inhibit specific K+ currents in neocortical cultures. Neurobiol Dis 13:177–190

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

We would like to thank Dr. David C. Osborn in the Microscopy Image and Spectroscopy Technology Laboratory in the Center for Nanoscience at University of Missouri-St. Louis for TEM imaging.

Conflict of interest

The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. Department of Chemistry and Biochemistry and Center for Nanoscience, University of Missouri-St. Louis, St. Louis, MO, USA

    Geeta S. Paranjape, Shana E. Terrill, Lisa K. Gouwens, Benjamin M. Ruck & Michael R. Nichols

  2. Department of Chemistry and Biochemistry, University of Missouri-St. Louis, One University Boulevard, St. Louis, MO, 63121, USA

    Michael R. Nichols

Authors
  1. Geeta S. Paranjape
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  2. Shana E. Terrill
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  3. Lisa K. Gouwens
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  4. Benjamin M. Ruck
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  5. Michael R. Nichols
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Corresponding author

Correspondence to Michael R. Nichols.

Additional information

Geeta S. Paranjape, Shana E. Terrill and Lisa K. Gouwens contributed equally.

This work was supported by Award Number R15AG033913 from the National Institute on Aging (MRN).

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Paranjape, G.S., Terrill, S.E., Gouwens, L.K. et al. Amyloid-β(1–42) Protofibrils Formed in Modified Artificial Cerebrospinal Fluid Bind and Activate Microglia. J Neuroimmune Pharmacol 8, 312–322 (2013). https://doi.org/10.1007/s11481-012-9424-6

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  • DOI: https://doi.org/10.1007/s11481-012-9424-6

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