Aggregation and fibril morphology of the Arctic mutation of Alzheimer’s Aβ peptide by CD, TEM, STEM and in situ AFM
Graphical abstract
AFM images of polymorphs of amyloid fibrils of the wild type and the Arctic mutation Alzheimer’s Aβ(1–40).
Introduction
Alzheimer’s disease (AD) is the most common age-related dementia whose hallmark is the abundance of amyloid plaques in the brain of AD patients. The principal constituent of these plaques are fibrils mainly composed of amyloid β-peptides most commonly as 40 or 42 amino acid long Aβ(1–40) and Aβ(1–42) variants. It is generally accepted that aggregation of Aβ-monomers or oligomers is involved in AD pathogenesis and there is increasing evidence that specific fibrillar species and, in particular, prefibrillar intermediates (oligomers) play a central role in the neurodegeneration (Selkoe, 1995, Lansbury, 1999, Dahlgren et al., 2002, Lashuel et al., 2002, Walsh et al., 2002, Petkova et al., 2005, Lal et al., 2007, Chimon et al., 2007, Inoue, 2008, Zheng et al., 2008, Small, 2009, Ono et al., 2009, Ahmed et al., 2010, Jang et al., 2010b, Sandberg et al., 2010), but as yet the precise mechanism is unknown. The structures of Aβ-monomers, dimers and oligomers have been visualized with high-resolution scanning tunneling microscopy at sufficient resolution to suggest the folding of the polypeptide chain (Losic et al., 2006). Nevertheless, different fibril morphologies can display distinguished molecular structures and neurotoxicity (Petkova et al., 2005, Petkova et al., 2002, Paravastu et al., 2008, Tycko et al., 2009, Miller et al., 2010). Supramolecular models for Aβ(1–40) and Aβ(1–42) have been obtained using structural constraints from STEM mass-per-length measurements, solid-state nuclear magnetic resonance spectroscopy (NMR) (Petkova et al., 2002, Petkova et al., 2005, Paravastu et al., 2008), as well as cryo-electron microscopy (Sachse et al., 2008, Schmidt et al., 2009). Although some features of the fibril structure obtained from these techniques are self-consistent, there are also differences in the model of fibril architecture determined from solid-state NMR and later from cryo-EM (Fändrich et al., 2011). From AFM measurements of Aβ bound to model phospholipid membranes Lal and co-workers have suggested that non-fibrillar Aβ oligomers may cause neuronal cell degeneration by formation of transmembrane cationic channels, which may disrupt the cellular Ca2+ homeostasis (Lal et al., 2007, Jang et al., 2010b). Already experiments on hypothalamic neurons (Kawahara et al., 1997) with the molecular chaperone αβ-crystallin (Stege et al., 1999) and PC12 cells (Chromy et al., 2003) verified that oligomeric Aβ intermediate globular assemblies are potent neurotoxins in the absence of any fibril formation. Sandberg et al., 2010, recently reported on a remarkably larger caspase-3/7 activity in a human neuroblastoma cell line, SH-SY5Y, incubated in the presence of cystein–cystein cross-linked model peptide Aβ(1–42)CC 100 kDa β-sheet oligomers, compared to amyloid fibrils. It is likely that different types of soluble amyloid oligomers have a common macroscopic structure and that they share a common mechanism of toxicity (Lashuel et al., 2002, Lashuel et al., 2003, Kayed et al., 2003). One of the most convincing pieces of experimental evidence for the ion-channel hypothesis came, however, from high-resolution atomic force microscopy (AFM) imaging of Aβ monomers incorporated in liposomes and lipid membranes (Rhee et al., 1998, Lin et al., 1999, Lin et al., 2001). In those experiments it has been shown that the peptide appears in globular structures, which do not form fibrils for an extended period of time. When incorporated into reconstituted membranes, many amyloid molecules form typical channel-like structures and elicit single ion-channel currents (Quist et al., 2005). AFM has further been used to study toxic effects on cells (Kawahara et al., 1997, Bhatia et al., 2000, Zhu et al., 2000). Since the oligomeric aggregates are responsible for AD toxicity, the structure and the whole pathway of aggregation kinetics from Aβ-oligomers/protofibrils to fibrils have been extensively investigated (Lansbury, 1999, Harper et al., 1997, Kirkitadze et al., 2001, Stine et al., 2003). Reviews of various experimental studies of amyloidosis of Alzheimer’s amyloid-β peptides and on supramolecular structure of amyloid fibrils can be found in a number of publications (Antzutkin, 2004, Tycko, 2006, Heise, 2008, Yang et al., 2010, Goldsbury et al., 2011, Fändrich et al., 2011).
The ‘Arctic’ E22G point mutation of Aβ(1–40) (arc-Aβ(1–40)) and Aβ(1–42) (arc-Aβ(1–42)) is a rare mutation found in a few families in northern Sweden, leading to an early onset (52–57 year-old) of Alzheimer’s disease (Nilsberth et al., 2001). A majority of mutations within the β-amyloid region of the amyloid precursor protein (APP) gene cause inherited forms of intracerebral hemorrhage. Most of these mutations may also cause cognitive impairment, but the Arctic APP mutation is the only known intra-β-amyloid mutation to date causing the more typical clinical picture of Alzheimer disease (Basun et al., 2008). It has been shown that the Arctic mutation carriers have lower plasma levels of Aβ than normal, while this mutation accelerates both Aβ oligomerization and fibrillogenesis in vitro (Dahlgren et al., 2002, Lashuel et al., 2003, Nilsberth et al., 2001, Paivio et al., 2004), and also in vivo (Nilsberth et al., 2001, Cheng et al., 2004, Englund et al., 2007). Testing “structure–toxicity” hypothesis Dahlgren et al., 2002, have found that an increase in the diameter of amyloid fibrils of arc-Aβ(1–40) is correlated with a significant decrease in viability of nerve cells in vitro.
The conformational transitions during aggregation of 25–30 μM Aβ(1–42), Aβ(1–40), arc-Aβ(1–40) and other mutations of Aβ have been studied in glycine buffer at pH 7.5 by Teplow and co-workers with circular dichroism (CD) (Kirkitadze et al., 2001). At a few days from the onset of aggregation the content of the α-helix secondary structure has increased at the expense of the initially predominantly random-coil conformation. Then, after reaching a maximum, the fraction of the α-helical component decreases with time, while the β-sheet component increases. The formation of amyloid fibrils has been detected by electron microscopy in the same samples over the same time intervals (Kirkitadze et al., 2001). Subsequently, arc-Aβ(1–40) aggregates have been studied by Lashuel et al., 2003, by electron microscopy. It has been observed that for this mutation both morphology and size distributions of Aβ protofibrils are different compared with those for wt-Aβ(1–40). Several morphologies of arc-Aβ(1–40) aggregates have been identified: (i) relatively compact spherical particles with a diameter of roughly 4–5 nm; (ii) annular pore-like protofibrils; (iii) large spherical particles of diameter 18–25 nm; and (iv) short filaments with a chain-like morphology (Lashuel et al., 2003). The conversion of arc-Aβ(1–40) protofibrils to fibrils proceeds more rapidly than that for protofibrils formed in mixed solutions of wt-Aβ(1–40)/arc-Aβ(1–40), which has been explained by a kinetic stabilization of the protofibrils in the course of co-incubation of arc-Aβ(1–40) with wt-Aβ(1–40) (Lashuel et al., 2003). Both conformations and the size distribution of arc-Aβ(1–40) aggregates have been also studied by Gräslund and co-workers, who have shown that protofibrils of this mutation are relatively stable oligomers with a large distribution of sizes ranging from 100 kDa (∼25-mer) to 3000 kDa (∼700-mer) with predominantly β-sheet secondary structural motifs (Paivio et al., 2004).
Molecular dynamics simulations of protofibrils of both arc-Aβ(1–40) and wt-Aβ(1–40) using a coarse grain model have revealed remarkable differences in the putative structure of oligomers formed by these two peptides (Fawzi et al., 2008). Despite an extensive number of above mentioned reports on arc-Aβ(1–40) several important issues are still left unexplored: (i) details of the morphological differences in polymorphs of fibrils of arc-Aβ(1–40), which grow and coexist in the same sample; (ii) mass-per-unit length of these fibrils, which is an important constraint needed for developing (yet unknown) supramolecular models for arc-Aβ(1–40) fibrils; (iii) structure and stability of transient aggregation intermediates; (iv) critical concentrations for arc-Aβ(1–40) fibril formation in different buffers at conditions close to physiological; (v) interaction of arc-Aβ(1–40) with phospholipid membranes; (vi) cellular toxicity of specific aggregation intermediates and different polymorphs of arc-Aβ(1–40) amyloid fibrils.
In the present work aggregation and polymorphism of the human arc-Aβ(1–40), is further studied in vitro using a variety of experimental techniques and compared with the behavior of the wild type Aβ(1–40) (wt-Aβ(1–40)) at similar conditions. Here, we visualize in detail aggregation events of both wt-Aβ(1–40) and arc-Aβ(1–40) with atomic force microscopy imaging, and complement these studies with both CD and ThT analyses. Furthermore, we present a systematic TEM, STEM and in situ AFM investigation of the detailed morphology of Aβ-fibrils in Tris buffer solutions and studies by AFM of structure of Aβ-oligomers and in-situ growth of amyloid fibrils adsorbed on a mica sample surface. To develop further the “structure–toxicity” concept, the precise measured EM and AFM structural parameters of different oligomers and polymorphs of arc-Aβ(1–40) fibrils can be employed for development of supramolecular models for these systems using structural constraints from solid-state NMR and other spectroscopic techniques and correlated further with data from neurotoxicity assays.
Section snippets
Sample preparation
Peptides (wt-Aβ(1–40) and arc-Aβ(1–40)) were synthesized, purified by HPLC and lyophilized (with special precautions to avoid formation of pre-aggregates) as previously described by (Antzutkin et al., 2000, Antzutkin et al., 2002, Antzutkin, 2004). A buffer solution was prepared dissolving 10 mM Tris, 5 mM EDTA, 10 mM KCl and 0.01 wt% NaN3 in doubly distilled water. pH of the initial buffer solution (100 mL, pH 8.75) was adjusted with 0.55 mL 0.1 M HCl(aq) and later (fine adjustments) with 0.05 M
Aggregation kinetics of arc-Aβ(1–40) and wt-Aβ(1–40): ThT fluorescence and circular dichroism measurements
Fig. 1 shows results of the thioflavin-T (ThT) fluorescence assay for both arc-Aβ(1–40) and wt-Aβ(1–40) peptides incubated at 50 μM in Tris buffer solutions at pH 7.4 and room temperature (ca 293 K) without agitation: an ‘S’-shaped dependence of fluorescence with time, after a lag period of ca 70 h, was found for arc-Aβ(1–40). This type of dependence is typical for self-catalytic processes such as aggregation. After reaching its maximum (at ca 120 h) the ThT fluorescence gradually decreased to ca
Conclusions
AFM characterization of Aβ mutations, supported by TEM, ThT fluorescence and CD data, is valuable approach for helping to elucidate AD pathogenesis. We confirm previous results, which have reported that the Arctic mutation of the Aβ(1–40) peptide is more ‘aggressive’ in its tendency to aggregate than the wild type Aβ(1–40) peptide. Not only does the intermediate phase of spherical aggregates appear at earlier times but also fibrils polymerize more rapidly from the onset of incubation for arc-Aβ
Acknowledgments
We thank Dr. Goldsbury for useful technical discussions regarding time-lapse imaging of fibril assembly and Dr. Mannequist for valuable ideas in the design of the tapping mode system. This work was supported by the Swedish Research Council (O.A., N.A. and G.G.) and from the Foundation in memory of J.C. and Seth M. Kempe (Grant Nos. JCK-2701 and JCK-2905, used for a stipend for A.F. a peptide synthesizer, HPLC and chemicals) but also for AFM support (SMK-2546). Alzheimer’s foundation in Sweden
References (85)
- et al.
Elasticity and adhesion force mapping reveals real-time clustering of growth factor receptors and associated changes in local cellular rheological properties
Biophys. J.
(2004) - et al.
Direct observation of Aβ amyloid fibril growth and inhibition
J. Mol. Biol.
(2004) - et al.
In-situ atomic force microscopy study of β-amyloid fibrillization
J. Mol. Biol.
(2000) - et al.
Aggressive brain amyloidosis in transgenic mice expressing human amyloid peptides with the Arctic mutation
Neurobiol. Aging
(2004) - et al.
Oligomeric and fibrillar species of amyloid-β peptides differentially affect neuronal viability
J. Biol. Chem.
(2002) - et al.
B-sheet structured β-amyloid(1–40) perturbs phosphatidylcholine model membranes
J. Mol. Biol.
(2007) - et al.
Protofibril assemblies of the Arctic, Dutch, and Flemish mutants of the Alzheimer’s Aβ(1–40) peptide
Biophys. J.
(2008) - et al.
Recent progress in understanding Alzheimer’s β-amyloid structures
Trends Biochem. Sci.
(2011) - et al.
Multiple assembly pathways underlie amyloid-β fibril polymorphisms
J. Mol. Biol.
(2005) - et al.
Amyloid fibril formation from full-length and fragments of amylin
J. Struct. Biol.
(2000)
Watching amyloid fibrils grow by time-lapse atomic force microscopy
J. Mol. Biol.
Amyloid structure and assembly: insights from scanning transmission electron microscopy
J. Struct. Biol.
Alternate aggregation pathways of the Alzheimer β-amyloid peptide: Aβ association kinetics at endosomal pH
J. Mol. Biol.
Dipolar recoupling NMR of biomolecular self-assemblies: determining inter- and intrastrand distances in fibrilized Alzheimer’s β-amyloid peptide
Solid State Nucl. Magn. Reson.
Observation of metastable Aβ amyloid protofibrils by atomic force microscopy
Chem. Biol.
Structural studies of soluble oligomers of the Alzheimer β-amyloid peptide
J. Mol. Biol.
β-Barrel topology of Alzheimer’s β-amyloid ion channels
J. Mol. Biol.
A left-handed 3(1) helical conformation in the Alzheimer Ab(12–28) peptide
FEBS Lett.
β2-Microglobulin and its deamidated variant, N17D form amyloid fibrils with a range of morphologies in vitro
J. Mol. Biol.
Mechanical manipulation of Alzheimer’s amyloid-β 1–42 fibrils
J. Struct. Biol.
Alzheimer’s disease amyloid β-protein forms Zn2+-sensitive, cation-selective channels across excited membrane patches from hypothalamic neurons
Biophys. J.
Identification and characterization of key kinetic intermediates in amyloid β-protein fibrillogenesis
J. Mol. Biol.
Amyloid-β ion channel: 3D structure and relevance to amyloid channel paradigm
Biochim. Biophys. Acta Biomembr.
Mixtures of wild-type and a pathogenic (E22G) form of Aβ(1-40) in vitro accumulate protofibrils, including amyloid pores
J. Mol. Biol.
High resolution scanning tunnelling microscopy of the beta-amyloid protein (Aβ1–40) of Alzheimer’s disease suggests a novel mechanism of oligomer assembly
J. Struct. Biol.
Aβ(1–40) fibril polymorphism implies diverse interaction patterns in amyloid fibrils
J. Mol. Biol.
Assembly of amyloid protofibrils via critical oligomers – a novel pathway of amyloid formation
J. Mol. Biol.
Characterization by atomic force microscopy of Alzheimer paired helical filaments under physiological conditions
Biophys. J.
Unique physicochemical profile of β-amyloid peptide variant Abeta(1–40)E22G protofibrils: conceivable neuropathogen in arctic mutant carriers
J. Mol. Biol.
Imaging real-time aggregation of amyloid beta protein (1–42) by atomic force microscopy
Peptides
Influence of residue 22 on the folding, aggregation profile, and toxicity of the Alzheimer’s amyloid beta peptide
Biophys. J.
The molecular chaperone αβ-crystallin enhances amyloid beta neurotoxicity
Biochem. Biophys. Res. Comm.
In vitro characterization of conditions for amyloid-β peptide oligomerization and fibrillogenesis
J. Biol. Chem.
Mass analysis of biological macromolecular complexes by STEM
Biol. Cell
Structural conversion of neurotoxic amyloid-β(1–42) oligomers to fibrils
Nat. Struct. Mol. Biol.
Multiple quantum solid state NMR indicates a parallel, not antiparallel, organization of β-sheets in Alzheimer’s β-amyloid fibrils
Proc. Natl. Acad. Sci. USA
Supramolecular structural constraints on Alzheimer’s β-amyloid fibrils from electron microscopy and solid-state nuclear magnetic resonance
Biochemistry
Amyloidosis of Alzheimer’s Aβ-peptides: solid-state nuclear magnetic resonance, electron paramagnetic resonance, transmission electron microscopy, scanning transmission electron microscopy and atomic force microscopy studies
Magn. Reson. Chem.
Alzheimer disease amyloid-β protein forms calcium channels in bilayer membranes: blockade by trimethamine and aluminium
Proc. Natl. Acad. Sci. USA
Clinical and neuropathological features of the Arctic APP gene mutation causing early-onset Alzheimer disease
Arch. Neurol.
Fresh and globular amyloid β protein (1–42) induces rapid cellular degeneration: evidence for AβP channel-mediated cellular toxicity
FASEB J.
Capturing intermediate structures of Alzheimer’s β-amyloid, Aβ(1–40), by solid-state NMR spectroscopy
J. Am. Chem. Soc.
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