Rapid Self-assembly of α-Synuclein Observed by In Situ Atomic Force Microscopy

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Abstract

Self-assembly of α-synuclein resulting in protein aggregates of diverse morphology has been implicated in the pathogenesis of Parkinson's disease and other neurodegenerative disorders known as synucleinopathies. Apart from its biomedical relevance, this aggregation process is representative of the interconversion of an unfolded protein into nanostructures with typical amyloid features. We have used in situ tapping mode atomic force microscopy to continuously monitor the self-assembly of wild-type α-synuclein, its disease-related mutants A30P and A53T, and the C-terminally truncated variant α-synuclein(1–108). Different aggregation modes were observed depending on experimental conditions, i.e. pH, protein concentration, polyamine concentration, temperature and the supporting substrate. At pH 7.5, in the absence of the biogenic polyamines spermidine or spermine, elongated sheets 1.1(±0.2) nm in height and presumably representing individual β-sheet structures, were formed on mica substrates within a few minutes. Their orientation was directed by the crystalline substructure of the substrate. In contrast, sheet formation was not observed with hydrophobic highly oriented pyrolytic graphite substrates, suggesting that negatively charged surfaces promote α-synuclein self-assembly. In the presence of spermidine or spermine 5.9(±1.0) nm high spheroidal structures were preferentially formed, sharing characteristics with similar structures previously reported for several amyloidogenic proteins and linked to neurotoxicity. α-Synuclein spheroid formation depended critically on polyamine binding to the C terminus, revealing a promoting effect of the C terminus on α-synuclein assembly in the bound state. In rare cases, fibril growth from spheroids or preformed aggregates was observed. At pH 5.0, fibrils were formed initially and incorporated into amorphous aggregates in the course of the aggregation process, providing evidence for the potential of amyloid fibril surfaces to act as nucleation sites in amorphous aggregation. This study provides a direct insight into different modes of α-synuclein self-assembly and identifies key factors modulating the aggregation process.

Introduction

The structural transition of the abundant protein α-synuclein from its monomeric form to aggregates rich in β-conformation has been implicated in the pathogenesis of Parkinson's disease (PD) and other neurological disorders.1., 2. Insoluble α-synuclein constitutes the main component of Lewy bodies and Lewy neurites, dense intra-neuronal inclusions of fibrillar and granular material that define PD neuropathologically.1 Three missense mutations in the α-synuclein gene (A30P, A53T and E46K) have been linked to rare early-onset familial forms of PD, providing further evidence of the relevance of α-synuclein for PD.3., 4., 60. Neuronal cell death seems to be a consequence of increased α-synuclein toxicity upon formation of aggregated structures, although the mechanism of toxicity remains unclear. Well-defined aggregation intermediates might account for much of the toxicity, a notion also suggested for other amyloidogenic proteins.2., 5., 6., 7., 8.

α-Synuclein belongs to the group of natively unfolded proteins lacking ordered secondary structure in vitro in physiological buffers.9 However, upon incubation at elevated temperatures with agitation, aggregates that share features typical of amyloid, e.g. fibrillar morphologies and high β-structure content, are formed and are reminiscent of the in vivo inclusions.10., 11., 12., 13. Electron paramagnetic resonance (EPR) and protease digestion studies concordantly report that the central 70–80 amino acid residues of the 140 amino acid protein constitute the aggregate core.14., 15. Whereas this region of the protein achieves a β-sheet conformation in the aggregated state, the C-terminal region comprising residues 103–140 remains unstructured.14 This is probably a result of electrostatic repulsion within the C terminus as 14 out of the 37 C-terminal amino acid residues are acidic.

Although the C terminus is not incorporated into the aggregate core, it modulates the aggregation process. C-terminal truncated fragments aggregate faster than full-length α-synuclein, revealing that the C terminus hinders aggregation.12., 16., 17. In addition, the C terminus can interact with cationic compounds such as metal ions by virtue of the accumulation of acidic residues, resulting in accelerated aggregation.6., 18., 19., 20., 21. The polyamines putrescine, spermidine and spermine, naturally occurring polycations, are particularly effective in the promotion of α-synuclein assembly.22 Their binding site has been localized to the C terminus (residues 109–140) by NMR.23

Other factors leading to an accelerated in vitro aggregation of α-synuclein are lower pH21., 24. and the disease-related A53T mutation.12., 25., 26., 27., 28. Studies of the aggregation kinetics of the A30P mutant are inconclusive, with reports of faster aggregation,26 similar aggregation speed,12 or slower fibrillation but faster monomer consumption relative to the wild-type protein.25., 27., 28.

Multimeric α-synuclein adopts a number of different morphologies, depending on aggregation incubation time and solution conditions. The earliest species detected by atomic force microscopy (AFM) demonstrate a bead-like structure with heights of 2–6 nm.13., 25., 27., 29. At later stages of the aggregation process, these globular structures are replaced by fibrillar species with distinctive morphologies. Apart from mature fibrils with heights of ∼10 nm, smaller fibrils with heights of approximately 5 nm are formed, and have been proposed to be protofilaments.13., 25. In addition, annular species with diameters of 7–10 nm are found in early, pre-fibrillar fractions of α-synuclein incubations.5., 30. Under conditions linked to strong shielding of the negative charges in the C terminus, such as low pH or high metal ion or polyamine concentrations, α-synuclein aggregates exhibit an amorphous morphology, although the staining characteristics of amyloid are retained.20., 21., 22.

The morphologies of different amyloidogenic peptides and proteins in the aggregated state show striking similarities, suggesting a common mode of assembly and potential toxicity.31 Recent studies indicate that the early, pre-fibrillar aggregation intermediates possess the highest toxicity.7., 8. The in vitro toxicity of such soluble oligomers from different peptides and proteins is inhibited by a structure-specific antibody, implying a common, structure-based mechanism of toxicity.8

The complexity of the aggregation process arising from the structural polymorphism of the diverse intermediates and the dependence on solution conditions implies that direct and continuous observation of the reaction would be very desirable. In situ tapping mode AFM under liquid is a powerful tool for continuous observation of amyloid assembly.32., 33., 34. The method enables the repeated imaging of mechanically delicate samples in physiological buffers and allows for selection of precisely defined solution conditions. In this study, we investigated the self-assembly of α-synuclein by in situ AFM.

Section snippets

Results

The following conditions were adopted for the AFM studies. (i) The liquid cell was filled with the buffer (typically 25 mM Tris–HCl (pH 7.5)) and additives of choice and an initial image of the surface was acquired to confirm the absence of contaminating particles. (ii) Monomeric (according to analytical gel-filtration) α-synuclein was injected into the fluid cell and the assembly process monitored by continuous acquisition of AFM scans for ∼45 minutes. (iii) The temperature was generally

Discussion

The data presented here demonstrate the applicability of in situ tapping mode AFM for observation of α-synuclein self-assembly. The method offers important advantages. First, it allows continuous monitoring of individual aggregate species. Second, as is recapitulated in Figure 7, it is capable of providing high-resolution images of pre-fibrillar spheroid aggregates as well as the elongated sheet structures with heights consistent with that of a single β-sheet. Third, imaging in fluid offers the

Preparation of wild-type, mutant and truncated α-synuclein

Recombinant human wild-type, A53T and A30P α-synuclein were expressed and purified as described21 using plasmid pT7-7 encoding for the proteins (courtesy of the Lansbury laboratory, Harvard Medical School, Cambridge, MA). The carboxy-terminally truncated α-synuclein fragment syn(1–108) was amplified by PCR from full-length wild-type α-synuclein and subcloned into pT7-7. The sequence was validated by DNA sequencing. Expression and purification of the truncated proteins was performed essentially

Acknowledgements

We thank Helmut Richmann for the construction of the tandem Peltier temperature controller. We are indebted to Herming Urlaub for the ESI-MS measurements. W.H. acknowledges support from the Stiftung Stipendien-Fonds des Verbandes der Chemischen Industrie and the Bundesministerium für Bildung und Forschung.

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