A major class of brain diseases called ‘synucleinopathies’, is characterised by the abnormal deposition of the protein a-synuclein (a-syn) in nerve cells (neurons). The most important member of this group is Parkinson’s disease where a-synuclein aggregates are found in dopamine-producing neurons.
These dopamine-producing neurons are gradually lost, eventually leading to the clinically observed inability to control movement. a-Syn in the presence of dopamine forms non-fibrillar oligomers, a molecule containing the two components, here denoted a-syn:dopamine.
These a-syn:dopamine oligomers have a variety of sizes (shown by electron microscopy), but there is no information about their supra-molecular structure and therefore, mode of association. Using small-angle X-ray scattering we could characterise shapes of a-syn:dopamine oligomers, an important part of understanding their physiological effects (e.g. neurotoxicity), and thus elucidating the role of dopamine and oligomeric a-syn in Parkinson’s disease, potentially leading to new preventive and therapeutic strategies.
Many neurodegenerative disorders, which include Alzheimer’s, Parkinson’s, Huntington’s and Creutzfelt-Jakob diseases, involve misfolding (abnormal structuring) and aggregation of certain proteins in the brain.
One of such proteins is a-syn whose normal function in a healthy brain has not yet been established, however its ability to form elongated polymers, so-called amyloid fibrils, was found associated with dementias (e.g. Parkinson’s disease, dementia with Lewy bodies and multiplesystematrophy).
Despite the presence of fibrillar material in the affected brain tissue, evidence suggests that the neurotoxic agents might be smaller soluble oligomers of a-syn. Dopamine is a neurotransmitter produced by particular neurons, whose death is a significant consequence of Parkinson’s disease.
In vitro, a-syn in the presence of dopamine forms small soluble oligomers which do not lead to fibrillar structure [1,2].
What we found
In our study we looked closely at untreated a-syn and small a-syn:dopamine species: monomer and trimer. We used small angle X-ray scattering n(SAXS), a technique that gives information on particle size and shape in solution.
While the size information is directly derived from the SAXS data, nshapes of molecules and their complexes are nobtained by computational modelling [3,4] and can be validated with other techniques, such as sedimentation velocity analysis, circular dichroism spectroscopy, mass spectrometry and electron spin resonance spectroscopy.
The size (expressed as ‘radius of gyration’, a different measure than linear dimensions) of a-syn monomer oxidised by dopamine (37 Å) was similar to that of the untreated monomer (36 Å) which coincides with the similarity of shapes shown in Figure 1A and B.
It was previously known that monomeric a-syn in solution is unstructured, while amyloid fibrils contain structural elements called b-sheets. We found that a-syn:dopamine trimers of ~ 50 Å have more b-sheet and turn structure than monomers.
A partly overlapping lateral (rather than end-to-end)arrangement of a-syn chains in the a-syn:dopamine trimer can be inferred from the size and a higher degree of structuring. Figure 2A shows the possible arrangements. Both monomers and trimers have nelongated ‘worm-like’ shapes, as illustrated in Figure 1, which are however more diverse for trimers than monomers.
It is possible that the trimers represent a mixture of species with different modes of assembly, see Figure 1C. Higher-mass oligomers of a-syn:dopamine progressively increased in size to 105 Å for a 26- mer. And as their size increased, these species more globular in shape, which is consistent with their images under electrone microscope [1].
Conclusions and outlook
In vitro, a-syn in the presence of dopamine forms soluble oligomers which do not lead to fibrillar structures and understanding this process is relevant for developing therapeutic and diagnostic strategies for Parkinson’s disease [5].
Our experiments provide further insight into the role of neurotransmitter dopamine in the development of Parkinson’s disease and other synucleinopathies and mechanisms of a-syn toxicity to brain cells.
Dopamine and/or its products (melanin) oxidize methionine residues in monomeric a-syn, which with time associates to form a-syn:dopamine oligomeric species. Our data indicate that inoligomers, a-syn is bound to polymeric dopamine (melanin) with four of its methionine residues oxidised.
On the basis of these and earlier data, we propose an oxidation-based mechanism of prevention of fibrillation by dopamine. Methionine oxidation in a-syn causes structural changes (including increased propensity for b-sheet formation) which allow cross-linking between chains and stabilization of the resulting oligomer by dopamine/ melanin (Figure 2B), also formed as a result of a redox process.
Despite these structural changes of a-syn chains in small oligomers, their partly overlapping lateral arrangement can prevent them from directly parallel association into (also b-sheetstructured) amyloid fibrils. In this way, dopamine may be responsible for toxic effects in Parkinson’s disease brain.
However, from the SAXS models, it is unclear which parts of a-syn interact with dopamine. Small angle neutron scattering (SANS) nondeuterated molecules. Thus, our next step will be to use deuterated dopamine in a SANS experiment on instrument Quokka at ANSTO in order to find its binding site on a-syn.
Authors
Agata Rekas1, Robert K. Knott1, Anna Sokolova1, Kevin J. Barnham2, Keyla Perez2, Colin L. Masters2, Simon C. Drew2, Roberto Cappai Cyril C. Curtain2 and Chi Le Lan Pham2
1ANSTO, 2University of Technology, Sydney, Australia and 3Novel Materials and Nanotechnology Laboratory, Valencia,Spain
References
- Cappai R, Leck SL, Tew DJ, Williamson NA, Smith DP, Galatis D, Sharples RA, Curtain CC, Ali FE, Cherny RA, Culvenor JG, Bottomley SP, Masters CL, Barnham KJ, Hill AF ‘Dopamine promotes alpha-synuclein aggregation into SDS-resistant soluble oligomers via a distinct folding pathway’, Faseb Journal, 19, (2005) 1377-9.
- Leong SL, Pham CL, Galatis D, Fodero-Tavoletti MT, Perez K, Hill AF, Masters CL, Ali FE, Barnham KJ, Cappai R, ‘Formation of dopamine-mediated alpha-synucleinsoluble oligomers requires methionine oxidation’. Free Radical Biology and Medicine, 46, (2009b) 1328-37.
- Svergun DI, ‘Restoring three-dimensional structure of biopolymers from solution scattering using simulated annealing’. Biophysical Journal, 76, (1999) 2879-2288.
- Konarev PV, Petoukhov MV, Volkov VV, Svergun DI ATSAS 2.1, a program package for small-angle scattering data analysis. Journal of Applied Crystallography, 39, (2006) 277-286.
- Conway KA, Rochet JC, Bieganski RM, Lansbury PT, Jr. ‘Kinetic stabilization of the a-synuclein protofibril by a dopamine-a synuclein adduct’. Science 294
Published: 15/09/2009