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Dopamine promotes -synuclein aggregation into SDS-resistant soluble oligomers via a distinct folding pathway

Roberto Cappai^*,,||,1, Su-Ling Leck, Deborah J. Tew^*,||, Nicholas A. Williamson, David P. Smith^*,||, Denise Galatis^*,||, Robyn A. Sharples^*,, Cyril C. Curtain^*,, Feda’ E. Ali^*,||, Robert A. Cherny^*,||, Janetta G. Culvenor^*,,||, Stephen P. Bottomley, Colin L. Masters^*,||, Kevin J. Barnham^*,|| and Andrew F. Hill^*,

^* Department of Pathology,
Centre for Neuroscience,
Department of Biochemistry and Molecular Biology,
Department of Microbiology and Immunology, The University of Melbourne, Victoria; and
^|| The Mental Health Research Institute of Victoria, Parkville Victoria 3052; and
School of Physics and Materials Engineering; and
Department of Biochemistry and Molecular Biology, Monash University, Victoria, 3168, Australia

¹Correspondence: The University of Melbourne, Department of Pathology, Victoria, 3010, Australia. E-mail: r.cappai{at}unimelb.edu.au

SPECIFIC AIMS

The pathological hallmark of Parkinson’s disease (PD) is the loss of dopamine producing neurons in the substantia nigra and the formation of intraneuronal Lewy bodies. Aggregated -synuclein (-SN) protein is the major component of the Lewy body. The aim of this work is to determine whether DA modulates the behavior of -SN by studying the effect of DA on -SN oligomerization. This would provide an important link between these two PD-associated molecules.

PRINCIPAL FINDINGS

1. Dopamine promotes -SN oligomerization into SDS-resistant soluble oligomeric species
The association of -SN aggregates within dopaminergic neurons in PD prompted us to investigate the effect of DA on -SN oligomerization. Incubating recombinant wild-type human -SN (14 µM) with increasing concentrations of DA for up to 3.5 h caused a time- and dose-dependent increase in SDS-resistant -SN oligomers. DA mediated -SN oligomers are rapidly formed and were readily detected after only 5 min with both 100 and 200 µM DA. While a 1:1 stoichiometry showed no detectable oligomer formation after 3.5 h, an overnight incubation yielded readily detectable DA mediated oligomers, indicating a 1:1 molar ratio can promote -SN oligomerization. The sizes of the -SN oligomers were consistent with assembly of -SN into dimers, trimers, tetramers, and higher molecular weight oligomeric species.

DA mediated -SN oligomers partitioned exclusively into the soluble fraction following centrifugation at 100,000 rpm (Fig. 1 a). In contrast, -SN in the absence of DA did not form oligomers and remained as a monomeric species in the soluble fraction. If -SN was aged for 6 days in the presence of DA, it remained as soluble SDS-stable oligomers (Fig. 1b ). However, aging -SN for 6 days in the absence of DA resulted in insoluble protein with a significant portion partitioned into the pellet fraction, which continued to migrate as a monomeric species on SDS-PAGE (Fig. 1b ). Therefore, DA clearly modulates the oligomerization of -SN into oligomers that are SDS resistant and soluble, in contrast to the SDS-sensitive insoluble aggregates that are formed when -SN was aged in the absence of DA.

View larger version (39K): [in this window] [in a new window]   Figure 1. Sedimentation analysis on the solubility of -SN oligomers formed in the presence of excess DA over 3.5 h (a) and 6 days (b). 14 µM -SN was incubated with 200 µM DA at 37°C, shaking at 1400 rpm. Incubated samples were centrifuged at 100,000 rpm for 30 min, obtaining soluble (S) and insoluble (P) fractions. Fractions were analyzed on 12% SDS-PAGE gel and stained with silver. -SN formed SDS-resistant oligomers in the presence of DA between 1 and 3.5 h and were soluble (a). After 6 days, -SN oligomers were still soluble but the -SN alone reaction did not form any oligomers and was also present in the pellet fraction.

2. DA-mediated -SN oligomers are thioflavin T negative and nonfibrillar
The kinetics of fibril formation was monitored using thioflavin T (ThT) fluorescence. Aggregation of -SN in the absence of DA showed a typical sigmoidal-shaped ThT fluorescence curve, with a lag phase for the first 24 h followed by an overall increase in reactivity after 6 days, indicating -SN amyloid formation. -SN, in the presence of DA displayed no significant increase in ThT fluorescence even after 6 days incubation. This indicated that the DA-mediated -SN oligomers are not amyloidogenic.

Electron microscopy showed that -SN aged for 6 days without DA formed 12 nm wide fibrils with structural characteristics typical of amyloid proteins. However, -SN incubated with DA for 6 days lacked elongated structured fibrils and were instead truncated oligomers, demonstrating that DA-mediated -SN oligomerization inhibited the formation of amyloidogenic fibrils.

3. DA alters the secondary structure of -SN
To determine the structural consequences of DA promoted oligomerization of -SN we studied the secondary structure of -SN by circular dichroism (CD). The CD spectrum of -SN in the absence of DA displayed a negative CD signal in the region of 200 nm, diagnostic of a protein undergoing rapid conformational exchange (i.e., random coil). Incubating -SN with increasing concentrations of DA caused a concomitant loss in random coil, with no increase in either -helix or ß-sheet content (Fig. 2 a). Therefore, DA induced a small amount of ordering of local structure. Titrating LUVs into an aqueous solution of -SN increased the -helical content to 64% (Fig. 2b ). The CD spectra of both the SDS and LUV titrations exhibit an isodichroic point, indicative of a two-state transition between the aqueous random coil and the lipid bound -helical forms of the protein. However, repeating the LUV titration in the presence of 200 µM DA reduced the helical content to only 25% and the isodichroic point was lost (Fig. 2c ). The shape and magnitude of the final spectrum indicated that the folding pathway for lipid bound -SN had been altered and that helical structure was inhibited by DA. In contrast, if -SN was first incubated in the presence of LUV, the addition of DA had only a small effect on the secondary structure of the protein by reducing the -helical content to 59%, suggesting that partitioning into the lipid bilayer protects the protein conformation (Fig. 2d ).

View larger version (26K): [in this window] [in a new window]   Figure 2. CD analysis of -SN plus DA and LUVs. A) Titration of increasing concentrations of DA, in increments of 25 µM up to 200 µM (indicated by the direction of the arrow), into an aqueous solution of 14 µM -SN. This resulted in a concomitant loss in random coil content in the absence of an increase in the CD signals for -helix or ß-sheet. B) Titration of increasing concentrations of LUVs in increments of 0.6 mM up to 4.2 mM (indicated by the direction of the arrows) into an aqueous solution of -SN resulted in the -helical content of the protein increasing. C) Titration of increasing concentrations of LUVs in increments of 0.6 mM up to 4.2 mM (indicated by the direction of the arrows) into an aqueous solution of -SN in the presence of 200 µM DA. (D): Comparing the CD spectra of -SN in aqueous solution (——), in 200 µM DA (— — —) in 200 µM DA, followed by addition of 8 mM LUV (— - - — - -) in 8 mM LUV (- - - - - -) and in 8 mM LUV to which 200 µM DA was added last (— - — - —). Incubation of -SN first in the presence of LUV followed by the addition of 200 µM DA, caused only a small effect on the secondary structure of the protein, reducing the -helical content to 59% suggesting that partitioning into the lipid bilayer protects the protein conformation.

4. Dopamine is an inhibitor of Fe³⁺-mediated -SN fibrilization
Fe³⁺ induces the fibrilization of -SN into ThT-positive species. To determine whether DA can affect Fe mediated -SN fibrilization, 14 µM -SN was incubated with either 200 µM DA, 14 µM Fe³⁺, both DA and Fe³⁺, or alone over a period of 6 days and ThT fluorescence was measured. -SN plus Fe³⁺ displayed a very rapid rate of fibrilization, 4-fold faster compared with -SN alone at day 1, which plateaued by day 3. The addition of DA to the -SN/Fe³⁺ reaction completely inhibited of ThT fluorescence. This clearly establishes that DA is a dominant modulator of -SN oligomerization into nonamyloid species.

CONCLUSIONS AND SIGNIFICANCE

We have identified a novel interaction between DA and -SN that may provide a physiological mechanism for the generation of -SN oligomers in vivo. DA promoted the dose-dependent formation of SDS-stable, soluble, and nonamyloidogenic -SN oligomers that are distinct from the typical fibrillar amyloid aggregates formed in the absence of DA (Fig. 3 ). This is a significant finding from the study and identifies DA having a key function in the initial phase of -SN aggregation distinct from the ability of DA to disassemble -SN fibrils into non-ordered aggregates as recently reported.

View larger version (15K): [in this window] [in a new window]   Figure 3. Model explaining the relationship between DA and -SN oligomerization. DA can act on the initial aggregation of -SN into soluble oligomers as opposed to insoluble amyloid fibrils. In addition, DA can act by disaggregating preformed -SN fibrils.

A pertinent finding of the current study was that -SN oligomers in the presence of Fe³⁺ and DA remained soluble and lacked ThT fluorescence after 6 days incubation. In contrast, -SN with Fe³⁺ alone formed insoluble amyloidogenic fibrils that were SDS sensitive and reactive. Therefore, DA has a specific dominant role in modulating the formation of soluble SDS-resistant -SN oligomers.

In an aqueous environment, -SN is essentially unstructured and displays a rapidly interconverting ensemble of conformations. Despite causing protein oligomerization, incubation of -SN with DA does not generate any defined elements of secondary structure, hence the negative response of these oligomers to thioflavin T. Instead, there is a slight reduction in the coil content, suggesting that any structural changes are small localized effects. The isodichroic point observed by CD when lipid membranes are titrated into aqueous -SN indicates that the change in protein conformation is a simple two-state transition. In the presence of DA, the folding pathway for the transition from coil to the membrane bound -helix is altered and a more complicated folding profile is evident, as shown by loss of the isodichroic point in the CD spectra. There is also a general loss in the helical content, suggesting that the folding into an -helical structure is inhibited.

Our findings suggest that excessive DA in the cytoplasm of neurons can lead to aggregation of -SN, supporting a link between a breakdown in DA homeostasis and PD. What is unknown is whether the DA-mediated -SN oligomers constitute a toxic species. This is important in relation to the emerging hypothesis that toxic-soluble oligomers are the pathogenic molecular species in a number of amyloid diseases, including the Aß peptide in Alzheimer’s disease (AD) and transthyretin in systemic amyloidosis. Our data would support a model linking a breakdown in DA homeostasis to the oligomerization of -SN over time, leading to the neurodegeneration associated with PD.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3437fje;

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This Article Full Text (PDF) Erratum All Versions of this Article: 19/10/1377 04-3437fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Cappai, R. Articles by Hill, A. F. Search for Related Content PubMed PubMed Citation Articles by Cappai, R. Articles by Hill, A. F.