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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online April 1, 2004 as doi:10.1096/fj.03-0770fje. |
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,2
* Department of Chemistry and Biochemistry, University of California, Santa Cruz, California, USA; and
The Parkinson’s Institute, Sunnyvale, California, USA
3Correspondence: Department of Chemistry and Biochemistry, University of California, Santa Cruz, CA 95064, USA. E-mail: enzyme{at}cats.ucsc.edu
SPECIFIC AIMS
Our aims were to 1) show that catecholamines such as dopamine and L-dopa inhibit formation of amyloid fibrils by Aß and 
-synuclein, and disaggregate existing fibrils of Aß or -synuclein, and 2) determine mechanisms of catecholamine inhibition of fibrillation and fibril disaggregation.
PRINCIPAL FINDINGS
1. Catecholamines (dopamine, L-dopa, epinephrine) inhibit fibrillation of Aß and -synuclein
Using a thioflavin T assay, light scattering, EM, and AFM, we show that micromolar concentrations of dopamine, L-dopa, norepinephrine, and epinephrine inhibit in vitro formation of amyloid fibrils of Aß and -synuclein. Amount of inhibition was dependent on concentration of dopamine or L-dopa. For -synuclein, size exclusion chromatography demonstrated that the major product of inhibition of -synuclein is oligomeric and involves covalent modification of the protein. CD and FTIR experiments showed that changes in conformation of soluble -synuclein during fibrillation (increased ß-structure) were absent in the presence of catecholamines. We compared effects of catecholamines on familial Parkinson’s disease -synuclein mutants, A53T and A30P, to wild-type -synuclein and found that the A53T variant was inhibited to a greater extent at lower dopamine concentrations whereas there was little difference between inhibition of A30P and wild-type.
2. Major active forms of dopamine and L-dopa in inhibiting fibrillation are products of oxidation
Catecholamines are very susceptible to oxidation, including formation of quinones and aminochromes. To separate the effects of catecholamines from those of their oxidative breakdown products, we compared inhibitory effects on -synuclein fibrillation of fresh and aged solutions as well as using anaerobic conditions. By using preparations of catecholamines that had been preincubated for 24 h, and by using anaerobic incubation conditions, we show that major inhibitory species are products of catecholamine oxidation. Dopaminochrome and adrenochrome were specifically shown to inhibit -synuclein fibrillation. Coincubation of antioxidants such as ascorbic acid or soluble forms of vitamin E had no effect on dopamine or L-dopa inhibition of fibrillation (ascorbic acid itself caused weak inhibition of fibrillation).
3. Catecholamines disaggregate fibrils of -synuclein and Aß
Addition of micromolar concentrations of dopamine or L-dopa to existing fibrils of -synuclein or Aß led to their disaggregation in a dose-dependent manner, as determined by decrease in thioflavin T (ThT) fluorescence (controls without dopamine showed no changes in ThT fluorescence) (Fig. 1
a, b). Light scattering was used to confirm dopamine-induced dissolution of fibrils. Scattering for the control was essentially unchanged over time, whereas presence of dopamine caused a substantial decrease in scattering, with a rate similar to that observed for decrease in ThT fluorescence for comparable conditions (Fig. 1c
). Analysis of pellets and supernatants by protein concentration and SDS PAGE showed that concentration of protein in the pellet decreased with time at a rate similar to that observed by the decrease in ThT fluorescence, confirming loss of fibrils.
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Direct confirmation of disaggregation of fibrils was obtained from AFM and EM images which showed loss of fibrils in the presence of catecholamines. Aqueous in situ AFM of a single fibril indicated that fibrils begin to disaggregate from the whole length of the fibril in the presence of dopamine, and not just at the ends (Fig. 2
). In absence of dopamine, fibrils were stable. AFM andHPLC experiments demonstrate that end products are oligomers.
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4. Catecholamines disaggregate -synuclein deposits in tissue samples
We also addressed the question of whether L-dopa would disaggregate -synuclein deposits in biological samples. In a recent study, we have shown that formation of -synuclein deposits can be induced in a model in which mice are exposed to the herbicide paraquat. A 20 min soaking of substantia nigra tissue from paraquat-treated mice results in significant loss of thioflavin S-reactive -synuclein (Fig. 2)
, presumably fibrillar -synuclein.
CONCLUSIONS AND SIGNIFICANCE
Aggregation of Aß and -synuclein are established as key factors in development of Alzheimer’s disease and Parkinson’s disease, respectively. Molecules which inhibit aggregation of these polypeptides may lead to therapies that prevent or control the corresponding diseases. Our data indicate that presence of micromolar concentrations of dopamine or L-dopa is sufficient to significantly inhibit fibril formation or disaggregate existing fibrils of Aß or -synuclein in vitro. Concentrations used in our experiments are physiologically relevant.
Our data indicate that the major active species in inhibition of fibrillation by catecholamines is probably a product (or products) of oxidative breakdown, and that inhibition most likely leads to accumulation of a stable oligomer. Catecholamine quinones are expected to be susceptible to nucleophilic attack via Michael addition or imine (Schiff-base) formation. These are likely to be main reactions leading to covalent modification of -synuclein via Lys side-chains under our experimental conditions.
Our findings have important implications for understanding protein deposition diseases, support the hypothesis that common molecular mechanisms may underlie development of pathological features of Parkinson’s and Alzheimer’s diseases, and suggest that common strategies of intervention could benefit patients suffering from these diseases.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0770fje; 
2 These authors contributed equally to this work.
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) µM dopamine, the number of fibrils decreased with increasing time of incubation (monitored by ThT). b) Preformed Aß fibrils (
). In the presence of 150 µM dopamine (o), the number of fibrils decreased with increasing time of incubation. c) As in panel a, but monitored by static light scattering:(•), in the presence of 150 µM dopamine (o), the number of fibrils decreased with increasing time. d) Decrease in number of fibrils as monitored by AFM in the presence of 150 µM dopamine; in absence of dopamine there was no change in coverage over time.
