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Single particle detection and characterization of synuclein co-aggregation
BBRC Biochemical and Biophysical Research Communications 333 (2005) 1202–1210 www.elsevier.com/locate/ybbrc
Single particle detection and characterization of synuclein co-aggregation Armin Giese a,*, Benedikt Bader a, Jan Bieschke a,1, Gregor Schaffar b, Sabine Odoy c, Philipp J. Kahle c, Christian Haass c, Hans Kretzschmar a a
c
Center for Neuropathology and Prion Research, LMU, Munich, Germany b Max-Planck-Institute for Biochemistry, Martinsried, Germany Laboratory of AlzheimerÕs and ParkinsonÕs Disease Research, Department of Biochemistry, LMU, Munich, Germany Received 31 May 2005 Available online 16 June 2005
Abstract Protein aggregation is the key event in a number of human diseases such as AlzheimerÕs and ParkinsonÕs disease. We present a general method to quantify and characterize protein aggregates by dual-colour scanning for intensely fluorescent targets (SIFT). In addition to high sensitivity, this approach offers a unique opportunity to study co-aggregation processes. As the ratio of two fluorescently labelled components can be analysed for each aggregate separately in a homogeneous assay, the molecular composition of aggregates can be studied even in samples containing a mixture of different types of aggregates. Using this method, we could show that wild-type a-synuclein forms co-aggregates with a mutant variant found in familial ParkinsonÕs disease. Moreover, we found a striking increase in aggregate formation at non-equimolar mixing ratios, which may have important therapeutic implications, as lowering the relative amount of aberrant protein may cause an increase of protein aggregation leading to adverse effects. 2005 Elsevier Inc. All rights reserved. Keywords: Synuclein; SIFT; Fluorescence correlation spectroscopy; Amyloid; Aggregation; Alzheimer; Parkinson; Lewy body; Dementia
All common neurodegenerative diseases are characterized by the formation and deposition of fibrillar aggregates of specific proteins such as tau protein and amyloid b-peptide in AlzheimerÕs disease, prion protein in prion diseases, and a-synuclein in ParkinsonÕs disease (PD) [1,2]. These protein deposits have become the mainstay in regard to diagnostic neuropathology [3]. Moreover, these protein aggregates seem to be key players in the pathogenesis of neuronal dysfunction and cell death [4–6]. However, whereas a number of mutations and biochemical modifications have been identified which render these proteins more susceptible to aggrega*
Corresponding author. Fax: +49 89 2180 78037. E-mail address: [email protected] (A. Giese). 1 Present address: The Scripps Research Institute, 10550 N. Torrey Pines Road, BCC 265 La Jolla, CA 92037, USA. 0006-291X/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.06.025
tion, the molecular processes involved in the formation of the specific protein aggregates in vivo are not well understood [7]. Initial evidence for a central role of a-synuclein in the pathogenesis of several neurodegenerative diseases came from the discovery of point mutations in the a-synuclein gene in families with autosomal-dominant familial PD [8,9]. Subsequently, a-synuclein has been identified as the major component of Lewy bodies and in Lewy neurites, which are the characteristic deposits in sporadic ParkinsonÕs disease, dementia with Lewy bodies, Lewy body variant of AlzheimerÕs disease, as well as of the glial cytoplasmatic inclusions that characterize multiple system atrophy [10–12]. Pathological deposition of aggregated a-synuclein was recapitulated in transgenic animal models [13–18]. Moreover, the recent identification of familial PD with triplication of the a-synuclein
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gene locus further corroborates the central role of a-synuclein in the pathogenesis of PD [19]. Studies on the aggregation properties of wild-type (wt) and mutated a-synuclein indicated alterations in the propensity to aggregate due to these mutations [20–22]. However, these studies were typically performed with purified proteins of one type. In contrast, patients affected by familial ParkinsonÕs disease with mutations in the a-synuclein gene are heterozygous for the mutation. Therefore, they can express both mutated and wt a-synuclein. Moreover, a recent report suggests that the ratio of the expression levels of mutated and wt a-synuclein determines severity and onset of disease in a surprising fashion, as relative under-expression of the mutation in lymphoblastoid cell lines was found to be associated with more severe manifestation of disease [23]. This leads to two important but as yet unanswered questions: (i) do mutant a-synuclein and wt a-synuclein co-aggregate into the same fibril? and (ii) does the molar ratio of the components influence aggregate formation? Individual co-aggregating protein species cannot be analysed separately with conventional methods used to study protein aggregation and amyloid formation such as Thioflavin T fluorescence, light scattering, or increase in proteinase K (PK) resistance, since these methods
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only measure the overall amount of aggregated protein. Consequently, these methods do not distinguish between a mixture of aggregates composed of different proteins and aggregates composed of a mixture of different proteins. We thus used a new approach based on single particle analysis by adapting a method derived from fluorescence correlation spectroscopy (FCS). FCS-derived methods allow efficient analysis of protein aggregation in neurodegenerative diseases such as prion diseases at the molecular level [24–30]. Conventional FCS is based on the analysis of fluctuations in fluorescence caused by the diffusion of fluorescently labelled molecules at nanomolar to picomolar concentrations through an open detection volume of approximately 1 fl defined by a focussed laser beam [27]. However, when analysing protein aggregation, the target particles are oligomers and multimers that can become highly labelled by binding multiple probe molecules such as monoclonal antibodies or monomers tagged with a fluorescent dye. Thus, target particles and unbound probe molecules can be separated easily by their relative molecular brightness. Using a two-colour scanning setup, we have developed a highly sensitive quantitative detection method termed ‘‘scanning for intensely fluorescent targets’’ (SIFT) [24,25]. Whereas scanning pri-
Fig. 1. Principle of aggregation analysis by dual-colour SIFT. Following excitation with two different laser lines, two different fluorophores (‘‘green’’ and ‘‘red’’) can be analysed simultaneously in the same focal volume in a confocal setup with single molecule sensitivity. The laser focus is moved through the sample by an optical scanning unit. Whenever an aggregate carrying multiple fluorescent labels passes the focus, this results in a short burst of high fluorescence intensity. Individual aggregates pass the focus at different points in time and can be analysed in regard to labelling ratio.
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marily increases detection efficiency, the use of two independent fluorescent probes allows for analysis of the molecular composition of target particles based on differences in labelling ratio. Because individual aggregates pass the detection volume at different points in time, samples containing a mixture of different types of aggregates can be analysed in regard to the number and relative abundance of different types of aggregates present in the sample (Fig. 1). We applied this technique to the analysis of co-aggregation in mixtures of wt and mutant a-synuclein.
Materials and methods Synuclein expression. The b-synuclein expression vector has been described previously [31]. The coding regions of human wt and A30P mutant a-synuclein [32] were amplified by polymerase chain reaction with primers 5 0 -TTCATTACATATGGATGTATTCATGAAAGG-3 0 and 5 0 -GGAATTCCATATGTTAGGCTTCAGGTTCGTAG-3 0 . Amplimers were subcloned into the NdeI site of pET-5a (Promega; Madison, WI) and constructs used to transform Escherichia coli BL21(DE3) pLys. All constructs were sequenced (Medigenomix, Germany). Bacterial cultures were induced with isopropyl-b-D-thiogalactoside for 4 h and lysed by freeze/thaw and sonication. After 15 min boiling, the heat-stable 17,000g supernatant was loaded onto Q-sepharose (Amersham Biosciences) and eluted with a 25–500 mM salt gradient. The pooled synuclein peak fractions were desalted by Sephacryl S-200 (Amersham Biosciences) gel filtration as described [14]. Fluorescent labelling. Protein labelling was done with amino-reactive fluorescent dyes Alexa Fluor-488-O-succinimidylester (Molecular Probes, USA) and Cyanin-5-O-succinimidylester (Amersham, USA), respectively. A mixture of 1 ll dissolved dye (2 mg/ml in DMSO) and 10 ll synuclein (2 mg/ml) was incubated overnight at 4 C. The reaction was stopped with NH4OH and unbound fluorophores were separated by centrifugation in Microspin columns (MoBiTec, Germany) filled with Sephadex G15 (Pharmacia Biotech, Sweden), equilibrated with 50 mM sodium phosphate buffer containing 0.01% NP40 at pH 6.9 (PN-buffer). Removal of unbound dye molecules and labelling efficiency were confirmed by FCS measurements. The typical labelling ratio achieved was approximately 2 dye molecules per synuclein molecule. Recombinant human prion protein (rPrP) was produced and labelled as described [26]. Aggregation assay. Preformed aggregates were removed from the synuclein stock solution (2 mg/ml) by ultrafiltration using a Nanosept 100K Omega filter (PALL, USA). Then, the stock solution was spiked with labelled synuclein at a molar ratio of approximately 1000:1 (unlabelled:labelled). If not otherwise specified, final synuclein concentration was 1 mg/ml and duplicates were used. Protein aggregation was achieved by incubation at 37 C in PN-buffer with constant agitation of 800 rpm in 200 ll reaction tubes (Kisker, Germany). To reduce evaporation in experiments with small sample volumes, 24-wellAssay-Chips (Evotec-Technologies, Germany) sealed with adhesive film were used in some experiments. In co-aggregation experiments, stock solutions of wt a-synuclein and A30P mutant a-synuclein were mixed after ultrafiltration in different molar ratios as indicated in Fig. 5 and diluted to a final total protein concentration of 1 mg/ml in PN buffer. Then a probe mix containing equimolar amounts of wt a-synuclein labelled with Alexa488 (‘‘green’’) and A30P mutant a-synuclein labelled with Cy5 (‘‘red’’) was added. Incubation was performed as described above. FCS/SIFT measurements and analysis. FCS and SIFT measurements were carried out on an Insight Reader (Evotec-Technologies, Germany) with dual-colour excitation at 488 and 633 nm, using a 40·
1.2 NA microscope objective (Olympus, Japan) and a pinhole diameter of 70 lm at FIDA setting. Excitation power was 100 lW at 488 nm and 150 lW at 633 nm. For SIFT measurements, scanning parameters were set to 100 lm scan path length, 50 Hz beamscanner frequency, 2000 lm positioning table movement, and five repeats of 20 s each. This is equivalent to approximately 10 mm/s scanning speed. The temperature for all measurements was 22 C. The fluorescence data were analysed by auto-correlation analysis using the FCSPPEvaluation software version 2.0 (Evotec-Technologies, Germany). Fluorescence intensity data were acquired with 40 ls bin width using the Insight Reader software. The frequency of specific combinations of ‘‘green’’ and ‘‘red’’ photon counts per bin was recorded in a two-dimensional intensity distribution histogram H I r ;I g with intensity values Ir, Ig from 0 to 255 photons/bin or, in some experiments, with Ir, Ig from 0 to 2000 photons/bin and a bin width of 150 ls [24] (Fig. 1). Data processing and the analysis of serial measurements were performed using a suite of software routines (XSMmanager). The intensity histogram was analysed as described previously [24,25] to detect quantitatively the signal of highly labelled targets over a fluorescent background of unbound probe molecules. Signal with fluorescent intensity in the green and the red detection channel (Ig, Ir) was cut off by a threshold value (T). To determine the number of high intensity bins (N), all frequencies hI r ;I g with ðI 2g þ I 2r P T 2 Þ were added. The number of high-intensity photons (P) was determined as the sum of hI r ;I g ðI r þ I g Þ with ðI 2g þ I 2r P T 2 Þ. Analysis was restricted to aggregates labelled by both the ‘‘green’’ and the ‘‘red’’ probe by excluding bins with an intensity ratio of Ir/Ig > 5:1 and Ir/Ig < 1:5, respectively. For each aggregation experiment the threshold value was calibrated using the measurement data at t = 0. In experiments on mixing ratios, wt and mutant synuclein (1:0, 5:1, 2:1, 1:1, 1:2, 1:5, and 0:1) were incubated in 3–4 separate wells during two series of experiments. Each well was measured five times and the average was then normalized for each series hP well i ¼ P well =P against the average signal P of all measurements from one series. Thioflavin T measurements. For fluorescence measurements quartz cuvettes (Perkin Elmer) with a 1 cm excitation path were used in a luminescence spectrometer LS-55 (Perkin Elmer) equipped with a Xelamp for excitation. The cuvettes were filled with 470 ll PN-buffer, 25 ll ThT stock solution (1 mM in water) and a 5 ll aliquot of the protein aggregation assay under constant stirring as described [33]. Fluorescence emission was measured at 490 nm. Electron microscopy. Synuclein samples were diluted in Hepes (pH 7.5) and adsorbed to copper grids (100 · 400 mesh; Plano, Germany), stained with 2% uranyl acetate, and viewed in a Philips EM420 electron microscope [34].
Results and discussion Highly sensitive detection of a-synuclein aggregates by SIFT For validation of the SIFT aggregation assay, we used a well-established in vitro model of a-synuclein aggregation [15]. After a lag phase of several days, incubation of recombinant a-synuclein at a concentration of 1 mg/ml resulted in the formation of aggregates with amyloid features, which could be detected by SIFT analysis as well as by a conventional Thioflavin T fluorescence assay (Fig. 2), consistent with a process of seeded aggregation, the standard model of amyloid formation [35]. The long lag phase indicates that the
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Fig. 2. Time course of a-synuclein aggregation. a-Synuclein was incubated at a concentration of 1 mg/ml. At various time points, aliquots of the same sample were analysed by (A) Thioflavin T fluorescence assay and (B) SIFT analysis. (A) The mean of duplicate measurements is given, (B) means ± SE of five measurements is shown. Serial dilutions of aliquots taken at day 7 as well as aliquots taken at day 0 and buffer samples were analysed by (C) Thioflavin T fluorescence and (D) SIFT analysis.
a-synuclein solution was essentially free of preformed aggregates at day 0, so that we were able to study true de novo aggregation. We attribute this to the fact that a-synuclein solutions were subjected to ultrafiltration before incubation, as we observed a shorter lag phase in experiments omitting this step (data not shown). In order to label a-synuclein aggregates for detection by our SIFT method, we spiked the a-synuclein solution with fluorescently tagged a-synuclein. As we used a-synuclein tagged with a ‘‘green’’ fluorescent dye (Alexa-488) and a-synuclein tagged with a ‘‘red’’ fluorescent dye (Cy5) simultaneously, incorporation of these probe molecules into newly formed aggregates resulted in particles that were highly labelled with both colours. For our experiments we used a molar ratio of labelled to unlabelled a-synuclein of approximately 1:1000 for two main reasons: (1) At significantly higher concentrations of labelled a-synuclein, the background fluorescence in the detection volume would interfere with aggregate detection. (2) At significantly lower molar ratios, the number of fluorescent labels per aggregate would be below our detection threshold, at least for smaller aggregates. In addition, we reasoned that the vast excess of unlabelled a-synuclein should reduce any potential interference of the fluorescent label with the aggregation process. In fact, in control experiments the amount of amyloid formation measured by Thioflavin T fluorescence in non-
spiked solutions of a-synuclein was similar to the amount found in spiked solutions. Furthermore, in control experiments, aggregate formation was also found in solutions containing only labelled a-synuclein, suggesting that fluorescent labelling did not result in a relevant change in aggregation properties. When we studied a-synuclein aggregation using SIFT, we found a similar time course as in the Thioflavin T measurements (Fig. 2), which further corroborates the validity of the results obtained by SIFT measurements. In order to compare SIFT measurements with the conventional Thioflavin T assay in regard to sensitivity, we studied a dilution series (Figs. 2C and D). Whereas with Thioflavin T only dilutions up to 1:10 were different from the background fluorescence of control measurements, we could clearly detect a-synuclein aggregates in dilutions up to 1:1000 of the same sample using SIFT analysis. This indicates that SIFT is about two orders of magnitude more sensitive than the conventional Thioflavin T assay. Specific formation of fibrillar aggregates Thioflavin contrast, our of multimeric of multimeric
T binds to amyloid-like structures. In SIFT assay is based on the detection aggregates. Whereas all amyloid consists aggregates, not all aggregates necessarily
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need to have the structural characteristics of amyloid. In order to corroborate that the aggregates measured by us using SIFT correspond to the amyloid-like fibrillar aggregates found in previous in vitro studies and in vivo, we combined SIFT measurements with PK digestion (Figs. 3A and B). Relative PK resistance has been described as a characteristic feature of pathological a-synuclein aggregates [15,36]. As PK had only a minor effect on the amount of aggregates detected by SIFT, we conclude that most of the signal is due to typical amyloid-like fibrillar aggregates. This is supported by EM studies performed in parallel experiments (Fig. 3C). To further validate that the aggregation process seen in our SIFT measurements corresponds to the formation of pathogenic fibrillar aggregates as seen in other studies, we added unlabelled b-synuclein to the mixture of unlabelled and labelled a-synuclein (Figs. 3D–F). bSynuclein has been previously shown to inhibit the aggregation of a-synuclein [37,38]. At a molar ratio of 1:1, b-synuclein inhibited the formation of large asynuclein aggregates almost completely. A significant inhibition was already seen at a molar ratio of 1:10. Thus, SIFT analysis provides a highly sensitive method to study protein aggregation and may become a powerful technique for high-throughput screening of anti-aggregative drugs.
Co-aggregation analysis by dual-colour SIFT In addition to sensitivity, dual-colour SIFT offers a unique opportunity to study co-aggregation processes. Because individual aggregates pass the detection volume at different points in time, the labelling ratio of two independent probes can be analysed for each aggregate separately in dual-colour measurements. Thus, the molecular composition of aggregates can be studied even in samples containing a mixture of different types of aggregates. To accurately model the situation in patients carrying an a-synuclein mutation linked to familial ParkinsonÕs disease, we analysed aggregate formation in mixtures of wt a-synuclein and a-synuclein carrying an A30P mutation. First, we addressed the question of whether these two molecules form separate fibrils or co-aggregate within a fibril. Following co-incubation, we only found aggregates yielding high fluorescence intensity both in the ‘‘green’’ and in the ‘‘red’’ channel (Fig. 4B), which were absent at the starting time of the incubation (Fig. 4A). This indicates that all aggregates were formed by co-aggregation of wt and mutant a-synuclein. In contrast, when wt a-synuclein spiked with ‘‘green’’ probe molecules and mutant a-synuclein spiked with ‘‘red’’ probe molecules were incubated separately and then analysed immediately after mixing, the different
Fig. 3. Specificity of a-synuclein aggregate formation. a-Synuclein was aggregated at 1 mg/ml for 7 days, diluted 1:100 with PN-buffer and incubated for 2 h (A) without or (B) with 1 lg/ml PK. a-Synuclein aggregates exhibit PK resistance. (C) Electron micrograph of a-synuclein aggregates obtained in an independent parallel experiment shows typical fibrillar aggregates. (D–F) a-Synuclein was incubated for 7 days in the presence of different concentrations of b-synuclein resulting in a dose-dependent inhibition of aggregate formation. Intensity histograms were recorded for 10 · 30 s with a bin width of 150 ls.
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Fig. 4. Synuclein co-aggregation analysis by dual-colour SIFT. (A) An equimolar mixture of wt a-synuclein spiked with wt a-synuclein-Alexa-488 (‘‘green’’) and A30P a-synuclein spiked with A30P a-synuclein-Cy5 (‘‘red’’) at a final total protein concentration of 1 mg/ml measured at day 0 exhibits only low fluorescence intensity in the ‘‘green’’ and ‘‘red’’ channel due to the stochastically fluctuating number of labelled monomers present in the detection volume at any time point. (B) After 7 days of incubation, co-aggregation of wt and mutant a-synuclein yielded large aggregates carrying multiple ‘‘green’’ and ‘‘red’’ labels on the same particle resulting in a corresponding number of bins with high fluorescence intensity in both the ‘‘green’’ and the ‘‘red’’ channel simultaneously. (C) When wt a-synuclein spiked with wt a-synuclein-Alexa-488 (‘‘green’’) and A30P a-synuclein spiked with A30P a-synuclein-Cy5 (‘‘red’’) were incubated in separate wells for 7 days and then mixed and measured, only pure wt aggregates and pure A30P aggregates are present that result in high intensity signal in only the green or only the red channel when passing the laser focus, respectively. (D) In a mixture of wt a-synuclein spiked with wt a-synuclein-Alexa-488 and prion protein labelled with Cy5 co-incubated for 7 days, pure prion protein aggregates can be detected in addition to more abundant particles consisting of aggregated a-synuclein and some bound prion protein. On the left, colour-coded 2D intensity histograms recorded for 5 · 20 s with a bin width of 40 ls are shown. On the right, a schematic representation of the types of particles present in the respective samples is given. The light grey circle indicates the open detection volume defined by the laser focus. , wt a-synuclein monomer; , wt a-synuclein monomer labelled with Alexa-Fluor-488; , a-synuclein A30P mutant monomer; , a-synuclein A30P mutant monomer labelled with Cy5; , prion protein monomer labelled with Cy5. Aggregates are symbolized by groups of monomers. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this paper.)
aggregate species carrying only one type of fluorescent label were clearly discernible (Fig. 4C). Furthermore, when we mixed another protein capable of forming amyloid-like aggregates, i.e., prion protein tagged with a ‘‘red’’ label, with a-synuclein spiked with ‘‘green’’ probe molecules, we found that at least two different types of aggregates were formed during co-incubation: in addition to a-synuclein aggregates that also contained prion protein molecules, pure prion protein aggregates were detected (Fig. 4D). Taken together, these findings show that mixtures of different aggregate species can form in co-incubation experiments with amyloidogenic proteins, and that these aggregates can be clearly separated from aggregates derived from coaggregation by dual-colour SIFT analysis. Ratio of wt and mutant a-synuclein determines aggregate formation In addition to the qualitative aspect of co-aggregation, we were interested in potential quantitative effects
of the interaction of wt and mutant a-synuclein. When we analysed mixtures of wt and mutant a-synuclein, we found a striking and unexpected increase in aggregate formation at specific molar ratios. Whereas aggregate formation in equimolar mixtures was in the range of what we found in solutions of either wt a-synuclein or mutant a-synuclein alone, ‘‘asymmetric’’ mixing ratios with a 2- to 5-fold excess of either of the two molecules favoured aggregate formation in our experiments. This resulted in an M-shaped dependence of aggregation on mixing ratio (Fig. 5). Comparing the results for the number of bins above cut-off level (Fig. 5A), primarily a measure of aggregate concentration, and the number of photons contained in bins above cut-off (Fig. 5B), primarily a measure of the amount of synuclein molecules contained in aggregates, indicates that the average size of the aggregates is unchanged. This suggests that the observed effect is mainly due to differences in the number of aggregates formed. Our findings in vitro may provide an explanation for a surprising observation in patients affected by
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are small oligomeric aggregation intermediates as indicated by several studies [39–42], these oligomers may differ in regard to structure, stability, and propensity to form larger aggregates depending on molar ratios of monomers [43]. Further evidence in this direction comes from our results using Thioflavin T fluorescence. Similar to the results of SIFT measurements, more aggregation was detected at ‘‘asymmetric’’ mixing ratios. However, whereas the amount of aggregates was the highest at mixing ratios of 1:5 and 5:1, the amount of amyloid-like structures was increased especially at a 2-fold excess of either of the two monomers. This indicates that the molar ratio has not only quantitative but also qualitative effects on the aggregation process, similar to the effects described for variations in buffer conditions [44]. Oligomers of particular molar ratios may be favored to be integrated into fibrils and to form amyloid whereas others may be excluded. Thus, varying mixing ratios may provide a means to specifically favour specific types of aggregates or intermediates that can than be used for further studies.
Conclusions
Fig. 5. Aggregate formation in mixtures of WT a-synuclein and A30P mutant synuclein depends on mixing ratio. Diagrams show the relative level of aggregate formation after 7 days of incubation in samples with different mixing ratios but same total protein concentration. Samples were analysed in parallel with different assays: (A) SIFT signal (N) indicates the number of bins above cut-off level. (B) SIFT signal (P) was generated by summing up the photon count from the ‘‘green’’ and ‘‘red’’ channel for all bins above cut-off level. (C) Thioflavin T fluorescence corresponds to the total amount of amyloid formation. Three to four independent experiments out of two separate series were normalized as described under Materials and methods. The mean and standard error is shown.
either an A30P or A53T mutation that was published recently. Corresponding to the levels of aggregate formation in our in vitro assay, only mild disease was found in individuals showing a roughly equimolar expression level of wt and mutant a-synuclein, whereas relative under-expression of the mutant at ratios of about 1:2 to 1:9 was associated with severe disease phenotype [23]. Currently, we cannot provide a comprehensive explanation for the striking difference between equimolar and ‘‘asymmetric’’ conditions at the molecular level. However, one could hypothesize that, if there
Formation of pathological amyloid aggregates in vivo takes place in a complex environment. Thus, inhibitory and amplifying effects from a variety of molecular interactions can affect this process. Co-aggregation of various species of amyloidogenic proteins may turn out to be an important determining factor for the manifestation of disease in humans [31]. We could show that in the case of a-synuclein, subtle variations in the molar ratio of different molecular species have a striking effect on aggregation. This may have important therapeutic implications for neurodegenerative aggregation diseases in general, as the lowering of the amount of aberrant protein variants may also have non-beneficial effects. SIFT analysis provides a powerful tool to study these co-aggregation processes, as—in addition to sensitivity—dual-colour analysis on the single particle level allows to study the molecular composition of aggregates even in samples containing a mixture of different types of aggregates. As SIFT measurements can be performed highly automated in homogeneous assay conditions with small sample volumes, this approach also provides a rapid and robust readout for high-throughput screening of anti-aggregative drugs [30].
Acknowledgments This work was supported by the Deutsche Forschungsgemeinschaft (Grant HA1737 and Special Program Grant SFB596) and by the European Commission (Grant QLK3-CT-2001-02345).
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Single particle detection and characterization of synuclein co-aggregation Armin Giese a,*, Benedikt Bader a, Jan Bieschke a,1, Gregor Schaffar b, Sabine Odoy c, Philipp J. Kahle c, Christian Haass c, Hans Kretzschmar a a
c
Center for Neuropathology and Prion Research, LMU, Munich, Germany b Max-Planck-Institute for Biochemistry, Martinsried, Germany Laboratory of AlzheimerÕs and ParkinsonÕs Disease Research, Department of Biochemistry, LMU, Munich, Germany Received 31 May 2005 Available online 16 June 2005
Abstract Protein aggregation is the key event in a number of human diseases such as AlzheimerÕs and ParkinsonÕs disease. We present a general method to quantify and characterize protein aggregates by dual-colour scanning for intensely fluorescent targets (SIFT). In addition to high sensitivity, this approach offers a unique opportunity to study co-aggregation processes. As the ratio of two fluorescently labelled components can be analysed for each aggregate separately in a homogeneous assay, the molecular composition of aggregates can be studied even in samples containing a mixture of different types of aggregates. Using this method, we could show that wild-type a-synuclein forms co-aggregates with a mutant variant found in familial ParkinsonÕs disease. Moreover, we found a striking increase in aggregate formation at non-equimolar mixing ratios, which may have important therapeutic implications, as lowering the relative amount of aberrant protein may cause an increase of protein aggregation leading to adverse effects. 2005 Elsevier Inc. All rights reserved. Keywords: Synuclein; SIFT; Fluorescence correlation spectroscopy; Amyloid; Aggregation; Alzheimer; Parkinson; Lewy body; Dementia
All common neurodegenerative diseases are characterized by the formation and deposition of fibrillar aggregates of specific proteins such as tau protein and amyloid b-peptide in AlzheimerÕs disease, prion protein in prion diseases, and a-synuclein in ParkinsonÕs disease (PD) [1,2]. These protein deposits have become the mainstay in regard to diagnostic neuropathology [3]. Moreover, these protein aggregates seem to be key players in the pathogenesis of neuronal dysfunction and cell death [4–6]. However, whereas a number of mutations and biochemical modifications have been identified which render these proteins more susceptible to aggrega*
Corresponding author. Fax: +49 89 2180 78037. E-mail address: [email protected] (A. Giese). 1 Present address: The Scripps Research Institute, 10550 N. Torrey Pines Road, BCC 265 La Jolla, CA 92037, USA. 0006-291X/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.06.025
tion, the molecular processes involved in the formation of the specific protein aggregates in vivo are not well understood [7]. Initial evidence for a central role of a-synuclein in the pathogenesis of several neurodegenerative diseases came from the discovery of point mutations in the a-synuclein gene in families with autosomal-dominant familial PD [8,9]. Subsequently, a-synuclein has been identified as the major component of Lewy bodies and in Lewy neurites, which are the characteristic deposits in sporadic ParkinsonÕs disease, dementia with Lewy bodies, Lewy body variant of AlzheimerÕs disease, as well as of the glial cytoplasmatic inclusions that characterize multiple system atrophy [10–12]. Pathological deposition of aggregated a-synuclein was recapitulated in transgenic animal models [13–18]. Moreover, the recent identification of familial PD with triplication of the a-synuclein
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gene locus further corroborates the central role of a-synuclein in the pathogenesis of PD [19]. Studies on the aggregation properties of wild-type (wt) and mutated a-synuclein indicated alterations in the propensity to aggregate due to these mutations [20–22]. However, these studies were typically performed with purified proteins of one type. In contrast, patients affected by familial ParkinsonÕs disease with mutations in the a-synuclein gene are heterozygous for the mutation. Therefore, they can express both mutated and wt a-synuclein. Moreover, a recent report suggests that the ratio of the expression levels of mutated and wt a-synuclein determines severity and onset of disease in a surprising fashion, as relative under-expression of the mutation in lymphoblastoid cell lines was found to be associated with more severe manifestation of disease [23]. This leads to two important but as yet unanswered questions: (i) do mutant a-synuclein and wt a-synuclein co-aggregate into the same fibril? and (ii) does the molar ratio of the components influence aggregate formation? Individual co-aggregating protein species cannot be analysed separately with conventional methods used to study protein aggregation and amyloid formation such as Thioflavin T fluorescence, light scattering, or increase in proteinase K (PK) resistance, since these methods
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only measure the overall amount of aggregated protein. Consequently, these methods do not distinguish between a mixture of aggregates composed of different proteins and aggregates composed of a mixture of different proteins. We thus used a new approach based on single particle analysis by adapting a method derived from fluorescence correlation spectroscopy (FCS). FCS-derived methods allow efficient analysis of protein aggregation in neurodegenerative diseases such as prion diseases at the molecular level [24–30]. Conventional FCS is based on the analysis of fluctuations in fluorescence caused by the diffusion of fluorescently labelled molecules at nanomolar to picomolar concentrations through an open detection volume of approximately 1 fl defined by a focussed laser beam [27]. However, when analysing protein aggregation, the target particles are oligomers and multimers that can become highly labelled by binding multiple probe molecules such as monoclonal antibodies or monomers tagged with a fluorescent dye. Thus, target particles and unbound probe molecules can be separated easily by their relative molecular brightness. Using a two-colour scanning setup, we have developed a highly sensitive quantitative detection method termed ‘‘scanning for intensely fluorescent targets’’ (SIFT) [24,25]. Whereas scanning pri-
Fig. 1. Principle of aggregation analysis by dual-colour SIFT. Following excitation with two different laser lines, two different fluorophores (‘‘green’’ and ‘‘red’’) can be analysed simultaneously in the same focal volume in a confocal setup with single molecule sensitivity. The laser focus is moved through the sample by an optical scanning unit. Whenever an aggregate carrying multiple fluorescent labels passes the focus, this results in a short burst of high fluorescence intensity. Individual aggregates pass the focus at different points in time and can be analysed in regard to labelling ratio.
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marily increases detection efficiency, the use of two independent fluorescent probes allows for analysis of the molecular composition of target particles based on differences in labelling ratio. Because individual aggregates pass the detection volume at different points in time, samples containing a mixture of different types of aggregates can be analysed in regard to the number and relative abundance of different types of aggregates present in the sample (Fig. 1). We applied this technique to the analysis of co-aggregation in mixtures of wt and mutant a-synuclein.
Materials and methods Synuclein expression. The b-synuclein expression vector has been described previously [31]. The coding regions of human wt and A30P mutant a-synuclein [32] were amplified by polymerase chain reaction with primers 5 0 -TTCATTACATATGGATGTATTCATGAAAGG-3 0 and 5 0 -GGAATTCCATATGTTAGGCTTCAGGTTCGTAG-3 0 . Amplimers were subcloned into the NdeI site of pET-5a (Promega; Madison, WI) and constructs used to transform Escherichia coli BL21(DE3) pLys. All constructs were sequenced (Medigenomix, Germany). Bacterial cultures were induced with isopropyl-b-D-thiogalactoside for 4 h and lysed by freeze/thaw and sonication. After 15 min boiling, the heat-stable 17,000g supernatant was loaded onto Q-sepharose (Amersham Biosciences) and eluted with a 25–500 mM salt gradient. The pooled synuclein peak fractions were desalted by Sephacryl S-200 (Amersham Biosciences) gel filtration as described [14]. Fluorescent labelling. Protein labelling was done with amino-reactive fluorescent dyes Alexa Fluor-488-O-succinimidylester (Molecular Probes, USA) and Cyanin-5-O-succinimidylester (Amersham, USA), respectively. A mixture of 1 ll dissolved dye (2 mg/ml in DMSO) and 10 ll synuclein (2 mg/ml) was incubated overnight at 4 C. The reaction was stopped with NH4OH and unbound fluorophores were separated by centrifugation in Microspin columns (MoBiTec, Germany) filled with Sephadex G15 (Pharmacia Biotech, Sweden), equilibrated with 50 mM sodium phosphate buffer containing 0.01% NP40 at pH 6.9 (PN-buffer). Removal of unbound dye molecules and labelling efficiency were confirmed by FCS measurements. The typical labelling ratio achieved was approximately 2 dye molecules per synuclein molecule. Recombinant human prion protein (rPrP) was produced and labelled as described [26]. Aggregation assay. Preformed aggregates were removed from the synuclein stock solution (2 mg/ml) by ultrafiltration using a Nanosept 100K Omega filter (PALL, USA). Then, the stock solution was spiked with labelled synuclein at a molar ratio of approximately 1000:1 (unlabelled:labelled). If not otherwise specified, final synuclein concentration was 1 mg/ml and duplicates were used. Protein aggregation was achieved by incubation at 37 C in PN-buffer with constant agitation of 800 rpm in 200 ll reaction tubes (Kisker, Germany). To reduce evaporation in experiments with small sample volumes, 24-wellAssay-Chips (Evotec-Technologies, Germany) sealed with adhesive film were used in some experiments. In co-aggregation experiments, stock solutions of wt a-synuclein and A30P mutant a-synuclein were mixed after ultrafiltration in different molar ratios as indicated in Fig. 5 and diluted to a final total protein concentration of 1 mg/ml in PN buffer. Then a probe mix containing equimolar amounts of wt a-synuclein labelled with Alexa488 (‘‘green’’) and A30P mutant a-synuclein labelled with Cy5 (‘‘red’’) was added. Incubation was performed as described above. FCS/SIFT measurements and analysis. FCS and SIFT measurements were carried out on an Insight Reader (Evotec-Technologies, Germany) with dual-colour excitation at 488 and 633 nm, using a 40·
1.2 NA microscope objective (Olympus, Japan) and a pinhole diameter of 70 lm at FIDA setting. Excitation power was 100 lW at 488 nm and 150 lW at 633 nm. For SIFT measurements, scanning parameters were set to 100 lm scan path length, 50 Hz beamscanner frequency, 2000 lm positioning table movement, and five repeats of 20 s each. This is equivalent to approximately 10 mm/s scanning speed. The temperature for all measurements was 22 C. The fluorescence data were analysed by auto-correlation analysis using the FCSPPEvaluation software version 2.0 (Evotec-Technologies, Germany). Fluorescence intensity data were acquired with 40 ls bin width using the Insight Reader software. The frequency of specific combinations of ‘‘green’’ and ‘‘red’’ photon counts per bin was recorded in a two-dimensional intensity distribution histogram H I r ;I g with intensity values Ir, Ig from 0 to 255 photons/bin or, in some experiments, with Ir, Ig from 0 to 2000 photons/bin and a bin width of 150 ls [24] (Fig. 1). Data processing and the analysis of serial measurements were performed using a suite of software routines (XSMmanager). The intensity histogram was analysed as described previously [24,25] to detect quantitatively the signal of highly labelled targets over a fluorescent background of unbound probe molecules. Signal with fluorescent intensity in the green and the red detection channel (Ig, Ir) was cut off by a threshold value (T). To determine the number of high intensity bins (N), all frequencies hI r ;I g with ðI 2g þ I 2r P T 2 Þ were added. The number of high-intensity photons (P) was determined as the sum of hI r ;I g ðI r þ I g Þ with ðI 2g þ I 2r P T 2 Þ. Analysis was restricted to aggregates labelled by both the ‘‘green’’ and the ‘‘red’’ probe by excluding bins with an intensity ratio of Ir/Ig > 5:1 and Ir/Ig < 1:5, respectively. For each aggregation experiment the threshold value was calibrated using the measurement data at t = 0. In experiments on mixing ratios, wt and mutant synuclein (1:0, 5:1, 2:1, 1:1, 1:2, 1:5, and 0:1) were incubated in 3–4 separate wells during two series of experiments. Each well was measured five times and the average was then normalized for each series hP well i ¼ P well =P against the average signal P of all measurements from one series. Thioflavin T measurements. For fluorescence measurements quartz cuvettes (Perkin Elmer) with a 1 cm excitation path were used in a luminescence spectrometer LS-55 (Perkin Elmer) equipped with a Xelamp for excitation. The cuvettes were filled with 470 ll PN-buffer, 25 ll ThT stock solution (1 mM in water) and a 5 ll aliquot of the protein aggregation assay under constant stirring as described [33]. Fluorescence emission was measured at 490 nm. Electron microscopy. Synuclein samples were diluted in Hepes (pH 7.5) and adsorbed to copper grids (100 · 400 mesh; Plano, Germany), stained with 2% uranyl acetate, and viewed in a Philips EM420 electron microscope [34].
Results and discussion Highly sensitive detection of a-synuclein aggregates by SIFT For validation of the SIFT aggregation assay, we used a well-established in vitro model of a-synuclein aggregation [15]. After a lag phase of several days, incubation of recombinant a-synuclein at a concentration of 1 mg/ml resulted in the formation of aggregates with amyloid features, which could be detected by SIFT analysis as well as by a conventional Thioflavin T fluorescence assay (Fig. 2), consistent with a process of seeded aggregation, the standard model of amyloid formation [35]. The long lag phase indicates that the
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Fig. 2. Time course of a-synuclein aggregation. a-Synuclein was incubated at a concentration of 1 mg/ml. At various time points, aliquots of the same sample were analysed by (A) Thioflavin T fluorescence assay and (B) SIFT analysis. (A) The mean of duplicate measurements is given, (B) means ± SE of five measurements is shown. Serial dilutions of aliquots taken at day 7 as well as aliquots taken at day 0 and buffer samples were analysed by (C) Thioflavin T fluorescence and (D) SIFT analysis.
a-synuclein solution was essentially free of preformed aggregates at day 0, so that we were able to study true de novo aggregation. We attribute this to the fact that a-synuclein solutions were subjected to ultrafiltration before incubation, as we observed a shorter lag phase in experiments omitting this step (data not shown). In order to label a-synuclein aggregates for detection by our SIFT method, we spiked the a-synuclein solution with fluorescently tagged a-synuclein. As we used a-synuclein tagged with a ‘‘green’’ fluorescent dye (Alexa-488) and a-synuclein tagged with a ‘‘red’’ fluorescent dye (Cy5) simultaneously, incorporation of these probe molecules into newly formed aggregates resulted in particles that were highly labelled with both colours. For our experiments we used a molar ratio of labelled to unlabelled a-synuclein of approximately 1:1000 for two main reasons: (1) At significantly higher concentrations of labelled a-synuclein, the background fluorescence in the detection volume would interfere with aggregate detection. (2) At significantly lower molar ratios, the number of fluorescent labels per aggregate would be below our detection threshold, at least for smaller aggregates. In addition, we reasoned that the vast excess of unlabelled a-synuclein should reduce any potential interference of the fluorescent label with the aggregation process. In fact, in control experiments the amount of amyloid formation measured by Thioflavin T fluorescence in non-
spiked solutions of a-synuclein was similar to the amount found in spiked solutions. Furthermore, in control experiments, aggregate formation was also found in solutions containing only labelled a-synuclein, suggesting that fluorescent labelling did not result in a relevant change in aggregation properties. When we studied a-synuclein aggregation using SIFT, we found a similar time course as in the Thioflavin T measurements (Fig. 2), which further corroborates the validity of the results obtained by SIFT measurements. In order to compare SIFT measurements with the conventional Thioflavin T assay in regard to sensitivity, we studied a dilution series (Figs. 2C and D). Whereas with Thioflavin T only dilutions up to 1:10 were different from the background fluorescence of control measurements, we could clearly detect a-synuclein aggregates in dilutions up to 1:1000 of the same sample using SIFT analysis. This indicates that SIFT is about two orders of magnitude more sensitive than the conventional Thioflavin T assay. Specific formation of fibrillar aggregates Thioflavin contrast, our of multimeric of multimeric
T binds to amyloid-like structures. In SIFT assay is based on the detection aggregates. Whereas all amyloid consists aggregates, not all aggregates necessarily
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need to have the structural characteristics of amyloid. In order to corroborate that the aggregates measured by us using SIFT correspond to the amyloid-like fibrillar aggregates found in previous in vitro studies and in vivo, we combined SIFT measurements with PK digestion (Figs. 3A and B). Relative PK resistance has been described as a characteristic feature of pathological a-synuclein aggregates [15,36]. As PK had only a minor effect on the amount of aggregates detected by SIFT, we conclude that most of the signal is due to typical amyloid-like fibrillar aggregates. This is supported by EM studies performed in parallel experiments (Fig. 3C). To further validate that the aggregation process seen in our SIFT measurements corresponds to the formation of pathogenic fibrillar aggregates as seen in other studies, we added unlabelled b-synuclein to the mixture of unlabelled and labelled a-synuclein (Figs. 3D–F). bSynuclein has been previously shown to inhibit the aggregation of a-synuclein [37,38]. At a molar ratio of 1:1, b-synuclein inhibited the formation of large asynuclein aggregates almost completely. A significant inhibition was already seen at a molar ratio of 1:10. Thus, SIFT analysis provides a highly sensitive method to study protein aggregation and may become a powerful technique for high-throughput screening of anti-aggregative drugs.
Co-aggregation analysis by dual-colour SIFT In addition to sensitivity, dual-colour SIFT offers a unique opportunity to study co-aggregation processes. Because individual aggregates pass the detection volume at different points in time, the labelling ratio of two independent probes can be analysed for each aggregate separately in dual-colour measurements. Thus, the molecular composition of aggregates can be studied even in samples containing a mixture of different types of aggregates. To accurately model the situation in patients carrying an a-synuclein mutation linked to familial ParkinsonÕs disease, we analysed aggregate formation in mixtures of wt a-synuclein and a-synuclein carrying an A30P mutation. First, we addressed the question of whether these two molecules form separate fibrils or co-aggregate within a fibril. Following co-incubation, we only found aggregates yielding high fluorescence intensity both in the ‘‘green’’ and in the ‘‘red’’ channel (Fig. 4B), which were absent at the starting time of the incubation (Fig. 4A). This indicates that all aggregates were formed by co-aggregation of wt and mutant a-synuclein. In contrast, when wt a-synuclein spiked with ‘‘green’’ probe molecules and mutant a-synuclein spiked with ‘‘red’’ probe molecules were incubated separately and then analysed immediately after mixing, the different
Fig. 3. Specificity of a-synuclein aggregate formation. a-Synuclein was aggregated at 1 mg/ml for 7 days, diluted 1:100 with PN-buffer and incubated for 2 h (A) without or (B) with 1 lg/ml PK. a-Synuclein aggregates exhibit PK resistance. (C) Electron micrograph of a-synuclein aggregates obtained in an independent parallel experiment shows typical fibrillar aggregates. (D–F) a-Synuclein was incubated for 7 days in the presence of different concentrations of b-synuclein resulting in a dose-dependent inhibition of aggregate formation. Intensity histograms were recorded for 10 · 30 s with a bin width of 150 ls.
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Fig. 4. Synuclein co-aggregation analysis by dual-colour SIFT. (A) An equimolar mixture of wt a-synuclein spiked with wt a-synuclein-Alexa-488 (‘‘green’’) and A30P a-synuclein spiked with A30P a-synuclein-Cy5 (‘‘red’’) at a final total protein concentration of 1 mg/ml measured at day 0 exhibits only low fluorescence intensity in the ‘‘green’’ and ‘‘red’’ channel due to the stochastically fluctuating number of labelled monomers present in the detection volume at any time point. (B) After 7 days of incubation, co-aggregation of wt and mutant a-synuclein yielded large aggregates carrying multiple ‘‘green’’ and ‘‘red’’ labels on the same particle resulting in a corresponding number of bins with high fluorescence intensity in both the ‘‘green’’ and the ‘‘red’’ channel simultaneously. (C) When wt a-synuclein spiked with wt a-synuclein-Alexa-488 (‘‘green’’) and A30P a-synuclein spiked with A30P a-synuclein-Cy5 (‘‘red’’) were incubated in separate wells for 7 days and then mixed and measured, only pure wt aggregates and pure A30P aggregates are present that result in high intensity signal in only the green or only the red channel when passing the laser focus, respectively. (D) In a mixture of wt a-synuclein spiked with wt a-synuclein-Alexa-488 and prion protein labelled with Cy5 co-incubated for 7 days, pure prion protein aggregates can be detected in addition to more abundant particles consisting of aggregated a-synuclein and some bound prion protein. On the left, colour-coded 2D intensity histograms recorded for 5 · 20 s with a bin width of 40 ls are shown. On the right, a schematic representation of the types of particles present in the respective samples is given. The light grey circle indicates the open detection volume defined by the laser focus. , wt a-synuclein monomer; , wt a-synuclein monomer labelled with Alexa-Fluor-488; , a-synuclein A30P mutant monomer; , a-synuclein A30P mutant monomer labelled with Cy5; , prion protein monomer labelled with Cy5. Aggregates are symbolized by groups of monomers. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this paper.)
aggregate species carrying only one type of fluorescent label were clearly discernible (Fig. 4C). Furthermore, when we mixed another protein capable of forming amyloid-like aggregates, i.e., prion protein tagged with a ‘‘red’’ label, with a-synuclein spiked with ‘‘green’’ probe molecules, we found that at least two different types of aggregates were formed during co-incubation: in addition to a-synuclein aggregates that also contained prion protein molecules, pure prion protein aggregates were detected (Fig. 4D). Taken together, these findings show that mixtures of different aggregate species can form in co-incubation experiments with amyloidogenic proteins, and that these aggregates can be clearly separated from aggregates derived from coaggregation by dual-colour SIFT analysis. Ratio of wt and mutant a-synuclein determines aggregate formation In addition to the qualitative aspect of co-aggregation, we were interested in potential quantitative effects
of the interaction of wt and mutant a-synuclein. When we analysed mixtures of wt and mutant a-synuclein, we found a striking and unexpected increase in aggregate formation at specific molar ratios. Whereas aggregate formation in equimolar mixtures was in the range of what we found in solutions of either wt a-synuclein or mutant a-synuclein alone, ‘‘asymmetric’’ mixing ratios with a 2- to 5-fold excess of either of the two molecules favoured aggregate formation in our experiments. This resulted in an M-shaped dependence of aggregation on mixing ratio (Fig. 5). Comparing the results for the number of bins above cut-off level (Fig. 5A), primarily a measure of aggregate concentration, and the number of photons contained in bins above cut-off (Fig. 5B), primarily a measure of the amount of synuclein molecules contained in aggregates, indicates that the average size of the aggregates is unchanged. This suggests that the observed effect is mainly due to differences in the number of aggregates formed. Our findings in vitro may provide an explanation for a surprising observation in patients affected by
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are small oligomeric aggregation intermediates as indicated by several studies [39–42], these oligomers may differ in regard to structure, stability, and propensity to form larger aggregates depending on molar ratios of monomers [43]. Further evidence in this direction comes from our results using Thioflavin T fluorescence. Similar to the results of SIFT measurements, more aggregation was detected at ‘‘asymmetric’’ mixing ratios. However, whereas the amount of aggregates was the highest at mixing ratios of 1:5 and 5:1, the amount of amyloid-like structures was increased especially at a 2-fold excess of either of the two monomers. This indicates that the molar ratio has not only quantitative but also qualitative effects on the aggregation process, similar to the effects described for variations in buffer conditions [44]. Oligomers of particular molar ratios may be favored to be integrated into fibrils and to form amyloid whereas others may be excluded. Thus, varying mixing ratios may provide a means to specifically favour specific types of aggregates or intermediates that can than be used for further studies.
Conclusions
Fig. 5. Aggregate formation in mixtures of WT a-synuclein and A30P mutant synuclein depends on mixing ratio. Diagrams show the relative level of aggregate formation after 7 days of incubation in samples with different mixing ratios but same total protein concentration. Samples were analysed in parallel with different assays: (A) SIFT signal (N) indicates the number of bins above cut-off level. (B) SIFT signal (P) was generated by summing up the photon count from the ‘‘green’’ and ‘‘red’’ channel for all bins above cut-off level. (C) Thioflavin T fluorescence corresponds to the total amount of amyloid formation. Three to four independent experiments out of two separate series were normalized as described under Materials and methods. The mean and standard error is shown.
either an A30P or A53T mutation that was published recently. Corresponding to the levels of aggregate formation in our in vitro assay, only mild disease was found in individuals showing a roughly equimolar expression level of wt and mutant a-synuclein, whereas relative under-expression of the mutant at ratios of about 1:2 to 1:9 was associated with severe disease phenotype [23]. Currently, we cannot provide a comprehensive explanation for the striking difference between equimolar and ‘‘asymmetric’’ conditions at the molecular level. However, one could hypothesize that, if there
Formation of pathological amyloid aggregates in vivo takes place in a complex environment. Thus, inhibitory and amplifying effects from a variety of molecular interactions can affect this process. Co-aggregation of various species of amyloidogenic proteins may turn out to be an important determining factor for the manifestation of disease in humans [31]. We could show that in the case of a-synuclein, subtle variations in the molar ratio of different molecular species have a striking effect on aggregation. This may have important therapeutic implications for neurodegenerative aggregation diseases in general, as the lowering of the amount of aberrant protein variants may also have non-beneficial effects. SIFT analysis provides a powerful tool to study these co-aggregation processes, as—in addition to sensitivity—dual-colour analysis on the single particle level allows to study the molecular composition of aggregates even in samples containing a mixture of different types of aggregates. As SIFT measurements can be performed highly automated in homogeneous assay conditions with small sample volumes, this approach also provides a rapid and robust readout for high-throughput screening of anti-aggregative drugs [30].
Acknowledgments This work was supported by the Deutsche Forschungsgemeinschaft (Grant HA1737 and Special Program Grant SFB596) and by the European Commission (Grant QLK3-CT-2001-02345).
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