Concept for Simultaneous and Specific in Situ Monitoring of Amyloid

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Concept for simultaneous and specific in situ monitoring of amyloid oligomers and fibrils via FRET Bruno Alies, Helene Eury, El Mokhtar Essassi, Genevieve Pratviel, Christelle Hureau, and Peter Faller Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac503509g • Publication Date (Web): 07 Nov 2014 Downloaded from http://pubs.acs.org on November 11, 2014

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Analytical Chemistry

Concept for simultaneous and specific in situ monitoring of amyloid oligomers and fibrils via FRET Bruno Alies a,b†, Helene Eury a,b, El Mokhtar Essassi c, Genevieve Pratviel *a,b, Christelle Hureau a,b, Peter Faller*a,b a

CNRS, LCC (Laboratoire de Chimie de Coordination), 205 route de Narbonne, BP 44099, F-31077 Toulouse Cedex 4 (France). b Université de Toulouse, UPS, INPT, F-31077 Toulouse Cedex 4 (France). c Laboratoire de Chimie Organique Hétérocyclique, Pôle de Compétences Pharmacochimie, Université Mohammed V-Agdal, Faculté des Sciences, Avenue Ibn Battouta, BP 1014, Rabat, Morroco ABSTRACT: Oligomeric species of amyloidogenic peptides or proteins are often considered as the most toxic species in several amyloid disorders, like Alzheimer or Parkinson's diseases, and hence came into the focus of research interest and as a therapeutic target. An easy and specific monitoring of oligomeric species would be of high utility in the field, as it is the case for thioflavin T fluorescence for the fibrillar aggregates. Here, we show proof of concept for a new sensitive method to increase specific detection of oligomers by two extrinsic fluorophores. This is achieved by exploiting a Förster resonance energy transfer (FRET) between the two fluorophores. Thus, a mixture of two extrinsic fluorophores, bis-ANS and a styrylquinoxalin derivative, enabled to monitor simultaneously and in situ the presence of oligomers and fibrils of amyloidogenic peptides. Thereby, the formation of oligomers and their transformation into fibrils can be followed.

Fluorescence is a very widespread spectroscopy to investigate processes in chemistry and biology. This highly sensitive method is compatible with physiological concentrations of biomolecules.1 In particular, fluorescence is widely used to monitor assembly and interactions of proteins.2 Protein/peptide aggregation into amyloid fibrils is regarded as a key factor in many diseases (Alzheimer’s, Parkinson’s, Diabetes type 2…).3 One of the most used techniques to follow the formation of fibrils is by the fluorescence of an extrinsic dye, mainly thioflavin T (ThT, Figure 1).4-7 Use of ThT is very convenient as it allows in situ measurement. There is a consensus that ThT does not modulate significantly the aggregation kinetics or structures when used at low concentrations.4-6,8 Moreover, ThT exhibits very low fluorescence in solution or in the presence of soluble amyloidogenic peptides/proteins and turns on upon binding to amyloid fibrils. This advantage has made steady state fluorescence measurements by ThT as probably the most often used technique in the area of amyloids and prions. However, during the last years a paradigm shift occurred from the fibrils to prefibrillar states, often called oligomers. It is indeed mainly thought that oligomers of amyloidogenic peptides/proteins are the most toxic species, and hence came into the focus of research and as a therapeutic target.9, 10 Thus, a fluorophore that would specifically turn on fluorescence upon binding to oligomeric forms and not to fibrils or monomers would be a real breakthrough and of very important utility in the field, from in vitro to in vivo measurements. However, so far, no compound specific for oligomeric species having the simplicity of ThT, i.e. extrinsic fluorophore (has just to be added), small size, cheap, little influence on aggre-

gation, strong turn on, etc. has been reported. This might be very difficult to achieve due to the similar secondary structure of oligomers and fibrils. Therefore, there is an important interest in developing other strategies to detect oligomers. Several such strategies were reported in the literature, but they are often based on covalent labelling or other constitutional changes of the amyloidogenic proteins and or need more sophisticated fluorescence measurements, e.g. anisotropy to distinguish oligomers from fibrils.11-14 Conformer sensitive methods work well for the conversion of unordered to β-sheet structure, but are often only partially selective to oligomers.15, 16 Extrinsic fluorophores can be used with the non-modified, native protein, but specificity against oligomers is difficult to obtain as well, in particular with small size molecules. 1anilinonaphthalene-8-sulfonate , 4-4-bis-1-phenylamino-8naphthalene sulfonate (bis-ANS, Figure 1), 4-(dicyanovinyl)julolidine , Nile Red and recently developed luminescentconjugated polymers are known to turn on fluorescence by binding to the oligomeric states but fluoresce also when bound to fibrils.12, 17-19 Indole-based compounds that only undergo a fluorescence quenching in the presence of prefibrillar structures, but not with fibrils, have been identified.5 Selectively recognition of early oligomers was achieved with a more elaborated, larger construct consisting of two peptides with a linker and a biotin-moiety.20 This brief survey shows that so far no method for oligomeric detection with the ease of ThT has been reported, although a variety of different strategies has been applied. Hence, further development of the known strategies or the design of new methods is needed.

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Herein, we develop a new concept to increase specific detection of oligomers by the use of a combination of extrinsic fluorophores. Indeed, by using a mixture of two fluorophores which can undergo FRET (Förster resonance energy transfer), we were able to monitor the time dependence of oligomers formation and simultaneous detection of fibrils in situ.

Scheme 1. Concept of FRET enhanced detection of oligomers (green emission) and fibrils (red emission) for amyloidogenic peptides/proteins. Two extrinsic fluorophores, namely 1 and 2, are present and the time course of aggregation of an amyloidogenic peptide is shown. At the beginning (t0) only monomers are present and no fluorophore binds and fluoresces. Later, at time t1 mostly oligomers are present, only fluorophore 1 binds and fluoresces. At the end (t2) fibrils are formed, both fluorophores (1 and 2) bind to the fibrils. Excitation of fluorophore 1 leads to FRET and emission from fluorophore 2. Consequently, the emission of fluorophore 1 is quenched. Note that the excitation is always at the same wavelength, i.e. for excitation of fluorophore 1. In the present study fluorophore 1 is bis-ANS and fluorophore 2 is SQ.

EXPERIMENTAL SECTION Peptide sample preparation of Aβ1-28. The peptide Aβ1-28 (sequence D-A-E-F-R-H-D-S-G-Y-E-V-H-H-Q-K-L-V-F-FA-E-D-V-G-S-N-K) was purchased from Genescript. The stock solutions of Aβ1-28 (∼1.2 mM) have been prepared by dissolving the peptide in Milli-Q water (resistivity = 18 MΩ cm−1) which resulted in a final pH of approx. 2. The pH of the solution was then adjusted to pH 12 by adding NaOH in order to monomerize the peptides. These stock solutions were stored at 253 K. The peptide concentrations were determined by using the molar extinction coefficient ε = 2400 M−1 cm−1 of the deprotonated tyrosine10 at 293 nm. Because deprotonated tyrosine does not absorb at 320 nm, absorption at that wavelength was subtracted in order to remove contributions from the baseline drifts. Chemicals. ZnSO4 monohydrate was purchased from Strem Chemicals. High quality (traces controlled) 4-(2hydroxyethyl)-1-piperazylethanesulfonic acid (HEPES) was

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purchased from Sigma-Aldrich. Thioflavin T (ThT) and 4-4bis-1-phenylamino-8-naphthalene sulfonate (bis-ANS) was brought from Acros. (E)-3-(4dimethylamino)styryl)quinoxalin-2-one or styryl-quinoxaline (SQ) was prepared as described previously. 21 Fluorescence measurements: Steady state fluorescence emission spectra were performed with a FluoroMax-4 spectrofluometer (Jobin-Yvon). Fluorescence kinetic has been measured by a FLUOstar Optima plate reader (BMG Labtech) on 96 well plates (Greiner). Kinetic measurement was carried at 25°C. To prevent evaporation of the sample, the UV-visible transparent and adhesive film was placed over the plate. Three replicate was made. Variations between replicate were negligible (< 5%), the average of them is shown in figures. Total volume in each wells was 200 µL. Condition concentration were [Aβ1-28] = 500 µM; [Zn(II)] = 250 µM ; [phosphate] = 10 mM pH = 7.4 ; [Fluorophore] = 10-30 µM ([ThT] or [SQ] or [Bis-ANS] or [SQ] and [Bis-ANS]). Filters for excitation and emission at 390 nm, 440 nm, 490 nm and 570 nm were used. Atomic force microscopy: Taping-mode AFM imaging was performed in air on a Smart SPM-1000 microscope (AISTNT, Novato, USA) equipped with a 100µm scanner. Sample solutions (5 times diluted with water (final concentration 100 µM peptide) after taken from the fluorescence measurement) were deposited on freshly cleaved mica and left for adsorption on the substrate for 10 min. They were then rinsed three times with deionized water to remove salts and loosely bound peptide and dried with compressed N2 before imaging. Commercial Si cantilevers (NanoWorld, Swizerland) with an elastic modulus of ~42 N m-1 were used. All images were acquired as 512 x 512 pixel images at a typical scan rate of 1.0 kHz with a vertical tip oscillation frequency of 250-350 kHz. Representative images of each sample were obtained by scanning at different locations. Other peptides: preparation of Aβ11-28, Aβ14-23 and Aβ140. The peptides were purchased from Genescript. The stock solutions of Aβ11-28 and Aβ14-23 (∼1.2 mM) have been prepared by dissolving the peptide in Milli-Q water (resistivity = 18 MΩ cm−1) which resulted in a final pH of approx. 2. The pH of the solution was then adjusted to pH 12 by adding NaOH in order to monomerize the peptides. These stock solutions were stored at 253 K. The peptide concentrations were determined by using the molar extinction coefficient ε = 390 M−1 cm−1 of the two phenylalanine at 258 nm. Because Phe does not absorb at 275 nm, absorption at that wavelength was subtracted in order to remove contributions from the baseline drifts. Around 8mg of Aβ1-40 was solubilized in 0.5 ml 50mM NaOH (very gentle vortexed to avoid bubbles and very shortly sonicated just to dissolve completely). After 15min of incubation at room temperature, the sample was passed over a Superdex 75 column (GE healthcare) connected to a FPLC (Akta) with 15 mM NaOH (no salt) as running solution. The peak corresponding to the monomer eluted at around 13 ml. The three or four most intense fractions (volume 0.5 ml) from the peak were centrifuged for 5 min at 20000g. Normally a small white deposit was observed. The supernatant was removed (volume 450 µl) from the different fraction and pooled. Then aliquots were frozen in liquid nitrogen and just thawed before the experiments (one thawed samples were not refrozen).


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RESULTS AND DISCUSSION

Table 1. Fluorescence property of compounds studies in presence of aggregates.

Concept is to monitor simultaneously oligomers and fibrils in situ Scheme 1 depicts the new concept for monitoring simultaneously oligomers and fibrils formation in situ. The concept is based on two fluorophores, 1 and 2 (in the present study bisANS and (E)-3-(4-(dimethylamino)styryl)quinoxalin-2-one (SQ), Figure 1). Fluorophore 1 (bis-ANS) turns on fluorescence when binding to oligomers and fibrils and fluorophore 2 (SQ) turns on when binding to fibrils only. A FRET from the first to the second fluorophore would allow the simultaneous and specific detection of oligomers and fibrils under the same excitation wavelength (i.e. excitation of fluorophore 1, BisANS). The choice of fluorophores. Bis-ANS is a common fluorophore to monitor hydrophobic surfaces in proteins. It has also been used to monitor peptide/protein aggregation.22 Bis-ANS becomes fluorescent when binding to oligomers but also to fibrils.12 A slightly higher fluorescence is observed for oligomers with respect to fibrils (Figure S1). Bis-ANS is classically excited at 350-400 and emission occurs at 485-515 nm. Thus, to observe FRET we replaced ThT (excitation and emission properties of which being not suited for coupling with bis-ANS) and we used a styryl-quinoxaline derivative (SQ, Figure 1) as fluorophore, which we recently synthesized and that showed ThT like activity, i.e. identical aggregation kinetics (Figure S2).21 SQ emits at 570 nm and shows an excitation maximum around 450 nm when bound to fibrils. Although not ideal, the spectral overlap of emission of bis-ANS with SQ excitation is high enough for a FRET, and hence could allow the proof of concept (see Table 1).

S

N N

N

N

Thioflavin T

N H

O

SQ O3S

SO3

H N

H N

bis-ANS

Figure 1: Structure of thioflavin T (ThT), SQ and bis-ANS.

Compound ThT SQ Bis-ANS

λexc (nm) 440 450 390

Bandwidth (nm) [a] 30 50 40

λemi (nm) 490 570 490

Bandwidth (nm) 40 80 80

[a] Given bandwidth corresponds to half height width of the band. Monitoring of oligomers and fibrils using FRET The well characterized peptide Aβ1-28 was used as leading amyloidogenic peptide, because we obtain highly reproducible aggregation kinetics from different preparations, as well as from different lots. Aβ1-28 consists of the first 28 amino acids of the amyloidogenic peptide Aβ related to Alzheimer’s disease. The main forms consist of 40 or 42 amino acids (Aβ1-40 or Aβ1-42). The aggregation of Aβ1-28 can be triggered by addition of Zn and hence is well controlled.23 The oligomers and fibrils formation has been followed, at several time points in the course of aggregation, by the simultaneous presence of bis-ANS and SQ (central panel in Figure 2). Bis-ANS was excited at 390 nm and hence emission could occur at 490 nm (direct emission from bis-ANS) or at 570 nm (via FRET between bis-ANS and SQ). In absence of SQ, intensity of the emission band observed at 490 nm increases with time, reaches a maximum after 10-20 minutes and then remains almost stable over time (left inset in Figure 2).† In presence of SQ, this band decreases with time until it disappears almost completely after 2h. Concomitantly with the decrease observed at 490 nm, an emission band appears and increases at 570 nm from 0 to 2h (central panel in Figure 2). This is in line with the concept described in scheme 1: prefibrillar states (oligomers) induce the fluorescence of bis-ANS and emission at 490 nm occurs. With time fibrils are formed that bind SQ as well, and excitation of bis-ANS leads to the emission of SQ via FRET. In the presence of bis-ANS only (Figure 2, left inset) no FRET can occur due to the absence of SQ and indeed, no decrease in the fluorescence emission of bis-ANS at 490 nm is observed from 10 min to 2h. Consequently bis-ANS only has a poor specificity towards oligomeric forms compared to fibrils and the presence of SQ is needed to observe the decrease at 490 nm. As a control, SQ only was also measured (Figure 2, right inset). Almost no fluorescence emission at 570nm is observed when irradiated at 390 nm (excited at wavelength for bis-ANS, i.e. FRET conditions), clearly showing that the presence of bis-ANS is necessary to observe the 570 nm emission. Hence, excitation at 390 nm of bis-ANS with recording the spectrum between 400 and 700 nm has the potential to monitor simultaneously the oligomeric forms (emission at 490 nm) and the amyloid fibrils (emission at 570 nm).



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change in the kinetics. In the presence of bis-ANS, SQ monitors faster amyloid fibril formation, indicating that bis-ANS accelerates the formation of fibrils. Note, that this is an advantage of the present new concept of two fluorophores with FRET, compared to the parallel measurement of two fluorophores in separate samples. In the latter case, if a fluorophore has an impact on the aggregation, the two samples cannot be compared directly anymore. In contrast, with the present concept, the two fluorophores being in the same sample, either fluorophore undergoes/monitors the same aggregation kinetic.

Figure 2: Emission spectra upon excitation at 390 nm of bis-ANS and SQ (red, main panel), bis-ANS only (blue, left inset), and SQ only (black, right inset) during aggregation of Aβ1-28, a t=0 (bold solid lines), 10, 20, 30, 60 (bold solid dark lines) and 120 min (dotted lines). The main panel (red) is in presence of bis-ANS and SQ. At the beginning, emission is at 490 nm, in line with emission of bis-ANS bound to oligomers. With time fibrils are formed, which bind bis-ANS and SQ. Thus emission of bis-ANS at 490 nm is diminishing at the expense of an increase in emission at 560 nm from SQ. This is in line with a FRET from bis-ANS to SQ. In the same experiment without SQ (left inset, blue), bis-ANS binds to oligomers and fluorescence is increasing. Then fibrils are formed,They bind bis-ANS as well and hence the emission of bis-ANS stays almost constant (As no SQ is present, FRET cannot occur). In the presence of SQ only (right inset, black), one would expect no emission at all, because excitation matches bis-ANS (and not SQ), which is absent. Indeed, the intensity is very low (magnified by 10 compared to the red specta). The residual fluorescence follows the kinetics of fibril formation (compared to the oligomer formation followed by bis-ANS only (blue)) as expected for SQ and is due the excitation in the tailing of the relatively broad SQ absorption. Experimental conditions: [Aβ1-28] = 500µM, [Bis-ANS]=[SQ]= 30µM, [Zn(II)]= 250µM, 10 mM phosphate buffer pH 7.4.

Fluorescence kinetics The same FRET phenomenon can also be demonstrated on the kinetic traces. The time course of the fluorescence in the simultaneous presence of bis-ANS and SQ is shown in Figure 3. Excitation of bis-ANS at 390 nm and emission of bis-ANS at 490 nm is shown in red. The bis-ANS fluorescence intensity increases at the beginning and reaches a maximum at about 30 min, monitoring formation of oligomeric species. Then, the fluorescence intensity decreases at 490 nm due to the FRET and disappeared almost completely. In contrast excitation of bis-ANS and emission at 590 mn of SQ (FRET, blue curve), showed a typical sigmoid and reflects the formation of amyloid fibrils. The black curve in Figure 3 shows the time dependent fluorescence of excitation and emission of SQ (450 nm and 570 nm, respectively) for comparison. As expected, SQ exhibits a sigmoidal curve similar to the one in the absence of bis-ANS (Figure S1 and S2). The only difference is a

Figure 3: Time-course measurement of Aβ1-28 aggregation monitored by the simultaneous presence of bis-ANS and SQ. The inset shows a comparison of emission of Bis-ANS in absence (dotted orange line) or presence of SQ (solid red line). Experimental conditions: [Aβ1-28] = 500µM, [Bis-ANS] = [SQ] = 30µM, [Zn(II)] = 250µM, 10 mM phosphate buffer pH 7.4

AFM of oligomeric and fibrilar forms In order to verify the aggregation state of Aβ1-28 as probed by FRET experiments, AFM images were recorded (Figure 4). Indeed at the maximum of the peak of bis–ANS fluorescence in presence of SQ (Figure 3 red curve at about 40 min) the image showed mostly oligomeric form and no mature fibrils (Figure 4, left). At the end of the kinetic at around 20 hours, typical amyloid fibrils were predominantly observed (Figure 4, right).


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Analytical Chemistry ThT, which has a good specificity toward amyloids. Thus bisANS might also interact with other aggregation states, like amorphous aggregates.The formation of amorphous or other aggregates in native Aβ1-40 could interfere with the FRET based oligomer detection. CONCLUSIONS

Figure 4: AFM images after 40 min (left) and 20 hours (right) incubation of Aβ1-28 in the presence of bis-ANS and SQ. Aggregation conditions like in Figure 3, then diluted 5x. [Aβ1-28] = 500µM, [Bis-ANS] = [SQ] = 30µM, [Zn(II)] = 250µM, 10 mM phosphate buffer pH 7.4. Fluorophore concentration dependence Another important point is the concentration of fluorophores. We varied the concentration of bis-ANS and SQ from 10 – 30µM (Figure 3, S3). Although FRET was observed for all the concentrations studied and simultaneous monitoring of oligomeric and fibrillar aggregates were possible, the strongest FRET effect was observed with 30µM SQ and bis-ANS (Table S1). In order to understand that we estimated the number of fluorophore binding sites by determining the number of ThT molecules bound to the fibrils formed (Figure S4) for Aβ1-28 aggregation under the present conditions. The concentration of ThT bound was estimated to be 70µM. This fits well with a maximal FRET effect of 30µM SQ and bis-ANS at 30µM, as almost all putative binding sites are occupied, which maximize the FRET. Application to other amyloidogenic peptides After showing that the concept works for Aβ1-28, we wondered whether it can be transposed directly to other peptides. First we used Aβ11-28, a peptide that aggregates reproducibly into amyloid fibrils.24, 25 Indeed, simultaneous detection of oligomeric and fibrillar species via FRET was observed, although the conditions were not optimized (Figure S5). Similarly, detection of oligomeric forms by FRET was also obtianed for the even shorter peptide Aβ14-23 (Figure S5).24 In contrast, for different native Aβ1-40 peptides simultaneous detection of oligomeric and fibrillar species via FRET was not detected. As Aβ1-40 aggregates at lower concentration (range of 5 - 50 µM) we adapted the concentration of fluorophores and screened several experimental conditions (like pH, concentration of peptides and or fluorophores, mutants, presence of metal ions etc.) but detection of FRET was always thwarted. This might be due to the following reasons: i) for longer Aβ1-40 peptides bis-ANS, SQ and ThT showed the same kinetics (Figure S6), suggesting that bis-ANS does not detect oligomeric species (only fibrils) or that the concentration of the oligomers is too low, ii) FRET was not observed maybe because the binding sites of the fluorophores are not close enough, or the affinity of the fluorophore is not strong enough. iii) bis-ANS is assumed to interact with hydrophobic patches of proteins in general. It is hence less specific compared to

We show proof of concept for a new principle of monitoring the kinetics of two successive conformational states, on the example of oligomer and fibrils formation of an amyloidogenic peptide. FRET of two extrinsic fluorophores was used to monitor simultaneously and in situ oligomer formation and their transformation into fibrils. Thereby the oligomer detection was enhanced, because bis-ANS alone did not distinguish between oligomers and fibrils, but became quite specific for oligomers in the presence of SQ (Figure 4). This new concept has the following advantages: i) only steady state fluorescence is needed, ii) due to the use of extrinsic fluorophores no derivatisation of peptide is needed iii) enhanced specificity for the oligomers iv) simultaneous observation of aggregation states at only one excitation wavelength and v) in situ measurement. Although shown that the concept works for Aβ1-28, Aβ1128 and Aβ14-23 and was successfully optimized for Aβ1-28, further work is needed to see if it is possible to apply the concept to detect oligomeric and fibrillar species simultaneously for full length Aβ1-40. This involves the search and synthesis of other fluorophores with high enough affinity to bind at low µM concentration and able to bind in proximity to each other in fibrils so that FRET can occur. Precedence that fluorophore binding sites can be close enough for FRET in full length amyloid proteins was reported for fibrils of α-synuclein and transthyretin, but was not used to monitor oligomeric forms.26, 27 Although bis-ANS has been reported to detect oligomeric species for several amyloidogenic proteins including Aβ12, 22, under our condition bis-ANS did not detect pre-fibrillar states in full length Aβ. Thus an improved fluorophore that recognizes and turn on fluorescence for oligomeric species of full length Aβ has the potential to make the FRET based oligomer detection possible. The present study provides the proof of concept of a new method to detect simultaneously oligomeric and fibrillar forms of amyloidogenic peptides by steady state fluorescence. This concept has the potential to be extended and adapted to other amyloidogenic peptides/proteins. In principle, it can be adapted beyond amyloidogenic proteins to systems that have successive conformational states, under the condition that appropriate fluorophores and binding sites are available.

ASSOCIATED CONTENT Supporting Information Supporting Information include figures S1 – S6 and table S1.This material is available free of charge via the Internet at http://pubs.acs.org

AUTHOR INFORMATION



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Corresponding Author

* E-mail [email protected]; [email protected]. Present Addresses

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†present address: Department of Chemistry, Duke University, Durham, NC 27708, USA.

Author Contributions

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The manuscript was written through contributions of all authors. / All authors have given approval to the final version of the manuscript.

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Notes The authors declare no competing financial interest.

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ACKNOWLEDGMENT Financial support from the “Region Midi-Pyrénées” (Research Grant APRTCN09004783) is acknowledged. We would like to thank Dr. Olivia Berthoumieu (LCC, Toulouse) for acquiring the AFM images and H. Benzeid (LCOH, Rabat) for the synthesis of SQ.

REFERENCES

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† The higher intensity of bis-ANS alone compared to bisANS with SQ at the beginning (0 min) is likely due to the fact that SQ absorbs at 390 nm, i.e. the wavelength of bis-ANS excitation (inner filter effect). Electronic Supplementary Information (ESI) available: [experimental section, Figures S1-S6 and table S1]. See DOI: 10.1039/b000000x/

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Table of content 162x126mm (96 x 96 DPI)


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Concept for Simultaneous and Specific in Situ Monitoring of Amyloid

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