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Sulindac Sulfide induces the formation of large Oligomeric Aggregates of the Alzheimer’s Disease Amyloid-# Peptide which exhibit reduced Neurotoxicity Elke Prade, Christian Barucker, Riddhiman Sarkar, Gerhard Althoff-Ospelt, Juan Miguel Lopez del Amo, Shireen Hossain, Yifei Zhong, Gerhard Multhaup, and Bernd Reif Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.5b01272 • Publication Date (Web): 22 Feb 2016 Downloaded from http://pubs.acs.org on March 1, 2016
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Sulindac Sulfide induces the formation of large Oligomeric Aggregates of the Alzheimer’s Disease Amyloid-β Peptide which exhibit reduced Neurotoxicity
Authors: Elke Prade1, Christian Barucker2, Riddhiman Sarkar1, Gerhard Althoff-Ospelt3, Juan Miguel Lopez del Amo4, Shireen Hossain2, Yifei Zhong2, Gerhard Multhaup2, Bernd Reif*1,5
Affiliations: 1
Munich Center for Integrated Protein Science (CIPS-M) at Department Chemie, Technische
Universität München (TUM), Lichtenbergstr. 4, 85747 Garching, Germany. 2
Department of Pharmacology & Therapeutics, McGill University, 3655 Promenade Sir-
William-Osler, Montreal QC, H3G 1Y6, Canada. 3
Bruker BioSpin, Silberstreifen 4, 76287 Rheinstetten, Germany.
4
Current address: CIC Energigune, Albert Einstein 48, 01510 Miñano, (Álava), Spain.
5
Helmholtz-Zentrum München (HMGU), Deutsches Forschungszentrum für Gesundheit und
Umwelt, Ingolstädter Landtstr. 1, 85764 Neuherberg, Germany. * Email:
[email protected] Version 11 date: 09.02.2016
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Abbreviations Aβ, amyloid-β; CMC, critical micelle concentration; CSP, chemical shift perturbation; GSM, γsecretase modulator; NSAID, nonsteroidal anti-inflammatory drug; PDSD, proton-driven spin diffusion; TEDOR, transferred echo double resonance.
Keywords Magic Angle Spinning (MAS) solid-state NMR · Amyloid fibrils · Non-Steroidal AntiInflammatory Drugs (NSAIDs) ·
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Abstract Alzheimer's disease is characterized by deposition of the amyloid β-peptide (Aβ) in brain tissue of affected individuals. In recent years, many potential lead structures have been suggested that can potentially be used for diagnosis and therapy. However, the mode of action of these compounds is so far not understood. Among these small molecules, the NonSteroidal Anti-Inflammatory Drug (NSAID) sulindac sulfide received a lot of attention. In this manuscript, we characterize the interaction between the monomeric Aβ peptide and the NSAID sulindac sulfide. We find that sulindac sulfide efficiently depletes the pool of toxic oligomers by enhancing the rate of fibril formation In vitro, sulindac sulfide forms colloidal particles which catalyze the formation of fibrils. Aggregation is immediate, presumably by perturbing the supersaturated Aβ solution. We find that sulindac sulfide induced aggregates are structurally homogeneous. The C-terminal part of the peptide adopts β-sheet structure, whereas the N-terminus is disordered. The salt bridge between D23 and K28 is present, similar as in the wild type fibril structure.
13
C-19F TEDOR experiments suggest that sulindac
sulfide co-localizes with the Aβ peptide in the aggregate.
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Introduction Deposits formed by insoluble aggregates of the amyloid-β (Aβ) peptide are the main constituents of amyloid plaques in Alzheimer’s disease (AD).1 However, soluble species of Aβ are correlated to disease symptoms associated with neurodegeneration and toxicity;2-4 Aβ peptides are proteolytically released by sequential cleavage of the amyloid precursor protein (APP) by the β- and γ-secretases.5, 6 This procedure yields Aβ fragments of varying lengths,7, 8
where Aβ1-40 and Aβ1-42 are detected as the main products under normal conditions.9, 10 In
addition to neurodegeneration, chronic inflammation occurs frequently in AD brains and contributes significantly to AD pathogenesis.11 Hence, there is a need for anti-inflammatory approaches to cure AD progression, such as administration of non-steroidal antiinflammatory drugs (NSAIDs) which have been demonstrated to be beneficial for the course of the disease.12
A number of NSAIDs have been identified as γ-secretase modulators (GSMs), which alter the distribution of generated Aβ peptides.13 By targeting the γ-secretase, the NSAID sulindac sulfide (Fig. 1A, left) decreases the levels of the disease-relevant Aβ1-42 in favor of production of shorter Aβ variants with decreased toxicity.14 Sulindac sulfide binds to the transmembrane sequence (TMS) of APP,15,
16
and this has been reported as an additional mechanism for
lowering Aβ1-42 levels.17 We have recently reported that sulindac sulfide intercalates specifically between the two β-strands formed in mature Aβ fibrils.18 Additionally, direct binding to Aβ monomers,17,
19
oligomers20 and fibrils19 has been reported, whereas other
reports refute a direct interaction.21 Similarly, there are controversial reports about the effect of sulindac sulfide on aggregation properties of Aβ.19, 22
Colloid formation is a common mechanism for promiscuous protein inhibition of various small molecules. The colloidal character of sulindac sulfide in aqueous solution can potentially result in non-specific amyloid binding21,
23, 24
and should be taken into account when
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assessing its influence on Aβ. Sulindac sulfide can be oxidized to its derivative sulindac sulfone (Fig. 1A, right). Although structurally similar, the two NSAIDs differ in their characteristics and effects on Aβ. In contrast to sulindac sulfide, sulindac sulfone does neither interact with APP-TMS17 nor with monomeric Aβ peptides,21 and does not form colloids in solution.21
Various small molecules interfere with aggregation of amyloids and constitute potential drug candidates. The molecular details of the involved interaction mechanisms remain unknown. Solid-state NMR is a particularly powerful technique to gain insight into amyloid-ligand interactions, and has been employed to study binding of Congo Red to HET-s fibrils,25 and interactions between Aβ and epigallocatechin gallate,26 curcumin,27,
28
metal ions,29,
30
and
sulindac sulfide.18 We have recently postulated that the monomeric entity of sulindac sulfide binds specifically to Aβ fibrils.18 In the current work, we report how sulindac sulfide colloids can sequester Aβ monomers non-specifically into off-pathway aggregates with reduced neurotoxicity. By solid-state NMR spectroscopy we characterize the structural elements present in the resulting aggregates.
Materials and Methods Aβ sample preparation Uniformly 15N or 15N-13C labeled Aβ1-40 peptide was obtained through recombinant expression into inclusion bodies of E.coli cells, followed by a washing protocol and purification using reverse-phase chromatography as previously described.31 Although the construct contains an N-terminal methionine, it shows the same biochemical properties as the wildtype peptide.32 Solutions of monomeric Aβ were prepared by initially dissolving the lyophilized peptide in 10 mM NaOH. To remove potential nucleation seeds, the solution was sonicated for 10 minutes in an ultrasonic bath, centrifuged for 10 minutes at 14’800 RPM and diluted in
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2X buffer (100 mM sodium phosphate + 100 mM NaCl buffer pH 7.3) to yield the respective Aβ concentration. For neurotoxicity assays, synthetic Aβ1-42 peptides (Peptide Specialty Laboratories, Germany) were monomerized and solubilized as described.33 Briefly, monomerized peptides were dissolved to 1 mg/ml in deionized water supplemented with ammonia to a final concentration of 0.13 % (measured pH 9.8). Preparation NSAID stock solutions Stocks of sulindac sulfide and sulindac sulfone were prepared in dimethyl sulfoxide (DMSO) or DMSO-d6 for solution-state NMR experiments. To prepare NMR samples, the respective amount of NSAID stock was added to a freshly prepared Aβ solution. Typically, the end concentrations of DMSO in aqueous solution did not exceed 1%. For size exclusion chromatography, sulindac sulfide was dissolved in methanol (final concentration 26.6%). Solution-state NMR sample preparation For all solution-state NMR experiments,
15
N or
15
N-13C labeled monomeric Aβ1-40 was
dissolved in buffer, mostly to concentrations of 50 µM or 100 µM. The respective amounts of sulindac sulfide or sulindac sulfone from DMSO-d6 stock solutions, or only DMSO-d6, were titrated to the peptide, and measured within 20 minutes. Solid-state NMR sample preparation A monomeric solution of 15N-13C labeled Aβ 1-40 at a concentration of 100 µM was incubated with a 5-fold molar excess (500 µM) and a 10-fold molar excess of sulindac sulfide (1 mM) quiescently at room temperature. Levels of Aβ remaining in solution was monitored by centrifuging a 200 µl sample, and measuring the absorbance of protein in the supernatant. After 24 hours of incubation, negligible amounts of Aβ remained in solution, and the Aβ aggregated by a 5-fold molar excess was sedimented into a 4.0 mm rotor, and the Aβ aggregated by a 10-fold molar excess of sulindac sulfide was sedimented into a 3.2 mm 6 ACS Paragon Plus Environment
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rotor. As a reference, Aβ was incubated with 1% DMSO quiescently for 8 days at room temperature. The resulting aggregate was sedimented into a 4.0 mm rotor. For all samples, approximately 10 mg of Aβ was used. Pure sulindac sulfide was measured in a 3.2 mm rotor. Solution state NMR measurements All solution state NMR experiments were measured on Bruker Avance III spectrometers, operating at a 1H Larmor frequency of 500 MHz and 600 MHz, both equipped with a triple resonance cryogenic probe, or 750 MHz equipped with a conventional triple resonance probe. Regular 1H-1D experiments were carried out using a watergate pulse sequence for solvent suppression.34 2D 1H-15N HMQCs were recorded employing the SOFAST HMQC pulse sequence.35 2D 1H-13C HSQCs were acquired using a watergate pulse sequence for water suppression and constant time to suppress homonuclear scalar couplings during the t1 period. All measurements were carried out at 277 K. The samples typically contained Aβ concentrations of 50 µM or 100 µM and 10% D2O. Solid-state NMR measurements An Aβ sample incubated with a 10-fold molar excess sulindac sulfide was measured on a Bruker Avance III narrow bore spectrometer operating at a 1H Larmor frequency of 750 MHz equipped with a triple resonance MAS probe for 1H,
13
C,
15
N based experiments. All
measurements were performed at 270 K and a MAS rotation frequency of 15 kHz for the 2D and 3D assignment spectra, and at 11 kHz for transferred echo double resonance (TEDOR)36, 37 experiments detecting
13
C-15N dipolar couplings. The Aβ aggregates induced
by a 5-fold molar excess of sulindac sulfide, as well as the reference Aβ were measured at 600 MHz equipped with a triple resonance MAS probe (1H,
13
C,
15
N) at a MAS rotation
frequency of 13 kHz and 275 K. In all experiments, 1H-13C magnetization transfer was achieved through cross polarization (CP).38 2D spectra were recorded using proton-driven spin diffusion (PDSD)39 for TEDOR for
13
13
C-13C magnetization transfer, with a mixing time of 50 ms, or
C-15N magnetization transfer. The 3D NCACX and NCOCX40 experiments were 7 ACS Paragon Plus Environment
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recorded using TEDOR for
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C -15N magnetization transfer, and a dipolar assisted rotational
resonance (DARR)39 mixing sequence.
3D TEDOR experiments based on a pulse sequence described by Jaroniec et al41 were carried out to detect the Aβ salt bridge and 19F atoms in the proximity of
13
C in the NSAID-Aβ
co-aggregates (supplementary Fig. 6).18 For the detection of the salt bridge, the TEDOR mixing times were set to 7.27 ms and 15.72 ms, respectively. All experiments involving
19
F
were recorded on a Bruker Avance III wide bore spectrometer operating at a 1H Larmor frequency of 600 MHz equipped with a triple resonance CP MAS probe (1H,
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C,
19
F). These
measurements were recorded at a MAS rotation frequency of 12.4 kHz and a temperature of 270 K with mixing times of 0.65, 1.29, 2.58, 5.16 and 7.74 ms. No t1 evolution time on was implemented. To exclude intramolecular
19
F
19
F-13C atoms of sulindac sulfide caused by
natural abundance, TEDOR spectra of pure sulindac sulfide (Fig. 7A, black) recorded under the same conditions were subtracted from the TEDOR spectra of Aβ incubated with sulindac sulfide (Fig. 7A, red) to yield intermolecular signals corresponding to
19
F dipolar interactions
to the protein only (Fig. 7A, green). The relative concentrations of sulindac sulfide in both samples were estimated by the signal-to-noise ratios of sulindac sulfide resonances at
13
C
chemical shifts of 104.86 ppm and 110.75 ppm. This approximation revealed that the pure sulindac sulfide sample contains double amounts of the NSAID, compared to the sample of Aβ incubated with sulindac sulfide. To take these differences into account, the pure sulindac sulfide reference spectrum was subtracted from the spectrum of Aβ incubated with sulindac sulfide with a ratio of 0.5. Data analysis and manipulation Solution-state NMR data was processed using NMRPipe.42 Integrals of NMR signals were calculated employing nmrglue.43 NMR spectra were assigned using CcpNmr Analysis44,
45
and secondary structure propensities were calculated employing TALOS+.46 Carbon 8 ACS Paragon Plus Environment
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secondary chemical shifts (∆δC) of Cα, Cβ and Cα-Cβ (ppm) were calculated according to the following equation: ∆δC = δCexperimental - δCrandom reported by Wishart et al.
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coil.
Random coil values were used as
Chemical shift perturbations (CSP) (∆δ (ppm)) were calculated
from 1H-15N HSQC spectra using the following equation: ∆δ = ½ [(δHsul – δHref)2+( 0.4(δNsul – δNref)) 2]1/2. Cell culture SH-SY5Y cells were purchased from DSMZ (ACC 209) and were routinely cultured as described.48 To determine Aβ toxicity in SH-SHY5 cells MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl-tetrazolium bromide) assay was performed as described.33 Aβ1-42 peptides at a concentration of 2.5 µM were pre-incubated with sulindac sulfide (40-fold molar excess) or with 1% DMSO for 0, 4 and 8 hours at room temperature and incubated with cells for 12 hours. Electron Microscopy (EM) Aβ1-42 peptides were dissolved to 50 µM and incubated for the indicated times at room temperature either in conjunction with sulindac sulfide (40-fold molar excess) or with 1% DMSO, respectively. Aliquots (5 µl) of matured peptide solutions were negatively stained with 2% aqueous uranyl acetate as described49 and applied to formvar-coated nickel grids. Micrographs were taken using a Philips CM120 electron microscope at 80 kV and a 1K CCD camera. Size exclusion chromatography (SEC) SEC was performed as described.48 Briefly, synthetic Aβ1-42 peptides (100 µM final concentration) were dissolved and mixed with sulindac sulfide (40-fold molar excess) or 1% DMSO before incubation for 0 hours, and 8 hours at room temperature. Oligomers were separated using a Superose 12 10/300 GL column (GE Healthcare, Germany) with PBS and a flow rate of 0.5 ml/min.
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Results Solution-state NMR experiments were employed to study the NSAID induced aggregation of Aβ. We monitored the NMR resonances of the peptide in the presence of increasing concentrations of sulindac sulfide (Fig. 1B, Fig. S1A), and found that Aβ solubility is greatly reduced by the NSAID. In the experiment, a solution containing 50 µM Aβ was titrated with sulindac sulfide. 1H amide Aβ resonances were integrated and plotted vs. the concentration of titrated sulindac sulfide in the sample. At a 5-fold molar excess of the sulindac sulfide (250 µM), only ~ 50% (27.2 µM) of Aβ remains in solution. At a 10-fold molar excess (500 µM), most of the peptide has aggregated, and the soluble population amounts to ~ 3% (1.6 µM). Experiments carried out subsequently indicate that the amount of soluble Aβ stays constant over time. In all titrations, precipitation is immediate within the deadtime of the experiment (the deadtime is on the order of 20 min). 2D 1H-15N HSQCs of 50 µM Aβ were recorded in the absence (black) and presence (red) of a 6-fold molar excess of sulindac sulfide (300 µM) (Fig. S1A). We find no specific CSPs for the backbone resonances in the presence of sulindac sulfide (Fig S2A). All backbone resonances decay uniformly and with comparable rates (Fig. S2B), suggesting that chemical exchange broadening is not causing the disappearance of the resonances. Similarly, no CSPs are observed for the side chain resonances of Aβ recorded in a 1H-13C HSQC, and the signal decays evenly for all signals (Fig. S3). The same set of experiments was repeated for Aβ in the presence of sulindac sulfone. Although the structure of this NSAID is highly similar to that of sulindac sulfide, it has been shown that sulindac sulfone behaves quite different in aqueous buffer.21 Again we monitored the solubility of Aβ (50 µM) upon addition of sulindac sulfone using solution-state NMR (Fig. 1B, Fig. S1A). We find that Aβ solubility is largely unaffected in the presence of sulindac sulfone. Fig. 1B (black symbols) represents the integrated Aβ amide resonances as a function of the concentration of sulindac sulfone in the sample. No Aβ aggregation was 10 ACS Paragon Plus Environment
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observed, and even at a 20-fold molar excess of the NSAID (1 mM), 97% (48.5 µM) of the peptide remains in solution. Similarly to sulindac sulfide, sulindac sulfone does not induce CSPs in Aβ backbone resonances (Fig. S1A).
Figure 1. Assessment of Aβ and NSAID solubility. (A) Chemical structures of sulindac sulfide (left) and sulindac sulfone (right). (B) Concentration of soluble Aβ as a function of the amount of sulindac sulfide (red) and sulindac sulfone (black) in the sample. (C) Concentration of soluble sulindac sulfide (red) and sulindac sulfone (black) as a function of the amount of titrated NSAID in the presence of Aβ () and in case of buffer only (*). The 1 respective solubilities were determined by integration of 1D- H resonances in the amide region (8.50 to 7.65 ppm) and by analysis of characteristic NSAID peaks (7.22 ppm and 7.08 ppm for sulindac sulfide, and 7.75 ppm and 7.52 ppm for sulindac sulfone). Corresponding NMR spectra are shown in Fig. S1. (D) Molar ratio of aggregated amounts of sulindac sulfide to aggregated amounts of Aβ (n) as a function of the amount of titrated sulindac 1 sulfide. The signal intensities were extracted from H-1D spectra.
To assess the solubility of the two NSAID molecules, we quantified NSAID intensities as a function of their concentration in the presence and absence of Aβ. For this purpose, we monitored two resolved aromatic resonances for sulindac sulfide and sulindac sulfone
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respectively. For sulindac sulfone alone in buffer (Fig. 1C, black symbols) the intensities increase linearly with the amount of titrated material, indicating that this molecule is highly soluble up to a concentration of 1 mM. By contrast, sulindac sulfide only shows this linear behavior for concentrations lower than ~ 400 µM (red). When higher concentrations are titrated to buffer, the NMR signal intensities do not increase further, indicating micelle formation. This effect is even more severe in the presence of Aβ, where a decrease of the amount of soluble sulindac sulfide (red) is observed at concentrations as low as ~ 100 µM, suggesting that the presence of Aβ promotes colloid formation of sulindac sulfide. On the contrary, the solubility of sulindac sulfone seems to be unaffected by the presence of Aβ (black). This behavior is also reflected in the line shapes of the respective 1D-1H NMR spectra (Fig. S1C). Fig. 1D shows the ratio of soluble sulindac sulfide to aggregated sulindac sulfide to Aβ vs. the amount of titrated sulindac sulfide. Initially, Aβ and sulindac sulfide do not interact. Above a critical micelle concentration (CMC) of around 150 µM, the molar ratio of sulindac sulfide and Aβ is approximately constant (6:1). Afterwards, this ratio is slowly increasing indicating presumably the formation of larger micelles. Apparently, Aβ and sulindac sulfide are co-aggregating, maintaining a constant ratio of sulindac sulfide and Aβ in the aggregate. In order to analyze the toxicity and aggregation behavior of Aβ1-42 co-incubated together with sulindac sulfide, we carried out biochemical assays (Fig. 2). Size-exclusion chromatography demonstrates immediate formation of a range of Aβ oligomers in the presence of sulindac sulfide or DMSO (Fig 2A). Aβ peptides were incubated for 0 and 8 hours in presence and absence of sulindac sulfide. Incubation with sulindac sulfide for 8 hours leads to a shift of Aβ populations from tetra-/hexamers to high-n oligomers (20-16mers) compared to control Aβ. These results were corroborated by EM investigations (Fig. 2B). Electron micrographs show that Aβ aggregation is enhanced when incubated with sulindac sulfide compared to controls. Analysis of Aβ aggregates reveals a significant increase in the size of oligomeric structures when co-incubated with sulindac sulfide after 8 hours (51 ± 2 vs. 71 ± 5 nm). Similarly, 12 ACS Paragon Plus Environment
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incubation for 24 hours and 7 days yields an increased amount of fibrillar aggregates. The viability of SH-SY5Y cells treated for 12 hours with pre-fomed Aβ oligomers with and without incubated sulindac sulfidewas assessed using a MTT assay (Fig. 2C). Aβ was pre-incubated for 0, 4 and 8 hours, respectively. Aβ-induced toxicity is significantly decreased after 8 hours of pre-incubation with sulindac sulfide in comparison to cells incubated with Aβ oliogmers formed in the presence of DMSO only.
Figure 2. Analysis of toxicity and aggregation behavior of Aβ β 1-42 co-incubated with sulindac sulfide. (A) SEC of Aβ peptides incubated for 0 and 8 hours in absence and presence of sulindac sulfide. At 0 hours, Aβ was mainly present as tetra-/hexamers. After 8 hours a slight increase of high-n oligomers (20-16mers) was detected, whereas Aβ co-incubated with sulindac sulfide leads to a decrease of tetra-/hexamers and an increase of higher oligomers (10x, 20-16x). (B) Electron micrographs of Aβ in absence and presence of sulindac sulfide after a
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incubation time of 8 and 24 hours and 7 days, respectively. Sulindac sulfide-incubated Aβ shows an increased propensity to form aggregates in comparison to controls. Aβ assembles into larger oligomeric structures when coincubated with sulindac sulfide after 8 hours compared to reference sample (51 ± 2 vs. 71 ± 5 nm). After 24 hours and 7 days, respectively, formation of an increased amount of Aβ fibrils peptides is observed. Scale bar: 100 nm. (C) Viability of SH-SY5Y cells after incubation with Aβ peptides. SH-SY5Y cells were treated for 12 hours with Aβ peptides pre-incubated with sulindac sulfide for 0, 4 and 8 hours, respectively to form oligomeric species. Aβ1-42 peptides pre-incubated with sulindac sulfide exert significantly less toxicity compared to pure Aβ (*p
