Lipid Coaggregates are Formed During Î±-Synuclein-Induced

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Protein:lipid co-aggregates are formed during #synuclein-induced disruption of lipid bilayers Andreas van Maarschalkerweerd, Valeria Vetri, Annette Eva Langkilde, Vito Foderà, and Bente Vestergaard Biomacromolecules, Just Accepted Manuscript • Publication Date (Web): 11 Sep 2014 Downloaded from http://pubs.acs.org on September 16, 2014

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Protein:lipid co-aggregates are formed during αsynuclein-induced disruption of lipid bilayers

Andreas van Maarschalkerweerd1, Valeria Vetri2*, Annette Eva Langkilde1, Vito Foderà1, and Bente Vestergaard1* 1

Department of Drug Design and Pharmacology, University of Copenhagen, Universitetsparken 2, 2100 Copenhagen, Denmark.

2

Dipartimento di Fisica e Chimica, Universitá di Palermo, Via Archirafi 36, 90123 Palermo, Italy

Abstract

Amyloid formation is associated with neurodegenerative diseases such as Parkinson’s disease (PD). Significant α-synuclein (αSN) deposition in lipid-rich Lewy bodies is a hallmark of PD. Nonetheless, an unraveling of the connection between neurodegeneration and amyloid fibrils, including the molecular mechanisms behind potential amyloid-mediated toxic effects, is still missing. Interaction between amyloid aggregates and the lipid cell membrane is expected to play a key role in the disease progress. Here, we present experimental data based on hybrid analysis of 2photon-microscopy, solution small angle X-ray scattering and circular dichroism data. Data show in

1 ACS Paragon Plus Environment

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real time changes in liposome morphology and stability upon protein addition, and reveal that membrane disruption mediated by amyloidogenic αSN is associated with dehydration of anionic lipid membranes and stimulation of protein secondary structure. As a result of membrane fragmentation soluble αSN:-lipid co-aggregates are formed, hence suggesting a novel molecular mechanism behind PD amyloid cytotoxicity.

Keywords 2-photon microscopy/α-synuclein/co-aggregate/Parkinson's disease/small angle X-ray scattering.

Introduction The aggregation process of peptides and proteins into highly structured amyloid fibrils is of wide interest, not least due to the implication of these phenomena in neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease (PD)1. A number of structurally distinct disease specific proteins and peptides have been observed to form amyloid fibrils2, yet the mature fibril aggregates share common structural features. It is speculated that such common structural motifs may consequently define a generic molecular mechanism of pathogenesis. Accumulating evidence suggests that amyloid-related toxicity is not directly associated with the presence of mature fibrils, but rather with the presence of a heterogeneous ensemble of aggregated structures generated during fibril assembly, which may modulate3 and potentially permeabilize cell membranes4, thereby initiating a common group of downstream pathologic processes1. Numerous studies investigate interactions of amyloidogenic species with cell membranes5-7, yet the molecular processes underlying cytotoxicity remain unclear. Complementary to the influence of amyloid species on membrane structure, membranes may act as active surfaces, (dis-)favouring aggregation prone


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conformations. This may be caused by direct interaction with protein hydrophobic or charged groups, by the accumulation of proteins in a crowded environment at liquid/lipid interfaces8 or by favouring nucleation, possibly via template assisted mechanisms9,

10

. The mutual influence of

protein and lipid structures is hence an important area of investigation to advance the understanding of amyloid related cytotoxic effects. The progressive neurodegeneration observed in PD has been related to α-synuclein (αSN) aggregation11. Native αSN is a 140 amino acid intrinsically disordered protein, abundant in presynaptic terminals of the human brain. αSN is observed to form amyloid fibrils both in vivo and in vitro, and a major hall-mark of PD is the deposition of αSN fibrils and lipids in Lewy bodies12. Several intermediate fibril species have been observed in vitro8,

13, 14

, most of which have been

suggested to play a role in PD pathogenesis. Different mechanisms for the cytotoxic effect are proposed, ranging from membrane disruption15, pore-like membrane permeabilisation14,

16, 17

or

more subtle effects such as membrane destabilization or membrane thinning4, 18, 19. The observation of deposited lipids within in vitro formed amyloid fibrils20 and in vivo human amyloid deposits12,

21

substantiates the importance of studying potential protein:lipid co-

aggregation. In vitro incorporation of lipids into or onto fibril structures has also been observed for αSN, an effect mediated by extraction of lipids from membranes during fibrillation15, 20. In many published studies, the effect of adding isolated or partially purified αSN fibrillar species are studied14,

20, 22, 23

. However, isolation of intermediate structural species may influence both the

structure of the species and the fibrillation kinetics. This suggests that the study should preferably be done without disturbing the fibrillation equilibrium, hence without isolating individual species from the solutions. Also, given the potential mutual influence of lipids and the fibril species, it is important to simultaneously monitor and characterize the biophysical and structural properties of both the lipid membrane and the aggregating protein. To do this, we combine small angle X-ray


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scattering (SAXS) with spectroscopy and 2-Photon Microscopy (2PM), using the fluorescent probe Laurdan. Laurdan is sensitive to the structural and biophysical environment of the membrane. It is a widely used dye for studying membrane order both in bulk (cuvette) and 2-photon-microscopy (2PM) experiments. Laurdan was successfully used in the study of protein:membrane interactions in systems ranging from solutions to live cells10. Bulk spectroscopic analysis of Laurdan emission has also been used for investigation of membrane interactions of several amyloid proteins and peptides, such as amyloid-β peptide24, lysozyme25, histidine-rich protein26 and β2-microglubin27 along with studies on αSN. Specifically Zhu et al. initially reported an increased fluidity of mixed anionic/zwitterionic membranes upon addition of protofibrils and fibrillated αSN28. Later reports by Jo et al. and Kamp et al. both described increased fluidity of synaptosomal, model lipid and bovine brain sphingomyelin membranes resulting from interaction with native αSN29, 30. In other cases, Laurdan has also been implemented in the study of amyloid(-like):membrane systems using 2PM3133

. By expanding the approach, combining Laurdan spectroscopy and 2PM with SAXS, we enable

direct observations in real time together with structural characterization of the process in solution. We observe that together with disruption of the liposomes, new species are formed. These species are in the sub-resolution range for 2PM, yet the overall significantly changed lipid environment reveals its existence both in bulk and in the microscopy analysis, and the new species are easily observed using SAXS. By a combination of 2PM, SAXS and circular dichroism (CD), we conclude that the new species are αSN:lipid co-aggregates. We reveal that the co-aggregates exhibit significant α-helical content and a dehydrated lipid environment. We speculate that the formation of this αSN:lipid co-aggregate could be of major importance for understanding the cytotoxic effect of αSN, and potentially other amyloid proteins.


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Materials and methods Materials 1-hexadecanoyl-2-(9Z-octadecenoyl)-sn-glycero-3-phosphocholine (POPC) and 1-hexadecanoyl2-(9Z-octadecenoyl)-sn-glycero-3-phospho-(1’rac-glycerol) (sodium salt) (POPG) were purchased from Avanti Polar Lipids. 6-dodecanoyl-2-dimethylaminonaphthalene (Laurdan), Calcein and Thioflavin T (ThT) were purchased from Sigma-Aldrich. Purification of αSN The expression construct was a kind gift from Bioneer, Hørsholm, Denmark. The Escherichia coli plasmid vector p-ET11-a was transfected into an Escherichia coli BL21 (DE 3) cell line for expression of αSN. The details on αSN purification are given in Supporting Information. Preparation of intermediate and fibril αSN (fibrillation assay) The lyophilized αSN was dissolved in a 20 mM PBS buffer with 150 mM NaCl, pH 7.4 prior to filtration using 0.20 µm spin filter (Millipore). The concentration was determined by A280 measured on a nanodrop UV-Vis spectrophotometer (Thermo Scientific) using an extinction coefficient of 5120 M-1cm-1 34. αSN fibrillation was induced above the protein super critical concentration14 at 12 mg/ml in 20 mM PBS buffer with 150 mM NaCl and 20 µM ThT (Sigma Aldrich) pH 7.4 in a Fluostar Optima plate-reader (BMG labtech) in 96 well optical bottom plates (Thermo Scientific). 150 µl volume samples were incubated at 37°C with orbital shaking (300 rpm, 2 mm) carried out in cycles of 280 sec followed by 80 sec pause with a 3 mm glass bead inside the sample35. ThT emission was used as indicator of the fibrillation state. The measurements were carried out under an excitation wavelength of 450 nm and the ThT emission was recorded at 480 nm (using 5 nm excitation and emission bandwidth). Intermediate samples were obtained by quenching aggregation kinetics in the early growth phase after approximately two hours. Quenching was achieved by withdrawing samples from the fibrillation assay. The stability of the intermediate sample was


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confirmed by SAXS (see suppl. Fig. S1). Fibril samples were obtained after longer incubation times and collected after the ThT fluorescence intensity reached a plateau value. Intermediate and fibrillar samples were prepared both in the presence and in the absence of ThT using the ThT fluorescence as internal clock for the progression of fibrillation assuming no significant ThT related impact on the fibrillation36. Liposomes preparation Giant vesicles (GV) and calcein encapsulating small unilamellar vesicles (SUV) were prepared essentially as described elsewhere37. The experimental details are provided in Supporting Information. SAXS data collection and primary analysis Native αSN was prepared at a concentration of 13.5 mg/ml from lyophilized powder, as described above, and characterized by SAXS. Details on the data acquisition and initial data processing are provided in Supporting Information. Prior to Ensemble Optimized Method (EOM) analysis, the Guinier range was determined in PRIMUS38. The valid Guinier range (sRg ≤ 1.3) determined the qmin of the data included in the analysis39. Data points until q=0.3 Å-1 were included. An initial estimate of the average mass of the solutes was made by extrapolating to the forward scattering intensity, I(0), and scaling with a BSA standard. EOM consist of two individual procedures performed by RANCH (random pool generation) and GAJOE (ensemble optimization and selection), respectively38, 40. Monomer and dimer pools of 10000 structures each were generated based on the primary structure of wild type αSN and the quasi Cα-Cα Ramachandran plot. The dimer pool was generated as an asymmetric assembly of two monomers with a contact point in the NACregion. The generic algorithm of GAJOE was used to select an optimized ensemble of structures from the generated pool that fits the experimental data. Constant subtraction was allowed in the


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fitting process, to correct for any possible systematic errors in the experimental data. A total of 1000 generations were completed with 100 repeats, using an ensemble size of 50. Fibrillated αSN and intermediate species were prepared in the platereader as described above at a concentration of 12 mg/ml before data collection (details on data acquisition and initial data processing are provided in Supporting Information). The composition of the intermediate sample was subsequently analyzed using OLIGOMER from the ATSAS program suite38, in which the scattering of the intermediate sample was fitted by a linear combination of the scattering from the native and fibrillated species, including or excluding the scattering from a wreath shaped oligomer14 as a third species, hence the volume fractions of the components are optimized to the best fit. The scattering profiles of POPG SUV and GVs were recorded before and after adding native αSN (molar lipid-protein ratio 50:1). The experimental details on the data collection are given in Supporting Information. The scattering from mixtures of αSN with liposomes were measured after mixing. The scattering profiles were subsequently fitted using OLIGOMER, as described above, with either two or three components. Here, input components were the experimental data curves measured on protein and lipid, prior to mixing, and the wreath shaped oligomer, respectively. Dye release assay Calcein release assays were carried out using a plate reader system (Fluostar, BMG Labtech) with 96-microwell polystyrene plates (NalgeNunc). Each well was filled with 150 µL of solution and three replicates for each sample were measured to determine the reproducibility of the results. αSN was mixed with SUVs in a 20:1 lipid-protein molar ratio. Control full release measurements were obtained at the end of each release kinetics by adding 25 µL 2.5% Triton-X (Sigma Aldrich) to each well. Experimental details are given in the Supporting Information. Circular dichroism


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The secondary structure of native and intermediate αSN was evaluated by means of CD both in the presence and in the absence of either POPC or POPG GVs. For the reported experiments, protein concentrations were 0.15 mg/ml. POPG and POPC GVs were added to the sample at a lipidprotein molar ratio of 20:1. The CD spectra of liposomes in the same conditions were subtracted as background. Details on the data recordings are given in the Supporting Information. Membrane fluidity measurements 6-dodecanoyl-2-dimethylaminonaphthalene (Laurdan) fluorescence and in particular its generalized polarization (GP) was used to investigate lipid bilayer properties during interaction with protein. Measurements were carried out at 25 ⁰C in semi-micro plastic cuvettes using a Jasco-FP8500 spectrophotometer equipped with a peltier thermostat. The stock solution of Laurdan-stained GVs was diluted to a concentration of 400 µM and left to equilibrate in the spectrophotometer for approximately 30 min or until the emission and the GP-ratio had stabilized. Emission spectra were recorded every 4 minutes at 370-620 nm with a bandwidth of 5 nm. The sample was excited at 375 nm and spectra were recorded with a response time of 1 s, data intervals of 0.5 nm, and a scan speed of 100 nm/min. If the sample was left untouched, precipitation of the liposomes would occur as evident from a gradual decrease in Raman scattering. The cuvette was gently shaken prior to all measurements to keep the sample uniformly dispersed. αSN was added to the cuvette after temperature equilibration in lipid-protein molar ratios of 20:1, 50:1, and 100:1 for native and fibrillated αSN and at 50:1 for intermediate αSN. Laurdan has a dipole moment which can reorient surrounding water molecules. Energy required from the excited state as a result of the reorientation cause a red shift of the fluorescence41. Indeed, it is possible to infer that no water is allowed in the densely packed lipid gel phases while higher hydration levels may occur in liquid crystalline phases. GP-ratios were calculated according to Parasassi et al. 42 as in equation 1 =

− (1) +


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where I440 and I490 are the emission intensities at 440 nm and 490 nm, respectively. The significance and the mathematical treatment of GP is described elsewhere41. The data were background subtracted using spectra of corresponding mixtures of PBS and HEPES buffers before normalizing the curves at 455 nm facilitating a visual interpretation of the relative shifts of 440 nm and 490 nm emission bands. The experiments were terminated after approximately 4 hours or when GP values were stabilized. Control measurements by direct Laurdan staining of structured and unstructured αSN showed only weak fluorescence which could not account for the changes of Laurdan emission. Laurdan alone did not present any significant fluorescent signal. In addition, the liposomes were shown to be intrinsically stable under the experimental conditions as the emission spectra and hence also the GP did not change during 3 hours of incubation. The observed changes in Laurdan emission can therefore be considered a direct result of the protein:lipid interaction. Control measurements were performed using Concanavalin A fibrils to investigate any effect of inert fibrils43. Concanavalin A fibrils were added to Laurdan-stained POPG GV in a lipid-protein ratio of 20:1. It was found that the emission of Laurdan did not change after several hours (Fig. S4). Addition of buffer alone did not significantly affect GP values in any of the lipid systems that were tested. 2-photon-microscopy measurements Aliquots of 200 µL Laurdan stained liposomes were deposited in Chambered Coverglass (LabTek IINunc). 512x512 pixels image stacks were sequentially acquired using a Leica TCS SP5 confocal scanning microscope with a 63x oil objective (Leica Microsystems, Germany) at a scanning frequency of 200 Hz under two-photon excitation at 780 nm (Spectra-Physics Mai-Tai Ti:Sa ultra-fast laser). Fluorescence signal was simultaneously recorded in two channels suitable for GP calculation41: 410-460 nm (blue channel) and 480-540 nm (green channel). Data were collected before and after addition of aliquots of protein solution at selected concentrations. GP data


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acquisition, significance and image analysis was previously described in the literature32. Cropped images (256x256 pixels) were analyzed using the SimFCS program (Laboratory for Fluorescence Dynamics, University of California, Irvine, CA) after calibration of the GP scale to obtain a GP=0.2 for a standard Laurdan solution in DMSO at 22°C32. The background was thresholded by intensity to avoid background fluorescence in images prior to addition of αSN32. Image analysis gives the histogram of the number of pixels with a given GP, the center of the histogram representing the average value of GP in the image. The addition of Concanavalin A fibrils did not alter the morphological characteristics of POPG GVs, it did not reduce the amounts of liposomes, and it did not cause any changes in GP as quantified by GP-intensity maps and histograms.

Results Structural analysis of αSN solution state Fibrillation of αSN was obtained by incubating freshly prepared αSN according to standard protocols44. Data reported in Fig. 1A show the time evolution of ThT fluorescence intensity whose growth can be ascribed to the growth of fibrillar species in solution36. The signal growth shows the typical sigmoidal profile with a lag-phase, growth phase and steady state. Aggregation was quenched (see Materials and Methods) at two different incubation times (2 h and 24 h) in order to obtain intermediate pre-fibrillar species and mature fibrils. Importantly, the intermediate sample was collected in the early stages of the elongation phase to ensure a high content of the intermediate oligomeric species which we have previously characterized structurally, and which has been hypothesized to be related to the cytotoxic effect in PD (see Fig. S1). In this sample native, intermediate and fibril species are expected to co-exist in a dynamic equilibrium14. With the aim of characterizing the structural features of the samples prior to mixing with lipid structures, we recorded solution SAXS data from native (non-incubated), intermediate and mature


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fibril samples (Fig. 1B). Native recombinant αSN consists mainly of intrinsically unfolded monomers, yet early oligomerization is commonly reported14,

34, 45

. The presence of even small

amounts of dimer or higher oligomers could potentially change the overall affinity towards the phospholipid membranes, hence requesting thorough pre-characterization of also the native samples. The homogeneity of a protein sample is commonly evaluated in SAXS by determining the average molecular weight using a reference (often Bovine Serum Albumin, BSA) or by absolute calibration46. In our case, basic SAXS analysis of the native samples indicates a molecular weight and radius of gyration, Rg, in accordance with a monomeric unfolded state. To assess the native state more thoroughly, the data were analyzed using the EOM, from the ATSAS software package40. The experimental scattering curves of native αSN were fitted using theoretical scattering curves generated from ensembles of randomly generated conformations of monomeric αSN, dimeric αSN, or a mixed pool of monomers/dimers (Fig.1C). The selected ensemble from the monomer pool yielded a nearly perfect fit, whereas sampling from the dimer pool alone did not provide a reliable fit. When fitting from a pool of both monomers and dimers, no dimer conformations were selected. The native sample is thus composed of monomers without any detectable amount of dimers or higher order species. Analysis of the SAXS data from the fibrillated samples clearly reveals the presence of high molecular weight aggregates in solution (Fig. 1B) as previously reported for the αSN fibril state14. Regarding the intermediate species, the forward scattering, I(0), and average Rg of the samples were confirmed to be well above those of the native samples (Table S1) hence inferring an increase in the average dimensions of the species present in solution. In order to test whether this average increase in dimensions relates to the presence of smaller intermediate oligomers, or simply to the presence of large fibrils in equilibrium with monomeric species, we attempted a fit of the SAXS data from the


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intermediate state with either two (monomer and fibril) or three (including intermediate oligomers) components. A linear combination of the monomer and fibril scattering curves results in large deviations between the fitted curve and the experimental data, whereas a linear combination, including the scattering profile of the wreath shaped oligomer previously published by us14, lead to a satisfying agreement with the experimental data (Fig. 1D). The volume fractions estimated by SAXS are 0.54 (monomers), 0.16 (oligomers) and 0.30 (fibrils). This strongly suggests that the presently investigated intermediate samples are directly comparable with those previously analyzed by us and that they contain relatively large amounts of intermediate oligomeric species, which are similar in shape to the wreath shaped oligomer. In conclusion, the SAXS-based characterization of native, intermediate and fibril samples reveals the presence of initially monomeric αSN (native samples), coexisting monomer/oligomer/fibril (intermediate samples) and, at late time points, predominantly fibrils (fibril samples). These three samples were then tested with respect to vesicle permeabilization/disruption potency. Vesicle disruption caused by native and intermediate αSN Fig. 2A shows the release of Calcein from POPG SUV as a function of time. No significant release from POPC SUV samples was observed with the experimental conditions investigated. These observations are in line with previous reports28, 47. As expected, the addition of αSN fibrils shows a drastically lower release than intermediate samples, but remarkably, in the current study, release from anionic liposomes is observed to the same extent and with compatible profiles when adding native or intermediate αSN samples (Fig. 2A). The Calcein release induced by native and intermediate samples reaches about 90% of the total Calcein content, thus indicating that within the time frame of the release assay, the majority of the lipid vesicles interact with αSN leading to the permeabilization/destabilization of the membrane and dye efflux. Interestingly, the release profiles induced by native and intermediate αSN samples cannot be fitted by a single exponential profile.


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This indicates that Calcein release induced by αSN both in native and intermediate states proceeds via a complex process, possibly involving several interconnected mechanisms. On the contrary, the temporal profile of the less efficient release induced by the fibril samples can be fitted using a single exponential. This suggests that a simpler mechanism, with a single mean energy barrier, underlies fibril-mediated disruption. Although the ThT-trace of the fibrillated sample has reached the steady-state, there might still be some residual non-fibrillated αSN left, which in theory might contribute to the induced calcein release.

αSN interaction reduces the overall membrane fluidity The average impact of αSN on the membrane order on both anionic and zwitterionic bilayers was further investigated by monitoring Laurdan fluorescence emission in cuvette experiments. Laurdanstained POPG and POPC GV were added to the three samples (native, intermediate and fibrils) and the changes in the emission spectrum of the dye were followed as a function of time. In Fig. 2B we show, as an example, the evolution of the Laurdan fluorescence emission spectrum over 4 h after addition of native αSN to a POPG GV sample. The normalized data show a gradual increase in intensity at 440 nm with a simultaneous decrease of intensity at 490 nm. This behavior can be ascribed to changes in Laurdan environment toward a lipid organization with an increased gel-like character, suggesting an enhanced compactness of the membrane. The spectral properties of Laurdan allow analyzing membrane structural and dynamical properties giving both bulk and topological information. In particular the emission spectrum of this dye is sensitive to membrane phases and is a reporter of both the presence of water molecules and their mobility. In particular, differences in Laurdan spectra in different membrane environments have been ascribed to the difference in water penetration in the membrane between the gel and liquid crystalline phases. As described in the methods, GP value is used to quantify relative changes in Laurdan emission


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spectrum and, as a consequence, it reflects the level of lipid packing. Changes in GP toward higher values can be interpreted as progressive changes of membrane fluidity toward ordered gel phase due to a progressive dehydration of lipid bilayer41, 42, 48, 49. Thus, the observed changes in our study can be interpreted as the result of bilayer dehydration41, 49. Similar changes in the Laurdan spectrum were observed after addition of αSN in the intermediate state, and only to a lesser extent when adding the fibrillar states to POPG GVs. On the contrary, no significant changes in the spectrum profile were observed when adding protein samples to POPC GVs (Fig. S2). Lipid charge is thus fundamental for the observed interaction. In order to have quantitative information on the time evolution of lipid organization after the addition of αSN in the different states, the generalized polarization (GP) ratio was estimated for the three samples42. The GP reports the relative changes in Laurdan emission. The Laurdan emission is characterized by distinct emission pattern related to the hydration state of the Laurdan dyes local environment (the lipid bilayer interior) commonly expressed as changes in membrane flexibility/rigidity. The variations of GP as a function of time after protein addition (relative to the GP value before protein addition) are reported in Fig. 2C showing a significant variation in the first 5 minutes after protein-addition. As it is evident, an increase in the GP value is observed in all samples, hence clearly indicating a decrease of membrane fluidity. The addition of αSN intermediate species produces a gradual growth of GP, while addition of native αSN produces a sudden GP increase (within the first 20 minutes), both reaching the same extent of change. Addition of fibril samples also causes a sudden GP increase, but, in line with the Calcein release data, only resulting in minor total changes in GP value. In Fig. 2D we report results of analogous experiments, varying the lipid/protein ratios. At increasing protein concentrations a larger GP growth is found as a function of time. In particular at lower concentrations (100:1 and 50:1 lipid/protein ratio) Laurdan GP grows to a plateau value, whilst at the higher protein concentration (20:1 lipid/protein ratio) we observe larger GP variation with a


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different temporal evolution that does not lead to a stable value within the explored time interval. Such behavior is observed only to a minor extent after addition of fibrils (Fig. S3). Control measurements were performed using Concanavalin A fibrils. These fibrils are expected to be inert and to not interact with lipids bilayers43. Accordingly, addition of Concanavalin A fibrils to Laurdan-stained POPG GVs in a lipid/protein ratio of 20:1 in exactly the same conditions did not result in any changes in the emission of Laurdan, even after several hours (Fig. S4). Lipid dehydration accompanies vesicle disruption The Laurdan cuvette experiments do not give specific information on possible changes in individual liposome morphology or on their effective disruption upon interaction with native or aggregated αSN species but they give bulk information on average membrane changes in the whole sample. We thus performed 2PM experiments, which allows imaging of lipid membranes using Laurdan and, through analyzing the fluorescence signals, obtaining GP measures41,

42

thereby

providing information on the physical characteristics of lipid bilayers and morphological changes simultaneously. This information can be obtained in real-time allowing monitoring of the kinetics of protein:membrane interactions. Importantly we note that 2PM does not require any treatment or fixation of the sample, and that this technique enables non-invasive dynamic studies with submicron resolution that can be performed also in live cell10, 32, 41. In Fig. 3A-D we report representative 2PM images acquired for Laurdan stained POPG GVs after addition of native αSN at 50:1 lipid/protein molar ratio. Transmission images acquired simultaneously (Fig. 3E-H) are also reported (Movie S1 and S2). As can be seen both in the fluorescence and transmission channels, two main effects are observed: i) a reduction of measured mean fluorescence intensity is observed in the whole image, and ii) this is accompanied by progressive disappearance of liposomes as a function of time. The decrease in liposome population continues until they are completely depleted. Control experiments with Concanavalin A fibrils, in


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exactly the same conditions, show that mean intensity, vesicle number and shape remain stable for analogous time intervals, indicating that the observed effects, including fluorescence intensity reduction, are caused by αSN interaction with GVs. Real time observation of the sample shows that many liposomes are disrupted within the first minutes after αSN addition. All liposomes in the sample are depleted within 4 hours. In these experiment series, the quantification of membrane fluidity is achieved by the GP analysis of 2PM measurements. The results are given as GP-maps at pixel resolution of corresponding images together with the corresponding GP histograms. Representative results of GP analysis of 256X256 images acquired as a function of time after native αSN addition are reported in Fig. 4. After protein addition liposome disruption and a sudden increase of average GP values is observed. Similar experiments were performed using intermediate samples, with comparable observations including rupture of liposomes and appearance of high GP pixels and with fibrillated samples were dramatic changes where not observed in the same timescale. The average GP value after addition of native, intermediate and fibrillated αSN is reported as a function of time in Fig. 5. In line with the cuvette experiments, an increase of average GP value is found for all samples. Again, the observed increase is much less evident after the addition of fibrils. It is also possible to analyze selected parts of the images containing single liposomes and hence to monitor GP changes as a function of time for structures that do not readily disappear. Repeated observations reveal that the GP values of liposomes that do not immediately disappear increase as a function of time (white arrows in Fig. 4B-D). In figure 4, a liposome disruption event is highlighted (red arrow) occurring in few minutes. The disintegration of a single liposome coupled with a significant increase of GP measured in its surroundings (compare red yellow pixel with black ones in neighbor area right after liposome disruption) is evident.


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These pixels are indicative of the presence of lipid membrane parts whose rigidity increases to the point that membrane is disrupted. High GP pixels in the surroundings contain lipids in the gel like phase sequestrated from disrupted membrane. Importantly as can be seen by comparing images in panel 4 B-D with data in panel A , pixels with elevated GP value appear as a function of time in the surrounding of liposomes that remain in the observed area.. Interestingly, these data indicates that the overall GP-increase measured in bulk experiments is the result of the progressive increase of GP in intact liposomes and by a generally increasing GP-background-like signal, which can be ascribed to a growing population of high GP species uniformly distributed in solution. These species have sizes below instrumental resolution. This suggests to us that lipids diffuse into solution after liposome rupture, organized in small rigid lipid clusters, potentially in complex with proteins. When adding αSN to POPC vesicles, neither morphological changes nor changes in the GP value could be observed. In conclusion, we monitor a clear disintegration of POPG GVs upon addition of monomeric or intermediate αSN, but much less so when adding fibrillated αSN to the liposome samples. An increase of GP values measured in the near vicinity of bursting lipid vesicles and in the whole image accompanying liposome disruption (and further supported by the integrated bulk experiments using fluorescence spectroscopy) shows that the lipid environment undergoes structural changes, resulting in smaller species with a more rigid and potentially dehydrated environment. These lipid species are smaller than the diffraction limited resolution in the 2PM experiment. SAXS, CD and 2PM based identification of a new protein:lipid complex The data in Fig. 6A show CD spectra measured for αSN in the presence of POPG GV starting from the native state. These data are in line with previous results showing that native monomeric αSN undergoes a transition from random coil towards α-helix when incubated with anionic bilayers50. Interestingly, in this experiment, the transition takes place within a time interval


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comparable to that of the initial vesicle disruption and the associated increased rigidity of the membrane environment. In concert, Calcein release, CD and 2PM/Laurdan spectroscopy data thus indicate that the formation of α-helical protein parallels both vesicle disruption and – importantly – also new physico-chemical properties of the lipid environment. The CD observations at late time points show that the protein structural changes remain after vesicle rupture. Without association to lipids, the protein is expected to remain in a random coil conformation. This hence suggests that the protein interacts with the disrupted lipids, specifically the formation of new oligomeric protein:lipid complexes. The same transition towards α-helix is observed for the protein sample at intermediate time point (Fig. S5A). No changes in secondary structure were detectable upon mixing native and intermediate αSN with POPC GV (Fig. S5B). With the aim of further exploring protein:vesicle interactions, SAXS experiments were performed on mixtures of POPG GV or SUV and αSN in the native state, immediately after adding protein. Analysis of data (Fig. 6B) was performed using the same approach as the one used for characterizing the molecular state of intermediate samples. We confirm the presence of a protein:lipid complex in the mixtures. Specifically, if the mixed samples would only contain a mixture of the individual protein and vesicle starting materials (i.e. protein and lipid do not interact), we should be able to fit the experimental data from the mixed sample as a linear combination of data collected on native αSN and vesicles, respectively. In Fig. 6B we report the poor result, after using such a two-component combination of native αSN and POPG SUV experimental signals (red line), to fit experimental data of the mixture (circles). The blue line reveals the fit with three components, including the wreath shaped oligomer as a tentative third species. The green line represents the residuals from the three component fits combined with the oligomer signal. This green line, hence, depicts a scattering curve, corresponding to the scattering which cannot be explained by the two starting materials, which then reflects the new protein:lipid


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aggregate. Several individual mixtures, including different protein batches and SUVs were analyzed and compared, resulting in essentially the same refined scattering curve (Fig. 6C). The GVs are not as homogeneous as the SUVs and thus less suitable for this approach, and a similar analysis (on a single batch) does result in a slightly different aggregate scattering signal, though clearly still revealing the presence of a newly formed additional species in solution (Fig. 6D). Importantly, this analysis strongly suggests that a specific interaction takes place in solution upon vesicle disruption, resulting in the formation of a specific protein:lipid aggregate. It is however not possible to firmly conclude, whether the novel protein:lipid aggregates are of a homogeneous or a heterogeneous nature, although the reproducibility of the residual scattering signal reveal that the same species are formed from several experiments.

Discussion Amyloid disease progression is a multifactorial phenomenon, influenced by factors as different as disease-specific protein point-mutations, mitochondrial dis-functionality and hampered chaperonemediated folding control. Whatever constitutes the biochemical background of the pathology, specific molecular interactions influence cell viability. In the case of PD, massive dopaminergic neuronal decay plays a significant role in the disease progression, and the formation of Lewy bodies, which are rich in αSN fibrillar aggregates, is a hallmark of the disease. However, several studies de-couple the presence of mature fibrillar aggregates and neuronal decay51-53 and it has long been debated whether a potential neurotoxic αSN species is an intermediate pre-fibrillar oligomer rather than the fibrils13, 14. In order to investigate potential cytotoxicity in vitro, the monitoring of Calcein release from loaded liposomes is a commonly used method16, 23, 54. This analysis however only provides indirect evidence for the underlying protein:lipid interaction and no direct mechanistic or structural insight into the alleged toxic interaction55. Here we provide such structural and mechanistic insight. We


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have combined 2PM investigations of an αSN:membrane system in concert with SAXS and CD based structural description of the components. Both native and intermediate αSN samples cause vesicle disruption The nature of the αSN:lipid interaction is strongly dependent on the structural state of αSN, and in order to investigate the influence of various fibrillating αSN states on lipid vesicles, it is essential to be able to characterize the individual states. In the native, pre-fibrillar state, even minor deviations from a completely monomeric population could potentially change the nature of the αSN:membrane interactions, hence, it is vital to obtain a full understanding of the native structural state. The native state of αSN in vitro is commonly assumed to be an intrinsically unfolded monomer, while under some conditions it has been reported to be a mixture of monomers and dimers13, 44, 45. The structural state of αSN in vivo is debated56-58 but in a more recent study, an unfolded conformation is confirmed by in-cell NMR59. Using SAXS analysis, we have thoroughly characterized the structural state of αSN in native, intermediate and fibril samples, hence the differences that are observed with respect to lipid interaction can be directly related to the occurrence of known structural species. We observed the interaction with model membranes of aggregating αSN at 3 different stages of supramolacular assembly with the aim of analyzing molecular mechanisms of membrane disruption in specific conditions. The result indicates that the structural effects on lipid model systems, induced by native and intermediate samples, are very comparable, if not indistinguishable. Calcein release profiles are identical, and both Laurdan bulk solution analysis and 2PM results indicate comparable changes when adding native or intermediate αSN samples to the liposomes. We wish to note that although the calcein release is observed on a longer time scale, this is not in contradiction to the observations in 2PM/CD. The experimental conditions differ significantly due to the choice of liposome dimensions, thus a direct comparison between these timescales is not possible. The calcein release


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experiments were performed on SUV and they give global information on large volume samples via the observation of dye diffusion, in an independent way of other presented data. These experiments not only allowed screening the model lipid systems and revealed the occurrence of liposome disruption. They also indicated (if coupled with Laurdan experiments) that although in different time scales the observed effects are quite general and not dependent on liposome size or membrane curvature. Our experimental setup differs from the previously published results in several aspects, which may cause some of the observed differences. The protein concentrations applied in the present Calcein release studies are higher compared to several previous studies, possibly explaining why monomers in some cases are reported not to cause a release14,

16

. Indeed, we observe a protein

concentration dependency in reported Laurdan spectroscopy analysis, which supports this suggestion. Secondly, when investigating the intermediate samples, we do not disrupt the delicate structural equilibrium of aggregating species by purifying individual species. While we still see an effect, in accordance with studies on purified oligomers13, 14, 23, it is noteworthy that we evidently also have a large pool of monomers in the intermediate samples, and that our native samples have a comparable effect on the liposome stability. These effects can be interpreted in different ways. One hypothesis could be that the native monomer itself has an influence on the vesicle state. This result being in line with recent reports in the field indicating that native protein and oligomers interaction with membranes follow the same pattern with small differences in the resulting membrane permeabilisation extent60. A potential conclusion based on these early observations would be that the presence of lipids under the conditions investigated leads to an equilibrium of the αSN monomer with a non-native monomeric or oligomeric species. This species may or may not correspond to the on-pathway oligomeric species observed during the previously reported SAXS-study14 but is unlikely to correspond to the


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isolated off-pathway species reported from other studies13, 23, 47. Nevertheless, given that the native and intermediate species reveal similar efficiency in inducing Calcein release, it is likely that the causative species differs structurally from the intermediate oligomer. Given the reports on the high membrane-disrupting activity of also isolated oligomers, this suggestion would hence imply that purified oligomers also are in equilibrium with this same non-native species, and that this so-far undescribed species causes the previously reported toxic effect. Laurdan monitors the lipid structural changes induced by αSN species and indicates the formation of novel lipid-containing structures The sensitivity of Laurdan to the packing of phospholipids and to the lipid bilayer hydration makes it an ideal tool for studying membrane dynamics42, 61. Our initial bulk analysis reveals that interaction between native/intermediate/fibril αSN and lipid membrane depends both on the protein aggregation state and on the protein concentration. Further, we observe that the lipid bilayer rigidity increases within a few hours after protein addition. In line with our observations, previous bulk studies reveal that addition of fibrillar aggregates of lysozyme induces GP increment, hence an increase in membrane rigidity25. In contrast, Zhu et al. reported a decrease in GP when αSN in different states was added to systems of mixed anionic and zwitterionic vesicles. This was accordingly interpreted as bilayer hydration28. Later two other studies showed blue shifts and hence an increase of GP in various αSN:lipid systems29, 30, However, none of these lipid systems compare with the purely anionic POPG bilayer examined here. Measurements presented in this work confirm that electrostatic interaction has a dominant role in αSN induced lipid bilayer changes. This suggests that also in a cellular environment charge effects, modulated by environmental conditions, may rule the lipid sequestration by amyloidogenic proteins. The mechanism of the alleged toxicity of various αSN species is commonly discussed in the literature with the pore-forming mechanism as the predominant theory14,

16, 17

. Laurdan bulk


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emission and Calcein release data are useful in terms of establishing a connection between bilayer dynamics and liposome stability, but they do not create a direct link to the changes in liposome morphology and population. This is obtained by our 2PM analysis. We see a dramatic decrease of liposome population after addition of αSN (Fig. 3A-D). The liposome burst is monitored in real time but importantly the measured fluorescence signal does not disappear as it would be expected if Laurdan was displaced to the solution. On the contrary, a diffuse fluorescence signal is observed with an overall increased GP value (Fig. 4). The 2PM data clearly show that the increase in GP is not only driven by structural changes in remaining intact membranes. The increase is greatly driven by the occurrence of residual lipids in mixture with Laurdan and protein, which hence reveals that lipids are organized in small rigid structures of a size below the instrumental resolution (app. 200 nm). 2PM analysis demonstrates the implicit error of considering changes in bulk solution as single events and how having direct local information is crucial for an exhaustive picture of the process. Importantly, on time-scales comparable to the 2PM results, CD monitors a transition of the protein secondary structural elements to a partial α-helical structure persisting after complete depletion of the liposome population, suggesting co-aggregation. This is also in accordance with the observations of the lipid structural changes being both time- and protein concentration-dependent (Fig. 6A). The combined results from CD and 2PM data point towards a newly formed protein:lipid structure with a more rigid lipid environment and a significant amount of α-helical αSN. Co-aggregation of especially anionic lipids and native αSN was recently described20 showing tangled fibril-like structures. Co-aggregation was described as a part of the fibrillation (above saturation) or as an association of lipids to the fibril surface (below saturation). In contrast we observe how novel soluble co-aggregates are formed. These co-aggregates, however, may form a template for further fibrillation, ultimately resulting in lipid-associated fibrils, in accordance with the recent study20. In another study, native αSN was reported to recruit lipids from supported


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bilayers during aggregation15. These observations potentially correspond to earlier AFM observations of αSN aggregating on lipid surfaces. The possibility of these structures to contain lipids was mentioned by the authors, but the nature of the aggregates was not further investigated62. Related studies of amyloid β2-microglobulin and islet amyloid polypeptide reported extraction of lipids from lipid-bilayers into or onto amyloid structures63,

64

. This resulted in defects and

remodeling of the bilayers associated with formation of new vesicle-like structures of unidentified composition. These findings support the relevance of considering the behavior of native αSN or other amyloid proteins in the presence of lipid, and not to assume that complex in vivo protein fibrillation phenomena can be adequately described exclusively by fibrillation at in vitro environments deprived of lipids. SAXS analysis shows the existence of novel αSN:lipid aggregates in solution The Laurdan 2PM investigations suggest that novel αSN:lipid aggregates are formed upon mixing protein samples and POPG GVs. At the same time, Calcein release assays performed using POPG SUVs reveal liposome destabilization, which assumingly reflects an associated phenomenon. Our further characterization of these co-aggregates are based on solution SAXS data, directly monitored from the undisturbed mixtures of protein and lipids. The ability to work on undisturbed equilibriums is a major advantage of this method65 which allows identification of dynamic species existing in solution equilibrium66, as previously applied in connection with amyloid fibrillation14, 67, 68

. The SAXS data obtained from mixtures of native αSN and POPG SUVs clearly indicated the

appearance of a new reproducible species. This species is structurally different from the wreathshaped oligomer previously observed during αSN fibrillation (hence resulting in distinctly different scattering curves). This is further underlined by the high α-helical content observed in this study, while the on-pathway oligomer is expected to be composed mainly of β-sheet14. The scattering


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curves corresponding to the protein:lipid co-aggregates were calculated from six repeated experiments using individually purified αSN protein pools, which yielded very similar curves (Fig. 6C), hence the arising co-aggregate is reproducible although it might still be heterogeneous. When the experiment was repeated with GVs, the resulting scattering curve, however, was different from the ones obtained from the POPG-SUV/αSN mixtures. The co-aggregate species may be heterogeneous, and the equilibrium with either GV or SUV POPG vesicles may induce different structural distributions of the αSN:lipid aggregates69. In addition, the larger heterogeneity of the GVs, in particular after partial disruption, may hamper a well-defined isolation of the scattering contribution from the novel species. Given this, it is not possible to provide a detailed structural analysis of the new co-aggregate based solely on the isolated scattering curve. SAXS data from homogeneous samples enable ab initio shape reconstruction38, but this is not possible if the scattering curve represents a mixture of species. However, we can unequivocally conclude that the mixing of αSN with lipid vesicles causes the formation of reproducible, co-aggregate structures. The global view of the data indicates that dominant forces in the observed phenomenon are of electrostatic and hydrophobic character. Due to long range electrostatic interaction, the αSN molecule will possibly accumulate at the membrane interface and the initially unstructured protein may interact with lipids forming α-helices, this process being driven by hydrophobic (entropic) interaction of hydrophobic residues with lipid acylchains. Lipid:protein association contributes in remodeling membrane structure leading to the exclusion of water molecules from the bilayer reducing membrane flexibility and altering the balance of liposome equilibrium causing its burst and diffusing in solution of rigid protein:lipid coaggregates. Recruitment of lipids from the cell membrane could potentially cause cytotoxicity and we hence hypothesize that co-aggregation of αSN and lipids could play a vital role in neurodegeneration as


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seen in PD. The results presented here hence provide evidence for the formation of an αSN:lipid coaggregate, in association with vesicle disruption. Whether other amyloid proteins possess the same ability remains to be investigated, but the method applied here can easily be adapted to other amyloid systems. It remains to be investigated how αSN or other amyloid proteins interact with more complex lipid bilayers, ultimately the neuronal surface. Laurdan based analysis does enable also live cell analysis10, which would be highly relevant for future analysis of the amyloid cytotoxic effect. Here, we suggest that recruitment of lipids from cell membranes into non-fibrillar co-aggregates constitutes a potential cytotoxic event either prior to or complementary to αSN oligomerization and fibrillation.

Conclusions Using a multiplexed approach resulting from the combination of imaging and SAXS experiments, we establish the existence of reproducible in vitro soluble co-aggregates, formed in context of amyloid mediated liposome rupture. We show that negatively charged lipid bilayers are dehydrated when incubated with native, intermediate or (to a lesser extent) fibrillated αSN, evidenced from the increase in Laurdan GP. By applying 2PM we are able to establish, that this increase of GP is not solely driven by dehydration of intact bilayers, but rather by the formation of new rigid species which are shown to be α-helical rich protein:lipid co-aggregates. These findings reveal how the non-fibrillated αSN species are active in membrane modulation and formation of co-aggregates. This nurtures the impression that the αSN-rich Lewy bodies are a deposit of inert products of the membrane disrupting event. The presented approach of combining advanced microscopy analysis with structural studies emphasizes the strong need to address complex systems with orthogonal methods. We hypothesize that the novel co-aggregated species is central for understanding the molecular mechanism behind the cytotoxic amyloid induced effect.


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Figure legends Figure 1. Early characterization of protein samples. (A) ThT fluorescence of fibrillating αSN. Circles indicate where intermediate (blue, early growth phase) and fibrillated green, (steady state) samples were withdrawn. Native samples (red circle) were withdrawn before initiating the assay. Error bars indicate the standard deviation on three replicates. (B) SAXS curves from native (red), intermediate (blue) and fibrillated αSN (green) samples show the gradual increase of average sizes during fibrillation. (C) EOM analysis of native αSN validating the monomeric state. The experimental curve (hollow circles) is shown with the fits using pools of monomers (purple; chi=1.048), dimers (pink; chi=2.078) or a combination of monomers and dimers (cyan; chi=1.085, no dimers selected respectively). The individual fits have been transposed for clarity. The experimental data are plotted with a constant subtraction, which was optimized during the fitting procedure (see Materials and Methods). (D) OLIGOMER analysis of SAXS data on intermediate αSN species shows that the wreath shaped oligomer is required to obtain an adequate fit to the experimental data. Intermediate scattering curve (hollow circles) is fitted to a form factor of monomer and fibril signal (red; chi=4.47) or monomer, wreath shaped oligomer and fibril signal (green; chi=1.59). Data are shown on a logarithmic scale to emphasize the difference in fitting quality. Figure 2. Analysis of protein:lipid interaction by means of fluorescence spectroscopy. (A) Calcein release from POPG SUVs caused by addition of native αSN (red), intermediate αSN (blue) and fibrillated (green) samples and corresponding release data for POPC SUVs with native (orange), intermediate (purple) and fibrillated (cyan) αSN. Lipid-protein molar ratio was 20:1. The standard deviation on three replicates was at all times below 7% (not shown). (B) Laurdan fluorescence emission (λex=380 nm) progressively changes after addition of native αSN to POPG GVs in a lipid-


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protein ratio of 20:1. The spectra are collected over a 4 h period. The arrows indicate changes in the fluorescence band shape as a function of time. (C) GP variation of Laurdan emission in bulk spectroscopic measurements as a function of time after adding native (red), intermediate (blue) and fibrillated (green) αSN to POPG GVs (molar ratio of 50:1) calculated with respect to GP-values for GV samples before addition of protein. (D) GP of Laurdan emission in bulk spectroscopic measurements after adding native αSN to POPG GVs lipid-protein molar ratios of 20:1 (circles), 50:1 (squares) and 100:1 (triangles). Figure 3. 2PM data of lipid-protein incubation. (A-D) 512x512 pixel representative images of Laurdan-stained POPG GVs showing fluorescence in a 410-460 nm band after addition of native αSN in a lipid-protein ratio of 50:1 after 2 min (A), 4 min (B) 6 min (C) and 8 min (D) with the corresponding images from the transmission channel in E-H showing gradual decrease of GV population. Figure 4. Data analysis of 2PM images. (A-D) 256x256 pixel GP-maps reporting GP-values of individual pixels before addition of native αSN (A) and after 2 min, 4 min and 6 min, respectively (B-D). The GP-value of the liposome marked with white arrows is 0.097 (B), 0.180 (C) and 0.221 (D). The red arrows in image B-D indicate a region of vesicle burst and subsequent occurrence of high GP species. The color bar indicates the GP value of each pixel ranging from -1 (blue) to +1 (red). (E-H) Corresponding histograms of the GP value distributions summarizing the GP values of each pixel. Figure 5. GP variation of Laurdan emission recorded with 2PM. Mean GP-value changes calculated from GP-maps after addition of native (red), intermediate (blue) and fibrillated (green) αSN to Laurdan-stained POPG GVs in lipid-protein molar ratios of 50:1 calculated with respect to the GP mean value measured before addition of protein.


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Figure 6. Incubation of native αSN with anionic liposomes leads to increasing α-helical content and structural indication of formation of co-aggregates. (A)

CD signal of native αSN before

(red) and after (0 h blue; 2 h green; 4 h orange) addition of POPG GVs in a lipid-protein molar ratio of 50:1. (B) Experimental SAXS data from a mixture of native αSN and POPG SUVs in a 50:1 molar ratio (open circles). OLIGOMER fits obtained using 2 components (native αSN and SUVs, red; chi=3.14) or 3 components (native αSN, SUVs and the wreath shaped oligomer, blue; chi=2.24). The sum of the oligomer signal and the residuals from the 3 component fit is shown in green. The curves have been transposed for clarity. (C) Sum of oligomer and residuals from 3 component fits performed on 6 experimental data sets of repeated native αSN:POPG SUV mixtures. (D) Experimental SAXS data from a mixture of native αSN and POPG SUVs in a 50:1 molar ratio (open circles). OLIGOMER fits obtained using 2 components; native αSN and SUVs (red; chi=3.93) or 3 components; native αSN, SUVs and the wreath shaped oligomer (blue; chi=1.86). The sum of the oligomer signal and the residuals from the 3 component fit is shown in green. The curves have been transposed for clarity.

Supporting information Supplementary experimental procedures for purification of αSN, liposome preparation, SAXS data acquisition, dye release assays and circular dichroism measurements. Table S1: Basic SAXS analysis of αSN. Figure S1: Quenching of intermediate samples from the early growth phase of αSN fibrillation. Figure S2: Bulk measurements of native αSN:POPC interactions. Figure S3: Data analysis of 2PM images of fibril αSN:POPG interactions. Figure S4: Concanavalin A control. Figure S5: CD of native and intermediate αSN with POPG and POPC GV. This material is available at free of charge via the internet at http://pubs.acs.org

Author information


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*CORRESPONDING AUTHORS: Bente Vestergaard Department of Drug Design and Pharmacology, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark Phone: +45 35336403 e-mail: [email protected] Valeria Vetri Dipartimento di Fisica e Chimica, Universitá di Palermo, Via Archirafi 36, 90123 Palermo, Italy Phone: +39 091 23891782 e-mail: valeria.vetriunipa.it

Author contributions All authors contributed to the experimental design. A.v.M. and V.V. conducted the research. A.v.M., V.V., A.E.L, and B.V. analyzed the data. All authors contributed to the preparation of the manuscript.

Acknowledgements The authors acknowledge the X33 and P12 beamlines at EMBL, Hamburg, and the I911-4 beamline at Max IV Laboratory, Lund, for beamtime and the beamline staffs are acknowledged for support. We thank Maurizio Leone and Valeria Militello for useful discussions and for kindly providing access to instrumentation at University of Palermo. We thank Bioneer, Hørsholm, Denmark, for providing the αSN expression construct. We thank Camilla Foged and Fabrice Rose


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Table of Content graphic (TOC)


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Lipid Coaggregates are Formed During Î±-Synuclein-Induced

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