Different Effects of Î±-Synuclein Mutants on Lipid Binding and

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Different effects of alpha-Synuclein mutants on lipid binding and aggregation detected by single molecule fluorescence spectroscopy and ThT fluorescence-based measurements Viktoria Ruf, Georg Sebastian Nübling, Sophia Willikens, Song Shi, Felix Schmidt, Johannes Levin, Kai Bötzel, Frits Kamp, and Armin Giese ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00579 • Publication Date (Web): 03 Jan 2019 Downloaded from http://pubs.acs.org on January 4, 2019

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ACS Chemical Neuroscience

Effects of pathogenic alpha-Synuclein mutants

Different effects of alpha-Synuclein mutants on lipid binding and aggregation detected by single molecule fluorescence spectroscopy and ThT fluorescence-based measurements Viktoria C. Ruf1*, Georg S. Nübling1, 2, Sophia Willikens1, Song Shi1, Felix Schmidt1, Johannes Levin2, 3, Kai Bötzel2, Frits Kamp4, and Armin Giese1* 1Center

for Neuropathology and Prion Research, Ludwig-Maximilians-University, Munich, Germany of Neurology, University Hospital Munich, Ludwig-Maximilians-University, Munich, Germany 3German Center for Neurodegenerative Diseases, Munich, Germany 4Biomedical Center, Metabolic Biochemistry, Ludwig-Maximilians-University, Munich, Germany 2Department

*Corresponding author [email protected] [email protected]

Abstract Six alpha-Synuclein point mutations are currently known to be associated with familial parkinsonism: A30P, E46K, H50Q, G51D, A53E and A53T. We performed a comprehensive in vitro analysis to study the impact of all aSyn mutations on lipid binding and aggregation behavior. Markedly reduced lipid binding of A30P, moderately attenuated binding of G51D and only very slightly reduced binding for the other mutants were observed. A30P was particularly prone to form metal ion induced oligomers, whereas A53T exhibited only weak tendencies to form oligomers. In turn, fibril formation occurred rapidly in H50Q, G51D and A53T, but only slowly in A30P, suggesting mutants prone to form oligomers tend to form fibrils to a lesser extent. This was supported by the observation that fibril formation of wild type aSyn, A30P and A53T was impaired in the presence of ferric iron. Additionally, we found the aggregation kinetics of mixtures of A30P or A53T and wt aSyn to be determined by the faster aggregating 1

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aSyn variant. Our results implicate differential mechanisms playing a role in aSyn pathology on the molecular level. This might contribute to a better understanding of Parkinson´s disease pathogenesis and provide potential links to develop prevention strategies and disease-modifying therapy.

Key words: Alpha-synuclein (α‐Synuclein), mutant, Parkinson´s disease, synucleinopathy, protein aggregation, protein‐lipid interaction

Graphical abstract

Introduction The aggregation of aSyn plays a central role in the pathogenesis of synucleinopathies like Parkinson´s disease (PD), Dementia with Lewy bodies (DLB) or Multiple System Atrophy (MSA). Point mutations, duplications and triplications of SNCA, the gene encoding for aSyn, lead to familial forms of PD

1-3.

So far, six missense mutations in the coding region of SNCA

have been identified in families with autosomal dominant forms of PD: A30P 4, E46K 5, 6, H50Q 7, 8,

G51D 9, 10, A53E 11, 12 and A53T 13. Additionally, two variants of unclear significance, A18T

and A29S, and very recently, a homozygous A53V mutation have been described

14, 15.

Clinically, patients with SNCA mutations develop progressive parkinsonism, however, with marked differences in terms of age of onset and clinical presentation of attendant symptoms

2


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(Table 1): While parkinsonian symptoms in patients with A30P, E46K and H50Q mutations manifest at higher age 1, 4, 13, carrier of the A53T, G51D or A53E mutation manifest at younger ages (early onset PD) 5, 7, 10, 11. Patients with the A53T mutation often display signs of associated cognitive impairment and dementia

13, 16,

whereas patients with the E46K or G51D mutation

frequently present with visual hallucinations

1, 5, 10, 17,

similar to Dementia with Lewy Bodies

(DLB). Furthermore, patients with the G51D and the A53E mutation feature clinically an MSAlike symptomatology and show oligodendroglial aSyn inclusions upon neuropathological examination, which are the characteristic hallmark of MSA pathology

10, 11.

Such distinct

clinical and neuropathological phenotypes in patients with particular SNCA mutations suggest different mechanisms acting on the molecular level in the pathogenesis of synucleinopathies, although the particular underlying pathomechanisms leading to cytotoxicity and cell death related to aSyn are still unclear. aSyn is a 140-amino-acid protein that is abundantly expressed in presynaptic nerve terminals

18.

It is intrinsically disordered in aqueous solution, however,

adopts an -helical structure upon binding to lipid membranes with high curvature and/or negatively charged lipids 19, 20. In contrast, misfolding of aSyn leads to formation of -sheet rich fibrillar aggregates in vitro as well as in vivo in the form of Lewy bodies. Metal ions have been shown to trigger the formation of oligomeric species and to accelerate the formation of fibrils under certain conditions 21-24. Notably, all SNCA mutations described so far, are located in the N-terminal part of aSyn, which can adopt -helix conformation in the presence of lipids (Figure 1), or -sheet conformation in fibrils, suggesting that the exchange of amino acids might influence the lipid binding and/or aggregation properties. Thus, the known pathogenic aSyn mutants display a suitable and valuable model to study the impact of single amino acid exchanges on its lipid binding and aggregation behavior. This may provide a better 3



understanding of the basic molecular processes in the pathogenesis of genetic as well as sporadic synucleinopathies. Several in vitro studies have already reported differences in the aggregation and lipid binding behavior of some aSyn mutants 25-30. However, a comprehensive comparison of all currently known pathogenic mutants tested in parallel in the same experimental conditions has not been published yet. Applying established single particle fluorescence techniques, we investigated binding of aSyn to small unilamellar lipid vesicles (SUVs) at nanomolar protein concentrations as well as metal ion induced oligomer formation. Additionally, we assessed the potential and kinetics of fibril formation of the different mutants using thioflavin T (ThT) fluorescence.

Figure 1. Three-dimensional lipid-bound -helical structure of aSyn. The mutations known to date are all located within the N-terminal helical part of the 140 amino-acid protein. The Cterminus remains unstructured both in -helical and ß-sheet variants of aSyn conformations (diagram generated using The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC)

4


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Table 1. Demographic, clinical and neuropathological phenotypes of patients with distinct SNCA mutations.

Results A30P and G51D show reduced binding to lipid membranes The interaction of aSyn and phospholipid membranes has been extensively studied and it was previously shown that aSyn has a high affinity to bind to lipid membranes in the gel state with high curvature and/or negatively charged lipids

20, 31, 32.

To compare the binding of the different

aSyn mutants to lipid membranes, 10 µM fluorescently labeled DPPC-SUVs (Dipalmitoyl-snglycero-3-phospho-choline small unilamellar vesicles) (488 nm, `green´) were co-incubated with 10-20 nM `red´ (647 nm) labeled mutant or wt aSyn. These concentrations were chosen because under these conditions distinct, but not yet saturated binding of aSyn to lipid vesicles is observed, so that differences in binding affinity can be optimally analyzed 32. Comparable amounts of the respective mutant were ascertained by assessing total fluorescence intensity (I. tot.) in the red channel for monomer only (Figure S1). Data are displayed in 2D-FIDA histograms, which depict the distribution of the number of photons that are detected within the bin length (40 µs) of SIFT (scanning for intensely fluorescent targets) measurements, but not average particle brightness

32, 33.

In these histograms bright bicolored particles, indicating

binding of red aSyn monomers to green lipid vesicles, result in data points around the bisectrix of the histogram, while monomeric aSyn and lipid vesicles result in data points close to the origin and along the x-axis, respectively (see Figure 2a for schematic representation). In the 2DFIDA histograms for different aSyn mutants, a clear reduction of bicolored particles, indicating decreased binding of aSyn to DPPC-SUV, was observed for A30P, whereas no obvious difference between wt aSyn and the other mutants could be seen (Figure 2a). Quantitative 6


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analysis of the particle brightness yielded an average number (n) of 6.4 wt aSyn monomers per vesicle, which was significantly reduced to 2.6 for A30P and 4.6 for G51D. For all other mutants, the analysis revealed slightly, however, not significantly reduced numbers of monomers per vesicle, varying between 5.2 and 5.8 (Figure 2b). The fraction of aSyn bound to vesicles was strongly diminished for A30P, i. e. about 8 % was bound to DPPC-SUV, compared to 23 % of wt aSyn. A moderate decrease of bound aSyn was observed for H50Q, G51D and A53E (16 %, 16 % and 14 %, respectively), whereas only a minor, not significant reduction was seen for E46K and A53T (each 18 %) (Figure 2c). The fraction of vesicles with bound aSyn was reduced at least moderately for all mutants, showing fractions of 28 %, 37 % or 40 % of vesicles with bound protein for A30P, G51D and A53E, respectively and 41 %, 43 % and 45 % for E46K, H50Q and A53T, respectively, compared to 65 % of vesicles with bound wt aSyn (Figure 2d).

7



Figure 2. Influence of single amino acid exchanges on lipid binding affinity. (a) Schematic depiction of vesicles and protein monomers in a 2D FIDA histogram (adapted from 32) and 2D FIDA histograms of aSyn and DPPC-SUV only and aSyn co-incubated with DPPC-SUV. For A30P, markedly less bicolored particles can be observed. (b) The number (n) of A30P and G51D monomers per vesicle was markedly decreased. (c) For all mutants, an at least slight reduction 8


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of the fraction of protein bound to vesicles was detected. (d) Additionally, the proportion of vesicles with bound protein was moderately to strongly decreased in different mutants. Error bars in (b), (c) and (d) indicate standard deviation (n ≥ 4), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Formation of metal ion induced oligomers is increased in A30P and reduced in A53T It was previously shown that physiological concentrations of trivalent metal ions, such as FeCl3 or AlCl3, induce the formation of distinct SDS-resistant aSyn oligomer species that resemble toxic oligomers found in vivo as they can insert into membranes and form pore-like structures 21, 32, 34, 35.

To characterize the propensities of different aSyn mutants to form oligomers induced

by trivalent metal ions, fluorescently labeled wt aSyn and aSyn mutants were incubated in the presence of 10 µM Fe3+ or Al3+, respectively, and SIFT measurements were performed. The number of monomers per oligomer, indicating the oligomer size, was obtained by FIDA analysis (i. e. ratio of oligomer brightness and monomer brightness, assuming no quenching), ranged between 60.2 and 78.1 monomers for Fe3+ and 64.3 and 75.3 monomers upon addition of Al3+, respectively, and was similar for wt aSyn and all mutants (Figure 3a, b). However, the calculated fraction of oligomerized protein per total protein revealed substantial differences: While up to 68 % (Fe3+) or 79 % (Al3+) of total A30P were found to have formed oligomers, only 17 % (Fe3+) or 27 % (Al3+) of total A53T showed oligomer formation, compared to 48 % (Fe3+) and 55 % (Al3+) of wt aSyn. The proportions of oligomers per total protein for H50Q, G51D and A53E were moderately reduced to 30-37 % (Fe3+) and 32-40 % (Al3+), whereas no difference compared to wt aSyn was observed for E46K (Figure 3c, d).

9



Figure 3. Differential effects of aSyn mutants on metal ion induced oligomer formation. (a, b) In all mutants, addition of 10 µM FeCl3 or AlCl3, respectively, induces the formation of oligomers of comparable size, indicated by the number (n) of monomers per oligomer. (c, d) However, the fractions of oligomerized protein per total protein display striking differences between the aSyn mutants: While A30P has a high tendency to form oligomers, the potential of A53T to form oligomers is low. H50Q, G51D and A53E show moderately decreased fractions of oligomers compared to wt aSyn. For E46K, no significant difference could be detected. Each single dot represents one independent experiment (n ≥ 4). Error bars indicate standard deviation, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fibril formation is slower in A30P and accelerated in A53T, H50Q and G51D To characterize the potential of aSyn mutants to form amyloid fibrils, fibril formation was induced by incubating monomeric aSyn at a concentration of 50 µM over 96 h at 37°C with 10


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continuous shaking at 1400 rpm. Fibril formation was confirmed for all aSyn mutants using electron microscopy (Figure 4c). To obtain further information about the aggregation kinetics of different aSyn mutants, we adapted the assay conditions to determine the ThT fluorescence automatically every hour over at least 150 hours using a microplate reader. Representative curves for the different mutants (Figure 4a). Quantification of the lag times, i. e. the time until the ThT fluorescence starts rising from the baseline, revealed a considerably prolonged lag time of 63 h for A30P, which did not reach a plateau of ThT fluorescence at least until 150 h of incubation, while very short lag times (5-6 h) were observed for H50Q, G51D and A53T, indicating delayed fibril formation of A30P versus accelerated fibril formation of H50Q, G51D and A53T. The accelerated aggregation of G51D compared to wt aSyn was supported by additional CD and Congo red absorption measurements (Figure S2). E46K showed a slightly increased average lag time of 42 h, whereas no significant difference could be detected for A53E (24 h), compared to wt aSyn (27 h) (Figure 4b).

11



Figure 4. Influence of aSyn mutants on fibril formation kinetics. (a) Representative curves of normalized aggregation kinetics of aSyn mutants monitored by ThT fluorescence over time. (b) Quantification of the lag phases indicates very slow fibril formation of A30P and very rapid fibril formation of H50Q, G51D and A53T. For E46K, a slight increase of the lag time was observed, A53E showed no difference of lag time compared to wt aSyn. Each single dot represents one independent experiment (n ≥ 5). Error bars indicate standard deviation, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (c) Representative EM images indicating fibril formation of all aSyn mutants after incubation of 50 µM monomeric aSyn for 96 h at 37°C under 1400 rpm (scale bar: 200 nm).

Fibril formation is impaired by ferric iron Since formation of metal ion induced oligomers and fibril formation seemed to be inversely correlated, we wondered if and to what extent metal ions would influence fibril formation. Therefore, we investigated fibril formation of A30P and A53T - the two mutants with the most significant difference compared to wt aSyn - in the presence of 100 µM, 300 µM and 1000 µM 12


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of ferric iron. The ThT fluorescence signals decreased with increasing concentrations of Fe3+ (Figure 5a-c), indicating less amyloid fibril formation. Regarding the lag times, at 100 µM of ferric iron, no differences between the lag times were seen. With 300 µM Fe3+, a trend towards slightly prolonged lag times, reaching statistical significance for A30P, could be observed. Upon addition of 1000 µM ferric iron, the lag phase of A53T was substantially prolonged to approximately 145 h, whereas no ThT signal was detected in wt aSyn or A30P after up to 200 h (Figure 5d).

Figure 5. Impact of ferric iron on fibril formation. (a-c) Representative kinetic curves of wt (a), A30P (b) and A53T (c) with increasing concentrations of ferric iron reveal decreasing ThT fluorescence with increasing concentrations of ferric iron, indicating less amyloid fibril formation. (d) Slight prolongation of lag times is observed with 100 µM and 300 µM ferric iron for all tested aSyn variants. Upon addition of 1000 µM ferric iron, fibril formation of wt aSyn and A30P is completely blocked and ThT fluorescence of A53T starts increasing not earlier than 145 h. Error bars in d indicate standard deviation (nwt = 6; nA30P = 4; nA53T = 4), *p < 0.05. 13



Aggregation kinetics in mixed preparations of A30P or A53T and wt aSyn are determined by the faster aggregating aSyn variant Since patients with SNCA mutations are with few exceptions heterozygous for the particular mutation, it would be assumed that they express both, wild type and the mutant protein. To investigate the effect of mixtures of wt aSyn and A30P or A53T, respectively, on fibril formation kinetics, we mixed either mutant at different ratios with wt aSyn retaining a total protein concentration of 50 µM (Figure 6). When A30P was mixed with wt aSyn, a marked reduction of the lag time compared to 100 % A30P was seen for all mixing proportions. In turn, compared to the lag time of pure wt aSyn, we were not able to detect a significant difference for any mixing ratio. Moreover, for A53T and wt aSyn, we observed a considerable decrease of lag times in all mixing ratios towards the typical lag time of A53T, being significant for all ratios as compared to wt aSyn.

Figure 6. Influence of mixing wt aSyn with A30P and A53T on aggregation kinetics. A30P and A53T were mixed with wt aSyn at different proportions. Lag times of mixtures seem to be obviously determined by the faster aggregating aSyn variant. Each independent experiment is represented by a single dot (n ≥ 3). Error bars indicate standard deviation, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0,0001.

14


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Discussion The observation that different SNCA mutations seem to modify the clinical phenotype in a distinct manner suggests different molecular mechanisms in the pathogenesis of the disease. Several studies have characterized individual aSyn mutants, however, a comprehensive and systematic analysis of all so far identified pathogenic aSyn mutants in parallel, enabling comparability between single mutants is lacking to date. In the present study, we analyzed all hitherto described pathogenic aSyn mutants with regard to lipid binding behavior and aggregation propensity under the same conditions. It was previously shown that aSyn binds with high affinity to negatively charged membranes and stressed gel-state membranes with high curvature 20, 32. Hereby, the N-terminal part of the otherwise natively unfolded protein

36

adopts an alpha-helical conformation, whereas the C-

terminal part remains unstructured. For our study, we chose the small unilamellar vesicles of DPPC, since it has been documented in detail that they bind aSyn with high affinity, also at room temperature and thus represent a valid model to study lipid binding of aSyn monomers 32. Interestingly, all mutations identified so far are found in the N-terminal part of the protein suggesting that membrane binding might be affected by single amino acid exchanges. In contrast to other studies, we quantified lipid binding by assessing the number of monomers per vesicle, the fraction of protein bound to vesicles and the fraction of vesicles with bound protein at the single particle level. Comparing the binding affinity of different mutants to DPPC-SUV, for which it was reported that wt aSyn has a high propensity to bind 32, we found that membrane binding of A30P was strongly and of G51D moderately reduced in terms of all three of the above-mentioned parameters, consistent with previous reports about decreased membrane binding affinity of A30P and G51D 30, 37-40. For A53E, we observed the fraction of protein bound

15



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to vesicles and the fraction of vesicles with bound protein to be diminished to a similar degree as with G51D, indicating attenuated lipid binding as proposed by Ghosh et al. 29, although the number of monomers per vesicle was not significantly reduced compared to wt aSyn. The number of monomers per vesicle of the remaining mutants did not differ significantly from wt aSyn, whereas the fractions of protein bound to vesicles and vesicles with bound protein were found to be mildly to moderately decreased. Thus, lipid interaction of E46K, H50Q, A53E and A53T appears indeed to be reduced in that less protein is bound to vesicles and less vesicles bind protein. However, since the number of monomers per vesicle is the same as for wt aSyn, a positive cooperative binding mode is suggested, where the binding of further monomers is facilitated once a single monomer has bound to a lipid vesicle. For G51D and especially A30P, this binding seems to be most strongly impaired. This may be due to a local disruption of helix formation caused by the amino acid substitution

30, 40.

This reduced interaction with lipid

membranes may represent a loss of physiological function which is likely to contribute to pathogenesis via impairment of vesicle trafficking and trafficking of aSyn itself and/or increasing the pool of unbound aSyn available for aggregate formation 38. To compare the oligomer formation of the different mutants, we used established methods to analyze oligomer formation induced by Fe3+ or Al3+ ions

21, 24.

Interestingly, consistent with

Stefanovic et al. 25, the calculated number of monomers per oligomer, i. e. the size of the formed oligomers, was similar for all mutants and wt aSyn. However, the relative amounts of aggregated protein substantially differed: While most of the applied A30P monomer appeared to have formed oligomers, only a low fraction of A53T monomers seems to form oligomers upon addition of trivalent metal ions. The fractions of aggregated protein of H50Q, G51D and A53E were moderately reduced, whereas no clear difference could be observed for E46K.

16


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Strikingly, the propensity to form fibrils in our ThT assay was inverse to the propensity to form oligomers: Although we were able to show that all mutants are qualitatively able to form fibrillary aggregates, fibril formation of A30P was substantially prolonged, whereas A53T, H50Q and G51D displayed considerably accelerated fibril formation compared to wt aSyn. Our findings for A30P, H50Q, E46K and A53T are well in line with publications from various groups 25, 26, 28, 41-46. For G51D, attenuated in vitro aggregation in terms of aggregation kinetics and maximum ThT fluorescence was proposed by two studies 26, 40. Under our assay conditions, G51D, similar to H50Q and A53T, readily formed fibrils. This difference may be explained by different assay conditions, in which the formation of aggregate species that are less well visualized by ThT fluorescence is favored, so that aggregation seems to be attenuated. As metal ion induced oligomer formation and fibril formation seem to be inversely favored by certain mutants, we performed further experiments to investigate the effect of ferric iron on fibril formation, expecting that (1) ferric iron would impair fibril formation by directing aggregation to a primarily oligomer forming pathway and (2) that fibril formation in a mutant less prone to form iron induced oligomers would be less impaired. Analyzing wt aSyn, A30P and A53T, we found that increasing amounts of ferric iron lead to a delay of fibril formation. Moreover, in the presence of 1 mM Fe3+, aggregation of wt aSyn and A30P could be completely blocked, whereas A53T was still able to form ThT positive aggregates, albeit after a prolonged lag phase. At the same time, we observed reduced ThT fluorescence levels, further indicating less fibril formation. Altogether, it appears that the underlying oligomer and fibril formation pathways in our experiments in fact counteract each other, meaning that for mutants prone to form metal ion induced oligomers, fibril formation is impeded and vice versa. Furthermore, in the presence of

17



trivalent metal ions, i. e. under conditions where oligomer formation is favored, fibril formation is impaired. Hence, from our data it appears that there exist different pathways of aSyn aggregation, which mutually counteract and are differentially preferred by different mutants, similar to the model proposed by Sierecki et al. 47. Alternatively, in seems possible that because of very efficient oligomer formation of A30P, the availability of free monomeric protein for fibrillar elongation is too low. Two recent studies using cryo-electron microscopy revealed the aSyn amyloid fibril structure to consist of two intertwined protofilaments 48, 49. Interestingly, all mutation sites except A30P are located in the interface between the protofilaments, what might be – at least one – reason for the different behavior of A30P compared to the other mutants. As heterozygous mutation carriers express both, wt and mutant aSyn, we analyzed fibril formation kinetics of mixtures of different proportions of wt aSyn and A30P or A53T and found that the lag time of the mixture was strongly determined by the faster aggregating aSyn variant and that even very small proportions of just 20 % of the faster aggregating aSyn variant could considerably shorten the lag phase. Our findings are particularly interesting regarding studies that report a substantially reduced expression of the mutant allele in lymphoblastoid cell lines of A53T and A30P patients 50-52, since according to our results, even very small amounts of the faster aggregating aSyn variant would be sufficient to substantially accelerate the formation of fibrils and potentially toxic aggregate species. However, molecular details of these interactions like the composition or the form and structure of the newly formed, presumably mixed aggregates, as shown by diverse groups 53, 54, remain to be elucidated by further studies.

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Conclusion We have performed the first comprehensive study systematically analyzing lipid binding and aggregation properties of all so far known pathogenic aSyn mutants in parallel under the same assay conditions. Our findings, summarized in Table 2, provide evidence for multiple, differentially preferred pathways playing a role in the pathogenesis of synucleinopathies, which obviously all result finally in an increased accumulation of toxic aSyn species and consecutive cell death. Moreover, different pathways might, at least in part, account for the clinical spectrum of synucleinopathies. In this way, the results of this study may help to better understand the pathogenesis of synucleinopathies and support the development of prevention and therapy strategies.

Table 2. Summary of physicochemical properties of different aSyn mutants compared to wt aSyn found in our study.

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Materials and methods Mutagenesis, purification and labeling of recombinant wild type aSyn The bacterial expression plasmid (pET-5a) containing full-length wt aSyn was previously described by Nuscher et al.

20.

Site-directed mutagenesis was performed on the pET-5a/a-

synuclein (136TAT) plasmid (wt-plasmid by Philipp Kahle, LMU Munich; the 136-TAC/TAT Mutation was inserted by Matthias Habeck, ZNP Munich) using primers causing single nucleotide exchanges as designated in 4, 5, 9-11, 13. PCR was conducted with Phusion Flash High-Fidelity PCR Master Mix (Thermo Fisher Scientific, Waltham, Massachusetts, USA) followed by Dpn I restriction enzyme digestion to degrade the original plasmid. After transformation into competent DH5 Escherichia coli (E. coli) the presence of the desired nucleotide exchange was verified by DNA sequencing (Eurofins Genomics). Wild type and the mutant recombinant aSyn proteins were obtained by expression in and purification from BL21(DE3) E. coli as specified in 20. Stock solutions of 1 mg/ml were prepared in 50 mM tris buffer, pH 7.0 and stored at -80°C. The inserted mutation of the recombinant protein was confirmed by Mass Spectroscopy (Sciex MALDI-TOF 4800) using α-Cyano-4-hydroxycinnamic acid as matrix. Protein labeling was performed with the amino-reactive fluorescent dye Alexa Fluor-647-O-succinimidylester (Thermo Fisher Scientific, Waltham, MA, USA) as previously described by Kostka et al.

21.

Briefly, recombinant protein and fluorescent dye at the ratio of 1:2 were incubated for 12 h at 4°C. Unbound fluorophores were separated by two filtration steps using PD10 columns (Amersham Biosciences, Munich, Germany) equilibrated with 50 mM Tris buffer, pH 7.0. 20


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Vesicle preparation Small unilamellar vesicles (SUVs) were prepared as described previously

32.

A green

fluorescent lipid dye, Bodipy-PE (Invitrogen, Carlsbad, CA, USA), was incorporated into the vesicles by mixing 0.1 % (w/w) with Dipalmitoyl-sn-glycero-3-phospho-choline (DPPC) (Avanti Polar Lipids, Alabaster, AL, USA) in chloroform. Following evaporation of the chloroform by a steam of N2 gas, the lipids were hydrated in 50 mM Tris-HCl, pH 7.0 at RT for 1 h. SUVs were made by sonication of a 200 µM lipid solution for 40 minutes at 45°C with 30 % power with a Ti-tip sonifier (Bandelin, Berlin, Germany). A vesicle diameter of 25-30 nm was confirmed with dynamic light scattering measurements using a High Performance Particle Sizer or a Nanosizer ZSP (Malvern Instruments, Herrenberg, Germany) (Figure S3). Vesicles were stored at room temperature and immediately utilized.

Vesicle binding studies and aggregation assay To characterize differences of aSyn mutants in terms of lipid binding and aggregation, single molecule spectroscopy techniques were applied using an Insight Reader (Evotec Technologies, Hamburg, Germany) with dual color excitation at 488 and 633 nm as described in

21, 32.

To

increase the sensitivity for rare particles like oligomers, samples were scanned by a horizontally moving focus (SIFT (scanning for intensely fluorescent targets)

33, 55).

For studying aSyn

membrane interactions, freshly prepared DPPC-SUV were diluted to a concentration of 10 µM (lipid concentration). Subsequently, labeled protein, diluted to a final concentration of 10-20 nM (10-20 particles per focus volume) in 50 mM Tris, pH 7.0, was added and incubated for 20 minutes at room temperature. Total sample volume was 20 µl. For the induction of oligomer 21



Page 22 of 36

formation, stock solutions of labeled protein were diluted in 50 mM Tris, pH 7.0 to a final concentration of 10-20 nM (10-20 particles per focus volume) and co-incubated with Fe3+ or Al3+ at a final concentration of 10 µM in a total sample volume of 20 µl for 20 minutes. At least three independent measurements of four replicates each were performed per measurement group. Measurement time was 10 seconds. All experiments were performed in 384 well glassbottom sample plates (Greiner Bioplates, Greiner, Solingen, Germany).

Analysis of membrane binding and protein aggregation Data of scanned measurements were directly visualized in 2D-FIDA (fluorescence intensity distribution ana-lysis) histograms (photon counting histograms), in which detected photons are summed over time intervals of constant length (bins; here: 40 µs) and the number of bins in which a specific number of photons was counted is plotted in a color encoded manner. Therefore, one datapoint in a 2D-FIDA histogram does not represent a single particle or the average brightness of detected particles. In addition, particle brightness (Q) and particle concentration (C) were determined by a modelbased mathematical fitting procedure using the software packages SIFT-2D and FCSPPEval Version 2.0 (Evotec Technologies, Hamburg, Germany) as previously described

56.

For the

characterization of membrane binding, particle brightness (QM/QV) of monomers and vesicles from wells with monomers or vesicles only, were determined separately in a one-componentfit using 2D-FIDA++ analysis software. Subsequently, particle brightness (QMV), total intensity (I.totMV) and concentration (CMV) of vesicles with bound protein as well as I.totM and I.totV, were determined by a three-component-fit, applying QM and QV from the previous calculations as fixed variables. The fraction (p) of protein bound to vesicles was determined by p =

22


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CMVQMV/I.totM. From p’ = CMVQMV/I.totV, the fraction of vesicles with bound protein could be calculated. The number (n) of single monomers per vesicle was derived from n = QMV/QM. Protein aggregation was analyzed using 1D-FIDA Fit. From a one-component-fit from a monomer control, QM, was determined. The particle brightness of oligomers (QO) was calculated in a two-component-fit with fixed QM. The fraction of aggregated protein results from p = COQO/I.tot. The number of monomers per oligomer is derived from QO/QM.

Fibril formation aSyn at a concentration of 50 µM along with 100 mM NaCl and 0.02% NaN3, pH 7.0 in a total volume of 1.4 ml was incubated at 37°C for 96 h under vigorous orbital shaking (1400 rpm) using an Eppendorf Thermomixer Comfort (Eppendorf, Hamburg, Germany)

57.

Fibril

formation was confirmed qualitatively by standard ThT measurements and electron microscopy. After freezing in liquid nitrogen fibrils were stored at -80°C.

Electron microscopy Electron microscopy was carried out as previously described 57. Briefly, undiluted fibril samples were treated with 1-2 % uranyl acetate. Microscopy was performed using a Libra 120 transmission electron microscope (Carl Zeiss Microscopy GmbH, Oberkochen, Germany).

Fibril formation kinetics To determine the aggregation kinetics in an automated manner, 96-well plates (Thermo-Fischer Scientific, Carlsbad, CA) containing 50 μM aSyn monomer of the different mutants in 50 mM Tris pH 7.0 and 30 µM ThT in a total volume of 140 µl were incubated at 37°C under constant 23



double-orbital shaking at 600 rpm (maximum shaking capacity) in a FLUOStar OPTIMA plate reader (BMG Labtech, Ortenberg, Germany). The formation of amyloid fibrils was monitored by ThT fluorescence, measured using an excitation wavelength of 440 nm. The emission intensity at 480 nm was recorded every hour. Lag times were determined in a blinded manner and defined by the last time point before the ThT signal increased compared to the base line. Congo red absorption spectra (340-700 nm) were measured with a Jasco 550 spectrometer (Jasco, Pfungstadt, Germany) in a cuvette with a 1 cm path length. For this purpose, 50 µM aSyn were incubated with 20 µM Congo red for two minutes at room temperature before the measurement was initiated.

Circular Dichroism spectroscopy CD spectra (190-260 nm) were recorded with a Jasco 810 spectropolarimeter (Jasco, Pfungstadt, Germany). A cuvette with a path length of 1 mm was filled with 200 µl of the aSyn solution. aSyn samples were diluted to a final concentration of 7 µM with distilled water.

Statistical analysis Statistical analysis was performed using Microsoft Excel 2016 (Microsoft Corporation, Redmond, WA, USA) and Prism (GraphPad Software, San Diego, CA, USA). Normal distribution was assumed, according to the central limit theorem, as means were calculated as means of means of at least three replicates per independent experiment. Homogeneity of variance was assessed using Levene´s test. Data were statistically compared using unpaired, two-sided Student´s t-test for data sets with equal variances and Welch´s t-test for data sets with

24


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unequal variances, respectively, with Bonferroni-Holm correction of p-values for multiple testing where applicable. Significance was determined at an -level of p < 0.05. Data are displayed as mean ± standard deviation.

Table of contents graphic Figure 1. Three-dimensional lipid-bound -helical structure of aSyn. Figure 2. Influence of single amino acid exchanges on lipid binding affinity Figure 3. Differential effects of aSyn mutants on metal ion induced oligomer formation. Figure 4. Influence of aSyn mutants on fibril formation kinetics. Figure 5. Impact of ferric iron on fibril formation. Figure 6. Influence of mixing wt aSyn with A30P and A53T on aggregation kinetics.

Supporting information Figure S1. Average particle brightness and number of aSyn monomers per focus volume. Depicted are the total intensitiy (I.tot.) of the red channel and the calculated number (n) of monomers per focus volume indicating comparable amounts of protein of each mutant were applied for co-incubation with DPPC-SUV. Figure S2. Congo red absorption and CD spectroscopy. Absorption spectra of Congo red (20 µM) of wt aSyn (a) and G51D mutant (b) aggregating at 37°C (conditions as in Figure 4 a, b), recorded a different time points. The kinetics of the increase in absorption at around 500 nm and the red shift of the maximum correspond to the kinetics recorded with CD spectroscopy (c and d) as well as the increase in ThT fluorescence (e). The changes in the shape of the CD spectra represent the transition of the protein in random coil towards ß-sheet conformation. Figure S3. Quality control of DPPC-SUVs by DLS. A vesicle diameter of 25-30 nm was confirmed with DLS measurements.

Acknowledgements We thank Brigitte Nuscher, Martin Bartels, Daniel Weckbecker, Michael Schmidt and Friederike Ruf for excellent technical support and helpful discussions.

Author information Viktoria Ruf, Armin Giese Zentrum für Neuropathologie und Prionforschung 25



Ludwig-Maximilians-Universität München Feodor-Lynen-Str. 23 D-81377 München Germany [email protected] Author Contributions Conceived and designed the experiments: VCR, SW, AG. Performed the experiments: VCR, SS, SW, FK, JL. Analyzed the data: VCR, GSN, SW, FK, AG. Contributed reagents/materials/analysis tools: VCR, SS, FS, KB, FK, JL, AG. Wrote the manuscript: VCR, FK, AG. Critical revision of the manuscript: all authors. Conflict of interest The authors declare no competing financial interest.

Funding Sources This work was funded exclusively by internal resources.

References 58-60

[1] Ibanez, P., Lesage, S., Janin, S., Lohmann, E., Durif, F., Destee, A., Bonnet, A. M., Brefel-Courbon, C., Heath, S., Zelenika, D., Agid, Y., Durr, A., and Brice, A. (2009) Alpha-synuclein gene rearrangements in dominantly inherited parkinsonism: frequency, phenotype, and mechanisms, Archives of neurology 66, 102-108. [2] Singleton, A. B., Farrer, M., Johnson, J., Singleton, A., Hague, S., Kachergus, J., Hulihan, M., Peuralinna, T., Dutra, A., Nussbaum, R., Lincoln, S., Crawley, A., Hanson, M., Maraganore, D., Adler, C., Cookson, M. R., Muenter, M., Baptista, M., Miller, D., Blancato, J., Hardy, J., and Gwinn-Hardy, K. (2003) alphaSynuclein locus triplication causes Parkinson's disease, Science (New York, N.Y.) 302, 841. [3] Olgiati, S., Thomas, A., Quadri, M., Breedveld, G. J., Graafland, J., Eussen, H., Douben, H., de Klein, A., Onofrj, M., and Bonifati, V. (2015) Early-onset parkinsonism caused by alpha-synuclein gene triplication: Clinical and genetic findings in a novel family, Parkinsonism & related disorders. [4] Kruger, R., Kuhn, W., Muller, T., Woitalla, D., Graeber, M., Kosel, S., Przuntek, H., Epplen, J. T., Schols, L., and Riess, O. (1998) Ala30Pro mutation in the gene encoding alpha-synuclein in Parkinson's disease, Nature genetics 18, 106-108. [5] Zarranz, J. J., Alegre, J., Gomez-Esteban, J. C., Lezcano, E., Ros, R., Ampuero, I., Vidal, L., Hoenicka, J., Rodriguez, O., Atares, B., Llorens, V., Gomez Tortosa, E., del Ser, T., Munoz, D. G., and de Yebenes, J. G. (2004) The new mutation, E46K, of alpha-synuclein causes Parkinson and Lewy body dementia, Annals of neurology 55, 164-173. [6] Pimentel, M. M., Rodrigues, F. C., Leite, M. A., Campos Junior, M., Rosso, A. L., Nicaretta, D. H., Pereira, J. S., Silva, D. J., Della Coletta, M. V., Vasconcellos, L. F., Abreu, G. M., Dos Santos, J. M., and SantosReboucas, C. B. (2015) Parkinson disease: alpha-synuclein mutational screening and new clinical insight into the p.E46K mutation, Parkinsonism & related disorders. [7] Appel-Cresswell, S., Vilarino-Guell, C., Encarnacion, M., Sherman, H., Yu, I., Shah, B., Weir, D., Thompson, C., Szu-Tu, C., Trinh, J., Aasly, J. O., Rajput, A., Rajput, A. H., Jon Stoessl, A., and Farrer, M. J. (2013) Alpha-synuclein p.H50Q, a novel pathogenic mutation for Parkinson's disease, Movement disorders : official journal of the Movement Disorder Society 28, 811-813. [8] Proukakis, C., Dudzik, C. G., Brier, T., MacKay, D. S., Cooper, J. M., Millhauser, G. L., Houlden, H., and Schapira, A. H. (2013) A novel alpha-synuclein missense mutation in Parkinson disease, Neurology 80, 1062-1064. [9] Lesage, S., Anheim, M., Letournel, F., Bousset, L., Honore, A., Rozas, N., Pieri, L., Madiona, K., Durr, A., Melki, R., Verny, C., and Brice, A. (2013) G51D alpha-synuclein mutation causes a novel parkinsonianpyramidal syndrome, Annals of neurology 73, 459-471.

26


Page 26 of 36

Page 27 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


[10] Kiely, A. P., Asi, Y. T., Kara, E., Limousin, P., Ling, H., Lewis, P., Proukakis, C., Quinn, N., Lees, A. J., Hardy, J., Revesz, T., Houlden, H., and Holton, J. L. (2013) alpha-Synucleinopathy associated with G51D SNCA mutation: a link between Parkinson's disease and multiple system atrophy?, Acta neuropathologica 125, 753-769. [11] Pasanen, P., Myllykangas, L., Siitonen, M., Raunio, A., Kaakkola, S., Lyytinen, J., Tienari, P. J., Poyhonen, M., and Paetau, A. (2014) A novel alpha-synuclein mutation A53E associated with atypical multiple system atrophy and Parkinson's disease-type pathology, Neurobiology of aging 35, 2180.e2181-2185. [12] Martikainen, M. H., Paivarinta, M., Hietala, M., and Kaasinen, V. (2015) Clinical and imaging findings in Parkinson disease associated with the A53E SNCA mutation, Neurology. Genetics 1, e27. [13] Polymeropoulos, M. H., Lavedan, C., Leroy, E., Ide, S. E., Dehejia, A., Dutra, A., Pike, B., Root, H., Rubenstein, J., Boyer, R., Stenroos, E. S., Chandrasekharappa, S., Athanassiadou, A., Papapetropoulos, T., Johnson, W. G., Lazzarini, A. M., Duvoisin, R. C., Di Iorio, G., Golbe, L. I., and Nussbaum, R. L. (1997) Mutation in the alpha-synuclein gene identified in families with Parkinson's disease, Science (New York, N.Y.) 276, 2045-2047. [14] Hoffman-Zacharska, D., Koziorowski, D., Ross, O. A., Milewski, M., Poznanski, J., Jurek, M., Wszolek, Z. K., Soto-Ortolaza, A., Slawek, J., Janik, P., Jamrozik, Z., Potulska-Chromik, A., Jasinska-Myga, B., Opala, G., Krygowska-Wajs, A., Czyzewski, K., Dickson, D. W., Bal, J., and Friedman, A. (2013) Novel A18T and pA29S substitutions in alpha-synuclein may be associated with sporadic Parkinson's disease, Parkinsonism & related disorders 19, 1057-1060. [15] Yoshino, H., Hirano, M., Stoessl, A. J., Imamichi, Y., Ikeda, A., Li, Y., Funayama, M., Yamada, I., Nakamura, Y., Sossi, V., Farrer, M. J., Nishioka, K., and Hattori, N. (2017) Homozygous alphasynuclein p.A53V in familial Parkinson's disease, Neurobiology of aging 57, 248.e247-248.e212. [16] Athanassiadou, A., Voutsinas, G., Psiouri, L., Leroy, E., Polymeropoulos, M. H., Ilias, A., Maniatis, G. M., and Papapetropoulos, T. (1999) Genetic analysis of families with Parkinson disease that carry the Ala53Thr mutation in the gene encoding alpha-synuclein, American journal of human genetics 65, 555558. [17] Kiely, A. P., Ling, H., Asi, Y. T., Kara, E., Proukakis, C., Schapira, A. H., Morris, H. R., Roberts, H. C., Lubbe, S., Limousin, P., Lewis, P. A., Lees, A. J., Quinn, N., Hardy, J., Love, S., Revesz, T., Houlden, H., and Holton, J. L. (2015) Distinct clinical and neuropathological features of G51D SNCA mutation cases compared with SNCA duplication and H50Q mutation, Molecular neurodegeneration 10, 41. [18] Iwai, A., Masliah, E., Yoshimoto, M., Ge, N., Flanagan, L., de Silva, H. A., Kittel, A., and Saitoh, T. (1995) The precursor protein of non-A beta component of Alzheimer's disease amyloid is a presynaptic protein of the central nervous system, Neuron 14, 467-475. [19] Bartels, T., Ahlstrom, L. S., Leftin, A., Kamp, F., Haass, C., Brown, M. F., and Beyer, K. (2010) The Nterminus of the intrinsically disordered protein alpha-synuclein triggers membrane binding and helix folding, Biophysical journal 99, 2116-2124. [20] Nuscher, B., Kamp, F., Mehnert, T., Odoy, S., Haass, C., Kahle, P. J., and Beyer, K. (2004) Alpha-synuclein has a high affinity for packing defects in a bilayer membrane: a thermodynamics study, The Journal of biological chemistry 279, 21966-21975. [21] Kostka, M., Hogen, T., Danzer, K. M., Levin, J., Habeck, M., Wirth, A., Wagner, R., Glabe, C. G., Finger, S., Heinzelmann, U., Garidel, P., Duan, W., Ross, C. A., Kretzschmar, H., and Giese, A. (2008) Single particle characterization of iron-induced pore-forming alpha-synuclein oligomers, The Journal of biological chemistry 283, 10992-11003. [22] Binolfi, A., Rasia, R. M., Bertoncini, C. W., Ceolin, M., Zweckstetter, M., Griesinger, C., Jovin, T. M., and Fernandez, C. O. (2006) Interaction of alpha-synuclein with divalent metal ions reveals key differences: a link between structure, binding specificity and fibrillation enhancement, Journal of the American Chemical Society 128, 9893-9901. [23] Santner, A., and Uversky, V. N. (2010) Metalloproteomics and metal toxicology of alpha-synuclein, Metallomics : integrated biometal science 2, 378-392. [24] Uversky, V. N., Li, J., and Fink, A. L. (2001) Metal-triggered structural transformations, aggregation, and fibrillation of human alpha-synuclein. A possible molecular NK between Parkinson's disease and heavy metal exposure, The Journal of biological chemistry 276, 44284-44296. [25] Stefanovic, A. N., Lindhoud, S., Semerdzhiev, S. A., Claessens, M. M., and Subramaniam, V. (2015) Oligomers of Parkinson's Disease-Related alpha-Synuclein Mutants Have Similar Structures but Distinctive Membrane Permeabilization Properties, Biochemistry 54, 3142-3150.

27



[26] Rutherford, N. J., Moore, B. D., Golde, T. E., and Giasson, B. I. (2014) Divergent effects of the H50Q and G51D SNCA mutations on the aggregation of alpha-synuclein, Journal of neurochemistry. [27] Rutherforda, N. J., and Giassona, B. I. (2015) The A53E alpha-synuclein pathological mutation demonstrates reduced aggregation propensity in vitro and in cell culture, Neuroscience letters. [28] Ghosh, D., Mondal, M., Mohite, G. M., Singh, P. K., Ranjan, P., Anoop, A., Ghosh, S., Jha, N. N., Kumar, A., and Maji, S. K. (2013) The Parkinson's disease-associated H50Q mutation accelerates alphaSynuclein aggregation in vitro, Biochemistry 52, 6925-6927. [29] Ghosh, D., Sahay, S., Ranjan, P., Salot, S., Mohite, G. M., Singh, P. K., Dwivedi, S., Carvalho, E., Banerjee, R., Kumar, A., and Maji, S. K. (2014) The Newly Discovered Parkinson's Disease Associated Finnish Mutation (A53E) Attenuates alpha-Synuclein Aggregation and Membrane Binding, Biochemistry. [30] Ysselstein, D., Joshi, M., Mishra, V., Griggs, A. M., Asiago, J. M., McCabe, G. P., Stanciu, L. A., Post, C. B., and Rochet, J. C. (2015) Effects of impaired membrane interactions on alpha-synuclein aggregation and neurotoxicity, Neurobiology of disease. [31] Davidson, W. S., Jonas, A., Clayton, D. F., and George, J. M. (1998) Stabilization of alpha-synuclein secondary structure upon binding to synthetic membranes, The Journal of biological chemistry 273, 9443-9449. [32] Hogen, T., Levin, J., Schmidt, F., Caruana, M., Vassallo, N., Kretzschmar, H., Botzel, K., Kamp, F., and Giese, A. (2012) Two different binding modes of alpha-synuclein to lipid vesicles depending on its aggregation state, Biophysical journal 102, 1646-1655. [33] Bieschke, J., Giese, A., Schulz-Schaeffer, W., Zerr, I., Poser, S., Eigen, M., and Kretzschmar, H. (2000) Ultrasensitive detection of pathological prion protein aggregates by dual-color scanning for intensely fluorescent targets, Proceedings of the National Academy of Sciences of the United States of America 97, 5468-5473. [34] Levin, J., Hogen, T., Hillmer, A. S., Bader, B., Schmidt, F., Kamp, F., Kretzschmar, H. A., Botzel, K., and Giese, A. (2011) Generation of ferric iron links oxidative stress to alpha-synuclein oligomer formation, Journal of Parkinson's disease 1, 205-216. [35] Schmidt, F., Levin, J., Kamp, F., Kretzschmar, H., Giese, A., and Botzel, K. (2012) Single-channel electrophysiology reveals a distinct and uniform pore complex formed by alpha-synuclein oligomers in lipid membranes, PloS one 7, e42545. [36] Weinreb, P. H., Zhen, W., Poon, A. W., Conway, K. A., and Lansbury, P. T., Jr. (1996) NACP, a protein implicated in Alzheimer's disease and learning, is natively unfolded, Biochemistry 35, 13709-13715. [37] Jo, E., Fuller, N., Rand, R. P., St George-Hyslop, P., and Fraser, P. E. (2002) Defective membrane interactions of familial Parkinson's disease mutant A30P alpha-synuclein, Journal of molecular biology 315, 799-807. [38] Jensen, P. H., Nielsen, M. S., Jakes, R., Dotti, C. G., and Goedert, M. (1998) Binding of alpha-synuclein to brain vesicles is abolished by familial Parkinson's disease mutation, The Journal of biological chemistry 273, 26292-26294. [39] Perrin, R. J., Woods, W. S., Clayton, D. F., and George, J. M. (2000) Interaction of human alpha-Synuclein and Parkinson's disease variants with phospholipids. Structural analysis using site-directed mutagenesis, The Journal of biological chemistry 275, 34393-34398. [40] Fares, M. B., Ait-Bouziad, N., Dikiy, I., Mbefo, M. K., Jovicic, A., Kiely, A., Holton, J. L., Lee, S. J., Gitler, A. D., Eliezer, D., and Lashuel, H. A. (2014) The novel Parkinson's disease linked mutation G51D attenuates in vitro aggregation and membrane binding of alpha-synuclein, and enhances its secretion and nuclear localization in cells, Human molecular genetics. [41] Chi, Y. C., Armstrong, G. S., Jones, D. N., Eisenmesser, E. Z., and Liu, C. W. (2014) Residue Histidine 50 Plays a Key Role in Protecting alpha-Synuclein from Aggregation at Physiological pH, The Journal of biological chemistry 289, 15474-15481. [42] Khalaf, O., Fauvet, B., Oueslati, A., Dikiy, I., Mahul-Mellier, A. L., Ruggeri, F. S., Mbefo, M., Vercruysse, F., Dietler, G., Lee, S. J., Eliezer, D., and Lashuel, H. A. (2014) The H50Q mutation enhances alphasynuclein aggregation, secretion and toxicity, The Journal of biological chemistry. [43] Porcari, R., Proukakis, C., Waudby, C. A., Bolognesi, B., Mangione, P. P., Paton, J. F., Mullin, S., Cabrita, L. D., Penco, A., Relini, A., Verona, G., Vendruscolo, M., Stoppini, M., Tartaglia, G. G., Camilloni, C., Christodoulou, J., Schapira, A. H., and Bellotti, V. (2014) The H50Q mutation induces a tenfold decrease in the solubility of alpha-synuclein, The Journal of biological chemistry. [44] Conway, K. A., Harper, J. D., and Lansbury, P. T. (1998) Accelerated in vitro fibril formation by a mutant alpha-synuclein linked to early-onset Parkinson disease, Nature medicine 4, 1318-1320.

28


Page 28 of 36

Page 29 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


[45] Conway, K. A., Lee, S. J., Rochet, J. C., Ding, T. T., Williamson, R. E., and Lansbury, P. T., Jr. (2000) Acceleration of oligomerization, not fibrillization, is a shared property of both alpha-synuclein mutations linked to early-onset Parkinson's disease: implications for pathogenesis and therapy, Proceedings of the National Academy of Sciences of the United States of America 97, 571-576. [46] Li, J., Uversky, V. N., and Fink, A. L. (2001) Effect of familial Parkinson's disease point mutations A30P and A53T on the structural properties, aggregation, and fibrillation of human alpha-synuclein, Biochemistry 40, 11604-11613. [47] Sierecki, E., Giles, N., Bowden, Q., Polinkovsky, M. E., Steinbeck, J., Arrioti, N., Rahman, D., Bhumkar, A., Nicovich, P. R., Ross, I., Parton, R. G., Bocking, T., and Gambin, Y. (2016) Nanomolar oligomerization and selective co-aggregation of alpha-synuclein pathogenic mutants revealed by singlemolecule fluorescence, Scientific reports 6, 37630. [48] Guerrero-Ferreira, R., Taylor, N. M., Mona, D., Ringler, P., Lauer, M. E., Riek, R., Britschgi, M., and Stahlberg, H. (2018) Cryo-EM structure of alpha-synuclein fibrils, eLife 7. [49] Li, Y., Zhao, C., Luo, F., Liu, Z., Gui, X., Luo, Z., Zhang, X., Li, D., Liu, C., and Li, X. (2018) Amyloid fibril structure of alpha-synuclein determined by cryo-electron microscopy, Cell research. [50] Kobayashi, H., Kruger, R., Markopoulou, K., Wszolek, Z., Chase, B., Taka, H., Mineki, R., Murayama, K., Riess, O., Mizuno, Y., and Hattori, N. (2003) Haploinsufficiency at the alpha-synuclein gene underlies phenotypic severity in familial Parkinson's disease, Brain : a journal of neurology 126, 32-42. [51] Voutsinas, G. E., Stavrou, E. F., Karousos, G., Dasoula, A., Papachatzopoulou, A., Syrrou, M., Verkerk, A. J., van der Spek, P., Patrinos, G. P., Stoger, R., and Athanassiadou, A. (2010) Allelic imbalance of expression and epigenetic regulation within the alpha-synuclein wild-type and p.Ala53Thr alleles in Parkinson disease, Human mutation 31, 685-691. [52] Markopoulou, K., Dickson, D. W., McComb, R. D., Wszolek, Z. K., Katechalidou, L., Avery, L., Stansbury, M. S., and Chase, B. A. (2008) Clinical, neuropathological and genotypic variability in SNCA A53T familial Parkinson's disease. Variability in familial Parkinson's disease, Acta neuropathologica 116, 2535. [53] Giasson, B. I., Uryu, K., Trojanowski, J. Q., and Lee, V. M. (1999) Mutant and wild type human alphasynucleins assemble into elongated filaments with distinct morphologies in vitro, The Journal of biological chemistry 274, 7619-7622. [54] Giese, A., Bader, B., Bieschke, J., Schaffar, G., Odoy, S., Kahle, P. J., Haass, C., and Kretzschmar, H. (2005) Single particle detection and characterization of synuclein co-aggregation, Biochemical and biophysical research communications 333, 1202-1210. [55] Giese, A., Bieschke, J., Eigen, M., and Kretzschmar, H. A. (2000) Putting prions into focus: application of single molecule detection to the diagnosis of prion diseases, Archives of virology. Supplementum, 161171. [56] Nubling, G. S., Levin, J., Bader, B., Lorenzl, S., Hillmer, A., Hogen, T., Kamp, F., and Giese, A. (2014) Modelling Ser129 phosphorylation inhibits membrane binding of pore-forming alpha-synuclein oligomers, PloS one 9, e98906. [57] Deeg, A. A., Reiner, A. M., Schmidt, F., Schueder, F., Ryazanov, S., Ruf, V. C., Giller, K., Becker, S., Leonov, A., Griesinger, C., Giese, A., and Zinth, W. (2015) Anle138b and related compounds are aggregation specific fluorescence markers and reveal high affinity binding to alpha-Synuclein aggregates, Biochimica et biophysica acta. [58] Seidel, K., Schols, L., Nuber, S., Petrasch-Parwez, E., Gierga, K., Wszolek, Z., Dickson, D., Gai, W. P., Bornemann, A., Riess, O., Rami, A., Den Dunnen, W. F., Deller, T., Rub, U., and Kruger, R. (2010) First appraisal of brain pathology owing to A30P mutant alpha-synuclein, Annals of neurology 67, 684689. [59] Zarranz, J. J., Fernandez-Bedoya, A., Lambarri, I., Gomez-Esteban, J. C., Lezcano, E., Zamacona, J., and Madoz, P. (2005) Abnormal sleep architecture is an early feature in the E46K familial synucleinopathy, Movement disorders : official journal of the Movement Disorder Society 20, 1310-1315. [60] Spira, P. J., Sharpe, D. M., Halliday, G., Cavanagh, J., and Nicholson, G. A. (2001) Clinical and pathological features of a Parkinsonian syndrome in a family with an Ala53Thr alpha-synuclein mutation, Annals of neurology 49, 313-319.

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Figure 1. Three-dimensional lipid-bound α-helical structure of aSyn. The mutations known to date are all located within the N-terminal helical part of the 140 amino-acid protein. The C-terminus remains unstructured both in α-helical and ß-sheet variants of aSyn conformations (diagram generated using The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC)


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Figure 2. Influence of single amino acid exchanges on lipid binding affinity. (a) Schematic depiction of vesicles and protein monomers in a 2D FIDA histogram (adapted from 32) and 2D FIDA histograms of aSyn and DPPC-SUV only and aSyn co-incubated with DPPC-SUV. For A30P, markedly less bicolored particles can be observed. (b) The number (n) of A30P and G51D monomers per vesicle was markedly decreased. (c) For all mutants, an at least slight reduction of the fraction of protein bound to vesicles was detected. (d) Additionally, the proportion of vesicles with bound protein was moderately to strongly decreased in different mutants. Error bars in (b), (c) and (d) indicate standard deviation (n ≥ 4), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.



Figure 3. Differential effects of aSyn mutants on metal ion induced oligomer formation. (a, b) In all mutants, addition of 10 µM FeCl3 or AlCl3, respectively, induces the formation of oligomers of comparable size, indicated by the number (n) of monomers per oligomer. (c, d) However, the fractions of oligomerized protein per total protein display striking differences between the aSyn mutants: While A30P has a high tendency to form oligomers, the potential of A53T to form oligomers is low. H50Q, G51D and A53E show moderately decreased fractions of oligomers compared to wt aSyn. For E46K, no significant difference could be detected. Each single dot represents one independent experiment (n ≥ 4). Error bars indicate stand-ard deviation, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. " "


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Figure 4. Influence of aSyn mutants on fibril formation kinetics. (a) Representative curves of normalized aggregation kinetics of aSyn mutants monitored by ThT fluorescence over time. (b) Quantification of the lag phases indicates very slow fibril formation of A30P and very rapid fibril formation of H50Q, G51D and A53T. For E46K, a slight increase of the lag time was observed, A53E showed no difference of lag time compared to wt aSyn. Each single dot represents one independent experiment (n ≥ 5). Error bars indicate standard deviation, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (c) Representative EM images indicating fibril formation of all aSyn mutants after incubation of 50 µM monomeric aSyn for 96 h at 37°C under 1400 rpm (scale bar: 200 nm).



Figure 5. Impact of ferric iron on fibril formation. (a-c) Representative kinetic curves of wt (a), A30P (b) and A53T (c) with increasing concentrations of ferric iron reveal decreasing ThT fluorescence with increasing concentrations of ferric iron, indicating less amyloid fibril formation. (d) Slight prolongation of lag times is observed with 100 µM and 300 µM ferric iron for all tested aSyn variants. Upon addition of 1000 µM ferric iron, fibril formation of wt aSyn and A30P is completely blocked and ThT fluorescence of A53T starts increasing not earlier than 145 h. Error bars in d indicate standard deviation (nwt = 6; nA30P = 4; nA53T = 4), *p < 0.05.


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Figure 6. Influence of mixing wt aSyn with A30P and A53T on aggregation kinetics. A30P and A53T were mixed with wt aSyn at different proportions. Lag times of mixtures seem to be obviously determined by the faster aggregating aSyn variant. Each independent experiment is represented by a single dot (n ≥ 3). Error bars indicate standard deviation, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0,0001.



80x40mm (300 x 300 DPI)


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Different Effects of Î±-Synuclein Mutants on Lipid Binding and

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