Author response: An engineered monomer binding-protein for α-synuclein efficiently inhibits the proliferation of amyloid fibrils (2019)

Article Figures and data Abstract Introduction Results Discussion Materials and methods Appendix 1 Appendix 2 Appendix 3 Data availability References Decision letter Author response Article and author information Metrics Abstract Removing or preventing the formation of α-synuclein aggregates is a plausible strategy against Parkinson’s disease. To this end, we have engineered the β-wrapin AS69 to bind monomeric α-synuclein with high affinity. In cultured cells, AS69 reduced the self-interaction of α-synuclein and formation of visible α-synuclein aggregates. In flies, AS69 reduced α-synuclein aggregates and the locomotor deficit resulting from α-synuclein expression in neuronal cells. In biophysical experiments in vitro, AS69 highly sub-stoichiometrically inhibited both primary and autocatalytic secondary nucleation processes, even in the presence of a large excess of monomer. We present evidence that the AS69-α-synuclein complex, rather than the free AS69, is the inhibitory species responsible for sub-stoichiometric inhibition of secondary nucleation. These results represent a new paradigm that high affinity monomer binders can lead to strongly sub-stoichiometric inhibition of nucleation processes. Introduction Cytoplasmic aggregates of the protein α-synuclein are the pathological hallmark of Parkinson’s disease (PD) and other synucleinopathies (Spillantini et al., 1997). Point mutations in the α-synuclein gene or triplication of the α-synuclein locus are associated with familial forms of PD, and the α-synuclein locus is a genetic risk factor for sporadic PD (Obeso et al., 2017). α-synuclein aggregate pathology was demonstrated to propagate from neuron to neuron (Desplats et al., 2009), and recent work has focused on understanding the cellular and molecular events in this process. From a therapeutic perspective, α-synuclein aggregation is thought to be the underlying cause of PD and remains the focus of causal therapeutic strategies. The link between α-synuclein aggregation and PD has been known for two decades (Spillantini et al., 1997; Conway et al., 1998); however, translation of this scientific discovery into a therapy has proven challenging. From the first description of small molecules that inhibit α-synuclein aggregation in 2006 (Masuda et al., 2006), the search for promising compounds has continued (Wagner et al., 2013; Tóth et al., 2014; Wrasidlo et al., 2016; Perni et al., 2017; Kurnik et al., 2018). While the first small molecules also inhibited the aggregation of tau and amyloid-β, more recent compounds bind α-synuclein more selectively and show reduced α-synuclein toxicity in mouse models of PD (Wrasidlo et al., 2016). We have taken a different strategy by engineering a protein, the β-wrapin AS69, to induce formation of a β-hairpin in monomeric α-synuclein upon binding (Figure 1a) (Mirecka et al., 2014). AS69 was selected by phage display (Mirecka et al., 2014) from protein libraries based on ZAβ3, an affibody against the amyloid-β peptide (Hoyer et al., 2008; Hoyer and Härd, 2008; Luheshi et al., 2010). AS69 thus not only binds α-synuclein with high and approximately constant affinity throughout the pH range most relevant for α-synuclein aggregation (Buell et al., 2014a; Figure 1b,c), but also induces a specific conformational change - akin to molecular chaperones (Muchowski and Wacker, 2005). Figure 1 Download asset Open asset AS69 binds to monomeric α-synuclein, inducing local folding of the region comprising residues 37–54 into a β-hairpin conformation. (a) Structural model of the AS69:α-synuclein complex based on NMR (pdb entry 4BXL) (Mirecka et al., 2014), generated with PyMOL (The PyMOL Molecular Graphics System, 1.2; Schrödinger, LLC.). AS69 (grey) is a disulfide-linked homodimer. α-Synuclein (orange) locally adopts β-hairpin conformation, while the remainder of the molecule, including the hydrophobic NAC segment (green), remains intrinsically disordered (Mirecka et al., 2014). Positions at which disease-related mutations have been identified are given in magenta. (b,c) The affinity of AS69 to α-synuclein at pH 7.4 (b) and pH 5.0 (c) analyzed by isothermal titration calorimetry (ITC) experiments. Titration of 420 μM α-synuclein into 47 μM AS69 in 20 mM sodium phosphate, 50 mM NaCl, pH 7.4 (b), or 320 μM α-synuclein into 32 μM AS69 in 20 mM sodium acetate, pH 5.0 (c), at 30 °C. The upper panels show the baseline-corrected instrumental response. The lower panels show the integrated data (filled squares) and the fit to a 1:1 binding model (continuous line). AS69 induces local folding of the region comprising residues 37–54 into a β-hairpin conformation in the otherwise intrinsically disordered, monomeric α-synuclein (Figure 1a). The critical role of this region for α-synuclein aggregation is indicated by the cluster of disease-related mutation sites (Figure 1a). Accordingly, modification of the local conformation by, for example, introduction of a disulfide bond strongly modulates aggregation (Shaykhalishahi et al., 2015). Sequestration of residues 37–54 of monomeric α-synuclein by AS69 inhibits the amyloid fibril formation of α-synuclein under conditions of vigorous shaking of the solution even at highly sub-stoichiometric ratios (Mirecka et al., 2014). Amyloid fibril formation, however, is not a one-step process but can be decomposed into different individual steps, including primary and secondary nucleation and fibril elongation. With vigorous shaking, for instance, primary nucleation can occur readily at the air-water interface (Campioni et al., 2014) and fibril fragmentation induced by the shaking amplifies the number of growth-competent fibril ends (Xue et al., 2009). To validate AS69 as a potential therapeutic agent, we therefore tested its biological effects in cellular and animal models, and found it to be a highly efficient inhibitor of α-synuclein aggregation and associated toxicity. In addition, we designed a set of experimental conditions to measure selectively the effect of AS69 on specific steps of α-synuclein aggregation. We found that AS69 is able to efficiently interfere with both the lipid-induced formation and the auto-catalytic amplification of α-synuclein amyloid fibril formation. These inhibitory effects on nucleation are observed even in the presence of a large excess of α-synuclein monomer, which is expected to sequester AS69 into inhibitor-monomer complexes. We show evidence that the secondary nucleation of α-synuclein can be inhibited by the α-synuclein-AS69 complex and, therefore the inhibitory effect of AS69 on this crucial step of aggregate amplification is unaffected by even large excess concentrations of free α-synuclein monomer. Results Co-expression of AS69 reduces visible α-synuclein aggregates in cell culture First, we explored the effect of the expression of AS69 on the viability of living cells and the association of α-synuclein in a cellular environment. In these model systems we not only expressed WT α-synuclein but also the A53T variant, which has been associated with familial PD and which produces aggregates more quickly than the WT protein (Conway et al., 1998; Flagmeier et al., 2016). We first used bimolecular fluorescence complementation (BiFC) to probe whether AS69 can interfere with formation of oligomeric α-synuclein species in living HEK293T cells (Falkenburger et al., 2016). Constructs of WT and A53T α-synuclein were tagged with the C-terminal segment of the fluorescent protein Venus (synuclein-VC) or with the complementary N-terminal segment of this protein (VN-synuclein) (Figure 2a). Neither of the two Venus fragments shows significant fluorescence by itself, but together they can generate a functional fluorescent protein (Bae et al., 2014) and hence function as a reporter for protein-protein interaction. We then transfected HEK293T cells with both synuclein-VC and VN-synuclein, in addition to AS69 (or LacZ as a control) and determined by flow cytometry the fraction of cells that displayed Venus fluorescence (Figure 2b, the raw data can be found in the table in Figure 2—source data 1). In the absence of AS69, the fraction of fluorescent cells was larger with the expression of A53T-α-synuclein than WT-α-synuclein (Figure 2b, p<0.05, two-way ANOVA). Co-expression of AS69 with both variants reduced the number and fraction of fluorescent cells (Figure 2b, p<0.05 for WT and p<0.01 for A53T, two-way ANOVA). AS69 did not, however, significantly affect the total quantity of α-synuclein in the cells, as determined from immunoblots (Figure 2c and d). This finding is consistent with the hypothesis that the effects of AS69 in this cellular model system result from inhibition of a direct interaction between α-synuclein molecules, and not from an enhanced clearance of α-synuclein. Despite the enhanced affinity for self-interaction which the fluorescence complementation tag might convey to α-synuclein compared to the untagged protein, the affinity for AS69 is high enough to sequester a significant proportion of the α-synuclein in living cells. Figure 2 with 1 supplement see all Download asset Open asset AS69 reduces aggregation of α-synuclein in cellular models. (a) Schematic representation of bimolecular fluorescence complementation where α-synuclein is tagged by either the C-terminal (VC) or the N-terminal (VN) fragment of the Venus protein. In dimers or larger oligomers of α-synuclein, the two Venus fragments can form a functional fluorescent protein. (b) The percentage of cells with BiFC fluorescence as determined by flow cytometry. HEK293T cells were transfected with α-synuclein (WT or A53T), fused to the VN or VC fragment and either LacZ (control) or AS69. Displayed are the results of n = 3 independent experiments and mean ± SEM. In each experiment, 75,000 cells were analyzed per group. Results were compared by one-way ANOVA, results of Sidak’s posthoc test depicted. (c) Immunoblot of lysates of cells transfected with EGFP-tagged α-synuclein and, in addition, AS69 or LacZ (control), developed with antibodies against α-synuclein (band just below 20 kDa, note that only the upper band reports α-synuclein, Dinter et al., 2016) and β-tubulin (band just below 50 kDa), the latter as a loading control. (d) Quantification of n = 4 independent blots as described in (c). Results were compared by t-test. (e) HEK293T cells were transfected with EGFP-tagged α-synuclein and the distribution of fluorescence was classified into the depicted groups. (f) Summarized results of n = 3 independent experiments with n = 300 cells classified per group in each experiment (mean ± SEM). Results were compared by two-way ANOVA and Sidak’s posthoc test. Figure 2—source data 1 Raw cell counts of cells from the three independent experiments shown in Figure 2b. The data can be found under the following link: https://osf.io/qs2yf/. https://cdn.elifesciences.org/articles/46112/elife-46112-fig2-data1-v4.xls Download elife-46112-fig2-data1-v4.xls Having established that α-synuclein and AS69 can interact in cells, we next probed the effects of AS69 on the formation of larger, optically visible aggregates of α-synuclein by transfecting HEK293T cells with A53T-α-synuclein tagged with enhanced green fluorescent protein (EGFP) as previously described (Opazo et al., 2008; Karpinar et al., 2009; Dinter et al., 2016; Figure 2e). The distribution of EGFP within transfected cells was classified as 'homogenous', 'containing particles' or 'unhealthy' (rounded cells that in time-lapse microscopy were observed to subsequently undergo apoptosis). Co-expression of AS69 with A53T α-synuclein led to an increase in the fraction of cells with a 'homogenous' distribution of EGFP and fewer cells showed α-synuclein particles relative to those cells without AS69 (Figure 2f). These findings indicate that the co-expression of AS69 reduces formation of visible aggregates in cultured human cells. Co-expression of AS69 rescues A53T α-synuclein-dependent phenotype in Drosophila melanogaster Subsequently, we tested the effects AS69 has in Drosophila melanogaster (fruit flies) expressing untagged A53T-α-synuclein in neurons (Figure 3). In the absence of AS69, these flies show a progressive reduction in the spontaneous climbing (i.e. neuronal impairment) between 15 and 25 days of age (Butler et al., 2012; Dinter et al., 2016; illustrated in Figure 3a). We then generated flies co-expressing either AS69 or GFP (as a control) with A53T α-synuclein in neurons. Flies expressing AS69 and A53T α-synuclein showed preserved climbing behaviour (Figure 3b, two-way ANOVA), demonstrating that neuronal expression of AS69 reduces the phenotype in this fly model of A53T α-synuclein toxicity. We further went on to determine whether or not the observed effect of AS69 on climbing behaviour could result from a reduction in the number of α-synuclein aggregates and used flies expressing in all neurons one copy of A53T-α-synuclein fused to VC, one copy of A53T-α-synuclein fused VN (Prasad et al., 2019), and, in addition, AS69 or 'always early RNAi' (see Materials and methods section) as a control. Aggregates of α-synuclein were quantified by a filter trap assay in which urea-treated lysates of fly heads were passed through a membrane and the quantity of α-synuclein aggregates retained in the membrane was detected by antibodies raised against α-synuclein (illustrated in Figure 3c). We found that the quantity of aggregates retained in the filter was significantly smaller in lysates from flies co-expressing AS69 and A53T-α-synuclein than in lysates from flies only expressing VN- and VC-tagged A53T-α-synuclein (Figure 3d and e). These findings confirm that AS69 reduces high molecular weight aggregates of α-synuclein in neuronal cells of Drosophila melanogaster. Figure 3 Download asset Open asset AS69 rescues the motor phenotype and reduces α-synuclein aggregation in Drosophila melanogaster. (a) Schematic representation of the climbing assay. The vials are tapped to move the flies to the base of the vial, and thereafter the flies climb towards the top of the vial; in this experiment the number of flies climbing 8 cm in 10 s was determined. (b) Performance in the climbing assay of Drosophila melanogaster expressing A53T-α-synuclein and either AS69 or GFP in neurons. At each time point, n = 30 flies were assayed per genotype; similar findings were observed for eight different lines expressing AS69. Results were compared by two-way ANOVA and Sidak’s posthoc test. (c) Schematic representation of the filter trap assay in which aggregates in the protein lysate are retained by a membrane, which is subsequently developed in the same manner as an immunoblot. (d) Results of the filter trap assay from lysates of control flies and flies expressing AS69 in addition to A53T-α-synuclein in all neurons. Two different quantities of the protein lysate were applied in each case, 5 and 25 μg. (e) Summary of the quantification of n = 3 dot blots as in (d). Only the 25 μg band was quantified. Results were compared by t-test. AS69 stoichiometrically inhibits the elongation of α-synuclein fibrils We next set out to elucidate the origin of the remarkable ability of AS69 to inhibit α-synuclein aggregate formation in cells and in vivo (Figure 2, Figure 3), and amyloid fibril formation in vitro (Mirecka et al., 2014). To this end, we performed a detailed mechanistic analysis, where we examined the effect of AS69 on the growth (Buell et al., 2014a), autocatalytic amplification (Buell et al., 2014a; Flagmeier et al., 2016) and lipid-induced formation (Galvagnion et al., 2015) of α-synuclein amyloid fibrils. We first carried out experiments in the presence of micromolar concentrations (in monomer equivalents) of pre-formed seed fibrils of α-synuclein at neutral pH under quiescent conditions (Figure 4a,b). We have shown previously that under these conditions only fibril elongation through addition of monomeric α-synuclein to fibril ends occurs at detectable rates (Buell et al., 2014a), and that the rate of de novo formation of fibrils is negligible. We therefore examined the effects of AS69 on fibril elongation and analyzed these data by fitting linear functions to the early stages of the aggregation time courses (see Appendix 1 for details of the analysis). The results indicate that fibril elongation is indeed inhibited by AS69 in a stoichiometric concentration-dependent manner (Figure 4c). In this experiment, both the seed fibrils and the AS69 compete for the monomeric α-synuclein and the relative affinities determine the kinetics and thermodynamics of the system. Figure 4 with 2 supplements see all Download asset Open asset AS69 inhibits α-synuclein fibril elongation. (a) Schematic representations of fibril elongation. (b) Change in ThT fluorescence when a 30 μM solution of monomeric α-synuclein was incubated in the presence of 5 μM pre-formed fibrils under quiescent conditions with increasing concentrations of AS69. (c) Relative rates of fibril elongation with increasing concentrations of AS69. The solid line corresponds to a prediction based on the affinity of AS69 for monomeric α-synuclein (240 nM, Figure 1b [Mirecka et al., 2014], see Appendix 1 for details). To obtain an estimate of the affinity of monomeric α-synuclein for the ends of fibrils, we performed elongation experiments at low monomer concentrations in the absence of AS69. We found evidence that the fibrils are able to elongate in the presence of 0.5 μM monomeric α-synuclein (see Appendix 1), providing an upper bound of the critical concentration (which is formally equivalent to a dissociation constant, see Appendix 1). Despite the similar affinity of monomeric α-synuclein for both fibril ends and AS69, the timescales of the two types of interactions are very different; monomeric α-synuclein was found to interact on a timescale of seconds with AS69, as seen by isothermal titration calorimetry (ITC) experiments (Mirecka et al., 2014 and Figure 1b and c), but to incorporate on a timescale of minutes to hours into free fibril ends (see Figure 4b and Buell et al., 2014a; Wördehoff et al., 2015). The slow kinetics of the latter process is partly because the number of fibril ends is much smaller than the number of monomers (Buell et al., 2014a), such that each fibril sequentially recruits many α-synuclein molecules. Therefore, the equilibrium between AS69 and α-synuclein should be rapidly established and perturbed only very slowly by the presence of the fibrils. Inhibition of fibril elongation is caused by monomer sequestration The initial fibril elongation rate as a function of AS69 concentration was found to follow closely the predicted concentration of unbound α-synuclein across the entire range of concentrations of AS69 used in this study, as shown in Figure 4c, where the solid line corresponds to the predicted elongation rate, assuming fibrils can only be elongated by unbound α-synuclein. The inhibition of fibril elongation can therefore be explained quantitatively by the sequestration of monomeric α-synuclein by AS69 and the assumption that the AS69:α-synuclein complex cannot be incorporated into the growing fibril. This conclusion is supported by the finding that the fibrils formed in the presence of increasing concentrations of AS69 are morphologically indistinguishable from the fibrils formed in the absence of AS69 (as judged from AFM images, see Figure 4—figure supplement 1). Our kinetic analysis of fibril elongation in the presence of AS69 does not, however, suggest a preferential interaction with fibril ends, as such an interaction can be expected to lead to a sub-stoichiometric inhibition of fibril elongation, which is not observed in our experiments. Indeed, the finding that the effect on elongation can be quantitatively described by considering only the interaction of AS69 with monomeric α-synuclein (Appendix 1) suggests a weak, if any, interaction of AS69 with fibrils. Furthermore, density gradient centrifugation (DGC) of samples containing only seeds and AS69 (Figure 5a and b) did not show AS69 to co-migrate with large species to any significant extent under conditions that favour elongation. In agreement with inhibition of fibril elongation by monomer sequestration, ZAβ3W, a binding protein for amyloid-β peptide (Grüning et al., 2013), which is a significantly weaker α-synuclein binder than AS69, correspondingly showed a considerably weaker inhibitory effect on α-synuclein fibril elongation (Figure 4—figure supplement 2). Figure 5 Download asset Open asset SDS-PAGE of density gradient centrifugation (DGC) experiments to probe the binding of AS69 to α-synuclein fibrils at pH 7.4 after elongation experiments. (a) 25 μM seeds, (b) 25 μM AS69 and 25 μM seeds, (c) 16.7 μM AS69fusASN, (d) 25 μM AS69fusASN and 25 μM seeds. AS69 sub-stoichiometrically inhibits amplification of α-synuclein fibrils These findings clearly demonstrate that AS69 inhibits fibril elongation in a stoichiometric manner through monomer sequestration. Consequently, inhibition of fibril elongation cannot explain the previously observed sub-stoichiometric inhibition of α-synuclein fibril formation by AS69 (Mirecka et al., 2014). We therefore performed seeded experiments under mildly acidic solution conditions in the presence of very low concentrations of pre-formed fibrils (nM monomer equivalents) under quiescent conditions (Figure 6a,b) (Buell et al., 2014a; Gaspar et al., 2017). those solution seeded aggregation has been shown to of two in addition to fibril elongation, secondary which the number of growth fibril ends, and Figure supplement 1b,c), which the aggregation rate by the number of fibrils through within aggregates (Buell et al., The de novo formation of amyloid fibrils through primary nucleation is if the solution is not and if are used (Figure supplement 1a). We that under these solution where only growth and secondary nucleation to the increase in fibril and the seeded aggregation is inhibited in a strongly sub-stoichiometric manner (Figure We these data to determine the rate of aggregation (see Appendix 2 for the from et (Figure on recent results on the of autocatalytic secondary nucleation of α-synuclein amyloid fibrils et al., we have the predicted inhibitory effect from monomer sequestration by AS69 in Figure (see Figure supplement 2 and Appendix 2 for details). We the of fibril elongation, monomer sequestration cannot explain the extent of even by assuming a very high of 5 (i.e. a of the rate of secondary nucleation on the of the free monomer which is not with recent that secondary nucleation of α-synuclein fibrils only on the concentration of free monomer et al., 2017). even in this the very inhibitory effect of low AS69 concentrations cannot be explained by monomer Figure with 3 supplements see all Download asset Open asset AS69 inhibits α-synuclein fibril (a) Schematic representation of fibril amplification through secondary nucleation Buell et (b) Change in ThT fluorescence when a μM solution of monomeric α-synuclein was incubated with increasing concentrations of AS69 in under quiescent conditions and (c) Relative rate of fibril amplification as a function of the concentration of AS69. The solid lines to based on the assumption that AS69 only through monomer sequestration, for different of the monomer of secondary nucleation (see Appendix 2 for details). inhibition of fibril amplification is not caused by interaction with the fibril We have previously been able to inhibition of the secondary nucleation of α-synuclein by the protein through for binding sites on the of the fibrils et al., 2016). we that AS69 is a significantly more efficient inhibitor of the autocatalytic amplification of α-synuclein amyloid fibrils than similar of inhibition is with a lower concentration This result is in the of the that AS69 binds efficiently to monomeric α-synuclein under both neutral and mildly acidic solution conditions (Figure 1b,c), we found evidence for a relevant direct interaction between the monomeric forms of and given the absence of any inhibitory effect of on the elongation of α-synuclein fibrils et al., 2016). Therefore, the of the AS69 bound within a complex with monomeric α-synuclein, AS69 is an efficient sub-stoichiometric inhibitor of the secondary nucleation of α-synuclein. This finding suggests that in addition to through for nucleation sites on the fibril AS69 or its complex with α-synuclein could interact with of the secondary nucleation process. To whether AS69 binds to the fibril under these secondary solution we performed experiments. in the density gradient of AS69 with fibrils, which direct interactions between these was (Figure AS69 was able to inhibit secondary nucleation through binding to the fibril in the presence of a large excess of monomer, its affinity to fibril to be much than to monomeric α-synuclein. This that under the conditions of the experiments which were performed in the absence of monomeric α-synuclein, all binding sites on the fibrils should be Therefore, the absence of detectable binding either a affinity for fibrils or a very low that is a very low density of binding sites for AS69 on the fibril Figure Download asset Open asset SDS-PAGE of density gradient centrifugation experiments to probe for binding of AS69 to fibril at pH (a) μM seeds, (b) μM AS69 and μM seeds, (c) μM AS69, μM seeds and μM monomer, and (d) μM AS69fusASN and μM seeds. AS69 binds to α-synuclein oligomers with affinity as to monomers We next tested whether binding of AS69 to oligomeric of α-synuclein could explain the efficient inhibition of secondary nucleation. The and of oligomeric on the to formation of amyloid fibrils any interaction between such species and AS69 to monomeric α-synuclein can be into oligomers that can be in because they not readily into amyloid fibrils et al., 2014). Despite it not that these species are fibril they are in and between monomeric and α-synuclein and hence can as a model for AS69 binding to α-synuclein et al., 2016) at neutral we were able to confirm the binding of AS69 to both monomeric (Figure supplement and oligomeric α-synuclein (Figure supplement and of the binding affinities 300 for monomeric and 30 for oligomeric The is in agreement with results from experiments under the same solution conditions (Figure 1b and et al., 2014), the affinity of AS69 to oligomeric α-synuclein has not previously been determined. The finding that AS69 is able to inhibit secondary nucleation in a highly sub-stoichiometric manner in the presence of a large excess of free monomer, to which it binds with high that the interactions of AS69 with aggregation be of significantly if they are to explain the the monomer the aggregation for AS69 because of the much lower concentration of the estimate (see Appendix 2 for suggests that the affinity of AS69 for aggregation to be of than to α-synuclein monomer to explain an inhibitory effect of the observed This affinity is indeed much than the affinity we have determined for an oligomeric of α-synuclein. complex of AS69 and α-synuclein efficiently inhibits secondary nucleation The analysis described in the that the complex could be the inhibitory The of this complex is even at low ratios of to interact with a fraction of aggregation is that while the AS69:α-synuclein complex is to incorporate into a fibril (see on the stoichiometric inhibition of fibril it can interact with oligomeric fibril