The C-terminal tail of α-synuclein protects against aggregate replication but is critical for oligomerization

Aggregation of the 140-residue protein α-synuclein (αSN) is a key factor in the etiology of Parkinson’s disease. Although the intensely anionic C-terminal domain (CTD) of αSN does not form part of the amyloid core region or affect membrane binding ability, truncation or reduction of charges in the CTD promotes fibrillation through as yet unknown mechanisms. Here, we study stepwise truncated CTDs and identify a threshold region around residue 121; constructs shorter than this dramatically increase their fibrillation tendency. Remarkably, these effects persist even when as little as 10% of the truncated variant is mixed with the full-length protein. Increased fibrillation can be explained by a substantial increase in self-replication, most likely via fragmentation. Paradoxically, truncation also suppresses toxic oligomer formation, and oligomers that can be formed by chemical modification show reduced membrane affinity and cytotoxicity. These remarkable changes correlate to the loss of negative electrostatic potential in the CTD and highlight a double-edged electrostatic safety guard.

the materials don't have a very strong physiological link, e.g. oligomers formed by freeze drying, synthetic lipid membranes and toxicity studies on SHSY5Y cells which are very different from dopaminergic neurons. However, the authors provide a robust analysis of the data they have provided which will aid understanding of the intramolecular interactions of aSN which promote/inhibit aggregation.
Major comment: 1) I strongly believe all ThT-based kinetic assays should be accompanied with data to show whether the ThT fluorescence signal is reflective of aggregation rate of the different species. There are a few papers now showing different ThT fluorescence intensities from different polymorphs of amyloids. The authors should provide some additional data to show that the ThT fluorescence reflects the fibril formation. For instance, quantification of the remaining monomer and/or of the formed fibrils can be done by quantification of protein concentration of either species, reversed phase chromatography, gel filtration, stained SDS-PAGE gels, performed at the end of the assays.
Minor comments: 2) Figure 1 -what does each point on the graph represent? 3) Figure 1 -Is aSN121 showing a single linear correlation? This graph looks more similar to aSN110 than the longer constructs. 4) Page 7, line 178 -are oligomers formed by rounds of freeze-drying physiologically relevant? 5) Page 7, line 181 -where in figure S5DE do the authors have the NaCl data? How does this differ to the PBS buffer (that I assume the other oligomers are in?). Buffer differences have been shown to be important for fibril polymorph formation, this could be interesting for oligomers too. 6) Page 8, line 186 -The peak size for the aSOs in Figure S5G don't look to give the same yield for 121 and 125 compared to 130 and 135. 7) Page 8, line 188-189 -It's not clear why ONE was used or how this is aiding oligomer formation of shorter fragments or why this is useful to investigate. 8) Page 9, line 216 -Is there a similar mechanism linking calcein release and cell toxicity? This seems a bit of a weak or presumptive link. 9) Discussion -What are the most abundant truncated species identified in brain tissue? Are there species identified in this work that promote fibrillisation of FL aSN, cell toxicity and calcein release that have also been identified in vivo and may thus be more important to investigate further? 10 In the paper entitled 'The C-terminal tail of α-synuclein protects against aggregate replication but is critical for oligomerization', the authors describe the results of a study aimed at determining effects of C-terminal truncations on the self-assembly properties of alpha-synuclein (aSyn), a protein that forms aggregates in the brains of individuals with Parkinson's disease (PD) and other synucleinopathy disorders. The investigators report that aSyn truncation variants with 121 or fewer residues have a substantially increased propensity to form amyloid-like fibrils, apparently because they more readily undergo secondary fibrillization as a result of enhanced fragmentation. An increased rate of fibrillization is also observed in mixtures of full-length aSyn with as little as 10% (mol/mol) of the aSyn-110 truncation variant. In contrast, C-terminal truncation interferes with aSyn oligomerization, and oligomers formed by truncation variants in the presence of the prooxidant 4-oxo-2-nonenal have a reduced propensity to permeabilize membranes and elicit cytotoxicity. The authors attribute these effects of the aSyn C-terminal tail to its electrostatic interactions with the N-terminal and NAC (central hydrophobic) domains, which favor oligomerization but interfere with the formation of amyloid-like fibrils. This paper is significant in terms of its relevance to molecular mechanisms involved in the pathogenesis of PD and other synucleinopathy disorders. In particular, the results are of a high impact because they yield important insights into structural features of the C-terminal domain of aSyn that modulate two key pathogenic events -the protein's conversion to potentially neurotoxic oligomers and the formation of aSyn amyloid-like fibrils that can spread throughout the brain. The findings reported here are novel and represent an important advance in the aSyn field.
Strengths of the paper in addition to its significance include the fact that it is well written and organized, and the authors have been thorough in terms of referencing the key literature of relevance to this study. The experiments have been carefully executed, yielding high-quality results, and the data processing and mathematical modeling are rigorous. On the other hand, there are several errors in the text and figures/figure legends, and parts of the paper are unclear. In addition, some of the authors' conclusions would be strengthened with additional discussion or by including additional data. These issues should be addressed in a revised version of the manuscript.
p. 5, line 110, 'high concentrations with a float slope'; replace 'float' with 'flat'. p. 6, lines 123-125, 'and the rate-limiting step shifts from monomer binding to the end of fibrils (which is dependent on [monomer]) to the conformational conversion of the monomer to the fibrillated state (which is not)'; it is unclear why the [monomer]-independent phase at high monomer concentrations is interpreted to reflect a conformational change of monomer to the fibrillated state (a component of the primary fibril elongation phase), as opposed to the secondary [monomer]-independent processes of 'fragmentation or saturated secondary nucleation' referred to earlier (lines 117-118). It would be helpful if this point were clarified. p. 6, lines 138-139, 'from plots of the initial rate of fibrillation against the total concentration of monomer and seed'; the plot in Fig. S4 shows only the seed concentration, not the total concentration of monomer and seed, on the x-axis (this point should be clarified).
p. 6, lines 142-143, 'To compare different constructs with different reaction orders of nucleation, we calculated the nucleation rate at 50 micromolar'; it would be helpful if a justification were provided for choosing 50 micromolar as the optimal concentration to account for different reaction orders.
p. 6, line 153, 'is around -0.5 for all mutants'; the value is closer to -0.3 to -0.4. p. 7, line 158, 'though an increase in the turnover rate on the surface of fibrils'; it would be helpful if this concept were explained in greater detail (turnover of what?) p. 7, lines 162-163, 'demonstrates the dominance of secondary processes which is most likely fragmentation'; additional detail should be provided to explain why the authors conclude that the stimulatory effect of agitation on the fibrillization rate reflects enhanced fibril fragmentation rather than turnover. p. 7, line 169, '60% aSN110 and higher showed the same behavior as 100% aSN110'; delete 'and higher' because 60% was the highest percentage of aSyn 110 tested in this experiment. p. 7, line 180, 'without any change in structure according to Small Angle X-ray Scattering'; refer to p. 10, lines 248-251, 'mutants containing disulfide bonds between residues 107 and 124 in CTD combined with disulfide bonds in the NTD-NAC region (residues 9, 42, 69 and 89) do not fibrillate; by contrast disulfide bonds in the NTD-NAC region alone are insufficient to inhibit fibrillation'; this statement should be reworded to indicate that intramolecular disulfide bonds linking cysteine residues introduced in the CTD and NTD-NAC regions were used in the study described in reference 51.  these experiments were carried out with full-length aSyn. Also, the paper would be strengthened by demonstrating that similar results are obtained when repeating these experiments with at least a subset of truncation variants (e.g. aSyn 125 and aSyn 130, to account for the pronounced difference in the secondary rate constants determined for these two variants in terms of a difference in the degree of fragmentation or secondary nucleation).    : it would be helpful to show the curve fits for aSyn 135, aSyn 130, and aSyn 125 for the secondary nucleation dominant and fragmentation dominant models separately, and to include MRE values on the plots as was done for aSyn WT. Also, the following statement in the legend, 'the models with saturation fit just as well', should be clarified (e.g. refer to 'the secondary nucleation dominant and fragmentation dominant models with saturation elongation, which produce a better fit with aSyn variants with more extensive C-terminal truncations (Fig. S3)'.       Supplement, p. 3, lines 83-87, 'Fibrils were sonicated to minimize light scattering and spectra were recorded from 250 to 185 nm in 1 mg/ml using 0.1 mm cuvette. All data are averages of three repeats with 0.5 nm steps per 0.5 s and 1 nm bandwidth. Thermal scans to monitor conformational changes upon vesicle binding were carried out as described. Briefly, a solution of 0.2 mg/mL monomeric aSN and 1 mg/mL DMPG was heated from 5 to 95 deg C at 1 deg C/min and ellipticity was monitored at 220 nm'; this text could be deleted because CD data are only shown for oligomeric aSyn in the absence of phospholipids ( The authors reveal the role of the C-terminal tail of αSN in the molecular mechanism of fibrillation and the formation and oligomeric species. Using fibrillation assay shows that removal of more than 15 residues strongly enhances fibrillation thanks to an increased rate of replication. By applying SAXS, they visualized αSN oligomers in solution, and they also show that αSN CTD truncations abolished oligomerization, and only more extensive, less stable oligomers are formed. Using calcein assays, they show that CTD truncations mutant are less disruptive towards lipid vesicles. These biophysical characterizations were linked with in-vitro studies using cell toxicity assays.
In summary, the article is very well written, easy to read, and complements the subject's bibliography well.
Response: We thank the reviewer for this generous assessment and for the constructive suggestions for improvement which we address below.
However, although the authors had a rigorous approach with the SAXS, fibrillation assay, calcein assays, and in vivo assays, I have some questions and remarks in my report.
Minor comments: 1) Please comment on the small primary nucleation rates for αSN115 shown in Fig. 2B.

Response:
We appreciate the reviewer's keen observation. The values for αSN115 do stand out.
Analysis is affected by the fact that the rates of primary and secondary nucleation are coupled to some extent in the fits, i.e. a decrease in primary rate can be compensated to some extent by an increase in secondary rate. This effect is apparent for SN115 to some extent. In addition, primary nucleation, due to the potential catalytic effects of plate surfaces and the air water interface, is more prone to experimental variation. Given these observations, we do not believe that the low primary rate of SN115 is indicative of a fundamentally different behaviour of SN115 to SN110 and SN121 but acknowledge the need to point this out. Accordingly, we note in the text now that "(we attribute the low kn value of αSN115 to a fitting artifact in which a low kn value is compensated by a higher Response: Duly corrected.

3) Please explain the meaning of [monomer]. Is there a reason not to use monomer instead?
Response: We use this term for text economy to indicate the concentration of monomer. To make it clear we explain this the first time we use it. 4) Page 7: Authors claim that secondary fibrillation is most likely controlled by "The fact that deficient concentrations of seeds (0.03%) strongly promoted the fibrillation demonstrates the dominance of secondary processes which is most likely fragmentation." Could the preparation of seeds by sonication lead to oxidation of αSN that may impact the fibrillation kinetics rather than assume the simple fragmentations?
Response: We would like to clarify that we use sonicated fibrils both for shaking and quiescent conditions (Fig. 2E); thus we carry out a comparative study using fibrils that have been sonicated to the same extent but are left to elongate under different conditions. However, it is a thoughtprovoking suggestion that sonication could lead to fibril oxidation and thus compromise seeding quality. We are not aware of any such studies in the literature and have not seen any evidence for this in our own studies with e.g. MALDI-TOF MS (though we must admit that we have not looked specifically for these modifications). We suspect that it will be difficult to gauge these effects as fibrils are typically protected against chemical insults better than monomers and the effects of oxidation may be difficult to discern within the usual errors. We are in fact embarking on a separate study to study the effect of sonication on fibril size distribution (using functional bacterial amyloid) and will bear this in mind in our analysis. Thank you for raising this! Representation of SAXS data with more detailed analysis of P(r) functions or models will avoid the comments like "similar shape, "similar modes of action in promoting interactions between different parts of αSN, "shorter constructs are more elongated with relatively bigger shells." Response: We appreciate the reviewer's thoughts on this and sympathize with the desire to provide more structural details. Unfortunately we find it difficult to advance further in our current analysis of the modelling. Our current models agree completely with our SAXS data and would therefore also be entirely consistent with p(r) functions from indirect Fourier transformation. Furthermore, please note that volumes of models are actually difficult to define as the shell of the oligomer has a lower density of protein than the compact core. We and others have tried to make ab-initio bead modelling of the SAXS data without any assumptions on the structure (cfr. Giehm et al. PNAS 2011) which leads to a similar structure as our model, namely an ellipsoidal shape. However, such modelling is done on a somewhat incorrect basis, since the SAXS intensity contains significant scattering from the random coil chains; this cannot be included properly as it requires an ensemble of particles to give the correct q dependence -and this is not possible with the methods currently available. The only way around this is to omit the high-q part of the SAXS intensities and then the models are of even lower resolution that usual for SAXS and cannot reproduce a core-shell structure. In a nutshell, these methods assumes compact globular structures and therefore cannot really investigate a core-shell structure. We therefore believe it is better to restrict ourselves to the current level of resolution. We checked the leftover monomer after fibrillation reach a plateau for all mutants at 70 µM concentration and now provide two new figures (Fig. S4 and Fig. S5). Fig. S4 shows that 66 to 85% of monomer are incorporated into fibrils for αSN FL to αSN 125 while almost the entire αSN population has been fibrillated for shorter mutants. Amylofit analysis involves normalizing the ThT fluorescence levels and therefore does not rely on absolute fluorescence values. Normalized data are perfectly adequate for Amylofit analysis provided most of the protein is aggregated. Thus we confirm that the ThT fluorescence time profiles actually reflects fibril formation and that the fibrils are similar in shape to full-length αSN (Fig. S5). To reflect this response, we now add that "SDS-PAGE analyses confirmed that the great majority of αSN had aggregated by the end of the reaction (Fig. S4). (Note that slightly incomplete levels of fibrillation will not affect the outcome of the analysis in Amylofit). TEM demonstrated very similar architecture for the fibrils formed by the different truncation variants (Fig. S5)."

Minor comments:
2) Figure 1 what does each point on the graph represent?
Response: This explanation is now added to the legend of the graph: "Each point represents the half-time of a ThT curve (i.e. the time to reach half the maximal fluorescence value) from a fibrillation experiment carried out at the indicated αSN concentration." 3) Figure 1 -Is aSN121 showing a single linear correlation? This graph looks more similar to aSN110 than the longer constructs.
Response: The reviewer is quite right. As we mention in the text, aSN121 is the first mutant (starting from αSNFL) that starts to deviate from its longer counterparts. This can be seen in double logarithmic plot with a broken linear correlation. Although αSN125 looks similar to shorter mutants in terms of fast aggregation and higher levels of secondary nucleation (fig 2, fig S2 and S3), it is not saturated in our measured concentrations and the double logarithmic plot shows no kink in its linear correlation (Fig. 1).

4) Page 7, line 178are oligomers formed by rounds of freeze-drying physiologically relevant?
Response: We originally checked different conditions to get a better yield of oligomer formation as it is normally very low. Every round of αSN monomer freeze-drying in water leads to further accumulation of oligomers. Even though the reason of higher yield oligomer formation as the result of freeze drying remains unclear (we assume a high energy barrier for oligomer formation that freeze drying helps monomer to cross), this method does not alter oligomer structure as shown by SAXS (fig S6 C). Therefore, we decided to test the formation of oligomer especially for shorter mutants with three times freeze-drying, considering that the formation of αSN oligomer happens in very low yield and varies from batch to batch (1 to 5% of monomer); furthermore shorter mutants lacking C-terminal Tyr residues are not detected so well due to reduced UV absorption. To make this point clear in the manuscript, we added a short description in the oligomer part in result section: "To test the general tendency of forming αSOs, we first optimized oligomerization conditions for αSN-FL by exploring parameters such as NaCl concentration, pH, shaking, multiple rounds of freeze-drying as well as the αSO inducing agent 4-oxononenal (ONE). Among these, multiple rounds of freeze-drying 44 and ONE modification 33 were most effective."

Response:
We have now added the following part to the discussion: "ONE is a product of peroxidation of polyunsaturated fatty acids present in the cell membrane 2 which has been reported to cross-link brain proteins 3 . In particularl, ONE can induce αSN oligomer formation which could be physiologically relevant considering the role of oxidative stress in PD 4,5 . Therefore, we investigated ONE-induced oligomer formation in CTD truncated mutants that do not form oligomers on their own." 8) Page 9, line 216 -Is there a similar mechanism linking calcein release and cell toxicity? This seems a bit of a weak or presumptive link.

Response:
We have now added the following at the beginning of the Results section on calcein assays: "There is evidence that permeabilization of cell membranes by αSOs leads to calcium ion influx and cell death [6][7][8] . As a simple model system to mimic this phenomenon, we determined the ability of these αSO constructs…" 9) Discussion -What are the most abundant truncated species identified in brain tissue? Are there species identified in this work that promote fibrillisation of FL aSN, cell toxicity and calcein release that have also been identified in vivo and may thus be more important to investigate further?
Response: Great point. In the Introduction we add that "Truncations at residues 103, 110, 113, 114, 115, 119, 122, 124, 125, 133, and 135 have been identified in vivo 9 . Those at 119 and 122 at most prevalent and occur at 20-25% abundance relative to full-length αSN." This is followed up by adding at the very end of the Discussion zone: "Several CTD truncated αSNs have been found in neural cells or Lewy bodies 10,11 Our work identifies a potential "danger zone" of particularly deleterious CTD truncations, namely a CTD truncation range from αSN 121 to αSN 125 which shows increased fibrillation compared to αSNFL while retaining the ability to form cytotoxic αSN oligomers on their own. In this context it is striking that the most common in vivo truncated forms of αSN are αSN119 and αSN122 9 . This highlights the deleterious consequences of biological processing of αSN." Response: We have tried to reformulate our thoughts to make it clearer and now provide the following section: "It is remarkable that ONE induces oligomer formation even in shorter constructs which are normally not able to form oligomer. Given that ONE-oligomers and unmodified oligomers have similar size and shape according to SAXS, we conclude that ONE helps otherwise unstable truncated oligomers form, instead of directing αSNs towards a different class of oligomers.
In other words, ONE could be seen as filling in CTD's role in stabilizing the oligomer. The basis for this remains unclear. One speculative possibility is that just as the CTD's anionic tail may reduce the electrostatic repulsion caused by the more cationic N-terminal region, similarly ONE may overcome the N-terminal positive charges either by modifying the Lys side chains or by crosslinking them to other parts of the protein. If inter-molecular interactions between CTD and NTD in the oligomeric state indeed helps form and stabilize the oligomers, then conversely, shortening of the CTD will decrease the frequency and strength of these interactions, leading to extended tails and therefore oligomers with bigger shells. However, there is a limit to the ability of ONE to compensate for truncation and eventually removal of sufficient residues (around αSN115) completely abolishes the ability to form oligomers without help from ONE." 11) Page 12, line 295-303 -Are changes in the charge of the protein or aSOs upon truncation likely to affect the interaction of aSN with the vesicles?
Response: To address this, we add the following to this section: "Only NTD and NAC regions are known to interact directly with vesicles in monomeric aSN 12,13 , but truncation of CTD may have indirect effects by altering the core-shell distribution within the oligomer." We then link this up with a discussion of the oligomer-membrane interactions known from the literature.

12) Supplementary Page 1, line 24 -What ThT concentration was used?
Response: Thank you, this is now indicated. 40 µM ThT was used for all experiments.
In the paper entitled 'The C-terminal tail of α-synuclein protects against aggregate replication but is critical for oligomerization', the authors describe the results of a study aimed at determining effects of C-terminal truncations on the self-assembly properties of alpha-synuclein (aSyn), a protein that forms aggregates in the brains of individuals with Parkinson's disease (PD) and other synucleinopathy disorders. The investigators report that aSyn truncation variants with 121 or fewer residues have a substantially increased propensity to form amyloid-like fibrils, apparently because they more readily undergo secondary fibrillization as a result of enhanced fragmentation. An increased rate of fibrillization is also observed in mixtures of full-length aSyn with as little as 10% This paper is significant in terms of its relevance to molecular mechanisms involved in the pathogenesis of PD and other synucleinopathy disorders. In particular, the results are of a high impact because they yield important insights into structural features of the C-terminal domain of aSyn that modulate two key pathogenic events -the protein's conversion to potentially neurotoxic oligomers and the formation of aSyn amyloid-like fibrils that can spread throughout the brain. The findings reported here are novel and represent an important advance in the aSyn field.
Strengths of the paper in addition to its significance include the fact that it is well written and Response: In the text we now replace total concentration with seed concentration, the only parameter that is varied in the experiment. As the concentration of monomer is constant (50µM), any changes in seed concentration reflect changes in total aSN concentration. Response: for simplicity we mention the γ value -0.5 because theoretically this value indicatesfragmentation or a saturated secondary nucleation. Smaller numbers show a probable saturation in elongation. We have now modified the text to read: "The scaling exponent γ …is comparable to -0.5, the value obtained from theory for a system with fragmentation or saturated secondary nucleation, for all mutants". Response: We thank the reviewer for prompting us to clarify this. We now clarify by adding "turnover rate, i.e. the rate of detachment of formed nuclei from the surface of fibrils and attachment of fresh monomer". 9. p. 7, lines 162-163, 'demonstrates the dominance of secondary processes which is most likely fragmentation'; additional detail should be provided to explain why the authors conclude that the stimulatory effect of agitation on the fibrillization rate reflects enhanced fibril fragmentation rather than turnover.

Response:
We cannot unambiguously conclude that fragmentation is the only secondary process.
Agitation has been shown to promote fragmentation in some systems, whereas its effect on increasing secondary nucleation may be reasonable but is, to our knowledge, not yet backed by experimental evidence in the context of amyloid formation. Thus, we argue in favor of fragmentation. We have now clarified this in the text where we have slightly toned down the emphasis on fragmentation to keep options more open and also emphasize that turnover increase is an option as well (though less likely). 10. p. 7, line 169, '60% aSN110 and higher showed the same behavior as 100% aSN110'; delete 'and higher' because 60% was the highest percentage of aSyn 110 tested in this experiment. Results section that these experiments were carried out with full-length aSyn. Also, the paper would be strengthened by demonstrating that similar results are obtained when repeating these experiments with at least a subset of truncation variants (e.g. aSyn 125 and aSyn 130, to account for the pronounced difference in the secondary rate constants determined for these two variants in terms of a difference in the degree of fragmentation or secondary nucleation).
Response: αSN mutant was specified in the legend. These data are for αSN121; however, similar results were observed for the other mutants that are added as figure S7. This is mentioned in the text now. 20. Fig. 2G: it should be indicated on the graph that the ratios in the legend refer to 'aSyn FL: aSyn 110'.
Response: Duly noted. We added a note to the legend indicating αSN FL:aSN 100. 21. Figs. 3, S6, S7 legends: the number of replicate data points and the method used to assess statistical significance should be indicated.
Response: Duly corrected. Fig. 3 and Fig. S6H legends: the error bars should be defined (e.g. SD or SEM).

22.
Response: Duly corrected. 23. Fig. 3D legend: specify that the number of monomers per oligomer for both kinds of oligomers increases with the extent of truncation.
Response: This is mentioned in the legend. 24. Fig. S2: it would be helpful to show the curve fits for aSyn 135, aSyn 130, and aSyn 125 for the secondary nucleation dominant and fragmentation dominant models separately, and to include MRE values on the plots as was done for aSyn WT. Also, the following statement in the legend, 'the models with saturation fit just as well', should be clarified (e.g. refer to 'the secondary nucleation dominant and fragmentation dominant models with saturation elongation, which produce a better fit with aSyn variants with more extensive C-terminal truncations (Fig. S3)'.
Response: We changed Fig. S2 accordingly and mentioned the saturation models in the legend. 25. Fig. S5C: the caption in the graph is difficult to read and should be enlarged.
Response: Duly corrected. 26. Fig. S5D, legend: the meaning of the asterisks written beside some of the pH values should be explained.
Response: Duly corrected. 29. Fig. S6B: a single label at the top of the graph should be used to indicate that the curves correspond to a set of ONE-treated aSyn oligomers, rather than repeatedly inserting 'ONE' in all of the curve labels below (currently, the curve labels are difficult to see because of the crowded nature of the graph).