Investigation of All Disease-Relevant Lysine Acetylation Sites in α-Synuclein Enabled by Non-canonical Amino Acid Mutagenesis

Reviewer #1 (Public review):

Summary:

This paper describes experiments with alpha-synuclein (aS) with acetylated lysines (acK) at various positions. Their findings on how to use non-canonical amino acid (ncAA) mutagenesis to generate aS with acetylated lysines are valuable. The paper then continues with a range of experiments to characterise the acetylated alpha-synuclein constructs at different positions, with the aim of providing insights into which sites are relevant to disease or their function inside cells. The paper concludes these experiments with the suggestion that inhibiting the Zn2+-dependent histone deacetylase HDAC8 to potentially increase acetylation at lysine 80 may have therapeutic benefit. However, the relevance of most of these experiments is unclear, mainly as the filaments that form from these constructs are different from those observed in human disease (but see below for more details). Moreover, using the recombinantly produced acetylated versions of alpha-synuclein to normalise mass-spectrometry data, the authors themselves report that acetylation of alpha-synuclein does not differ between individuals with Parkinson's disease or healthy controls.

Strengths:

The authors report difficulties with chemical synthesis, and then decide to make these constructs using non-canonical amino acid (ncAA) mutagenesis, which seems to work reasonably well (yields vary somewhat). In the Conclusion section, the authors report that they used these recombinant proteins to obtain quantitative insights into the levels of acetylation of lysines in individuals with PD versus healthy controls, for which they find no significant differences. This part of the work is valuable.

Weaknesses:

The authors then use circular dichroism to show that aSyn with acK at position 43 has less alpha-helical content. From this result, they deduce that "only this site could potentially perturb aS function in neurotransmitter trafficking", but no experiments on neurotransmitter trafficking were performed.

Subsequently, they measure the aggregation speed of the variants in seeded aggregation experiments with preformed fibrils (PFFs) from WT aSyn, and conclude that acK at positions 12, 43, and 80 yields slower aggregation. They reach similar conclusions when measuring seeded aggregation in primary cultures. As far as I understand it, the seeding experiments in cells use seeds that are assembled from partially acetylated alpha-synuclein, but that are made of non-acetylated wildtype alpha-synuclein, and the alpha-synuclein that is endogenous in the cells is also non-acetylated (or at least not beyond what happens in these cells at endogenous levels). It is therefore unclear how the cellular seeding experiments relate to the in vitro aggregation assays with (partially) acetylated substrates. Anyway, both aggregation experiments ignore that the structures of aSyn filaments in Parkinson's disease (PD) or multiple system atrophy (MSA) are different from those formed in these experiments, and that, therefore, the observed aggregation kinetics are likely irrelevant for the speed with which disease-relevant filaments form in the brain.

NMR and FCS experiments show that acK at positions 12 and 43 may reduce binding to vesicles, which then leaves only acK80.

Finally, the authors describe the cryo-EM structure of mixtures of acK80:WT aSyn filaments, which are predominantly made of WT aSyn, with a previously described structure. Filaments made of only acK80 aSyn have a modified arrangement of this structure, where the now neutral side chain of residue 80 packs inside a hydrophobic pocket. The authors discuss differences between the acK80 structures and those of other structures from in vitro assembled aSyn filaments, none of which are the same as those observed from PD or MSA brains, nor are any attempts made to transfer observations from the in vitro experiments to the structures of disease. The relevance of the cryo-EM structures for human disease, therefore, remains unclear.

The Conclusion on p.20 mentions an interesting and valuable result: the authors used the acetylated recombinant proteins to determine the extent of acetylation within human protein samples by quantitative liquid chromatography MS (SI, Figures S41-S49). Their conclusion is that "The level of acetylation was variable - no clear trend was observed between healthy control and patients - nor between patients of different diseases (SI, Table S4, Supplementary Data 1)" This result implies that acetylation of aS is not directly related to its pathogenicity, which again adds doubts on the disease-relevance of the results described in the rest of the paper.

Reviewer #2 (Public review):

Summary:

Shimogawa et al. studied the effect of lysine acetylation at different sites in the alpha-synuclein (aS) sequence on the protein-membrane affinity, seeding capacity in the test tube and in cells, and on the structure of fibrils, using a range of biophysical methods. They use non-canonical amino acid (ncAA) mutagenesis to prepare aS lysine acetylated variant at different sites.

Strengths:

The major strength of this paper is the approach used for the production of site-specific lysine acetylated variants of aS using ncAA mutagenesis, as well as the combination of a range of biophysical methods together with cellular assays and structure biology to decipher the effect of lysine acetylation on aS-membrane binding, seeding propensity, and fibril structure. This approach allowed the author to find that lysine acetylation at positions 12, 43, and 80 led to lower seeding capacity of aS in the test tube and in cells, but only acetylation at lysine 80 did not affect aS-membrane interaction. These results suggest that lysine acetylation at position 80 may be protective against aggregation without perturbing the proposed functional role of aS in synaptic plasticity.

Weaknesses:

SDS is not a good membrane model to investigate the effect of lysine acetylation on aS membrane-binding because it is a harsh detergent and solubilizes membranes. Negatively charged vesicles or vesicles made of a mixture of lipids mimicking the lipid composition of synaptic vesicles are more accepted in the field to study aS-membrane interactions. The authors used such vesicles for the FCS experiments, and they could be used for the initial screening of the 12 lysine acetylated variants of aS.

It would help the reader to have the experimental details (e.g., buffer, protein/lipid concentrations) for the different assays written in the figure legend.

The authors use an assay consisting of mixing 10% fibrils + 90% monomer to investigate the effect of lysine acetylation on aS. However, the assay only probes fibril elongation and/or secondary processes. The current wording can be misleading, and the term aggregation could be replaced by seeding capacity for clarity. For example, the authors state that lysine acetylation at sites 12, 43, and 80 each inhibits aggregation, but this statement is not supported by the data. Instead, the data show that the acetylation at these sites slows down the fibril elongation and thus decreases the seeding capacity of aS fibrils. In order to state that lysine acetylation has an effect on aS aggregation, fibril formation, the author should use an assay where the de novo formation of fibrils is assessed, such as in the presence of lipid vesicles or under shaking conditions.

It is not clear from the EM data that the structures of the different lysine acetylated variants are different, unlike what is stated in the text.

Reviewer #3 (Public review):

Shimogawa et al. describe the generation of acetylated aSyn variants by genetic code expansion to elucidate effects on vesicle binding, aggregation, and seeding effects. The authors compared a semi-synthetic approach to obtain acetylated aSyn variants with genetic code expansion and concluded that the latter was more efficient in generating all 12 variants studied here, despite the low yields for some of them. Selected acetylated variants were used in advanced NMR, FCS, and cryo-EM experiments to elucidate structural and functional changes caused by acetylation of aSyn. Finally, site-specific differences in deacetylation by HDAC 8 were identified.

The study is of high scientific quality, andthe results are convincingly supported by the experimental data provided. The challenges the authors report regarding semi-synthetic access to aSyn are somewhat surprising, as this protein has been made by a variety of different semi-synthesis strategies in satisfactory yields and without similar problems being reported.

The role of PTMs such as acetylation in neurodegenerative diseases is of high relevance for the field, and a particular strength of this study is the use of authentic acetylated aSyn instead of acetylation-mimicking mutations. The finding that certain lysine acetylations can slow down aggregation even when present only at 10-25% of total aSyn is exciting and bears some potential for diagnostics and therapeutic intervention.

Author response:

We thank you for your efforts in reviewing our manuscript. We sincerely appreciate that the reviewers were all enthusiastic about our comparison of native chemical ligation (NCL) and non-canonical amino acid (ncAA) mutagenesis methods for installing acetyl lysine (AcK) in alpha-synuclein, as well as the wide variety of biochemical experiments enabled by our ncAA approach. We respond to the critiques specific to each reviewer here.

Reviewer #1:

Expressed concern that in vitro studies of effects on membrane binding were not followed up with neurotransmitter trafficking experiments. While we certainly think that such studies would be interesting, they would presumably require the use of acetylation mimic mutants (Lys-to-Gln mutations), which we would want to validate by comparison to our semi-synthetic proteins with authentic AcK. Such experiments are planned for a follow-up manuscript, and we will investigate the reviewer’s suggested experiment at that time.

Reviewer #1 Noted that the method of in vitro seeding really reports on the impact of acetylation on the elongation phase of aggregation. We will clarify this in our revisions. They also expressed concern that this was different than the role that acetylation would play in seeding cellular aggregation with pre-acetylated fibrils. We will also acknowledge and clarify this in our revisions. Having the monomer population acetylated in cells presents technical challenges that might also be addressed with Gln mutant mimics, and we plan to pursue such experiments in the follow-up manuscript noted above.

Reviewer #1 Criticized the fact that the pre-formed fibrils used in seeding would not have the same polymorph as PD or MSA fibrils derived from patient material. They were also critical of how our cryo-EM structure of AcK80 fibrils related to the PD and MSA polymorphs. Finally, while the reviewer liked the MS experiments used to quantify acetylation levels from patient samples, they felt that our findings then threw the physiological relevance of our structural and biochemical experiments into question. We believe that all of these critiques can be addressed by clarifying our purpose. We are not necessarily trying to claim that our AcK80 fold is populated in health or disease, but that by driving Lys80 acetylation, one could push fibrils to adopt this conformation, which is less aggregation-prone. A similar argument has been made in investigations of alpha-synuclein glycosylation and phosphorylation. Our results in Figure 9 imply that this could be done with HDAC8 inhibition. We will revise the manuscript to make these ideas clearer, while being sure to acknowledge the limitations noted by Reviewer #1.

Reviewer #2:

Expressed concern over our use of SDS micelles for initial investigation of the 12 AcK variants, rather than the phospholipid vesicles used in later FCS and NMR experiments. We will note this shortcoming in revisions of our manuscript, but we do not believe that using vesicles instead would change the conclusions of these experiments (that only AcK43 produces an effect, and a modest one at that).

We will add additional detail to the figure captions, as requested by Reviewer #2.

Reviewer #2 shared some of the concerns of Reviewer #1 regarding the distinctions of which phase of aggregation we were investigating in our in vitro experiments. As noted above, we will clarify this language.

Finally, Reviewer #2 stated that “It is not clear from the EM data that the structures of the different lysine acetylated variants are different.” We feel that it is quite clear from structures in Figure 8 and the EM density maps in Figure S38 that the AcK80 fold is indeed different. Although the overall polymorphs are somewhat similar to WT, the position of K80 clearly changes upon acetylation, altering the local fold significantly and the global fold more moderately.

Reviewer #3:

Found the results convincing, including the potential therapeutic implications. The only concern noted was that they found the difficulties in semi-synthesis of AcK-modified alpha-synuclein surprising given that it has been made many times before through NCL. Indeed, our own laboratory has made alpha-synuclein through NCL, and the yields reported here are in keeping with our own previous results. However, since NCL did not give higher yields than ncAA methods, and it is significantly easier to scan AcK positions using ncAAs, we felt that ncAAs are the method of choice in this case. We will clarify this position in the revised manuscript.

In conclusion, on behalf of all authors, I again thank the reviewers for both their positive and negative observations in helping us to improve our manuscript. We will revise it to strive for greater clarity as we have noted in this letter.