Widespread nuclear lamina injuries defeat proteostatic purposes of α-synuclein amyloid inclusions

  1. Shemin Mansuri
  2. Aanchal Jain
  3. Richa Singh
  4. Shivali Rawat
  5. Debodyuti Mondal
  6. Swasti Raychaudhuri

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Abstract

Biogenesis of inclusion bodies (IBs) facilitates protein quality control (PQC). Canonical aggresomes execute degradation of misfolded proteins while non-degradable amyloids sequester into insoluble protein deposits. Lewy bodies (LBs) are filamentous amyloid inclusions of α-synuclein, but PQC benefits and drawbacks associated with LB-like IBs remain underexplored. Here, we report that crosstalk between filamentous LB-like IBs and aggresome-like IBs of α-synuclein (Syn-aggresomes) buffer the load, aggregation state, and turnover of the amyloidogenic protein in mouse primary neurons and HEK293T cells. Filamentous LB-like IBs possess unorthodox PQC capacities of self-quarantining α-synuclein amyloids and being degradable upon receding fresh amyloidogenesis. Syn-aggresomes equilibrate biogenesis of filamentous LB-like IBs by facilitating spontaneous degradation of α-synuclein and conditional turnover of disintegrated α-synuclein amyloids. Thus, both types of IB primarily contribute to PQC. Incidentally, the overgrown perinuclear LB-like IBs become degenerative once these are misidentified by BICD2, a cargo-adapter for the cytosolic motor-protein dynein. Microscopy indicates that microtubules surrounding the perinuclear filamentous inclusions are also distorted, misbalancing the cytoskeleton-nucleoskeleton tension leading to widespread lamina injuries. Together, nucleocytoplasmic mixing, DNA damage, and deregulated transcription of stress chaperones defeat the proteostatic purposes of the filamentous amyloids of α-synuclein.

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  1. Version published to 10.1242/jcs.261935
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    Reply to the reviewers

    We thank the reviewers for thorough reading and for providing useful suggestions to improve our manuscript. We find two major issues indicated by the reviewers.

    1. Lack of pathophysiological relevance to attract a broader readership – to address, we have stained brain slices of PD patient’s with p129-Syn and Lamin B1 antibodies. Microscopy images show extensive lamina damages in the patient brain slices which contain p129-Syn positive inclusions. These images are now included in the current revision of the manuscript as __ 6C-D__. We think that these results in the pathologically relevant systems will now establish a connection between lamina defects with neurodegeneration in PD and will be attractive for a broader audience.

    Experimental issues as indicated by major and minor points – majority of the points have been addressed in the current revision attached herewith. Given opportunity to submit a full revision, we shall incorporate more experiments to address all the points in the final revised manuscript.

    Point by point response to reviewer’s concerns:

    Reviewer 1

    R1: The work by Mansuri and collaborators reports that LB-like filamentous inclusions of α-Synuclein are able to associate with and perturb the nuclear lamina due to an unbalanced mechanical tension between cytoskeleton and nucleoskeleton. Consequently, lamina-injuries are proposed as a major driver of proteostasis sensitivity in cells with LB-like Syn-IBs.

    It is a complex work, in which a range of different cellular, biochemical and molecular techniques have been used. Readers of the paper (including the undersigned) will be wondering if a similar behaviour occurs in pathological systems, such as iPSC derived dopaminergic neurons arising from patients carrying the synuclein pathological mutations reported in this work.

    Response: We thank the reviewer for bringing out the lack of pathophysiological relevance in our manuscript. To address, we imaged post-mortem thalamus sections of a Parkinson’s Disease (PD) patient (BioChain Institute Inc., USA Cat# T2236079Par) and a control (BioChain Institute Inc., USA Cat#T2234079). Our experiments clearly show extensive lamina deformities in the patient brain (Fig. 6C-D) and connects with neurodegeneration in a pathological system.

    Major points

    R1: Authors should explain why there is a so high amount of p129-Syn in unseeded neurons (Fig. 1Ai, Fig. S1Bi): "p129-Syn was distributed throughout the neuron cell body and projections including light staining in the nucleus", as its accumulation is typical of PD-like α-syn aggregates. Similarly, unseeded neurons labeled with p129-Syn in Fig. 1Ai, Fig. 1Bi, and Fig. S1Bi and Fig. S1Ci are very different each other. Why? As neurons are unseeded, the pathological signature of PD-like α-syn aggregates should be very low or absent in all cases.

    Response: We agree with the reviewer that very low amount of p129-Syn should be present in unseeded neurons. We standardized microscopy parameters using fields that contained neurons with both large LB-like perinuclear IBs and smaller peripheral Syn-filaments. We used Leica SP8 confocal microscope. Argon laser power was kept constant at 30% of full potential while Smart Gain was titrated to visualize the smaller filaments. For example, the smaller filaments were not clearly visible in Annexure Figure 1Ai when Smart gain was 690V. Smaller filaments were prominent when the Smart Gain was increased to 848V (Annexure Figure 1Aii, included with the revision plan attached herewith). We also observed light intra-nuclear staining of p129-Syn at 848V Smart Gain when we zoomed the arrow indicated nucleus in Fig. 1Aii shown below as Annexure Figure 1Aiii. Accordingly, we used Smart Gain: 650-850V in all the images presented in the manuscript. Brightness and contrast are now adjusted for all the images prepared for the revised manuscript for the optimum view of the immunostaining. All the raw image files will be submitted to https://www.ebi.ac.uk/biostudies in due course.

    In order to rule out imaging artefacts at the higher Smart Gain (650V – 850V), we performed a control experiment without adding primary antibody against p129-Syn during immunostaining. Secondary antibodies were added and the Smart Gain was ~950-1000V during imaging. The light staining of p129-Syn as visible in Fig. 1Ai and 1Bi in the revised manuscript were not visible in this experiment (Annexure Figure 1B).

    A table indicating the Smart Gain for all the images is included in the revised manuscript as__ Methods Table S5 - Laser Intensity.__

    Reviewer 1 has also pointed out the difference in staining of p129-Syn in Fig. 1Ai and Fig. 1Bi. For Fig.1Ai, Rabbit monoclonal (p129-Syn (MJF-R13 (8-8), epitope: phosphoserine 129, cat# ab168381), and for Fig. 1Bi Mouse monoclonal (P-syn/81A, epitope: phosphoserine 129, cat# ab184674) were used. This information is now included in the figure legends. The difference in the staining pattern is due to the use of the different primary and secondary antibodies.

    Lastly, we want to emphasize that the staining pattern seen in unseeded neurons () are not the typical PD-like Syn-aggregates but the soluble p129-Syn that is yet to be incorporated into the amyloid-filaments. p129-Syn ((antibody MJF-R13 (8-8)) staining pattern in 1Ai is continuous in the projections and light dotted in the periphery and inside nucleus. These dots also accumulate on the Microtubule Organizing Centre (MTOC) indicating the presence of aggresome-like inclusion bodies in the neurons. The staining pattern in 1Bi (antibody P-syn/81A) is dotted throughout. In both the cases, the continuous or dotted staining were not observed after seeding. The continuous staining at the projections seen in 1Ai is broken into smaller filaments in 1Aii (indicated by arrowheads). The broken filaments are much more increased in number and length in Fig 1Bii and the staining-intensity prominently increased. Accumulation of multiple larger filaments into perinuclear LBs is typical PD-like (Fig. 1Bii, yellow arrowhead).

    The continuous staining and the broken staining patterns at the projections are also visible in the zoomed out MIP images presented in S1Bi and ii, respectively. The increase in fluorescence intensity of p129-Syn staining is prominent between S1Ci and ii indicating accumulation of p129-Syn in the form of large amyloid filaments in seeded neurons.

    We now discuss the staining patterns in the revised manuscript. Please see pages 4-7.

    R1: Authors should try to perform a more accurate quantification of the various colocalizations reported along the manuscript, i.e. by reporting the Pearson correlation coefficient or the Mander's overlap coefficient.

    Response:As suggested by the reviewers, Pearson’s co-localization coefficient values have been added separately for all figured showing co-localization in Supplementary note: Colocalization figures and table.

    Minor points

    R1: In Fig. S1B the red fluorescent signal arising from γ-tubulin staining is not visible in the merged picture.

    Response: Fig. S1B are the zoomed out MIP images of Fig. 1A. γ-Tubulin stains centrosome as tiny dots at the perinucleus in one of the z-sections of the MIP. To visualize these tiny dots in the MIP images, we have 1) optimized the brightness contrast of the MIP images and 2) provided a separate channel for γ-tubulin (arrowheads). These corrections are included in the revised version.

    R1: Page 6: results of Fig. S1D-E should be explained properly (CALNEXIND and CMX-Ros staining).

    Response:As suggested, we revised this part in Page 7.

    R1: Fig. 2A: the indication of SNCA in western blotting is not proper, as in this experiment you evaluated the protein level, so it is better to report "α-syn";

    Response:We agree with the reviewer. SNCA in western blots has been changed to α-Syn all the figures and figure legends.

    R1: Fig. S2B: there is great variability in the number of SNCA(A53T)- EGFP and SNCA(DM)-EGFP cells with IBs during the course of PFF-incubation, so that authors did not reveal any significant difference. I think it is not completely correct to emphasize this data at page 9, lanes 12-13;

    Response:We agree with the reviewer that the difference in number of SNCA(A53T)-EGFP and SNCA(DM)-EGFP cells with IBs was not statistically significant. Yet, we always observed aggressive biogenesis LB-like IBs in SNCA(DM)-EGFP cells. The statement in the manuscript is now corrected as per the reviewer’s suggestion (Page 9).

    __R1:__Did authors reveal any cytotoxicity upon Congo Red treatment at the indicated concentrations (Fig. S2G)?

    Response: Previously, Congo Red incubation was found to be non-toxic for neuronal cells even at 350 µM (PMID: 7991613). We have now performed MTT assay after Congo red treatment in our cells. The graph is now included as S2H. We did not observe any difference in cell viability even after treating the cells with the highest dose (100 µM) used in the experiment.

    R1: I have concerns about the percentages reported in Fig. S2G: the percentage of cells with filaments in the absence of Congo Red is apparently too low as compared to the previously reported percentages.

    Response:The reviewer is right. Number of Syn-filament containing cells varies between experiments because of ‘age’ of the recombinant amyloid seeds, different batches of seed preparation etc. We are repeating this experiment to increase the biological N. Results will be included and discussed in the final revised version

    R1: Fig. S2G: I also believe that authors should report representative images of cells treated with Congo Red, in which Syn-filament biogenesis is prevented;

    Response:As instructed by reviewer, the images are included in Fig. S2G.

    R1: Fig. 2Eiii: The stick arrowhead seems to indicate a separate blob that is not so red: authors should consider to show separated channels and not only the merged picture (as in Fig. S3).

    Response:We agree with the reviewer that the blob is not so red. We could not accommodate the separate channels in the main figure because of space constraint. Therefore, we presented the separate channels in Fig. S3A. Now we are including the stick arrowhead also at Fig. S3A.

    R1:Page 10: authors should explain why they performed the LC3 staining;

    Response:Previous reports indicated association of LC3B with α-Synuclein inclusions in neurons (PMID: 21412173, 31375560). Therefore, we also stained our cells with LC3 antibody. The references are now incorporated in Page 10.

    R1: Why in Fig.2i, SNCA(DM) the ubiquitin signal is pink and not red?

    Response:The blue of the DAPI is slightly overlapping with the ubiquitin staining at the aggresomes as these bodies are perinuclear making it appear pink. Separate channels are provided in Fig. S3E.

    R1: Fig. 3, western blotting: as I previously reported, I think it would be better to write "total α-syn" instead of SNCA. Fig. 3D: is should be useful to explain properly the content of the soluble and insoluble fractions.

    Response:We agree with the reviewer. SNCA in western blots has been changed to α-Syn all the figures and figure legends.

    R1: Explain in the legend of Fig. 4 what is h2b tdTOMATO

    Response:We thank the reviewer for pointing out the lack of information. This is now included with a reference in the revised manuscript.

    Significance

    R1: Overall this is interesting to read, a lot of data are presented, demonstrating a new potential phenomena that would be important to a specialized audience in the field of synuclein misfolding, aggregation and cellular toxicity.

    Response: We have now included immunofluorescence images of post-mortem thalamus sections of a Parkinson’s Disease (PD) patient (BioChain Institute Inc., USA Cat# T2236079Par) and a control (BioChain Institute Inc., USA Cat#T2234079). Our experiments clearly show lamina deformities in patient brain (Fig. 6D). We think that these experiments will highlight the pathophysiological relevance of the manuscript to make it appropriate for a wider audience.

    Reviewer 2

    __R2:__The present paper titled "Nuclear-injuries by aberrant dynein-forces defeat proteostatic purposes of Lewy Body-like Inclusions" provides an in details and compelling study about the formation of aggregates of SNCA in presence of PFFs, which other proteins play a role in the formation of this inclusions, and which pathways are the major players. They study provides many well-done experiments to highlight the composition and the process formation of these aggregates. unfortunately I think the study is lacking in connecting these events with neurodegeneration. how do all the pathways study impact viability and functionality of neurons and other disease relevant cells like astrocytes and microglia? it is thus a work which mainly focuses on the pathways leading to the formation of inclusions leaving untouched the question of how this might impact the disease. This does not take away the value of the findings but it should be taken in consideration when deciding which journal to submit.

    Response:We thank the reviewer for the encouraging words and also for bringing out the lack of pathophysiological relevance in our manuscript. To address, we have performed immunofluorescence experiments with post-mortem thalamus sections of a Parkinson’s Disease (PD) patient (BioChain Institute Inc., USA Cat# T2236079Par) and a control (BioChain Institute Inc., USA Cat#T2234079). Our results show extensive lamina deformities in patient brain (Fig. 6C-D) connecting neurodegeneration in PD with lamina injuries.

    Further, although we found that LB-containing primary neurons and Hek293T cells do not show any loss in cell viability as estimated by LDH and MTT assays respectively (Fig 4A-B), they show sensitivity to additional stresses. LB-like IB containing Hek293T cells were unable to trigger stress response pathways and were vulnerable to heat stress. These results were already included in the earlier version of the manuscript (Fig. 4H-I). We now estimated sensitivity of neurons in presence of additional stress. We have subjected LB-containing neurons and control neurons to heat stress and estimated induction of Hsp chaperones by western blot and quantitative mass spectrometry. Preliminary results (included herewith) indicate that Hsp-upregulation is defective in neurons with LB-like IBs. These results are now included as Figure 4J-M in the attached revised manuscript. Repeat experiments with quantitative mass spectrometry will be included in the final revision.

    R2: I have a few suggestion for each figure which will not take much time, energies or expenses but that would overall make the paper easier to read and digest.

    R2::Fig 1: quantification of aggregates dimension, number and colocalization score with p62 (Pearson)

    Response:Co-localization score with p62 is included in the current revision (Supplementary note: Colocalization figures and table). Quantification of aggregate dimension, number etc. in neurons have been already documented by Mahul-Mellier et al. (PMID: 32075919). We are following the same protocol and therefore did not repeat the counting for neurons. However, if the reviewer thinks that its mandatory, we shall do that and include with full revision.

    __R2:__Fig 2: aesthetic comment: the way to read the figure should be consistent throughout the figure. they should be assembled either all in vertical or all in horizontal.

    Response:We tried. We find the current organization is the best fit to accommodate all panels.

    R2: Fig 3: 3E better to put an image without nocodazole to visualize the difference

    Response:The control image is now added in Fig. 3E.

    R2: 3D probe WB also for SNCA

    Response:Sorry for the confusion. The western blots in 3D are probed for both total Synuclein and p129-Syn. As suggested by the first reviewer, we have also changed SNCA to α-Syn which indicates the total Synuclein protein level.

    R2: 3K this WB needs quantification to backup the statement made

    Response:We are repeating this experiment. Results will be included and discussed in the final revised version.

    R2: 3I check the - and + for PFF and doxy. I believe they are wrong

    Response:We have rearranged the figure. The scheme in Fig. 3I (now Fig. 3H) is correct but we have made it simpler to avoid confusion.

    R2: Fig 4: missing IF of peri nuclear IBs with HS

    Response:The images are now included as Fig. S4E and discussed in page 19.

    R2: Fig 5: quantification of H2BTdTom exit from the nucleus

    Response:We have performed this experiment as a supporting evidence of the nuclear damage in presence of LB-like IBs. We have quantified the damages in Fig. 5A and D. We have also performed quantitative mass spectrometry to show nuclear entry of associated organelle proteins (Fig. S5G). We think, quantifying the H2BTdTom exit will not be a significant value addition to the manuscript.

    R2: Fig 6: some neurons with large PFF seems very unhealthy. is it possible to quantify neuronal viability may not with MTT which is not suited for single cells analysis?

    Response:The reviewer correctly pointed out that neurons with large LB-like IBs seemed unhealthy which was confirmed by ƴH2AX staining indicative of extensive DNA damage in Fig. 6B.

    R2: maybe it would be nice to have a WB with soluble and insoluble SNCA and p129 with ciliobrevin D with and without PFF. Ciliobrevin D might also impact degradative systems as demonstrated by the EHNA compound (PMCID: PMC5584856).

    Response:We have performed the dynein experiments to figure out the role of cytoskeleton-nucleoskeleton tension in the lamina injuries in LB-like inclusion containing cells. However, we think that the reviewer has correctly pointed out that dynein may have a direct role in degrading Synuclein by either autophagy or proteasome. Given the results of the suggested experiments are not going to change the final conclusion of the manuscript, we propose to limit ourselves in discussing this possibility and citing the paper in the current revised version of the manuscript (page 29).

    Significance

    R2: As already stated above, the experiments are correctly performed and the evidence are well-presented and demonstrated. the realm that this paper falls into is not though neuroscience. The aim of this paper is to study the formation of inclusions regardless of their impact on disease-relevant cell type functions. the presented experiments are numerous and even though the message is pretty clear some figure might be too crowded to correctly convey the message (see fig 3). some of these findings even tough with much less details were already suggested by other papers (PMCID: PMC5584856) in which the importance of the dynein was studied in the context of the communication between autophagy and proteasome. I think adding this angle with few experiments might add a little bit more relevance but it is also true that this paper has already a lot of data.

    Response:Thank you very much for the encouraging comments

    R2: the type of audience for this paper I think is a very specialized audience which is interested in molecular mechanisms of inclusions formation and protein-protein interaction. as a final statement the paper is beautifully done and is relevant but it lacks the translational angle.

    Response:We again thank the reviewer for reminding the lack of pathophysiological relevance. We have now included microscopic images of brain slices of PD patients with extensive lamina defects (Fig. 6D) and think this will attract a broader audience.

    R2: my field of expertise is neuroscience. I have expertise in bimolecular techniques as well as cellular techniques to study neurodegenerative diseases

  6. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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    Referee #2

    Evidence, reproducibility and clarity

    The present paper titled "Nuclear-injuries by aberrant dynein-forces defeat proteostatic purposes of Lewy Body-like Inclusions" provides an in details and compelling study about the formation of aggregates of SNCA in presence of PFFs, which other proteins play a role in the formation of this inclusions, and which pathways are the major players. They study provides many well-done experiments to highlight the composition and the process formation of these aggregates. unfortunately I think the study is lacking in connecting these events with neurodegeneration. how do all the pathways study impact viability and functionality of neurons and other disease relevant cells like astrocytes and microglia? it is thus a work which mainly focuses on the pathways leading to the formation of inclusions leaving untouched the question of how this might impact the disease. This does not take away the value of the findings but it should be taken in consideration when deciding which journal to submit.

    I have a few suggestion for each figure which will not take much time, energies or expenses but that would overall make the paper easier to read and digest.

    Fig 1: quantification of aggregates dimension, number and colocalization score with p62 (Pearson)

    Fig 2: aesthetic comment: the way to read the figure should be consistent throughout the figure. they should be assembled either all in vertical or all in horizontal.

    Fig 3: 3E better to put an image without nocodazole to visualize the difference 3D probe WB also for SNCA 3K this WB needs quantification to backup the statement made 3I check the - and + for PFF and doxy. I believe they are wrong

    Fig 4:missing IF of peri nuclear IBs with HS

    Fig 5: quantification of H2BTdTom exit from the nucleus

    Fig 6: some neurons with large PFF seems very unhealthy. is it possible to quantify neuronal viability may not with MTT which is not suited for single cells analysis

    Fig 7: maybe it would be nice to have a WB with soluble and insoluble SNCA and p129 with ciliobrevin D with and without PFF. Ciliobrevin D might also impact degradative systems as demonstrated by the EHNA compound (PMCID: PMC5584856).

    Significance

    As already stated above, the experiments are correctly performed and the evidence are well-presented and demonstrated. the realm that this paper falls into is not though neuroscience. The aim of this paper is to study the formation of inclusions regardless of their impact on disease-relevant cell type functions. the presented experiments are numerous and even though the message is pretty clear some figure might be too crowded to correctly convey the message (see fig 3). some of these findings even tough with much less details were already suggested by other papers (PMCID: PMC5584856) in which the importance of the dynein was studied in the context of the communication between autophagy and proteasome. I think adding this angle with few experiments might add a little bit more relevance but it is also true that this paper has already a lot of data.

    the type of audience for this paper I think is a very specialized audience which is interested in molecular mechanisms of inclusions formation and protein-protein interaction. as a final statement the paper is beautifully done and is relevant but it lacks the translational angle.

    my field of expertise is neuroscience. I have expertise in bimolecular techniques as well as cellular techniques to study neurodegenerative diseases

  7. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    The work by Mansuri and collaborators reports that LB-like filamentous inclusions of α-Synuclein are able to associate with and perturb the nuclear lamina due to an unbalanced mechanical tension between cytoskeleton and nucleoskeleton. Consequently, lamina-injuries are proposed as a major driver of proteostasis sensitivity in cells with LB-like Syn-IBs. It is a complex work, in which a range of different cellular, biochemical and molecular techniques have been used. Readers of the paper (including the undersigned) will be wondering if a similar behaviour occurs in pathological systems, such as iPSC derived dopaminergic neurons arising from patients carrying the synuclein pathological mutations reported in thins work.

    There are some concerns that should be addressed by the authors.

    Major points

    • Authors should explain why there is a so high amount of p129-Syn in unseeded neurons (Fig. 1Ai, Fig. S1Bi): "p129-Syn was distributed throughout the neuron cell body and projections including light staining in the nucleus", as its accumulation is typical of PD-like α-syn aggregates. Similarly, unseeded neurons labelled with p129-Syn in Fig. 1Ai, Fig. 1Bi, and Fig. S1Bi and Fig. S1Ci are very different each other. Why? As neurons are unseeded, the pathological signature of PD-like α-syn aggregates should be very low or absent in all cases.
    • Authors should try to perform a more accurate quantification of the various colocalizations reported along the manuscript, i.e. by reporting the Pearson correlation coefficient or the Mander's overlap coefficient. Minor points
    • In Fig. S1B the red fluorescent signal arising from γ-tubulin staining is not visible in the merged picture.
    • Page 6: results of Fig. S1D-E should be explained properly (CALNEXIND and CMX-Ros staining).
    • Fig. 2A: the indication of SNCA in western blotting is not proper, as in this experiment you evaluated the protein level, so it is better to report "α-syn";
    • Fig. S2B: there is a great variability in the number of SNCA(A53T)- EGFP and SNCA(DM)-EGFP cells with IBs during the course of PFF-incubation, so that authors did not reveal any significant difference. I think it is not completely correct to emphasize this data at page 9, lanes 12-13;
    • Did authors reveal any cytotoxicity upon Congo Red treatment at the indicated concentrations (Fig. S2G)?
    • I have concerns about the percentages reported in Fig. S2G: the percentage of cells with filaments in the absence of Congo Red is apparently too low as compared to the previously reported percentages.
    • Fig. S2G: I also believe that authors should report representative images of cells treated with Congo Red, in which Syn-filament biogenesis is prevented;
    • Fig. 2Eiii: The stick arrowhead seems to indicate a separate blob that is not so red: authors should consider to show separated channels and not only the merged picture (as in Fig. S3).
    • Page 10: authors should explain why they performed the LC3 staining;
    • Why in Fig.2i, SNCA(DM) the ubiquitin signal is pink and not red?
    • Fig. 3, western blotting: as I previously reported, I think it would be better to write "total α-syn" instead of SNCA.
    • Fig. 3D: is should be useful to explain properly the content of the soluble and insoluble fractions.
    • Explain in the legend of Fig. 4 what is h2b tdTOMATO.

    Significance

    Overall this is interesting to read, a lot of data are presented, demonstrating a new potential phenomena that would be important to a specialized audience in the field of synuclein misfolding, aggregation and cellular toxicity.

  8. Version published to 10.1101/2021.10.29.466393 on bioRxiv