Brain-derived and in vitro-seeded alpha-synuclein fibrils exhibit distinct biophysical profiles

Reviewer #1 (Public Review):

SUMMARY:

Parkinson's disease (PD) and other synucleinopathies, including Parkinson's Disease Dementia (PDD), Dementia with Lewy Bodies (DLB), and Multiple System Atrophy (MSA), pose significant challenges for early diagnosis, as their clinical manifestations often emerge after substantial neurodegeneration has occurred. In this context, the Alpha-Synuclein Seeding Amplification Assay (SAA) has garnered considerable attention for its potential as a diagnostic tool, capable of detecting pathological forms of alpha-synuclein (αSyn) even before the onset of classical clinical symptoms and signs. The assay exploits αSyn's intrinsic property to convert healthy forms into pathological ones, subsequently amplifying these pathological forms for visualization. This study aims to investigate the efficacy of SAA in accurately identifying subtypes of synucleinopathies, including PD, PDD, DLB, and MSA. To achieve this, the results from the patient brain-derived αSyn SAA are compared with those obtained through conformational stability assays, immunolabeling, and electron microscopy. Study shows that brain-derived αSyn fibrils exhibit significant differences across various synucleinopathies in their conformation, biochemical profile and phosphorylation patterns. Importantly, the SAA method appears to fall short in capturing these distinctions.

The study's findings are highly relevant given the rapidly advancing landscape of utilizing the SAA for the diagnosis and differentiation of various forms of PD and synucleinopathies using patient biofluids. It is somewhat surprising that the authors primarily characterize SAA as a research tool without delving into its potential as a biomarker detection assay, especially in the context of the field's excitement about its diagnostic applications. Additionally, a missed opportunity lies in not referencing a recent study that employed SAA successfully to diagnose PD and subtype the condition using a vast sample size. To further strengthen the results, the inclusion of healthy control brains in the biochemical and immunostaining/immunoblot experiments would provide more robust comparisons. Overall, the authors have conducted their experiments diligently, and their study offers valuable insights that align with the ongoing efforts to enhance early diagnosis and subtype differentiation in the domain of synucleinopathies.

STRENGTH:

The strengths of this research article are indeed notable and contribute to the credibility and significance of the study:

Important Research Question: The study addresses a crucial question in the field of neurodegenerative diseases by evaluating the effectiveness of the αSyn SAA in diagnosing and differentiating synucleinopathies. This question is of significant clinical and scientific interest.
Comprehensive Introduction: The article provides a thorough and well-structured introduction to the topic with an illustration, setting the stage for the research. It ensures that readers, including those unfamiliar with the subject matter, can grasp the context and significance of the study.
Use of Patient Brain Tissue: The use of patient-derived brain tissue samples from various synucleinopathies, including PD, PDD, DLB, and MSA, enhances the clinical relevance and applicability of the findings.
Replication and Statistical Significance: Conducting the experiments six times for each sample demonstrates the rigor of the study and the robustness of the results, and increases the confidence in the conclusions drawn.
Clarity in Experimental Results and Discussion: The authors have presented the experimental results in a clear and understandable manner. I was personally impressed by images showing twisted and straight conformations of αSyn, as well as immunogold labeling for phosphorylation of αSyn, which aids in conveying the findings effectively to the readers. The results clearly show distinct differences in the characteristics of αSyn fibrils across different synucleinopathies. It also highlights the more aggressive seeding capacities and higher biochemical stability of αSyn in PDD and DLB patients, offering valuable insights into the pathophysiology of these conditions. The authors also clearly show that SAA fails in differentiating the disease types within the synucleinopathies.
Clinical relevance: The study underscores the importance of considering complementary diagnostic methods alongside SAA for a more comprehensive understanding of synucleinopathy subtypes. The study might also play an important role in potential FDA approval of SAA as a diagnostic tool for synucleinopathies, especially for PD.
These strengths collectively make the study a valuable contribution to the field of neurodegenerative diseases, shedding light on the limitations and potential applications of SAA in the diagnosis and differentiation of various synucleinopathies.

WEAKNESS:

While this study is overall robust, there are several aspects that could further enhance the quality and interpretation of the findings.

Clinical Data on Patient Brain Samples: The inclusion of specific details such as post-mortem intervals and the age at disease onset for patient brain samples would be valuable. These factors could significantly affect the quality of the tissues and their relevance to the study. Moreover, given the large variation in disease duration between PD and PDD, it's important to consider disease duration as a potential confounding factor, especially when concluding that PDD patients have a more severe form of synucleinopathy compared to PD.
Inclusion of Healthy Controls in Multiple Tests: Given the importance of healthy controls in scientific studies, especially those involving human brain samples, the authors could consider using healthy controls in more tests to strengthen the robustness of the findings. Expanding the use of healthy controls in biochemical profiling and phosphorylation profiles would provide a better basis for comparison and clarify the significance of results in a disease context.
This will help the authors to elaborate on the interpretation of results, for example, in Figure 3, where the authors claim that PD brains show mostly monomeric αSyn forms (line 119 and 120, and also in 222 and 223). Whether it implies the absence of alpha-syn pathology in PD brains? If there are differences from healthy controls? What are these low molecular weight bands (<15kD) (line 125-126) and whether they are also present in healthy controls? Also, we do not have a perfect pS129-specific (anti-p𝛼Syn) antibody. They are known for non-specific labeling. Investigating the phosphorylation levels in healthy controls and comparing them to PD brains, especially considering the predominance of monomeric (healthy αSyn?) in PD brains, would help clarify the observed changes.
Age of Healthy Controls: Providing information about the age at death for healthy controls is crucial, as age can impact the accumulation of αSyn. Also include if the brain samples were age-matched, or analyses were age-adjusted.
Braak Staging Discrepancy: The study reports the same Braak staging for both PD and PDD, despite the significant difference in disease duration. Maybe other reviewers with clinical experience might have a better take on this. This observation merits discussion in the paper, allowing readers to better understand the implications of this finding.
Citation of Relevant Studies: The paper should consider citing and discussing a recent celebrated study on PD biomarkers that used thousands of cerebrospinal fluid (CSF) samples from different PD patient cohorts to demonstrate the effectiveness of SAA as a biochemical assay for diagnosing PD and its subtypes (https://doi.org/10.1016/S1474-4422(23)00109-6).
In summary, these suggestions aim to enhance the study's quality and the clarity of its findings, ultimately contributing to a more comprehensive understanding of synucleinopathies and the diagnostic potential of SAA.

Reviewer #2 (Public Review):

Most neurodegenerative diseases are characterized by the self-templated misfolding of a particular protein in a manner that enables progressive spread throughout the central nervous system. In diseases including Parkinson's disease (PD) and multiple system atrophy (MSA), the protein alpha-synuclein misfolds into unique shapes, or strains, which use this self-replicating mechanism to encode disease-specific information. Previous research suggests that a major contributor to the lack of successful clinical trials across neurodegenerative diseases is the lack of disease-relevant strains used in preclinical testing. While MSA patient samples are known to replicate efficiently in cell and mouse models of disease, Lewy body disease (LBD) patient samples do not. To overcome this obstacle, the seeding amplification assay (SAA) uses recombinant alpha-synuclein to amplify the misfolded protein structure present in a human patient sample. The resulting fibrils are then widely used by many laboratories as a model of PD. In this manuscript, Lee et al., set out to compare the strain properties of alpha-synuclein fibrils isolated from LBD and MSA patient samples with the resulting amplified fibrils following SAA. Using orthogonal biochemical and structural approaches to strengthen their analyses, the authors report that the SAA-amplified fibrils do not recapitulate the disease-relevant strains present in the patient samples. Moreover, their data suggest that regardless of which strain is used to seed the SAA reaction, the same strain is generated. These results clearly demonstrate that the SAA-amplified material is not disease-relevant. SAA fibrils are broadly used in academic and pharmaceutical laboratories. They are used in ongoing drug discovery efforts and recombinant fibrils broadly inform much of what is known about alpha-synuclein strain biology in LBD patients. The implications of the reported work are, therefore, expansive. These findings add to the growing ledger of reasons that the use of SAA fibrils in research should be halted until improved methods for amplification with high fidelity are developed.

Reviewer #3 (Public Review):

Summary:

This interesting manuscript presents a comparison of biophysical properties, TEM appearances, and phosphorylation patterns of brain-derived synuclein fibrils from 3 subjects each with Parkinson Disease (PD), Parkinson Disease with Dementia (PDD), Dementia with Lewy bodies (DLB) and Multiple System Atrophy (MSA), the effects of studying these brain-derived fibrils in a Seeding Aggregation Assay (SAA), and a comparison of the seeded and resultant fibers. The results are not unexpected.

Strengths:

The work explores an important question. Namely, what is the fidelity of synuclein fibrils produced during an SAA reaction to the starting material if that material has been extracted from the brains of deceased patients with synucleinopathies.

Weaknesses:

The work suffers from several methodological flaws

The experiments are missing two important controls. 1) what to fibrils generated by different in vitro fibril preparations made from recombinant synclein protein look like; and 2) the use of CSF from the same patients whose brain tissue was used to assess whether CSF and brain seeds look and behave identically. The latter is perhaps the most important question of all - namely how representative are CSF seeds of what is going on in patients' brains?

In their discussion the authors do not comment on the obvious differences in the conditions leading to the formation of seeds in the brain and in the artificial conditions of the seeding assay. Why should the two sets of conditions be expected to yield similar morphologies, especially since the extracted fibrils are subjected to harsh conditions for solubilization and re-suspension.

Finally, the key experiment was not performed - would the resultant seeds from SAA preparations from the different nosological entities produce different pathologies when injected into animal brains? But perhaps this is the subject of a future manuscript.

Furthermore, the authors comment on phosphorylation patterns, stating that the resultant seeds are less heavy phosphorylated than the original material. Again, this should not be surprising, since the SAA assay conditions are not known to contain the enzymes necessary to phosphorylate synuclein. The discussion of PTMs is limited to pS-129 phosphorylation. What about other PTMs? How does the pattern of PTMs affect the seeding pattern.

Lastly, the manuscript contains no data on how the diagnostic categories were assigned at autopsy. This information should be included in the supplementary material.