Exogenous Amyloid Sequences: Their Role in Amyloid-Beta Heterotypic Aggregation

Summary/Background:

Amyloid-β (Aβ) peptides aggregation is related to the formation of amyloid plaques in the brain of individuals with Alzheimer’s Disease (AD)¹. Aβ peptides arise from sequential cleavage of the amyloid precursor protein (APP) by β- and γ-secretases through the amyloidogenic pathway^2,3. These peptides exist in several isoforms, mainly Aβ40 and Aβ42, which differ in their length and aggregation properties. While Aβ40 is the most abundant form, Aβ42 contains two additional hydrophobic residues at the C-terminus, making it more prone to self-aggregation and neurotoxicity^4,5. The increased Aβ42/Aβ40 ratio promotes the formation of toxic oligomers and amyloid plaques, key pathological hallmarks of Alzheimer’s disease^6,7.

Aβ peptides are present in equilibrium between the central nervous system (CNS) and the circulation^8,9. Disruption of the blood–brain barrier can lead to Aβ accumulation within the CNS, promoting its aggregation and contributing to the onset of AD^10,11. Aβ aggregation may occur through interactions between different peptide isoforms or between Aβ peptides and other molecules, such as RNA or α-synuclein^12–14. Growing evidence has revealed interactions between CNS molecules and the microbiota^15,16. In this context, a previous work from the authors of the preprint highlighted here demonstrated that Aβ peptides engage with microbiota-derived molecules, indicating that microbial products have the potential to shape the aggregation pathway¹⁷. In the present work, the authors sought to determine how amyloid peptides derived from bacteria potentially present in the intestinal microbiota modulate Aβ42 aggregation and propagation, and how they affect cross-seeding events with Aβ40-derived seeds.

Significance/Why I think this preprint is important:

This study provides clear evidence that amyloid-like peptides from bacteria, including those that may be present in the gut microbiota, can influence multiple steps of Aβ aggregation relevant to Alzheimer’s disease. By revealing that these exogenous peptides can enhance or suppress Aβ42 aggregation and affect both direct and cross-seeding with Aβ40, the work highlights the multiple mechanisms by which microbial molecules can influence Aβ behavior. Importantly, the finding that peptides such as RI5 and P9 generate Aβ40 aggregates that delay Aβ42 fibril formation points to inhibitory effects that could be explored as therapeutic strategies. These results suggest that specific molecular features of microbial peptides can modulate amyloid growth in both harmful and potentially protective ways. Overall, the study broadens our understanding of how gut-derived molecules may shape Aβ dynamics and underscores their potential as targets for future interventions.

Key findings:

Exogenous peptides derived from bacteria seed Aβ42 aggregation

The authors selected ten bacterial prion-like peptides, each 21 amino acids in length, which had been previously reported to interact with Aβ40 peptides, to serve as exogenous seeds¹⁷. ThT fluorescence measurements were used to follow Aβ42 aggregation in the presence or absence of the exogenous seeds (preprint Figure 2). Each seed produced a distinct kinetic signature, generally accelerating fibril formation relative to Aβ42 alone. These data imply that gut microbiota–derived circulating molecules may seed Aβ42 aggregation.

Aβ42 aggregates formed with exogenous peptides accelerate further aggregation

Further experiments were carried out to assess the seeding capacity of Aβ42 aggregates formed either alone or in the presence of the exogenous amyloid peptides, using Aβ42 peptides as substrates (preprint Figure 3). Aggregation reactions showed a similarly accelerated lag phase when Aβ42 aggregates generated in the presence of exogenous peptides were used as seeds, as well as when Aβ42-alone aggregates were applied. However, the aggregation profiles varied depending on the specific exogenous peptide: for most of them, the secondary nucleation phase was further accelerated compared to reactions seeded with Aβ42 alone. These findings suggest that the fibrils formed in the presence of exogenous peptides have different properties and can affect several steps of the host protein aggregation process. This means that the exogenous peptides may act early in the aggregation pathway or later, making the overall aggregation process faster.

Exogenous peptides modulate Aβ cross-seeding and aggregation kinetics

In addition, the authors tested whether Aβ40 aggregates produced alone or in the presence of the exogenous peptides could cross-seed and accelerate Aβ42 aggregation (preprint Figure 4). Among the exogenous peptides tested, only HP1 accelerated the lag phase relative to pure Aβ40 aggregates. In contrast, Aβ40 aggregates generated with RI5 or P9 exerted an inhibitory effect on Aβ42 aggregation, leading to a slower aggregation profile. Importantly, HP1, which originates from the H. pylori cytotoxin, has been linked to Alzheimer’s disease and was reported in the authors’ previous study to significantly compromise cognitive function in C. elegans.

Exogenous amyloid sequences modulate Aβ seeding pathways

The findings presented in this preprint demonstrate that exogenous amyloid sequences can influence both direct seeding and cross-seeding events between Aβ isoforms. These effects vary among the different molecules (figure 5), indicating that their specific molecular properties shape both the magnitude and the direction of their influence on the aggregation process. Such insights may contribute to the development of future therapeutic strategies.

Comments/Questions:

Major comments:

1. While the authors suggest that exogenous molecules can influence later stages of aggregation, even after they are no longer present (page 8, 2^nd paragraph), this point could be clarified further. It is not entirely clear from the data how this conclusion was reached. Providing additional explanations or referencing specific experimental results would strengthen this interpretation.
2. The authors present interesting data using different Aβ isoforms, which aggregate differently depending on the exogenous amyloid peptide. It could be very helpful to include the amino acid sequences of both isoforms, as done for the exogenous peptides. This would make it easier for readers who are less familiar with Aβ sequences and structures to follow the results. For example, when discussing the importance of residues 41 and 42 during the lag phase of aggregation (page 10, 1^st paragraph), having the sequences visible would provide helpful context and enhance understanding.
3. It may be helpful for the authors to clarify how the exogenous peptides interact with Aβ. Do the resulting aggregates consist solely of Aβ, or do they contain a mixture of Aβ and the exogenous peptides?
4. Given that gut-derived molecules are physically distant from the CNS, where might such interactions realistically occur in vivo? It would be valuable to discuss on whether, and in which physiological context, Aβ could plausibly encounter gut-derived peptides such as HP1.

Minor comments:

1. Including page numbers throughout the document is recommended.
2. In page 2, paragraph 3, line 6, replace “withing” with “within”.
3. When mentioning the group’s previous work using elegans (page 3), it is not immediately clear to the reader that this study was conducted by the authors themselves. Reorganizing this part of the text to make the connection more explicit would help readers better follow the continuity of the research.
4. On page 11, the authors mention that preprint Figure 5 includes a column showing statistical significance; however, the p-values are not clear. It might be helpful to either add p-value information in the figure legend or adjust the text so that it accurately reflects the figure.
5. The authors’ previous work cited in this preprint is listed as another preprint. However, I noticed that the data has already been published. It might be helpful to update the reference to the published version, which could provide readers with a completer and more accessible source.

 

References

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Language refinement was supported using ChatGPT (OpenAI), which assisted in improving clarity and readability.

Tags: aβ aggregation, aβ peptide, microbiota

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