PREreview of Horizontal and vertical transmission of transgenerational memories via theCer1transposon

We, the students of MICI5029/5049, a Graduate Level Molecular Pathogenesis Journal Club at Dalhousie University in Halifax, NS, Canada, hereby submit a review of the following BioRxiv preprint:

Horizontal and vertical transmission of transgenerational memories via the Cer1 transposon. Rebecca S. Moore, Rachel Kaletsky, Chen Lesnik, Vanessa Cota, Edith Blackman, Lance R. Parsons, Zemer Gitai, Coleen T. Murphy. bioRxiv 2020.12.28.424563; doi: https://doi.org/10.1101/2020.12.28.424563

We will adhere to the Universal Principled (UP) Review guidelines proposed in:

Universal Principled Review: A Community-Driven Method to Improve Peer Review. Krummel M, Blish C, Kuhns M, Cadwell K, Oberst A, Goldrath A, Ansel KM, Chi H, O'Connell R, Wherry EJ, Pepper M; Future Immunology Consortium. Cell. 2019 Dec 12;179(7):1441-1445. doi: 10.1016/j.cell.2019.11.029.

SUMMARY:  C. elegans can learn to avoid pathogenic P. aeruginosa by processing bacterial small RNAs in intestinal epithelial cells, transmitting this genetic information to neurons to achieve avoidance behavior and germ cells to vertically transmit avoidance behavior to ~4 future generations. Here, Dr. Coleen Murphy’s team demonstrate that this genetic information is encapsulated by capsids encoded by the Cer1 retrotransposon. In addition to vertical transmission, Cer1 capsids are also required for horizontal transmission to naïve animals. Thus, C. elegans has usurped a retrotransposon to enable transgenerational immune memory.

OVERALL ASSESSMENT: 

STRENGTHS:  The preprint is clearly and concisely written, and the data is clearly presented. It addresses a fascinating topic of broad interest. In general, the experimental approaches are sound and the author’s conclusions are well-supported by the data. 

WEAKNESSES:  The manuscript could be further strengthened by conducting biochemical/genetic analysis of VLPs (which is likely to be quite technically challenging, as discussed by the authors). The authors should also consider placing these findings in the context of recent studies from Maria-Carla Saleh’s lab on vertical transmission of immune memory in flies, as there could be some relevant parallels between these two models. 

DETAILED U.P. ASSESSMENT:

OBJECTIVE CRITERIA (QUALITY)

1.  Quality: Experiments (1–3 scale) SCORE = 1.5

●      Figure by figure, do experiments, as performed, have the proper controls?

[Note: in this section, the class discusses proper controls, but also uses the ‘figure-by-figure' opportunity to discuss rationale and approaches]

o   Figure 1: We didn’t find any issues with the controls, although there was some discussion about the rationale for using the F2 generation as substrate for horizontal transfer experiments. Why F2 and not one (or a few) of the other generations? Is it a pragmatic choice, to avoid longer or larger experiments that might test multiple generations? 

▪       The students noted the difference in aversion behavior between progeny that are lawn trained vs P11 RNA trained (compare Fig 1 B and 1C). Could the authors comment on these differences? It would have been nice to have seen F2 aversion from lawn-trained worms, similar to the experiment shown in Fig 1C using P11. 

▪       In Figure 1K the authors tested if the worms could also learn to avoid P. florescens and S. marcescens, but the rationale for the choice of these bacteria specifically was unclear. We know that C. elegans avoids P. aeruginosabecause the bacteria can cause disease; would the worm have a reason to avoid P florescens and S marcescens if they do not cause disease? 

▪       The authors could provide more detailed methodology about the Choice Index assay. 

o   Figure 2: There was some discussion about the use of electron microscopy to identify VLPs. It is likely that Fraction 6 is quite heterogeneous, and additional particles beyond VLPs may be present, including non-VLP particles that might be expected to stain with an anti-Cer1 antibody. Could the authors address this possibility or cover it in the Discussion?

o   Figure 3: There was no issue with controls, but the class thought that the logical leap from Figure 2 to focus specifically on Cer1 was not well explained. 

o   Figure 4: OK

o   Figure 5: Panel A is confusing without either a visual schematic of when RNAi is administered or a better explanation within the figure legend.

o   Figure 6: OK

o   Figure 7: OK (but this figure prompted a discussion about matricide/cannibalism and potential for horizontal transfer of immune memory in nature).

●      Are specific analyses performed using methods that are consistent with answering the specific question?

o   The experimental approaches were generally sound.

●      Is there the appropriate technical expertise in the collection and analysis of data presented?

o   Appropriate technical expertise was demonstrated. 

●      Do analyses use the best-possible (most unambiguous) available methods quantified via appropriate statistical comparisons?

o   There is no mention of what statistical tests were performed to support the data, figure by figure. The methods mentions that Prism8 was used for statistics but does not say what tests were performed. This should be addressed.

●      Are controls or experimental foundations consistent with established findings in the field? A review that raises concerns regarding inconsistency with widely reproduced observations should list at least two examples in the literature of such results. Addressing this question may occasionally require a supplemental figure that, for example, re-graphs multi-axis data from the primary figure using established axes or gating strategies to demonstrate how results in this paper line up with established understandings. It should not be necessary to defend exactly why these may be different from established truths, although doing so may increase the impact of the study and discussion of discrepancies is an important aspect of scholarship.

o   The experimental foundations of this study are generally solid. The authors cited the proper literature and presented strong data that was sufficient to support their conclusions.  

2. Quality: Completeness (1–3 scale) SCORE = 1

●      Does the collection of experiments and associated analysis of data support the proposed title- and abstract-level conclusions? Typically, the major (title- or abstract-level) conclusions are expected to be supported by at least two experimental systems.

o   Generally, the data supports the conclusions as stated in the title and abstract. 

o   The class had some reservations about the evidence provided for a functional role for Cer1 VLPs specifically rather than other types of extracellular vesicles that might also contain Cer1 antigen. 

●      Are there experiments or analyses that have not been performed but if ‘‘true’’ would disprove the conclusion (sometimes considered a fatal flaw in the study)? In some cases, a reviewer may propose an alternative conclusion and abstract that is clearly defensible with the experiments as presented, and one solution to ‘‘completeness’’ here should always be to temper an abstract or remove a conclusion and to discuss this alternative in the discussion section.

o   N/A

3. Quality: Reproducibility (1–3 scale) SCORE = 1

●      Figure by figure, were experiments repeated per a standard of 3 repeats or 5 mice per cohort, etc.?

o   Sufficient numbers of animals and biological and technical replicates were performed to provide a high-confidence dataset. 

●      Is there sufficient raw data presented to assess rigor of the analysis?

o   Yes

Are methods for experimentation and analysis adequately outlined to permit reproducibility?

o   The methods are generally clearly described (as judged by this group of non-experts). The authors may consider providing clearer descriptions of statistical tests, as mentioned above. 

●      If a ‘‘discovery’’ dataset is used, has a ‘‘validation’’ cohort been assessed and/or has the issue of false discovery been addressed?

o   N/A

4. Quality: Scholarship (1–4 scale but generally not the basis for acceptance or rejection) SCORE = 1.5

●       Has the author cited and discussed the merits of the relevant data that would argue against their conclusion?

o    Yes

●       Has the author cited and/or discussed the important works that are consistent with their conclusion and that a reader should be especially familiar when considering the work?

o    The authors may consider discussing this recent paper, which describes mechanisms of RNA-mediated vertical transfer of immune memory in Drosophila melanogaster and Aedes egyptii. 

▪        Mondotte JA, Gausson V, Frangeul L, Suzuki Y, Vazeille M, Mongelli V, Blanc H, Failloux AB, Saleh MC. Evidence For Long-Lasting Transgenerational Antiviral Immunity in Insects. Cell Rep. 2020 Dec 15;33(11):108506. doi: 10.1016/j.celrep.2020.108506. PMID: 33326778; PMCID: PMC7758158.

o    No one in the class has first-hand experience with C. elegans research, so we would have benefitted from additional information about the frequency and circumstances of matricide/cannibalism in C. elegans or other roundworms. This would provide helpful context about opportunities for horizontal transfer of immune memory between worms in nature.

●       Specific (helpful) comments on grammar, diction, paper structure, or data presentation (e.g., change a graph style or color scheme) go in this section, but scores in this area should not to be significant bases for decisions.

o    The writing was clear and concise. The lean writing style did not hinder reader comprehension. We really liked the first sentence of the abstract, which puts the study in much broader context and ‘hooks’ the reader. ‘Animals face both external and internal dangers: pathogens threaten from the environment, and unstable genomic elements threaten from within.”

o    Consistency in Choice Index y-axis labelling throughout would be helpful. 

o    Clear figure labeling to indicate that OP50 is E. coli would be helpful (there was some confusion about that)

o    In Figure 1J, the students were confused about the ‘N2’ label. 

o    In Figure 7, the students suggested that consistency in labeling either ‘Hawaiian’ or ‘CB4856’.

MORE SUBJECTIVE CRITERIA (IMPACT)

1.Impact: Novelty/Fundamental and Broad Interest (1–4 scale) SCORE = 1

●      A score here should be accompanied by a statement delineating the most interesting and/or important conceptual finding(s), as they stand right now with the current scope of the paper. A ‘‘1’’ would be expected to be understood for the importance by a layperson but would also be of top interest (have lasting impact) on the field.

o    The key finding relates to the Cer1 capsids serving as the vehicle for both vertical and horizontal transfer of immune memory. The concept of the helpful capsids could be understood by a layperson because they are conceptually simple (i.e. a pathogen-derived ‘box’ that has been usurped by the host and now helps to transmit immune memories).

●      How big of an advance would you consider the findings to be if fully supported but not extended? It would be appropriate to cite literature to provide context for evaluating the advance. However, great care must be taken to avoid exaggerating what is known comparing these findings to the current dogma (see Box 2). Citations (figure by figure) are essential here.

o   There was broad agreement that this manuscript represents a considerable advance without further extension.

o   The authors draw a parallel between their system and the Arc retrotransposon of flies that supports the plasticity of the neuromuscular junction. This prompted additional discussions about other relevant models like the mechanisms of RNA-mediated vertical transfer of immune memory in Drosophila melanogaster and Aedes egyptii, as described above.

o   If ‘helpful’ retrotransposons involved in immunity are subsequently discovered in other eukaryotes, these studies should be considered foundational.

o   There was some discussion about the proposed RNA-seq experiment on VLPs. The class acknowledged the technical challenges of generating sufficient VLP material for an RNA-seq experiment, but some students thought that this experiment may be required to satisfy peer reviewers. 

2.Impact: Extensibility (1–4 or N/A scale) SCORE = N/A

●  Has an initial result (e.g., of a paradigm in a cell line) been extended to be shown (or implicated) to be important in a bigger scheme (e.g., in animals or in a human cohort)?

●  This criterion is only valuable as a scoring parameter if it is present, indicated by the N/A option if it simply doesn’t apply. The extent to which this is necessary for a result to be considered of value is important. It should be explicitly discussed by a reviewer why it would be required. What work (scope and expected time) and/or discussion would improve this score, and what would this improvement add to the conclusions of the study? Care should be taken to avoid casually suggesting experiments of great cost (e.g., ‘‘repeat a mouse-based experiment in humans’’) and difficulty that merely confirm but do not extend (see Bad Behaviors, Box 2).

o    N/A

o    Even though this is clearly an ‘N/A’ situation, the class discussed the paper in the context of the Drosophila Arc retrotransposon, and remains intrigued about whether retrotransposons may perform similar functions in other eukaryotes.