Differential Expression of Endogenous Retroviruses and Inflammatory Mediators in Female and Male Offspring in a Mouse Model of Maternal Immune Activation
, Anna Maria Tartaglione 2,†, Martina Giudice 1, Erica D’Avorio 1, Vita Petrone 1, Nicola Toschi 3,4, Flavia Chiarotti 2, Martino Tony Miele 1, Gemma Calamandrei 2, Enrico Garaci 5,6, Claudia Matteucci 1, Paola Sinibaldi-Vallebona 1,7, Laura Ricceri 2,‡ and Emanuela Balestrieri 1,*,‡Round 1
Reviewer 1 Report
The paper starts with an in-depth review of current literature regarding environmental risk factors for neuropsychiatric conditions, focusing mostly on the correlation between the expression of Endogenous Retroviruses (ERVs) and the severity of certain conditions, as well as suggested underlying mechanisms.
The main goal in the paper is to analyze multiple parameters (genetic, inflammatory etc.) in both male and female mice as related to maternal immune activation following exposure to polyinosinic:polycytidylic acid (Poly I:C).
In the results section, the tables have very low resolution.
The authors found a significant effect for prenatal exposure to Poly I:C on ERVs, related genes and inflammatory effectors. Interestingly, these findings seem to have been specific to the prefrontal cortex and the hippocampus, as similar findings were not observed in the blood. The authors mention the slow cell turnover in the brain as a probable explanation for these findings.
The authors then go on to discuss the importance and relevance of specific findings, such as expression levels of Syn-B, ING3, transposable elements and others. In addition, they discuss similarities between current findings and previous ones, in which a different model has been used (e.g. valproic acid).
Another discussion takes place regarding the correlation between ERVs and interferons: the authors discuss current findings and compare them to previous ones, while suggesting plausible hypotheses for the observed interactions and their effect on the development/progression of CNS diseases.
Finally, the authors discuss sex differences found in the study: females presented with a significantly higher levels of ERVs, related genes and inflammatory effectors in the prefrontal cortex. An interesting discussion about the meaning of these findings, along with comparison to previous findings, takes place.
The paper is well written, comprehensive and sound.
Author Response
We thank the reviewer for the careful revision and for the comments. We will upload a new version of the tables at better resolution.
Reviewer 2 Report
The paper by Cipriani and coworkers is a continuation of their previous work on retrotransposon and related gene expression in autism animal models. Previously, they studied this question in the VPA and BTRB autism models, and the current work is with mice born to polyIC treated mothers. The work is a detailed analysis of transcriptional profiles of select transposon elements and related genes, as well as inflammatory effectors by real time RT PCR in prefrontal cortex, hippocampus and blood. The expression of many of these genes are upregulated, as expected, although no specific cell types can be identified by the approach used. Also, there is no information of whether the level of inflammatory cytokines are actually increased at the protein level. The main finding is that prenatal exposure upregulates the expression of transposon genes, both in the PFC and hippocampus, while blood levels are unchanged. They previously reported that expression of these genes in blood cells (as well as in brain) is altered during early development, suggesting that the continuous dysregulation of these genes in brain could be due to the slower turnover of cells in the brain, which is certainly the case with neurons.
Presentation of the data is straightforward in column graphs. It is not clear if the PCA analysis adds anything significant to the results and it is overtly complicated. It might be useful to compare the individual PCAs across tissues, but there is no metric to show if there is a significant difference.
Overall, the work is descriptive with little insight into the underlying mechanism. Limitations include the candidate gene approach and the use of bulk brain tissue, while unbiased transcriptomic profiling, some at the single cell level, is quite common.
Additional comments:
1. Line1 elements are technically not ERVs
2. It would be useful to discuss possible mechanisms underlying the co-expression of genes in the PFC and HPC.
3. Why and how ING3 is downregulated, while most of the genes are upregulated?
4. Could it be that upregulation of MORC is due to compensation?
Author Response
We thank the reviewer for the careful revision and for the comments that helped us to improve the manuscript.
Presentation of the data is straightforward in column graphs.
We represented our data as boxplots to clearly describe the real range of data distribution (first/third interquartile range with median value) also in line with non-parametric statistical analyses performed on the same data. We added these details in figure legends.
It is not clear if the PCA analysis adds anything significant to the results and it is overtly complicated. It might be useful to compare the individual PCAs across tissues, but there is no metric to show if there is a significant difference.
Overall, the work is descriptive with little insight into the underlying mechanism. Limitations include the candidate gene approach and the use of bulk brain tissue, while unbiased transcriptomic profiling, some at the single cell level, is quite common.
We thank the reviewer for raising this point. However, we had specified in the discussion (now highlighted on page 11), relevance of our PCA results.
We believe that PCA results strengthen conventional non parametric results and provide an indication that ERV expression is differently regulated in blood and brain. We agree with the reviewer that future investigations, including single cell -omic approaches could give a more detailed picture of the altered expression profile.
We added this limitation in the conclusion section (highlighted paragraph page 16).
Additional comments:
- Line1 elements are technically not ERVs
We thank the reviewer, we modified the text accordingly
- It would be useful to discuss possible mechanisms underlying the co-expression of genes in the PFC and HPC.
We thank the reviewer for raising this point, we added a paragraph in the discussion mentioning the involvement of microglia activation as one of the mediators of MIA effects in these brain regions (now highlighted on page 10).
- Why and how ING3 is downregulated, while most of the genes are upregulated?
We thank the reviewer, we better clarified this point in the discussion section (page 10)
- Could it be that upregulation of MORC is due to compensation?
We thank the reviewer and as stated in the discussion, it is plausible to speculate that despite its role as as epigenetic silencer of transposable elements, its high expression could be related to the fact that it may not be involved as a “gatekeeper” of ERV expression in the central nervous system (page 10).
Author Response File: 
Author Response.pdf
