Peer review of: Impaired peroxisomal import in Drosophila oenocytes causes cardiac dysfunction by (2020)

Reviewers' comments:
Reviewer #1 (Remarks to the Author):
Huang and colleagues present compelling and interesting data exploring the effects of Oenocyte- mediated inflammation in the age-related decline of cardiac function in Drosophila. The authors show that increased oxidative stress in aging oenocytes results in the JNK/Kay-mediated expression of Upd3 and subsequent activation of JAK/STAT signaling in cardiomyocytes, and that this non- autonomous signaling mechanism promotes cardiac arrhythmias. The authors further establish that the cause for the elevated ROS in aging oenocytes is the impairment of peroxisomal import, and, accordingly, show that factors that promote peroxisomal import in oenocytes can rescue age-related cardiac arrhythmias.
The work is expansive and logically presented, the data are overall of good quality and properly interpreted, and the manuscript is well - written and should be of interest to a broad audience. The topic addressed, namely how aging causes systemic inflammation and how dysfunction in specific tissues promotes dysfunction in others is of particular interest to the aging community and the presented data make a significant contribution to our understanding of the loss of homeostasis of the systemic milieu. It is likely that these findings can also be translated to vertebrate/human cases of cardiac dysfunction.
I have a few minor comments that the authors should address before the paper is published:
- The authors use STAT nuclear localization as a readout for STAT activity, but the images are not very clear, and it seems difficult to clearly establish nuclear localization of STAT in oenocytes. There are very good STAT reporters (10XSTAT-GFP and 2XSTAT-GFP) for flies, and the authors should use these to validate their results. Alternatively, one could also use qPCR to establish expression of SOCS36E, a target gene for STAT in flies.
- Figs. 3 H and I are not informative and should be taken out of the manuscript. If STAT activation in cardiomyocytes by oenocyte-expressed Upd3 can be clearly established (see above), then this experiment is unnecessary.
- A critical control for many of the presented data is to show that the PromE and PromEGS drivers are indeed excluded from cardiomycocytes. The authors should show convincing evidence (images or western blots) showing that the drivers are only expressed in oenocytes, not the heart.
Reviewer #2 (Remarks to the Author):
Summary: The submitted article, “Impaired peroxisomal import in Drosophila hepatocyte-like cells induces cardiac dysfunction through the pro-inflammatory cytokine Upd3” by Huang et al, uses the Drosophila genetic model to examine the possibility that liver-like oenocytes impact cardiac function in the fly. They convincingly show that aging induced cardiac arrhythmia and paraquat induced ROS stress can be blocked by oenocyte-specific over expression of SOD. They identify age-dependent increases in UPD3 expression in oenocytes and show that the effects on UPD3 are mediated by signaling through the JNK pathway. The authors show UPD3 from oenocytes is secreted into the hemolymph and is taken up by the heart. They also show that UPD3 induces JAK-STAT signaling in the heart. Consistent with their observations, oenocyte-specific KD of UPD3 prevented both the age- dependent increase in cardiac expression of STAT and cardiac arrhythmias. Their data supports the notion that it is a decrease in peroxisome import via PEX5 and not impaired mitochondrial function in oenocytes that is the cause of the increased UPD3 signaling. These findings are novel and extremely interesting as they suggest that non-cardiac peroxisomes play important non-autonomous roles in heart function via IL6/UPD3-mediated inter-organ communication.
The authors provide a significant amount of good data to support these conclusions. The data are overall compelling but there are some controls that are missing and see comments below.
Major Points-
1. The RU-based Gene induction experiments are good but missing key controls, as feeding with RU by itself has some cardiac effects. The driver lines should be outcrossed to flies from the RNAi or OE genetic backgrounds and treated with and without RU to control for the effect of drug treatment and driver genetic background. Stock centers have the background insertion lines that should be used for these controls, (eg. the appropriate control for Fig. 5f would be a cross between the RNAi background line and the PromE GS line with the progeny being fed +/- RU).
2. For the experiments to identify the mediators of Pex5 – mediated PIS, the authors indicate that Kay is involved but did not test Jra, the other part of the AP-1 complex, why not? The case would be stronger if Jra KD had the same effect as Kay KD.
3. Lines 210-11 : Interesting, free GFP proteins were also found in the hemolymph, which may be due to a cleavage of the C-terminus of Upd3 occurring after its secretion. Is UPD3 normally cleaved in the C terminus? What is the basis for this statement?
4. Fig. 3h - It is unclear how much hemolymph was loaded onto the gels. Were hemolymph extractions pooled or was the entire 0.5ul extracted from a single fly loaded into a single well? Data should be quantified and normalized to a loading control. What is the “Non-specific” band?
Minor Points-
1. For all figures, please clarify the genotypes of the “CNTRL” flies are (see also comment above).
2. Document contains typos and errors in English language usage that should be corrected.
2. Fig. 1 a - statistical tests used on these data should be a one-way ANOVA (3 genotypes).
3. Fig. 1 e,f&h - statistical tests used on these data should be a two-way ANOVA (2 genotypes and 2 conditions). Typos in legend for (f) and (g).
4. Fig. 2 a – It seems that the Venn diagram is from ref 13. This should be stated in the legend as well as ages of flies and some detail of the paraquat treatment from that study (how long, how much).
5. Fig. 2b – t-test is not appropriate; there are multiple genotypes and two treatments. 6. Fig. 2c – t-test not appropriate (3 genotypes and 2 treatments)
7. Fig. 2e – graph not consistent with others.
8. Fig. 2f – t-test not appropriate (3 genotypes and 2 treatments)
9. Fig. 2 I – what is the age of the flies being tested?
10. Fig. 3b – need to explain how “near cardiomyocyte nucleus” was determined. 11. Fig. 3e – t-test not appropriate (3 genotypes and 2 treatments)
12. Fig. 4h – what do the dashed lines represent?
13. Fig. 4i – one way ANOVA is needed
14. A control line has been included for Fig. 5 f & h but it is not clear to this reviewer what the genotype(s) is (are). If it is the oenocyte specific KD of Pex 5 by itself that is not a control per se (see Major point 1).
Reviewer #3 (Remarks to the Author):
The link between altered ROS homeostasis/peroxisomal function in hepatocyte-like oenocytes and cardiac aging is studied in a Drosophila model. In general, this is an extensive study that is well conducted with many knockdown/rescue setups. It is also appreciated that the authors sought for mechanistic insights underlying the increased expression of the inflammatory messenger Upd3 in oenocytes. The insights generated are novel and illustrate that peroxisome dysfunction in one tissue may affect distant tissues through the release of cytokines.
However, there are some discordances and shortcomings with regard to the primary cause of Upd3 upregulation in oenocytes and the ‘exclusivity‘ of Upd3, which need to be resolved.
From Fig2b it is clear that knockdown of at least 8 different genes can decrease PQ induced arrythmicity. In addition, Upd3 is secreted at high levels from other tissues. Still, oenocyte Upd3 is selected as the single cytokine mediating aging- and stress-induced cardiac arrhythmicity. Was an overexpression of Upd3 in other tissues at young ages performed?
The relation between the different paradigms in oenocytes causing arrythmicity i.e. increased oxidative stress due to knockdown of catalase or SOD1 and the impaired import of peroxisomal proteins due to knockdown of Pex1, Pex5 is not clear. It is claimed that the knockdown of these Pex genes impacts on ROS metabolism but ROS levels were not determined. Importantly, it should be clarified why knockdown of Pex19 does not induce the same effects as compared to Pex1 and Pex5. This is incomprehensible as peroxisomal metabolism is obliterated with all Pex knockdowns. Furthermore, a potential involvement of altered lipid metabolism was disregarded but this was not thoroughly investigated. Dhapat was knocked down as a way to reduce plasmalogen levels but it was not assessed whether the levels of these lipids were effectively reduced in this time span. Phosphomevalonate kinase was suppressed but the relevance of this is obscure as the role of peroxisomes in cholesterol synthesis is still being debated (Wanders and Waterham, Ann rev biochem, 2006). Notably, the potential importance of α- and β-oxidation, both mentioned by the authors as essential functions of peroxisomes in the introduction and discussion, and of particular importance in tissues with high lipid metabolism such as oenocytes, was not addressed. This should also be assessed. This is essential because it was previously shown that suppression of the peroxisomal β-oxidation enzyme ACOX1, induces inflammatory genes including IL6 (El Hajj et al, Endocrinology 2012). This paper should be referred to.
The inclusion of the Pex1 G843D fibroblast line to validate the findings in the oenocytes is a strength of the manuscript. A drawback is that the analysis was limited to IL6 and P-JNK. At least, the consequences on ROS levels should also be included for a better correlation with the fly data. It should be clarified whether this is a homozygous mutant line.
PMP70 staining is used to identify peroxisomes. Although this is one of 3 ABC transporters that is often expressed in the peroxisomal membrane, this can not be considered as a marker for peroxisomes because its abundance in peroxisomes can vary according to the cell type. It is advised to identify peroxisomes using antibodies directed to PEX proteins, for example PEX14. Also, better use the terminology ABCD3 than PMP70.
Minor comments
Fig 5b: ICC of P-JNK is shown to be increased in Pex5 RNAi but it would be better to confirm this by western blotting
What is meant with the term peroxikinies??
In figure 8m a scheme is shown summarizing the data after overexpression of Pex5 in oenocytes, but it would be better to have a more general graph in which all data are compiled.
Throughout the manuscript there are many grammatical/typographical errors (plural (e.g. peroxisomes instead of peroxisome) – tenses – verbs)