Cholesterol mediated ferroptosis suppression reveals essential roles of Coenzyme Q and squalene

Recent findings have shown that fatty acid metabolism is profoundly involved in ferroptosis. However, the role of cholesterol in this process remains incompletely understood. In this work, we show that modulating cholesterol levels changes vulnerability of cells to ferroptosis. Cholesterol alters metabolic flux of the mevalonate pathway by promoting Squalene Epoxidase (SQLE) degradation, a rate limiting step in cholesterol biosynthesis, thereby increasing both CoQ10 and squalene levels. Importantly, whereas inactivation of Farnesyl-Diphosphate Farnesyltransferase 1 (FDFT1), the branch point of cholesterol biosynthesis pathway, exhibits minimal effect on ferroptosis, simultaneous inhibition of both CoQ10 and squalene biosynthesis completely abrogates the effect of cholesterol. Mouse models of ischemia-reperfusion and doxorubicin induced hepatoxicity confirm the protective role of cholesterol in ferroptosis. Our study elucidates a potential role of ferroptosis in diseases related to dysregulation of cholesterol metabolism and suggests a possible therapeutic target that involves ferroptotic cell death.


Referee expertise:
Referee #1: Cholesterol metabolism, ferroptosis Referee #2:lipid metabolism, ferroptosis Referee #3: lipid metabolism Reviewers' comments: Reviewer #1 (Remarks to the Author): Sun et al demonstrates a causal link between cholesterol and ferroptosis susceptibility as well as its clinical relevance.Although the notion that mevalonate pathway regulates ferroptosis susceptibility has been well recognized, potential interests exist regarding the its interplay with cholesterol homeostasis and its implication in hepatoxicity.Overall, this study is well designed, yet several aspects need to be better interpreted.Major comments: 1. Figure 1a: The levels of lipid peroxidation should be provided to demonstrate that the cells were really protected from ferroptosis.2. Figure 2d, e: Whether the background lipid increase was corrected by FL2 channel when detecting lipid peroxidation using C11 BODIPY staining?3. Figure S2e, f: The legend should be carefully checked.Cysteine starvation induced a nearly 90% of cell death (Figures S1e, S1h, S2c), while this was not observed in Figure S2e, f. 4. Figure 2a-c: Why did RSL3 induce a discrete frequency of ferroptosis.5. Figures 3, 4: CH&Desmo also significantly attenuated ferroptosis of CoQ10-depleted or FSP1deleted cells.Does that mean CH&Desmo inhibit ferroptosis independently of FSP1-CoQ10 axis?In Figure 4, FDFT1 deletion alone was sufficient to abrogate the protective effect of CH on ferroptosis.The contribution of FSP1-CoQ10 axis remains doubtful.6. Appropriate statistical analysis should be used, such as one/two-way ANOVA and multiple comparison for three or more groups.Minor comments: 1.Some grammatical mistakes such as "that involve" in Abstract, "array of" in Introduction, "are able to", "independent on each other" in Results and etc.The manuscript should be thoroughly checked.In this manuscript, Sun et al. suggest that cholesterol inhibits ferroptosis via CoQ10 and squalene metabolism and further show that cholesterol diet can protect from hepatotoxicity.Overall, the majority of experiments are well-designed, well-conducted, and clear enough to support the author's claim.There are only minor basic issues.

Supplementary Figures particularly Figures S1 and S5
1.The data suggest that the levels cholesterol rather than its intermediates themselves eventually affect CoQ10 and squalene metabolism to inhibit ferroptosis.However, the intermediate metabolism seems to be focused in the manuscript and supplementary figures, so a graphic summary of ferroptosis inhibition by cholesterol would be helpful for readers.2. Why several intermediates for cholesterol synthesis are dispensable for ferroptosis suppression?Did not they contribute to total cholesterol levels?3.If desmosterol inhibits ferroptosis via cholesterol synthesis, the inhibitory effect of desmosterol should be tested in DHCR7 KO cells 4. In addition, as the authors suggest, the possible additional mechanism by which 7DHC inhibits ferroptosis via direct inhibition of autoxidation may be indicated in the figure.Furthermore, if possible, direct observation of 7DHC-mediated inhibition of autoxidation is required (Supp Fig. S3d, related to page 6). 5.The levels of cholesterol in cells treated with MꞵCD should be provided.Because the direct observation of altered cholesterol levels upon avasimible and MꞵCD is critical, please provide these data (supplementary fig.2d) in the main figure.6.The involvement of CoQ10 in CH-mediated ferroptosis suppression is still unclear.First, recycling of CoQ10 by FSP1 may be much more critical for ferroptosis suppression rather than the levels of CoQ10.Partial inhibition of ferroptosis by CH in FSP1 KO cells may suggest the requirement of FSP1 for CHmediated ferroptosis suppression but it also imply that CH can also inhibit ferroptosis in the absence of FSP1.Could the author see the inhibition of ferroptosis by supplementing CoQ10 that can lead to 2 fold increase?This can be just discussed.7. Fig. 5a, Lipo may be corrected to Lipro-1.

Reviewer #3 (Remarks to the Author):
This study is interesting.I went through the paper.Please consider the following comments to improve the quality of your paper.
1. Figure 1a: The levels of lipid peroxidation should be provided to demonstrate that the cells were really protected from ferroptosis.
RESPONSE: we agree with the reviewer's comments and apologize for the lack of this piece of data.We have performed BODIPY 581/591-C11 staining and microscopy imaging after various sterols treatment (for both original and newly tested sterols).This data is now added as new Figure 1a-c    Figure 2d figure 2e 3. Figure S2e, f: The legend should be carefully checked.Cysteine starvation induced a nearly 90% of cell death (Figures S1e, S1h, S2c), while this was not observed in Figure S2e, f.RESPONSE: we thank the reviewer for careful reading.We apologize for this carelessness.We carefully checked our lab note.In Figure s2c, cysteine starvation was 24h instead of 10h.In Figure s2e, f, cysteine starvation lasted for 17h instead of 27h.The legend was rewritten.
4. Figure 2a-c We presented a diagram below to further explain our data in Figure 4 (numbers next to CoQ10 or squalene indicate relative cellular levels of these two molecules, 1 denotes basic levels under normal condition): 6. Appropriate statistical analysis should be used, such as one/two-way ANOVA and multiple comparison for three or more groups.RESPONSE: we thank the reviewer for this critical point.We have made corrections.Please see below and new descriptions in the Statistics and Reproducibility section (line 764).
All comparisons were tested using unpaired two-tailed Student's t-test or one/two-way ANOVA with Tukey's post hoc test, with a confidence interval (CI) of 95%.P ≤ 0.05 was considered statistically significant.
Minor comments: 1. Some grammatical mistakes such as "that involve" in Abstract, "array of" in Introduction, "are able to", "independent on each other" in Results and etc.
The manuscript should be thoroughly checked.

RESPONSE:
We thank the reviewer for pointing out these grammatical mistakes.These mistakes were corrected.Reviewer #2:

Supplementary Figures particularly Figures S1 and S5
1.The data suggest that the levels cholesterol rather than its intermediates themselves eventually affect CoQ10 and squalene metabolism to inhibit ferroptosis.However, the intermediate metabolism seems to be focused in the manuscript and supplementary figures, so a graphic summary of ferroptosis inhibition by cholesterol would be helpful for readers.
RESPONSE: we thank the reviewer for this wonderful suggestion.We have added a graphic summary as new Figure 6.  as measured by mass-spectrometry assay (new Figure S3k).Importantly, it also reduced RSL3 induced cell death (new Figure 3k), suggesting that increasing cellular CoQ10 content can inhibit ferroptosis.
Our hypothesis that CoQ10-FSP1 pathway plays a role in the action of CH&Desmo could further be supported by the result that inhibition of FDFT1 by TAK-475 or knockout of FDFT1 has no effect on ferroptosis (Figure 4j, k), which could be explained by decreased levels of squalene but increased levels of CoQ, with a net "0" effect on ferroptosis.
We presented a diagram below to further explain our hypothesis (numbers next to CoQ10 or squalene indicate relative cellular levels of these two molecules, 1 denotes basic levels under normal condition): should be better cited and arranged to easily follow the results.3. Figures1, 5: What are Fer-1 and Lipo?Reviewer #2 (Remarks to the Author): and Figure s1a-c (line 150 and 152 in the manuscript).

2 .
Figure 2d, e.However, the original quantitative data was not corrected by FL2 channel (red fluorescence channel).We have now replaced this quantitative data after correcting FL2 channel as new Figure 2d, e (right panels) respectively.
Figure3k), suggesting that increasing cellular CoQ10 content can inhibit ferroptosis.3. CH&desmo treatment increased cellular CoQ10 content by around 50%, 2folds more than that of after adding 250nM exogenous CoQ10 (Figure3b).4. Inhibition of FDFT1 by TAK-475 or knockout of FDFT1 has no effect on ferroptosis (Figure4j, k), which could be explained by decreased levels of squalene but increased levels of CoQ, with a net "0" effect on ferroptosis.
figure s5, we have moved original Figure s5b, c to Figure s5a, b respectively,

2 .
Why several intermediates for cholesterol synthesis are dispensable for ferroptosis suppression?Did not they contribute to total cholesterol levels?RESPONSE: we thank the reviewer's comments.To answer this question, we measured intracellular cholesterol levels by filipin staining followed by flow cytometry analysis in the presence of various sterol intermediates.Desmosterol and 7-DHC indeed increased intracellular cholesterol, possibly because these two sterols are direct precursors to cholesterol (New Figures2h), whereas other intermediates showed marginal or no effect on cellular cholesterol levels.However, inhibiting DHCR24 to block cholesterol biosynthesis from Desmosterol did not influence the effect of Desmosterol on ferropotosis inhibition (Figure2f-h).As for 7-DHC, Angeli et al have shown that inactivation of DHCR7 that converts 7-DHC to cholesterol did not impact the anti-ferroptotic effect of 7-DHC (doi:10.21203/rs.3.rs-943221/v1(2021)).(line204-211) New text added in the manuscript:As upstream precursors to cholesterol, excess intermediates of cholesterol biosynthesis may enhance cholesterol biosynthesis, we thus tested this possibility by measuring intracellular cholesterol levels in the presence of sterol intermediates.Desmo and 7-DHC significantly increased cellular cholesterol levels as measured by flow cytometry, slightly less than that of CH supplementation.Latho also induced a marginal increase of intracellular cholesterol levels, whereas Zymo and Lano had no effect (Supplementary Fig. Figure3).To address the issue whether the levels of CoQ10 is important, we performed new experiments to investigate whether exogenous CoQ10 could inhibit ferroptosis, as suggested by the reviewer.Indeed, addition of 250nM exogenous CoQ10 led to around 25% increase of intracellular CoQ10 content