Phytoene and Phytoene-Rich Microalgae Extracts Extend Lifespan in C. elegans and Protect against Amyloid-β Toxicity in an Alzheimer’s Disease Model
, Antonis A. Karamalegkos 2, Paula Mapelli-Brahm 1, Marina Ezcurra 2,*,† and Antonio J. Meléndez-Martínez 1,†
Round 1
Reviewer 1 Report
The study deals with the bioactivity of phytoene-rich carotenoid extracts from the micro-algae Dunaliella bardawil and Chlorella sorokiniana, and pure phytoene,in C. elegans. Microalgae extracts, as well as phytoene protect against oxidative stress and amyloid-β toxicity in a model of Alzheimer’s disease and extend lifespan.
Specific points
-Table 1-Carotenoid content was quantified by HPLC analysis and showed high levels of phytoene present in the extracts. How do the other carotenoids contribute to bioactivity.
- Juglone is used to generate superoxide anion radicals but this phenolic compound is also an antioxidant. How is it considered to evaluate the capacity of phytoene-rich extracts and phytoene to increase resistance to oxidative stress?(figure3)
- Several studies using C. elegans have demonstrated that carotenoid-rich extracts from plant sources not only protect against oxidative stress but increase lifespan. The information gained in the present report is largely incremental.
Specific points
-Table 1-Carotenoid content was quantified by HPLC analysis and showed high levels of phytoene present in the extracts. How do the other carotenoids contribute to bioactivity.
- Juglone is used to generate superoxide anion radicals but this phenolic compound is also an antioxidant. How is it considered to evaluate the capacity of phytoene-rich extracts and phytoene to increase resistance to oxidative stress?(figure3)
Author Response
Comment 1: Several studies using C. elegans have demonstrated that carotenoid-rich extracts from plant sources not only protect against oxidative stress but increase lifespan. The information gained in the present report is largely incremental.
Response 1: We thank the reviewer for pointing out that the novelty of our work has not been made clear in the manuscript. The reviewer correctly points out that carotenoid-rich extracts have been shown to have protective effects, but no study has looked at the carotenoid phytoene specifically. To our knowledge there are no studies demonstrating that phytoene has protective bioactivity in a whole animal model. We have made changes to the Abstract, Introduction to make the novelty of our work clearer.
Comment 2: Several studies using C. elegans have demonstrated that carotenoid-rich extracts from plant sources not only protect against oxidative stress but increase lifespan. The information gained in the present report is largely incremental.
Response 2: The main novelty of our work is the finding that phytoene is protective. This has not been shown before and is different from mixed carotenoid extracts extracted from plants. Moreover we demonstrate potential for carotenoids extracted from microalgae rather than terrestrial plants. This is an important distinction because of the growing demands on agricultural land and the need to identify alternative sources of nutritious and healthy foods. We have made these points clearer as outlined in the previous response.
Comment 3: Table 1-Carotenoid content was quantified by HPLC analysis and showed high levels of phytoene present in the extracts. How do the other carotenoids contribute to bioactivity.
Response 3: As the reviewer points out, the microalgae extracts contain a pool of carotenoids, of which the majority have demonstrated bioactivity. The goal of this specific study was to investigate if phytoene specifically has positive health effects, as this has not been shown in the past. Although it would be interesting to test how the different individual carotenoids in the pool compare in terms of effect on health, this is beyond the scope of this specific study, and something we would be interested in doing in the future.
Comment 4: Juglone is used to generate superoxide anion radicals but this phenolic compound is also an antioxidant. How is it considered to evaluate the capacity of phytoene-rich extracts and phytoene to increase resistance to oxidative stress?(figure3)
Response 4:
The reviewer points out that juglone can act both as an oxidant and antioxidant. As juglone contains a hydroxyl group, the compound is classified as a phenolic compound and on this basis it is assumed it has antioxidant properties. However, its structure also contains a quinone moiety, which is a redox factor that can produce reactive oxygen species. In the literature there is very limited evidence for antioxidant activity in vitro or vivo (see Ahmad et al 2019). In contrast it has been demonstrated that at the concentrations used in C. elegans juglone assays, the compound is highly toxic and generates ROS (see Chobot and Hadacek 2009) and exposure to juglone is a widely used and accepted way to measure resistance to oxidative stress in C. elegans (see Senchuk et al 2017).
Reviewer 2 Report
Review on the manuscript of Morón-Ortiz Á et al.: “Phytoene and phytoene-rich microalgae extracts extend lifespan in C. elegans and protect against amyloid-β toxicity in an Alzheimer’s disease model”.
In this manuscript, the Authors explored the anti-aging effects of microalgal extracts containing the carotenoid phytoene in the C. elegans animal model. The Authors show that Chlorella sorokiniana and Dunaliella bardawil extracts enriched in phytoene have anti-ageing properties, as they protected against oxidative damage and amyloid-β42 proteotoxicity, and extended the animal’s lifespan.
Overall, I find this topic to be of great interest, as carotenoids are increasingly recognized for their importance in maintaining health and preventing chronic diseases due to their antioxidant, anti-inflammatory, and immune-supporting properties. Therefore, a thorough understanding of their biological effects is crucial, as they can potentially enhance human well-being and overall health. I believe the manuscript effectively addresses the questions posed by the Authors. However, I have listed below some issues that I hope the Authors will find useful in addressing.
1 - For this study, the Authors used microalgae extracts from Dunaliella bardawil and Chlorella sorokiniana. Can the Authors provide a justification for choosing these two particular microalgae over others? This information can easily be added at the end of the introduction.
2 - For the development assays, “thirty adult C. elegans were placed on experimental plates and allowed to lay eggs for 2 hours. After 52-54 hours, the number of animals at L4 and adult stages was counted to evaluate the effects on the developmental rate”. At 20 ºC, the C. elegans lifespan is about 3.5 days. Therefore, considering that the experiments were performed at a temperature of 20 ºC (as provided in the manuscript), no adult animals should be found after 54 hours (2,25 days). This idea is supported in subsection 2.10 “Oxidative Stress Assays”, where the Authors indicate that “approximately 60 hours after egg laying, 50 nematodes at the L4 stage…”. Can the authors clarify this point? I also consider that the best way to evaluate animal development would be by measuring the worm length in images captured at a specific timepoint.
3 - For the statistical analysis of the data shown in figure 3, the Authors used the Two-Way ANOVA. In my opinion, the statistical analysis should be performed with the Fisher’s exact test. However, the statistics represented in Figure 3 are relative to DMSO, which serves as the true control of the experiment. The data shown in Figure 3 shows that 1 μg/mL and 2 μg/mL of C. sorokiniana and D. bardawil extracts, as well as phytoene standard, speed up animal development (resulting in a higher percentage of animals at the adult stage). However, in the text, the Authors indicate that “extracts at concentrations of 0.2 μg/mL, 1 μg/mL, and 2 μg/mL were tested and no differences were found between the extracts and the controls. Phytoene at the same concentrations did not affect development either”. Can the authors clarify this point and revise the manuscript to align with the results shown in the figures?
4 - For the statistical analysis of the data shown in Figure 4 (indicated as figure 3 - survival data), the Authors used a two-way ANOVA. However, since there is only one variable (the treatment condition), a One-way ANOVA is the appropriate method. Can the Authors clarify this point? The representation of the statistics with capital letters (without any indication of their meaning) is not consistent with the other figures, where asterisks are used. I strongly recommend that the Authors standardize the way the statistics are presented. In addition, it is not clear to which group (DMSO or OP50) the comparisons are being made.
5 In the experiment shown in Figure 4, it is evident that no negative control (without juglone) was used. One would expect that juglone alone could cause a more pronounced reduction in survival.
Minor points:
1 - I recommend that the Authors reduce the number of keywords to a maximum of ten, as specified in the journal's template.
2 - In the Materials and Methods section, subsection 2.3 Phytoene Microalgae Enrichment, can the authors provide information on how long the incubation with norflurazon lasted?
3 - I recommend that the Authors insert Figure 1 after it is first cited in the text.
4 - On line 207, “Figure 1” should be “Figure 3”.
5 - The figure corresponding to the oxidative stress/survival data should be Figure 4. Please also correct this information in the text accordingly.
6 - The figure corresponding to the proteotoxicity data should be Figure 5. Please also correct this information in the text accordingly.
7 - The figure corresponding to the lifespan data should be Figure 6. Please also correct this information in the text accordingly.
8 - In Figure 6 (lifespan data), it is unclear what the significance indicated by the symbol “#” signifies.
Author Response
Comment 1: For this study, the Authors used microalgae extracts from Dunaliella bardawil and Chlorella sorokiniana. Can the Authors provide a justification for choosing these two particular microalgae over others? This information can easily be added at the end of the introduction.
Response 1: We thank the reviewer for the comments on the manuscript and the helpful suggestions. To clarify the use of the specific speices we have added the following justification to the end of the introduction: "For the present study, Dunaliella bardawil and Chlorella sorokiniana were selected. These are species from two microalgae genus that are exploited commercially and widely studied for their ability to accumulate carotenoids. More specifically, both species have proven amenable for phytoene accumulation."
Comment 2: For the development assays, “thirty adult C. elegans were placed on experimental plates and allowed to lay eggs for 2 hours. After 52-54 hours, the number of animals at L4 and adult stages was counted to evaluate the effects on the developmental rate”. At 20 ºC, the C. elegans lifespan is about 3.5 days. Therefore, considering that the experiments were performed at a temperature of 20 ºC (as provided in the manuscript), no adult animals should be found after 54 hours (2,25 days). This idea is supported in subsection 2.10 “Oxidative Stress Assays”, where the Authors indicate that “approximately 60 hours after egg laying, 50 nematodes at the L4 stage…”. Can the authors clarify this point? I also consider that the best way to evaluate animal development would be by measuring the worm length in images captured at a specific timepoint.
Response 2: The reviewer asks for clarification regarding developmental assays, we are happy to provide this. At standard conditions and 20 ºC, wildtype C. elegans have a lifespan of 2-3 weeks, not 3.5 days. The developmental assay measures the proportion of animals that have developed from egg to the last larval stage (L4) or adulthood (sexual maturity) after 52-54 hours. After reaching this stage, animals will go and live for an additional 2-3 weeks.
The second question relates to how to best measure development. The reviewer suggests to measure size instead of developmental stage. Size is not an appropriate measure of developmental rate because there are many conditions in which the animals are smaller in size without affecting development (reaching sexual maturity). This is true across species, size is not necessarily a requirement for sexual maturity.
Comment 3: For the statistical analysis of the data shown in figure 3, the Authors used the Two-Way ANOVA. In my opinion, the statistical analysis should be performed with the Fisher’s exact test. However, the statistics represented in Figure 3 are relative to DMSO, which serves as the true control of the experiment. The data shown in Figure 3 shows that 1 μg/mL and 2 μg/mL of C. sorokiniana and D. bardawil extracts, as well as phytoene standard, speed up animal development (resulting in a higher percentage of animals at the adult stage). However, in the text, the Authors indicate that “extracts at concentrations of 0.2 μg/mL, 1 μg/mL, and 2 μg/mL were tested and no differences were found between the extracts and the controls. Phytoene at the same concentrations did not affect development either”. Can the authors clarify this point and revise the manuscript to align with the results shown in the figures?
Response 3: We thank the reviewer for the insightful comments. We have addressed the reviewer’s concerns and performed the suggested statistical analysis using Fisher’s exact test as recommended. Consequently, Figure 3 has been updated to reflect the new statistical analysis. The results section (3.3, lines 238–241) has been revised to align with the updated figures. Specifically, we have clarified that extracts at concentrations of 0,2 μg/mL, 1 μg/mL and 2 μg/mL of C. sorokiniana and D. bardawil, as well as 1 μg/mL and 2 μg/mL of phytoene standard, do indeed speed up animal development, resulting in a higher percentage of animals at the adult stage, which was not stated in the previous version of the manuscript.
Comment 4: For the statistical analysis of the data shown in Figure 4 (indicated as figure 3 - survival data), the Authors used a two-way ANOVA. However, since there is only one variable (the treatment condition), a One-way ANOVA is the appropriate method. Can the Authors clarify this point? The representation of the statistics with capital letters (without any indication of their meaning) is not consistent with the other figures, where asterisks are used. I strongly recommend that the Authors standardize the way the statistics are presented. In addition, it is not clear to which group (DMSO or OP50) the comparisons are being made.
Response 4: We have re-analyzed the data using a One-way ANOVA, as suggested, given that there is only one variable. The results of this updated analysis are now reflected in Figure 4. To standardize the representation of statistical significance, we have replaced the capital letters in Figure 4 with asterisks, consistent with the notation used in our other figures. Additionally, the figure legend has been updated to clearly indicate that comparisons are made relative to the DMSO control group.
Comment 5: In the experiment shown in Figure 4, it is evident that no negative control (without juglone) was used. One would expect that juglone alone could cause a more pronounced reduction in survival.
Reponse 5: The reviewer asks regarding a non-juglone control in Figure 4. We assume this refers to Figure 3 in which the juglone assay data is shown. We did not include a non-juglone control because the question we are asking is if phytoene and the phytoene-rich extracts protect against oxidative stress. Including a non-juglone control will indeed show that juglone is toxic, but this has been shown extensively in other publications.
The reviewer comments that juglone alone causes a more pronounced reduction in survival. We have indeed included juglone alone in the experimental conditions. ‘OP50’ refers to animals cultivated in standard conditions exposed to juglone but not supplemented with DMSO, phytoene or extracts. We have added more detail in the figure legends to make the control conditions clearer.
Comment 6: I recommend that the Authors reduce the number of keywords to a maximum of ten, as specified in the journal's template.
Response 6: We have reduced the number of keywords according to journal specifications.
Comment 7: In the Materials and Methods section, subsection 2.3 Phytoene Microalgae Enrichment, can the authors provide information on how long the incubation with norflurazon lasted?
Response 7: Incubation lasted 48-72 hours, this has been added to the Methods section.
Comment 7: I recommend that the Authors insert Figure 1 after it is first cited in the text.
Reponse 7: We have moved Figure 1 accordingly.
Comment 8: On line 207, “Figure 1” should be “Figure 3”.
Response 8: We have corrected this error.
Comment 9: The figure corresponding to the oxidative stress/survival data should be Figure 4. Please also correct this information in the text accordingly.
We have corrected this error.
Comment 10: The figure corresponding to the proteotoxicity data should be Figure 5. Please also correct this information in the text accordingly.
We have corrected this error.
Comment 11: The figure corresponding to the lifespan data should be Figure 6. Please also correct this information in the text accordingly.
We have corrected this error.
Comment 12: In Figure 6 (lifespan data), it is unclear what the significance indicated by the symbol “#” signifies.
Response 12: # means p = 0.0399, comparing phytoene-enriched D. bardawil and phytoene-enriched C. sorokiniana extracts, this has now been added to the figure legend.
Round 2
Reviewer 1 Report
.The reviewer is satisfied with the revised version of the manuscript. All comments and concerns have been addressed carefully and thoughtfully.
.The reviewer is satisfied with the revised version of the manuscript. All comments and concerns have been addressed carefully and thoughtfully.
Author Response
Comment: The reviewer is satisfied with the revised version of the manuscript. All comments and concerns have been addressed carefully and thoughtfully.
Response: We thank the reviewer for their valuable input on the manuscript and are pleased the reviewer is satisfied.
Reviewer 2 Report
Second review on the manuscript of Morón-Ortiz Á et al.: “Phytoene and phytoene-rich microalgae extracts extend lifespan in C. elegans and protect against amyloid-β toxicity in an Alzheimer’s disease model”.
In this manuscript, the Authors explored the anti-aging effects of microalgal extracts containing the carotenoid phytoene in the C. elegans animal model. The Authors show that Chlorella sorokiniana and Dunaliella bardawil extracts enriched in phytoene have anti-ageing properties, as they protected against oxidative damage and amyloid-β42 proteotoxicity, and extended the animal’s lifespan.
This is the second version of the manuscript following peer review. After carefully reading the revised manuscript and the Authors' reply letter, I found some issues that remain insufficiently clarified. Therefore, I have listed below some points that I hope the Authors will find useful in addressing.
1 - For the development assay, when I said “At 20 ºC, the C. elegans lifespan is about 3.5 days”, I meant to say “At 20 ºC, the C. elegans life cycle is about 3.5 days”. Apologies for the mistake. Therefore, considering that the experiments were conducted at 20 ºC (as stated in the manuscript), no adult animals should be present after 54 hours (2.25 days). This is supported by subsection 2.10 “Oxidative Stress Assays”, where the Authors indicate that “approximately 60 hours after egg laying, 50 nematodes at the L4 stage…”. Could the authors clarify this point?
2 - In response to my suggestion, “I also consider that the best way to evaluate animal development would be by measuring the worm length in images captured at a specific time point”, the Authors clarified that “size is not an appropriate measure of developmental rate because there are many conditions in which the animals are smaller in size without affecting development (reaching sexual maturity). This is true across species; size is not necessarily a requirement for sexual maturity”. I totally agree that different strains have different sizes when reaching sexual maturity. For example, lon-1(e185) worms are much longer than wildtype, whereas dpy-11(e224) worms are much smaller, even at the same developmental stage (adulthood, for example). However, the Authors used the same strain (N2) for this assay. Therefore, the size of the worms is directly comparable, and a reduction in size reflects a defect in development.
3 - In the experiment shown in Figure 4, the data description is not clear. It appears that all conditions include juglone. If this is the case, it is evident that no negative controls (without juglone) are represented (OP-50 without juglone and DMSO without juglone). Therefore, the Authors need to include these two negative controls. Alternatively, if the first two bars are the negative controls, then the controls for OP-50 plus juglone and DMSO plus juglone are missing. Thus, the graph should represent four controls: OP-50 and DMSO, both with and without juglone.
None.
Author Response
Comment 1: For the development assay, when I said “At 20 ºC, the C. elegans lifespan is about 3.5 days”, I meant to say “At 20 ºC, the C. elegans life cycle is about 3.5 days”. Apologies for the mistake. Therefore, considering that the experiments were conducted at 20 ºC (as stated in the manuscript), no adult animals should be present after 54 hours (2.25 days). This is supported by subsection 2.10 “Oxidative Stress Assays”, where the Authors indicate that “approximately 60 hours after egg laying, 50 nematodes at the L4 stage…”. Could the authors clarify this point?
Response 1:
We thank the reviewer for clarifying their point regarding the development assay and for their input on this point. After double checking our method protocols we realise there is typo in the manuscript and that the developmental assay was conducted at 62-64 hours after the eggs were laid, not 52-54 hours.
We have utilized Wormatlas (a publicly funded C. elegans resource database) to double check the developmental timings for wildtype C. elegans and they are as follows:
In utero development (before egg is laid): 2.5h
Ex utero development (after egg is laid but before hatching): 9h
1st larval stage L1: 12h
2nd larval stage L2: 8h
3rd larval stage: 8h
4th larval stage (before adult molt): 10h
Young adult to sexually mature adult: 8h
(see https://www.wormatlas.org/hermaphrodite/introduction/mainframe.htm)
When the developmental assay is set up, adult mothers are allowed to lay eggs during a 2 hour window and then removed and the remaining eggs are used to measure development. The assay starts after eggs have been laid, meaning that after 9+12+8+8+10+8 = 55 h animals will reaching adulthood. The timings are at 22C, at 20C the development will be slightly slower. This means that after 62-64 hours a mixed population of L4 and adults will be obtained, which is reflected in our data in Figure 3. We have corrected this error in the Results and Methods.
We are aware that the C. elegans lifecycle is referred to as being 3.5 days, which can be misleading in terms of the exact timings from egg to adult. We hope this clarifies the timings used and results we obtained for both the development and oxidative stress assays, and thank the reviewer again for spotting the error.
Comment 2: In response to my suggestion, “I also consider that the best way to evaluate animal development would be by measuring the worm length in images captured at a specific time point”, the Authors clarified that “size is not an appropriate measure of developmental rate because there are many conditions in which the animals are smaller in size without affecting development (reaching sexual maturity). This is true across species; size is not necessarily a requirement for sexual maturity”. I totally agree that different strains have different sizes when reaching sexual maturity. For example, lon-1(e185) worms are much longer than wildtype, whereas dpy-11(e224) worms are much smaller, even at the same developmental stage (adulthood, for example). However, the Authors used the same strain (N2) for this assay. Therefore, the size of the worms is directly comparable, and a reduction in size reflects a defect in development.
Response 2: We agree with the reviewer that growth and body size can be used as a measure of development, and that wildtype animals in cultivated in standard conditions reach specific sizes at each developmental stage and would like to alter out statement above regarding size not being an appropriate measure; body size can indeed be used as a measure of development. In this study however we were specifically interested in the timing of sexual maturity. This is because size although affected by developmental stage, can also be altered by other factors such as diet, affecting the size of cells and the amount of lipids stored. In the Results section we spell out that we are measuring timing to reach adulthood specifically: "We conducted developmental assays to detect any effects on the development of larvae into adulthood’. We have added information in the Methods to clarify this point.
Carotenoids have been reported to reduce obesity and lipid storage in C. elegans and other species (Hernandez-Cruz et al 2023), thus it is not unlikely that the extracts and phytoene used in this study might alter body composition and body size without affecting time to reach sexual maturation. We will indeed investigate the effects of the microalgae extracts and phytoene on lipid storage in a separate project, where we will also measure body size. We thank the reviewer for this suggestion.
Round 3
Reviewer 2 Report
None.
Third review on the manuscript of Morón-Ortiz Á et al.: “Phytoene and phytoene-rich microalgae extracts extend lifespan in C. elegans and protect against amyloid-β toxicity in an Alzheimer’s disease model”.
In this manuscript, the Authors explored the anti-aging effects of microalgal extracts containing the carotenoid phytoene in the C. elegans animal model. The Authors show that Chlorella sorokiniana and Dunaliella bardawil extracts enriched in phytoene have anti-ageing properties, as they protected against oxidative damage and amyloid-β42 proteotoxicity, and extended the animal’s lifespan.
This is the third version of the manuscript following peer review. After carefully reading the revised manuscript and the Authors' reply letter, I found that one issue remains unclarified form the beginning of the review process, which I mention below:
1 - In the experiment shown in Figure 4, the data description is not clear. It appears that all conditions include juglone. If this is the case, it is evident that no negative controls (without juglone) are represented (OP-50 without juglone and DMSO without juglone). Therefore, the Authors need to include these two negative controls. Alternatively, if the first two bars are the negative controls, then the controls for OP-50 plus juglone and DMSO plus juglone are missing. Thus, the graph should represent four controls: OP-50 and DMSO, both with and without juglone.
Author Response
Comment 1: In the experiment shown in Figure 4, the data description is not clear. It appears that all conditions include juglone. If this is the case, it is evident that no negative controls (without juglone) are represented (OP-50 without juglone and DMSO without juglone). Therefore, the Authors need to include these two negative controls. Alternatively, if the first two bars are the negative controls, then the controls for OP-50 plus juglone and DMSO plus juglone are missing. Thus, the graph should represent four controls: OP-50 and DMSO, both with and without juglone.
Response 1: We apologise if not having addressed this comment previously. We followed standard protocols to measure juglone sensitivity. As the question asked is if a compound/extract alters the sensitivity to juglone, all experimental conditions include exposure to juglone, comparing survival when phytoene/extracts are present or absent. The experiment is designed to evaluate if phytoene/extracts have a protective effect against juglone.
In figure 4 juglone was added to all conditions including OP50 and DMSO controls. Although we do not disagree that a control without juglone could have been added for clarity, this is outside standard proceedures and standard protocols. I have included a few references below to clarify that we followed standard procedure when conducting the assay. In addition, our survival assay (Figure 6) shows no deaths in any conditions (without juglone obviously) during the first few days of adulthood. We hope this satisfies the reveiwer in terms of the robustness of the experiment in testing senstivity to juglone.
https://bio-protocol.org/en/bpdetail?id=2086&type=0
https://www.nature.com/articles/s41467-021-22561-x#Sec2
https://www.nature.com/articles/srep21611#Sec9
