SRS microscopy identifies inhibition of vitellogenesis as a mediator of lifespan extension by caloric restriction in C. elegans
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Abstract
The molecular mechanisms of aging are not fully understood. Here, we used label-free Stimulated Raman scattering (SRS) microscopy to investigate changes in proteins and lipids throughout the lifespan of C. elegans. We observed a dramatic buildup of proteins within the body cavity or pseudocoelom of aged adults that was blunted by interventions that extend lifespan: caloric restriction (CR) and the reduced insulin/insulin-like growth factor signaling (IIS) pathway. Using a combination of microscopy, proteomic analysis, and validation with mutant strains, we identified vitellogenins as the key molecular components of the protein buildup in the pseudocoelom. Vitellogenins shuttle nutrients from intestine to embryos and are homologous to human apolipoprotein B, the causal driver of cardiovascular disease. We then showed that CR and knockdown of vitellogenins both extend lifespan by >60%, but their combination has no additional effect on lifespan, suggesting that CR extends the lifespan of C. elegans in part by inhibiting vitellogenesis. The extensive dataset of more than 12,000 images stitched into over 350 whole-animal SRS images of C. elegans at different ages and subjected to different longevity intervention will be a valuable resource for researchers interested in aging.
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This beautiful study highlights the power of SRS microscopy for discovering and following up on important observations. If addressed, I have a couple of questions that would provide clarity to the reader.
The inset graph showing the normalized pure spectra of proteins and lipids is critical for understanding the protein and lipid channels used throughout. I see where the normalization is explained in the methods, but I didn't find the details about how those curves were obtained. What was used for the pure protein and lipid samples? How many wavenumbers were sampled (just the 5 with the tick marks on the x-axis)?
In Figure 1, it would be helpful to add markings to point out the additional structures described in the figure caption for non-experts in C. elegans.
In Figure 2, the inset really demonstrates the protein buildup in the …
This beautiful study highlights the power of SRS microscopy for discovering and following up on important observations. If addressed, I have a couple of questions that would provide clarity to the reader.
The inset graph showing the normalized pure spectra of proteins and lipids is critical for understanding the protein and lipid channels used throughout. I see where the normalization is explained in the methods, but I didn't find the details about how those curves were obtained. What was used for the pure protein and lipid samples? How many wavenumbers were sampled (just the 5 with the tick marks on the x-axis)?
In Figure 1, it would be helpful to add markings to point out the additional structures described in the figure caption for non-experts in C. elegans.
In Figure 2, the inset really demonstrates the protein buildup in the pseudocoelomic space. Did you notice that that region on the worm was particularly striking across treatments? Would comparing the quantification in those FOV show a more pronounced effect than the entire worm?
The schematic showing the experimental design in Fig 1G. is helpful, but I didn't see the reasoning in the text to explain why the daf-2 mutants weren't exposed to the calorie restriction treatment like for ceh-60 worms.
In Figure S1, were the protein or lipid quantifications ever normalized to the area of the worm?
What do you make of the decreased protein buildup in ceh-60 CR worms as compared to ceh-60 fed (Fig. 6F). I see that there was no additive effect of CR on the mutants in terms of lifespan, but I found it interesting that it did look significantly lower in this plot.
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