Lipids Leading to Longer Lives

Lipids for Life

95 SH-directed inhibitors were screened for their effect on C. elegans lifespan. Screening was done in 96-well plates and compounds were generally screened at 50 μM with a few exceptions that were screened from 5–25 μM due to lower solubility. Approximately 10 animals were incubated per well in liquid culture throughout the assay. Data for representative hit compounds (red dots) are shown as average values from multiple replicates (n = 3 independent experiments, shown on same vertical line, where black horizontal dash marks the average lifespan change for each hit compound).

Paper: Chen AL, Lum KM, Lara-Gonzalez P, et al. Pharmacological convergence reveals a lipid pathway that regulates C. elegans lifespan. Nat Chem Biol. 2019;15(5):453–462. doi:10.1038/s41589-019-0243-4. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6548519/

Recent studies in the field of Biochemistry have started developing a library of various small molecules that have been able to increase the life expectancy of C. elegans. C. elegans have been the model of choice for these previous trial-and-error based experiments primarily due to the ease at which these animals can be used in a lab setting and their relatively short, natural life spans of only a couple weeks1,2. This work has become an exciting subject of study when taken in context of other recent work that has identified fundamental signaling pathways in C. elegans that are conserved even in humans which implies the possibility that some of these small molecules may also have positive effects in humans3,4. There had been no previously published research into the actual mechanisms and pathways associated with these identified life extending molecules nor how these molecules could be scaled up to work in a human model.

            Chen et al. sought to find the most effective and universally applicable of these identified small molecules and then develop a mechanism of proposed action as to why it is exerts it effect and how it could work in a human system. To do this, Chen et al. first performed a general for active serine hydrolase (SH) enzymes, one of the most universally prevalent enzyme families, in a C. elegans model using a biotinylated fluorophosphonate probe targeting the conserved active site in all members of the SH family. After narrowing down the list of SH enzymes prevent in C. elegans, Chen et al. proceeded to test 95 known SH-inhibitor molecules for its effect on lifespan and showed that the carbamate JZL184 increased lifespan by ~45% (results shown in figure).

            JZL184 was primarily known as an irreversible inhibitor to mammalian monoacylglycerol lipase (MAGL) which is primarily known for degrading 2-Arachidonoylglycerol (2-AG) in humans; this enzyme however, that lacks any orthologous equivalent in C. elegans. Luckily, this inhibitor is known to conveniently bind to SH enzymes which allowed Chen et al. to perform simple binding assays with the inhibitor and previous identified active SH enzymes to find that the primary target of JZL184 in C. elegans is Fatty acid amide hydrolase-4 (FAAH-4), a variant of FAAH which is known to degrade a small fraction of 2-AG’s in humans. Chen et al. then go on to prove the relationship between JZL184, FAAH-4, and lifespan by knocking out the FAAH-4 gene and found that the knockout of the hydrolase produced similar effects as treatment with JZL184 and that JZL184 treatment didn’t produce a compounded life extended effect when FAAH-4 was knocked out, showing a dependence between the two.

            This paper is primarily interested covering the possible pharmacological benefits of this JZL184 molecule, the experiment also reveals the importance of the still relatively novel, Cannabinoid system. 2-AG, a derivative of Arachidonic acid as the name implies, is a major agonist for the Cannabinoid receptor type 1 (CB1) and the primary ligand for CB2, the only two receptors in the system. CB2 is known for its presence in immune cells and association with cytokine release but these effects seem to be fairly unimportant in terms of the extended lifespan seen in the tested C. elegans as it is unlikely that pathogens would present in a lab setting. The more interesting target of 2-AG is the CB1 which is found in the central and peripheral nervous system and is associated with diminished cellular uptake of calcium in neurons which in-turn then lowers the release of neurotransmitters into the synapse in all nervous systems5. A colloquial example of the effects of this receptor is that THC, the active ingredient in marijuana, is known to be a major ligand for the CB1 receptor.

            Any number of speculations could then be made about what the results of this research could mean for future research on the area and for the public at large. Should the media pick this up as “proof” for the literal life altering effects of smoking weed? No, no probably not. There are any number flaws in that conclusion and still requires many more years of research in the field to even reach a point where that question can be asked. It does, however, bring up several interesting points about the relevance of CB1 and, particularly for this class, the effects of 2-AG. While covering PUFAs in class, we ended up talking about the relationship between omega-3 and omega-6 fatty acids in a fairly one-dimensional way: omega-3s are for anti-inflammatory response and are good and omega-6s are for inflammatory response and are bad. This paper adds another, fairly important, dimension to this argument by showing that some omega-6 derivatives have a positive and even anti-excitatory properties.

            Chen et al. developed a pathway to describe, to a certain extent, the mechanism behind their initial findings: an increase in available JZL184 irreversibly inhibits the function of FAAH-4, preventing it from degrading 2-AG, in-turn increasing levels of 2-AG which then upregulates the activity of CB1 receptors and that downregulates synaptic activity. The particular value of this result is one that Chen et al. emphasize several times throughout the paper is that they believe that this proposed pathway is also highly conserved in humans so that the research done in C. elegans could have major implications in the field of healthcare3,4.

            While I didn’t cover everything that Chen et al. published doing in their experiments to develop this pathway so you will primarily just have to take my word for it that there are still some holes and other results that can still be looked into further. They found that FAAH-4KD C. elegans showed an increase in food consumption which was then counteracted by treatment with JZL184 and that this inhibitor also associated fairly will with the SH enzyme Y53F4B.18 and that knockout trials without that SH also showed minor increases in lifespan. Chen et al. certainly go well and beyond showing the importance on 2-AG in lifespan but there is still room for far more research into the related molecules and in the many branches present in their proposed lifespan increasing pathway.

Citations:

  1. Evason K, Huang C, Yamben I, Covey DF & Kornfeld K Anticonvulsant medications extend worm life-span. Science 307, 258–262, doi:10.1126/science.1105299 (2005).
  2. Petrascheck M, Ye X & Buck LB An antidepressant that extends lifespan in adult Caenorhabditis elegans. Nature 450, 553–556, doi:10.1038/nature05991 (2007).
  3. Kenyon C, Chang J, Gensch E, Rudner A & Tabtiang R A C. elegans mutant that lives twice as long as wild type. Nature 366, 461–464, doi:10.1038/366461a0 (1993).
  4. Hamilton B et al. A systematic RNAi screen for longevity genes in C. elegans. Genes Dev 19, 1544–1555, doi:10.1101/gad.1308205 (2005)
  5. Mackie, K. “Mechanisms of CB1 Receptor Signaling: Endocannabinoid Modulation of Synaptic Strength.” International Journal of Obesity 30, no. 1 (April 2006): S19–23. https://doi.org/10.1038/sj.ijo.0803273.

22 thoughts on “Lipids Leading to Longer Lives”

  1. Clever use of alliteration in the title by the way Ethan. Science surrounding longevity is truly one of my favorite topics to read about, whether it be related to humans or another organism. It affects all of us, and (on rare but exciting occasions) the implications of this type of research can be used to inform the way we conduct ourselves on a daily basis, both dietarily and physically. C. elegans is a super cool organism as well, and has been instrumental in our piecing together of the roles of things such as insulin signaling in animal development and longevity (although I may be a tad biased since my lab uses the worm). I noted the approach the authors take here was reminiscent of one of the journal club papers we looked at in 341 (i.e. the ABPP), but I am interested to know whether they looked at anything beyond serine hydrolase enzymes in terms of their impact on longevity, or at least what lead them to narrow their focus on these enzymes. This seems like an inherent limitation of this type of screen. Furthermore, I wonder why the authors chose to downplay the significance of 2-AG’s function with respect to the CB2 receptor in favor of that of the CB1 receptor. Although I agree that the threat of pathogens is rare for worms in a sterile laboratory setting, I am almost MORE interested in thinking about this pathway in terms of its immunomodulatory potential, especially given the known impact of global inflammation levels on human health and longevity. Do they make any mention of whether agonism of this particular endocannabinoid receptor (CB2 that is) causes release of immunostimulatory cytokines that promote inflammation, or does this type of endocannabinoid signaling suppress an immune response? Either way, I could certainly see this having significant bearing on the organism’s lifespan for better or for worse.

    1. In terms of the Authors focus regarding this pathway I must admit that I got ahead of myself, drawing conclusions of my own based on their work and other resources around it, and in doing so focused a significant section of my review on the CB receptor system to help form a pleasing basis for me to use to digest the paper. The Author’s interest in this paper stopped promptly at 2-AG level in this pathway, only mentioning CB receptors a handful of times and only ever in reference to 2-AG. The importance of CB1 over CB2 was one that I, somewhat unfairly, fabricated based on the conditions in the lab and in no way reflect the views that are expressed in the paper nor probably the most scientifically sound views. From what I can tell, there is less understood about the CB2 receptor other than it resembles a CB1 receptor, is expressed somewhat ubiquitously but in low concentrations on non-immune cells, and that it can inhibit the signal transduction pathway associated with cytokine release. I agree that down regulation of an immune based inflammatory response (especially in a nonpathogenic setting) would to a very direct and logical way to increase lifespan in the context of this research. ultimately, I must admit that the focus on CB1 was a choice I made to reach a broader audience but I wouldn’t argue against it not being the most scientific focus.

  2. Hey Ethan! Thanks for such a great summary of the highlights from the Chen et al. article. It’s exciting to think about the implications of the work they did, both in terms of increasing C. elegan lifespan for experimental purposes and the possible clinical applications for humans using the conserved pathway. You mentioned in almost a tongue-in-cheek way that the media could try to spin these findings into radical statements about the health benefits of marijuana, and while I also feel that the research can’t match those headlines just yet, I am definitely interested in just how the work you summarized will reach the clinic. As you said, JZL184 isn’t naturally occurring within C. elegans, but its presence produced an increase in their lifespan. However, since it does exist in mammalian systems, I’m interested to see if the same pathway can be manipulated for things like anti-anxiety medications– especially because medical CBD is gaining popularity for this purpose. We already know that JZL184 decreased synaptic activity in the C. elegans, so, this “anti-excitatory” property of the omega-6 derivative (as you called it) seems to point to a neurological application, wherein the same pathway could strengthen the inhibitory properties in the amygdala to help treat anxiety. I don’t have very much neuro-knowledge, but that’s where my thoughts wandered first while reading your review. If you have any thoughts about the viability of JZL184 or this pathway in medications for mental health (or literally anything else) I’d love to hear your opinion!

    1. To be honest, I haven’t thought too much about the neurological effects this pathway could have in humans due to the limitations of my own knowledge and research in the field but I agree that the possible cognitive regulatory effects of this pathway would probably be the most efficient route to take after this work with regard to medicine in humans. My initial thoughts regarding the viability of JZL184 as a drug primarily relate to how well the dose can be localized to areas of the brain. It’s difficult to predict what the effects of inhibiting the activity of MAGL would be even if the inhibition was isolated to the brain; however, this study does at least raise the possibility that an increase in monoacylglycerols like 2-AG could produce a positive effect on humans.

  3. Great job giving us the main takeaways from the Chen et al. paper. I noticed that in your figure it shows there were 95 small molecules tested for longevity effects in C. elegans. Most either had a negative or relatively neutral effect on lifespan, which should be expected for introducing foreign substances into an organism. The diagram shows a similar trend to point mutations, in which most changes are either harmful or neutral to the organism. However, there were a couple dozen which significantly increased longevity, including JZL184. My question is, did you find any studies that investigated the mechanisms for other lifespan-increasing molecules? There even seems to be one which increased lifespan more than JZL184. I understand the authors chose JZL184 because it is involved in a conserved pathway in humans, but I’d be interested to see the results of other C. elegans studies of this type.

    1. In terms of the red Hit-compound that is shown above JZL184 on the diagram, the authors make no mention of any compound that exceeds a 60% lifespan increase so it was my interpretation that the dot there was something else. The authors did briefly touch on other molecules tested like WWL154 that also were an analogue part of this pathway but didn’t yield the same high results as JZL184 did. In terms of what I was able to find it seems that SH inhibitors are the current hot topic in C. elegans lifespan regulation so most work only revolves around testing similar small molecules like JZL184 and WWL154.

  4. Great post Ethan! I think you did a fantastic job picking out the key points from the Chen et. al article, and I would agree with the previous commenters about how exciting this sort of research is, and am very curious to see what could come next as a result of these findings. My main question that I have after reading through is about how JZL184 and some of the other small molecules are actually able to increase the lifespan of C. elegans. The authors don’t seem to really touch upon the pathways involved with some of the other small molecules (and Brian brought up a good point about how this is likely because JZL184 is involved in a conserved pathway in humans), but I’m still unsure of the JZL184 pathway’s capabilities. It’s clear that the main result of the introduction of JZL184 in C. elegans is the up-regulation of CB1 receptor activity, which in turn down regulates synaptic activity. How is this down regulation capable of increasing the lifespan of C. elegans (even if by only a few days)?

    1. Unfortunately I really don’t have a solid answer as to how this actually happens but I believe I can speculate a bit with what I know about the pathway and some general evolutionary biology ideas. There is a good chance that not all systems in the body are necessarily worried on increasing the natural lifespan of the organism so that this JZL184 pathway is part of a bigger regulatory system that balances ability to respond effectively to stimulus against maintaining the greatest possible lifespan of the organism. You could argue that this experiment could only be reproduced in a lab setting because this molecule that may just sedate the organism and slow down their natural life cycle wouldn’t actually end up increasing the average lifespan of the population because more organisms die of outside influences. I understand where your question is coming from since this idea of a miracle molecule that just increases life for free doesn’t really hold up after thinking about what these experiments are really testing and what else must be going on in nature for this not to have just evolved on its own in the first place.

  5. Hey Ethan, amazing job summarizing some important points of this Chen et al. paper and providing plenty of your own thoughts as well. Increasing lifespan is always a highly desirable topic and attracts many individuals. The fact that the carbamate JZL184 was seen to increase lifespan by almost 50% is mind blowing. This is less impactful for something like C. elegans with a relatively low lifespan, but that would be significant if you could increase human lifespan by even a fraction of that amount. The fact that JZL184 is only one of many compounds that increased lifespan by a decent amount, it makes me wonder how many potential compounds there are to increase lifespan in humans. Of course, much more research needs to be done because messing with pathways can always be risky due to unforeseen consequences. I’m curious at how well this will translate to larger organisms. I’d be interested in seeing this experiment performed on other animals with longer lifespans to see potential long-term consequences. I’m also worried about harmful neurological effects that may surface. Nonetheless, this paper still excellently shows the importance that 2-AG has on lifespan.

    1. I agree completely that one of the most important parts of future work will involve scaling finding up to more complex organisms and it is really difficult to predict what direction that kind of research with take. While the upside of looking at this SH pathway certainly is that it is largely conserved in humans as well, there are so many different factors between C. elegans and humans that I would be surprised if by the time research getting to human testing phase it uses a small molecule that is anything like JZL184 just due to how much change and work this research will have to trudge through from here on out.

  6. I really like how you broke down this paper and made it easy to follow with their science and conclusions and the results they present are very interesting and could possibly lead to a breakthrough in understanding aging in C. elegans which might then be applied to humans down the line. The one thing I find really interesting about this paper is that seem to state how they discovered the pathway which leads to aging in C. elegans and how the main target of JZL184 is very different from humans than in C. elegans. They go into how they identified FAAH and more specifically FAAH-4 as the target by first running a BLAST and finding there isn’t an enzyme like MAGL in C. elegans, but they never explain why they started this BLAST to begin with and why they started looking at JZL184’s targets, so is this something the group focuses on? The paper also concludes with the idea that this supports there is a pathway which is responsible for aging in both C. elegans and mammals and though it obviously won’t translate nicely to humans that this is a start in understanding. I know C. elegans are often used in biological studies, but this seems to be a rather large leap especially because the pathway is different and even this research shows that they’re very different. Perhaps this pathway is simpler than in humans, but even the authors address complications, so how easily do you think this translates or is this more of a stretch that could be true but is much more complicated than how the authors conclude?

    1. My interpretation of the order in which they actually developed this focus on JZL184 and FAAH-4 is that they first found that JZL184 was the most effective at increasing lifespan and then performed some sort of binding assay with JZL184 and all of SH they found to be expressed in C. elegans and then the FAAH-4 enzyme came up as the primary target. I do agree that the authors kind of brush over the actual degree to which FAAH-4 resembles the MAGL analogue in humans that they had in mind so its hard to imagine that in human systems the analogue to the SH they spent so much time examining might not actually be the most effective regulating target. The authors do mention some work they did with other SHs that showed a high affinity for JZL184 like Y53F4B.18 which also yielded promising results when put through the same tests as the FAAH-4. This leads me to believe that even with all differences in scaling these results up to a human model, JZL184 will probably still be able to inhibit the enzymatic activity of some hydrolyses that will likely provide some sort beneficial effect on human lifespan to some capacity.

  7. Hi Ethan! I think you did a great job of explaining the goals and experiments of this paper and relating it back to topics we have learned in class. I am a little confused about how the 95 SH-inhibitors were chosen by the authors? Perhaps I just missed it in your explanation, but is it just because those are all of the known inhibitors and testing all of them just led to a more impactful paper?

    I was shocked after reading how the authors discovered that JZL184 is responsible for ~45% lifespan increase. Of course, C. elegans are one of the more simple organisms to use for experiments such as this, but do you think that testing this experiment in human cells would greatly differ because of the complexity that is inherently present in these cells or is the similarity in the C. elegans and human genome sufficient enough for any further conclusions?

    1. With regard to the 95 SHs tested, the paper spends much more time going into their process which wasn’t reflected in my summery of the paper, basically the authors first tested for the presence of 200+ known SH enzymes from some library in C. elegans and then came up with only about 95 of them that were expressed in C. elegans naturally. In terms of how translational this research really is will probably depend on more than just the conserved regions between human and C. elegan genomes, that the authors show is certainly present, because of just different the factors surrounding a normal humans life and a lab bound C. elegan.

  8. Ethan, this was such a cool paper. I am personally interested in the biochemistry of longevity as it is such a complex network and it appears that almost everything has an effect on lifespan. That being said, I am curious if the observations made in this paper is a sort of misstep of biology from our perspective. I can’t help but wonder if the authors simply stumbled upon an “intentionally” selected for regulatory mechanism through which C.elegans limits its life span as to not compete with younger generations. To me this seems more realistic than the alternative that the inhibition of FAAH-4 drastically increases longevity for X,Y,Z reason. It seems to me that this would be an extremely selective adaptation if that were the case. I would be curious to see how control worms compete with ∆FAAH-4 knock outs. Proof that the control condition is desired could elude to this being an isolated trait not conserved in Human biology.
    I was immediately interested in the translational potential for this treatment, however, upon a quick BLASTp it appears that the C.elegans protein only shares 32-34% homology with human fatty acid amide hydrolases. Obviously Humans and C.elegans are extremely diverse and evolutionarily speaking are quite distant, however, if a mouse equivalent of JZL184 were developed how would you design a study to evaluate both the efficacy of the treatment, if it exists, and the mechanism through which the inhibition of their target would mediate such increase in longevity? Obviously, the physiology of humans is much more complex, and due to this, I find it difficult to believe that disruption of monoacylglycerol lipase activity would promote such drastic increases in longevity in humans. I am also curious about your thoughts on transcriptomics analysis of the worm transcriptome to identify differential gene regulation in the presence of the inhibitor. I am curious as to what pathway dysregulation might be at play.

    1. I do agree that a part of biology is based in death and that this pathway is originally meant for senescence so then disrupting it may not be so much increasing the “lively hood” of the C.elegans but simply restricting a system meant to bring about a natural death. Human society has vastly out paced human evolution which is why we find ourselves so maladjusted to our current environment in a number of ways this means that, in my opinion, humans have reached a point that is above natural biological evolution. In this context, the difference between a miracle drug and something that merely inhibits our natural death timer is only academic. Unfortunately I can only speculate how this research could be effectively transferred to a mouse model because they same procedure would require so much more time to undertake in longer living animals, especially if the drug is just too effective. Ultimately, there is no way to be sure this research could lead to any increase in human lifespan but I do have some confidence in it at least leading to some increase in quality of life for some people.

  9. Hey Ethan, I really enjoyed reading your post, and I think you did a great job selecting highlights from this rather lengthy paper! I especially like the connection you made to class concerning omega-3 and omega-6 fatty acids. It really reinforces the point that a balance between omega-3 and omega-6 derivatives is necessary, despite the list of negative effects that we saw associated with omega-6 derivatives. My question for you is about the section of the paper where they discuss FAAH-4 deletion C. elegans. They say that lifespan was increased in these animals, and that the addition of JZL184 further increased lifespan. Maybe I missed something in my read of the paper, but my initial thought was that JZL184 may inhibit some other similar enzyme that could cause this lifespan increase. But, after reviewing an earlier passage the authors show that it does not inhibit other amidases. Do you think that JZL184’s partial inhibition of Y53F4B.18 has an effect on lifespan? I am having trouble thinking of reasons why JZL184 would increase lifespan in the absence of FAAH-4. Thanks!

    1. Yes, I didn’t really explain the work they did with of SHs very well in my review but they also ended up finding that JZL184’s does help to increase lifespan by inhibiting Y53F4B.18 as well as FAAH-4 by showing a compounding effect when both those SHs are knocked out and assumed that JZL184’s effect on Y53F4B.18 was at least partially responsible for the difference in lifespan they noted between faah-4 and JZL184 supplemented C. elegans. Another thing that further complicated the pretty picture the authors try to wrap-up their paper with is the strange increase of appetite that was noted in faah-4 C. elegans but once they were given JZL184 the increase in appetite was lost, so it seems like there really is a lot more going on behind the scenes in this pathway that we have yet to really figure out.

  10. Hi Ethan!

    Great post and really interesting paper! First, I just want to say that I think it is really amazing that techniques like ABPP make studies like this possible – where the authors are able to take this almost global approach to their question. It feels very thorough. I also thought it was cool that the authors were able to use a pharmacological similarity to deduce an unexpected functional or metabolic similarity in the C. elegans FAAH-4 and human MAGL proteins. This ends up being a major point in their discussion, though it seems to be a finding they almost stumbled upon in attempting to answer a less specific question about longevity. The authors suggest that this could be used more intentionally as a method for identifying orthology across species. Did you come across any other papers that have done this since this publication? Or do you know if they were the first to do this?

    On a separate note, towards the beginning of the paper, the authors mention that conventional libraries are composed primarily of reversible inhibitors rather than the irreversible library used in this paper. While I certainly understand the experimental value of irreversible inhibition, do you think that there would be any advantage to reversible inhibition of these targets? Or is irreversible inhibition preferable because the effects are longer-lasting? The authors emphasize the importance of this distinction in facilitating data collection, but not necessarily in principle.

    1. As far as I have seen, there has not been any more noteworthy publications branching off from the paper in the 8ish months its been published. In terms of FAAH-4, it was known for a fair while to work in a similar way to MAGL as its iso-forms are expressed in humans and were known to act in a similar way to MAGL just on a much lower scale. I believe the finding that FAAH-4 is significant in the C. elegans to the point of fulfilling the equivalent role of MAGL is a novel finding in this paper. Through the authors experiments it looked like loss of FAAH-4 didn’t cause any observable issues in the C. elegans which leads me to believe that there might not be too much of an issue with significant inhibition in a human system. With that in mind, I find that irreversible inhibition would make for a far better drug as it could be given at a much lower and controlled doses and be a far more effective inhibitor of FAAH-4 while not potentially disrupting other natural systems in the body.

  11. This is a very intriguing post Ethan. Prolonging one’s life might be one of the most interesting and prevalent goals in human history. It’s also pretty astounding that supplementing a single small molecule inhibitor could reliably increase an organisms lifespan by almost 1.5 times. One thing that confused me was the decision to begin the experiment by testing with Serine Hydrolase enzymes in the first place. Is there any reason why they chose SH inhibitors to test in the first place, other than the fact that they are prevalent in C. elegans? I was also confused as to how exactly C. elegans ultimately had their lives extended by JZL184. The effect of its inhibition of FAAH-4 on the pathway was a decrease in synaptic activity. Perhaps this is more of neurological or biological question, but I wasn’t exactly sure how this would translate to a longer lifespan in the organism.

    1. I believe the authors started looking into SH enzymes because they were a family of enzymes that were known to be fairly well conserved between humans and C. elegans and there was already a great deal of work done on the topic that they were able to work with. Ultimately, the authors don’t really start to address where this life increasing ability is actually coming about within this paper so all i can really do is speculate on the topic. From what I can tell, the effects of JZL184 primarily lead to a slowing down of the C. elegans systems, decreasing the speed at which it can perform processes. It is possible that this slow-down mechanism can also delay the C. elegans natural aging cycle so that they simply take longer to reach the end of their natural life cycle.

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