 Okay, welcome back everybody, this will be our third and final session of this special symposium, I think we have five speakers in this session, so up first it is really my pleasure to introduce my good friend Michael Polymenus, Michael is at the Department of Biochemistry and Biophysics at Texas A&M University and he is going to be talking about work that that his lab has been doing on aging and yeast, coupling one-carbon metabolism with longevity. All right, you can see me and I presume it looks good. Okay, good, let me get my laser pointer. All right, so I would also like to thank the organizers for the chance to present our work. In this talk, I will mostly tell you about our work on the two paralogues that I have somal protein, RPL-22 in yeast, specifically how changes in methionine and Syrian metabolism explain the different phenotypes of these paralogues, which include longevity. In unpublished work, I will also tell you how we have followed up these results to learn about the more general role of this metabolic pathway, one-carbon metabolism in longevity. So the work has been a collaboration between my lab, Brian Kennedy's lab, first of the back and now in Singapore and Matt's lab at the University of Washington. So here, I just collected all the available data for ribosomal protein mutant phenotypes from yeast to humans. The second column shows the number of RP genes for which mutations exist and the third column, the number of distinct phenotypes that arise from this mutation. So there are many, many phenotypes. However, I'll also note that yeast does not have more ribosomal proteins. It just has more genes that encode them because most RP genes in yeast are duplicated, encoding highly similar paralogues. The last column shows the most common phenotypes in each species. So the number of phenotypes that arise from RP mutations is very large, as I said from here in hundreds, but the most common ones are manifestations of hypo proliferation, such as decreased proliferation. If you read these descriptions here, smaller cells, organs or organisms. So however, as I said, the spectrum is very large and in yeast loss of the one of the two paralogues can lead to very specific phenotypes. For example, losing RPL22a sensitizes cells to reactive oxygen species. They have defects, alterations in cell cycle progression, and of course, they live longer. In this case, we mean replicative longevity, so they divide more times. None of that happens if you lose the other paralogues, the RPL22a, even though the two of them are actually very similar, the only different at a few amino acids. So we want to know why that is. You actually probably see here. All right. So first, we isolated ribosomes from asynchronously growing cells and looked at the relative abundance of RPL22 by mass spectrometry in wild type cells and cells that lack one of the two paralogues or both. The double mutant is alive, so RPL22 is one of the few ribosomal proteins that's actually not essential. But although it is alive, the double mutant divides slower than either of the symbols. Loss of the RPL22b doesn't really affect the doubling time that much. It's the same as wild type, but RPL22a does. So wild type cells then, if you look at the relative abundance of the two proteins, have a lot more RPL22a than b, so here in pink. Now if you delete RPL22a, they try to make it up and they make more b, they don't quite catch up. In the double mutant here, the levels you see are obviously the error, the noise in our measurements here. So that's the relative abundance in cells of the two proteins. What happens to the overall protein synthesis capacity in these mutants? So to answer, we quantify the incorporation of a methionine analog, HPG methionine, into newly synthesized proteins. Through click chemistry now, if then HPG fluoresces, and then you can quantify the incorporation by simply looking at cells under the fluorescence microscope zone here, or by quantifying the data with flow cytometry. So protein synthesis is lower in RPL22a cells by about 50% compared to wild type cells. The double mutant is also lower, and there is no drop in protein synthesis in RPL22b cells. So how does this drop now in protein synthesis lead to all the specific phenotypes that we talked about about RPL22a? So first we look at the changes in the steady state mRNA levels in RPL22a versus B mutants. There were not many mRNAs affected as shown here on the heat map. You see many rows because we did this in a cell cycle dependent manner. We isolated all these libraries. We made all these libraries throughout the cell cycle starting in early G1 all the way down to mitosis. These were highly synchronous cells. And so every row is a different cell cycle point starting again early G1 all the way to mitosis. So very few targets, less than 100, and the only gene ontology that wasn't reached is this one, glucose metabolic process, which really says that they expressed less genes that are related, key metabolic enzymes that are related with glycolysis. So that in the RPL22a cells. So lower glycolysis in RPL22 mutant cells. But then we really cared about the translation only control the mRNAs. And for that we did ribosome profiling from the exact same libraries throughout the cell cycle. And we compared the relative translation on efficiency between RPL22a and RPL22b mutants. Again, there were very few genes that were affected about 80 or so. And most were downregulated you see here. And the only gene ontology meaning sorry downregulate RPL22a mutants. And the only gene ontology that was significantly repressed was this one sitting family amino acid metabolic process. And so in the diagram at the bottom here any all the blue gene names highlighted here all of them were repressed translationally in RPL22a cells compared to the parallel mutants. So here is where we got a little excited because we saw met three which at the time was the only one known in this from this list that was known when deleted the cells live longer. But then all the other phenotypes if you see what they do they are actually part of the larger group of one carbon metabolism. And they are needed to make a variety of things that you need to grow and divide including nucleotides amino acids and also lipids not shown here. So that could explain very well the the defecting growth and and cell division and in the cell cycle phenotype which we had seen. And or for example they could also explain the the effect in the in the with a reactive oxygen species response because of course you need the same pathway to make glutathione. And we we confirmed that the levels for example of met three are lower in RPL22a cells compared to B cells and actually the drop that we saw in this immunobloids was about the same as we saw with the translation efficiency from the Aragoson profiling experiment. So this is kind of interesting also because it ties with RNA-seq data that you saw in the previous slide of course serine which is the input of one carbon in this folate-based metabolism comes from glycolysis through three phosphoglycerate. So they seem to tie together now. All right then we looked at metabolite levels from asynchronous cells in this case and we compared the RPL22 versus RPL22B mutants. And so this was untargeted metabolomics and then we the metabolites whose levels change significantly we use them as input in the Metaboanalyst platform and identify the relevant pathways for enrichment. And the results were actually very concordant with what we saw with RNA-seq and ribosec profiling. So glycolytic intermediates again were significantly down in RPL22a mutants compared to wild type or RPL22B. You can see three phosphoglycerate which as I said is where you make seeding from. That was down in RPL22a cells and also we saw a bunch of amino acids here including glycine and serine metabolism that we saw previously or methametabolism. So it all made sense. We wanted to be sure so we did some targeted amino acid measurements and some here on the right and again glycine was down and serine was up. And that's kind of interesting because it's actually important because a low glycine to serine ratio is diagnostic for a low flux into one carbonyl metabolism. So that was consistent. We also found that tryptophan levels were lower and you can ask me about that later. All right so MET3 was a known longevity gene. What about if the whole idea that this all this gene expression changes and metabolite changes a heat to one carbonyl metabolism there may be other genes in the pathway could also be longevity genes. And sure enough here's shown two of them as HM2 and NAD17 in the in the replicative lifespan assaying. So that was good and in fact there are more over the years that actually some done recently and some that Matt and Brian have been doing for many years. So anything you see here shown in bold when you delete it in yeast increases lifespan and the in capitals are the equivalent names in animals in mice. So this is again I hope I have convinced you at this point that we really identified why RPL22a cells live longer and all these phenotypes that you see here can be explained simply by changes by downregulating to one carbonyl metabolism. And they also point to something about one carbonyl metabolism. The more you think about it I mean it's really an excellent platform to integrate nutrient status protein synthesis and cell divisions and longevity. So if you look at the if you compile all the data not only from yeast but also from worms again whatever you see here in bold has already been shown to be a longevity mutation always loss of function. So the others doesn't mean they are not some of them have not been tested the ones that are not involved. So really there is quite a few genes here in the pathway and for many of them there are actually chemical inhibitors and including this one of the highlight here the target for DHFR which of course is methotrexate. And just to to remind you how methotrexate works what I didn't show in the previous slide is that and I didn't mention is that the useful form of forlate for all these reactions is actually tetrahedral forlate and all of them use that. But in this reaction where you make a thymidylate the tetrahedral forlate gets oxidized to dihydrofolate which has to be reduced again by DHFR to make THF. So it's this reaction that methotrexate blocks and in effect causes a global forlate deficiency so THF levels you know are down for all of the all these reactions. So we added with test virus concentrations of low dose methotrexate in both yeast so here n worms and in both cases we found that low doses and we're talking about one micro molar extended longevity in both systems. This is a replicative longevity and in worms it's actual chronological lifespan. So that was exciting what about if it's true for yeast and worms what about mice we really want to do that we have not done that so what you see here is a meta-analysis of public data. Methotrexate has been around for 70 years but it has not been tested in longevity but there have been many toxicity studies that have been done decades ago including for long-term low dose exposure to methotrexate. Again at high doses methotrexate is used to treat cancer but at low doses it's actually still a first-line treatment for rheumatoid arthritis. So there is a lot of data around and from all the experiments done in the 70s with methotrexate given to mice from week seven to week 120 there was no toxicity associated with these doses they up to 20 parts per million in the food and here I plotted the data and in five so in all these conditions and in five out of eight treatments that they had used in all in these old toxicity studies the lifespan was actually increased and in the example you saw here it was significantly increased the p-value is less than 0.05 based on the low grant test. So what is actually astonishing about these old experiments is that the treatment they started treatment very early in life at week seven and it is known that for such an agent like such as methotrexate the toxicity is actually very high early in life in fact the LD 50 at week 10 is almost an order of money to higher than week 50 or something if you give it. So one wonders what will happen if you actually start even later in midlife and see if you say even a more pronounced effect. But at this point I will close and I highlight again what I stated about the ribosomal protein mutants we think the one carbon metabolism fully explains the longevity of VARPIL 22A mutants and other phenotypes. I showed you some initial evidence for about genetic and chemical interventions in the same pathway and I will close with a realization that whether we like it or not we are all on the anti intervention to prevent because you know the U.S. and many other countries they supplement staple foods with folates and that makes sense because folate deficiency during pregnancy leads to birth defects. So that explains why the fortification of let's say flour with folate is beneficial but if we are even remotely correct maybe there is somebody thinking that needs to be done about you know later in life effects from a public health point of view. So again thank you for listening I'll be happy to take any questions. Thanks Michael that was great and you know I mean I've always been sort of confused why the folate cycle and methionine cycle keep coming up over and over and over in these genetic screens in yeast and C. elegans in particular for longevity factors and so it's nice to start to see this maybe moving towards a more mechanistic understanding of what's going on. So there are a few few questions in the chat so Alex Chen is asking doesn't giving massive doses of B. vitamin significantly affect 1C metabolism and he also asks whether studies have been done on folate inhibition and lifespan. So okay I'll start with the second one so folate inhibition and lifespan as far as I know methotrexate again this is the slides I saw in the end it has not been done so specifically for other antifolates I don't know I don't think so I have not seen them. In people that have been observational studies so people who take it for you know rheumatoid arthritis they do appear to have reduced mortality especially from cardiovascular disease there was a big clinical trial that was done to see if that correlation holds. If you have another if you had a heart attack methotrexate will not inhibit you will not help you from not getting another one so whether you have an overall benefit I don't know at this point but that's clearly something we want to do but I don't think it has been done in longevity studies I have not seen it I would love to see it and I forgot about the first question about vitamin. I think the question is more whether high doses of folate will affect 1C metabolism and Alex also asks whether it might have an impact on lifespan extension from CR so sort of going the other direction so the other direction I'm sorry and I was going no no he asked about both but I think that was the other half of the question so I don't know actually if anybody has supplemented fully with folate and then see what happens I don't I have not seen it but I don't know I could be wrong so any standard diet has folates as far as I know even in mice I mean it is folate rich to fully test it I mean to go extra I don't know I have not seen it so I was that's it I don't know that's the answer it's an interesting question and just to give a little bit of context so there is data in C. elegans on on folate restriction by inhibiting folate production of the microbiome of the microbiome yes folate and that does extend lifespan yes the other sort of interesting connection here is David Gems published a paper that metformin's mechanism of action in worms at least is through folate restriction and I think there's a little bit of data in in mammalian cells that metformin affects one carbon metabolism although people there have proposed amp kinase as the mechanism so again there are all these connections and it's really neat to start to see start to see some some some mechanistic insight um Jess has a question uh about why it is mechanistically do you have any idea why it is that in the the RPL 22a mutant that you get differential expression of these one C metabolism genes is it a translational mechanism what's going on there okay so I on that one I'm biased because uh yes I think we do get mostly translational control uh this is that that was the output of the assay because we use ribosome profiling directly to measure that which directly reflects translational in physics how that happens um I I don't know about half of them have upstream open rendering frames we want to test them in more detail to see why what's the actual mechanism that's that's a big thing that my love wants to do but I don't have any more information about the precise mechanisms but I do think it's a it's a about the cis elements or if there are even any other mRNA binding proteins unbind in a particular way and then keep it some of them but not others uh we want to know but I do believe it's it's translational in the case of RPL 22a and and another the the second part of Jess's question is is there any connection between uh one C enzymes and autophagy or this RPL 22 folate mechanism and autophagy that you know about I I don't know I I was saying I I don't know something to be studied in the future okay uh we're at time so thank you Michael that was great thank you thank you all the questions um so our next uh presentation this is this will be a video recording because our speaker uh couldn't be here uh in person this so we will have a video presentation by Nicole Jenkins who is at the Melbourne Dementia Research Center uh University of Melbourne Australia there will be no questions after the video presentation so we'll just go right to the next speaker and Nicole's talk title is changes changes and ferris iron and glutathione promote ferroptosis and frailty in aging C elegans good afternoon my name is Nicole Jenkins I'm from the Melbourne Dementia Research Center in Australia I would like to thank the senior editors Jess and Matt for putting this special issue and symposium together I'm sorry that the time zone differences mean that I cannot join you in person today I'd like to begin by acknowledging the senior authors on the paper my mentors Gavin McColl and Ashley Bush as well as all of the other authors who contributed to this research our group is particularly interested in the impact of iron on aging iron is a trace metal found in all animals and is essential for life with all the eukaryotes exploiting its redox chemistry for various activities such as dna replication and growth our previous researchers caused us to question whether an early developmental dependence on iron for reproduction and cellular biochemistry represents an ancient and conserved liability in late life iron exists in cells primarily as either fe 2 plus known as ferrous iron or fe 3 plus known as ferric iron labour iron in the ferrous form is a potent oxidant which can cause substantial stress to the cell unregulated redox cycling between fe 2 plus and fe 3 plus is a known overt cellular stress this kind of biochemistry is mitigated by a range of storage and chaperone pathways to ensure that the labile or reactive iron is kept to a minimum intracellular iron concentration increases over lifetime in many species including humans and in particular within the brain this accumulation of iron may be an underlying contributor to the progression of age-related conditions such as Parkinson's and Alzheimer's diseases the recently described iron dependent cell death pathway known as fereptosis may be the missing link between oxidative stress aging and neurodegenerative diseases fereptosis has been widely studied in cell model systems with a focus on cancer biology fereptosis kills malignant cells but may also be inappropriately activated in conditions such as ischemic injury and neurodegeneration this cell death mechanism is executed by phospholipid hydro peroxides induced by either iron dependent laboxygenases or by radical mediated auto oxidation that is catalyzed by iron under homeostatic conditions the fereptotic signal is terminated by glutathione peroxidase 4 so known as gpx4 a phospholipid hydro peroxidase that uses glutathione as a cofactor while the signaling that regulates fereptosis is being studied in depth the role of iron load in this death signal remains to be characterised we wondered if the age-related increase in ferrous iron may also result in fereptosis during aging could late life iron dyshomistasis be sufficient to trigger fereptosis in late life and what role this might have in aging to study this we use the nematode model system of senorabditis elegans for those unfamiliar with this system many essential characteristics that are central to human biology exist in c elegans in addition many insights into the biology of aging had their genesis in the study of this model during aging c elegans exhibit characteristics of senescence seen in higher organisms including slowed movement and muscle wasting our laboratory has previously demonstrated that c elegans accumulates iron over adult lifetime we have done this using a number of analytical techniques including several synchrotron based imaging methods we've also shown that ferrous iron the substrate for fereptosis increases during aging by the research we have detailed in our elife paper we first began by modelling acute fereptosis in c elegans to do this we use the chemical compound dm which conjugates glutathione causing acute depletion glutathione is suggested to be the dominant coordinating ligand for cytosolic ferrous iron and is also the substrate used by gpx4 to clear the lipid peroxides that induce fereptotic cell death we found that treating young animals with different doses of dm reduced glutathione levels in a dose dependent manner we also observed a dose dependent effect on survival with approximately 50 of animals dying within 24 hours of exposure to 10 millimolar dm these results confirmed that treatment with dm represents an acute stress that reduces glutathione levels and causes death we then examined the changes in glutathione levels and survival after dm exposure during aging as the animals age the glutathione levels decreased these older animals with reduced glutathione were significantly more sensitive to dm mediated glutathione depletion these results suggested that older animals may be sensitive to cell death by fereptosis to explore this further we've employed two interventions the first intervention was to use sih an iron collator to reduce the labour iron pool importantly sih binds to iron in a redox silent manner so it should reduce oxidative stress within the cell the second intervention was to inhibit fereptosis using lipox statin a peroxyl radical trapping fereptosis inhibitor when we looked at dm treating animals we found that pretreatment with either compound improved dm resistance of adult c elegans with a more market effects in under sih treatment this increased survival in sih treated animals may be due to the increased basal levels of glutathione in these animals which is also preserved after dm treatment because we were interested to know if we're observing fereptosis we wanted to determine whether individual cell death precedes organismal death to do this we use propitium iodide to visualise moribund cells in vivo propitium iodide is a fluorescent intercalating agent that binds to dna but cannot cross the membrane of live cells making it possible to identify the nuclei of recently dead or dying cells this is shown in the image of the worm at the top of the slide when we treated young animals with dm and propitium iodide we observed death of individual cells within the animals prior to organismal death animals pretreated with both sih and lipox statin exhibited lower levels of cell death we also examined animals for evidence of cell death during aging we observed cell death in four six and eight day old adults this cell death was significantly attenuated by both lipox statin and sih treatment our results with cell death and compound treatment were also supported by changes in lipid peroxidation markers which i do not have time to discuss today lowering cellular iron suppressors fereptosis but the peroxyl radical trapping fereptosis inhibitors such as lipox statin are not expected to change iron levels we examined the impact of sih and lipox statin interventions on iron levels over lifespan in live c elegans using synchrotron based x-ray fluorescence microscopy the details of which can be found in our paper using these imaging methods we found that both total iron and arial density increased with age and control animals as expected based on our previous work sih dramatically reduced the arial density of iron and also reduced the variance within populations but this effect was not seen in lipox statin treated animals determining the level of ferrous versus ferric iron in a living organism is quite a challenge but can be determined using a technique known as phi zones that we have refined in our laboratory to measure the speciation of iron in c elegans zones refers to x-ray absorption near edge spectroscopy using fluorescence detection for visualization and directly assesses the in vivo coordination environments of metal ions in biological specimens since fe2 plus in the low bile ion pool is the specific substrate for fereptosis and we have previously reported that this rises with aging in c elegans we investigated both dm treatment and the impact of our interventions during aging the phi zones allowed us to evaluate steady state iron speciation to determine the ratio of fe2 plus to fe3 plus we found that dm increased the proportion of ferrous iron we also observed an age related increase in ferrous iron this age related increase in the fe2 plus fraction was normalized to that of a young animal by both lipox statin and sih treatments we've demonstrated the effects of both sih and lipox statin in adult animals we confirmed the expected effects of reduction of iron in the case of sih and of reducing cell death and lipoproxidation for both sih and lipox statin consistent with fereptosis but what about lifespan as you can see from the results presented on this slide both sih and lipox statin increased lifespan with an average median increase of 100 percent and 70 percent respectively however both of these interventions appear to affect the animals in different ways with markedly different hazard rates observed relative to control populations and also between the two different treatments what is difficult to convey in a paper is just how different the treated animals looked and behaved the sih treated animals appeared quite robust and vigorous just prior to death whereas lipox statin treated animals seem to be aging in a similar manner to control animals at an early age and then exhibited mid-life vigor which can be seen with the respective hazard rates in the other graph on this slide because of the large increase in lifespan that we observed and the striking differences in behaviour we saw in aging animals we looked at how our interventions compared to previous results in aging by employing the temporal scaling analysis published by Nicholas Straustrup and colleagues in their elegant paper from 2016 we thank these authors for sharing their data and methods with us to enable us to do this analysis these authors found that diverse interventions including temperature oxidative stress and disruptions of insulin like signaling pathways and other genetic interventions all altered lifespan by temporal scaling which they defined as an apparent stretching or shrinking of time for an intervention to extend lifespan by temporal scaling it must alter all physiological determinants of the risk of death to the same extent throughout adult life our results indicated that neither sih nor lipox statin altered lifespan by temporal scaling this suggests that neither intervention acts as a global regulator of aging but perhaps affects late life frailty instead it's worth noting that other interventions that were reported to affect lifespan outside this temporal scaling model by the previous sources had market effects on development and fitness which were not readily apparent in our animals interventions that increase lifespan in sea elegans often do so at the detriment of fitness and health span we made a number of comparisons regarding the fitness related traits such as body size fertility and movement between treated and controlled populations we found that iron collation with sih surprisingly resulted in an increase in the size of the animals with these populations continuing to grow compared to control populations indicating improved vigor to a later age in contrast lipox statin treated animals were not different to control animals with respect to size with respect to fertility the results shown on this slide indicate that early fertility is not affected by either iron collation or fereptosis inhibition however lipox statin does significantly reduce overall reproductive output relative to untreated animals whilst sih has no effect on the overall number of progeny produced the effects of both interventions on movement parameters were also assessed since peak motile velocity has been previously demonstrated to correlate strongly with sea elegans health span and longevity and may be considered to be the best estimate of health span in this model system as expected control animals showed a steady decline in maximum velocity as they aged treatment with sih and lipox statin markedly improved the maximum velocity of aging animals with increases also seen in distance traveled and mean velocity in summary the results that I've discussed today indicates that both lipox statin and sih protect against acute glutathione depletion with respect to iron levels only sih limits age related iron increase but both lipox statin and sih limit the increase of ferrous iron with age treatment to reduce iron and inhibit fereptosis both significantly increase lifespan but our treatments with sih and lipox statin seem to act at specific life phases rather than resulting in global regulation of aging our results indicate the targeting iron can improve health span with minimal fitness trade-offs limiting fereptosis in adult animals promote healthy aging and also has the potential in the treatment of age-related diseases and again I would like to thank the co-authors of this work listed here and also the Australian Research Council University Melbourne and Miller Foundation who have provided funding for this thank you all right and thank you to Nicole Jenkins for preparing that that video now we will return to our live programming so next up is Hosni Sharif McGill University in Canada and I see Hosni there and and the title of his talk is going to be senolytics to reduce pain and degeneration in human invertebral discs intervertebral discs I'm sorry hello everyone thank you for having me today and I'll talk about the role of senolytics in disc degeneration and low back pain I'll try to introduce low back pain for those who are not familiar with so it's a global age-related health problem approximated with intervertebral disc degeneration and experimented by about by 80% of individuals at some time in their life and it's the number one cause of years living with disabilities low back pain despite this problem it is little known about the cellular and molecular mechanism leading to painful degeneration leaving surgical removals and vertebral fusion in end stage of the disease as the most common treatment the person on the coast in reduced quality of life and in economic health to help their system are enormous and exceed as an example in US 100 billion per dollar so here is here is an image of the vertebral disc non-degenerate the red is the vertebral body and yellow is the two discs commonly the disc is divided into the same zone of cartilaginous template not shown here but the most important for us here is the outer annulus vertebrosis that will be labeled AF through the representation and the central gelatinous Np which is the old arc with characteristic different cell population with different biochemical and bio-mechanical properties with an increasing proteas protection and planetary factor discs start to losing cell proteoblackin collagen network and mechanical property this can i interrupt you for a second i'm sorry your voice is kind of muffled is it possible to speak directly into the microphone yes so i'm trying is it better now is it any better it's better yes thank you yeah okay i have to raise my voice i think so yeah uh the stress of the major catabolism when it's over catabolism when it's overcome the anabolism it leads to more production of those cytokine chemical and disc degeneration and here it's an image of a disc degeneration we found also an increased number of senescent cell in the general disc it is report our reduced cell senescent just faculty for people then so it's a reversible cell cycle arrest that can be coded either for duplicative senescent case of aging by telomere attrition or by stress inducing premature senescence which is in the case of our disc our most common that is coded by external or stimuli like oxidative stress you know that's the agent so the cell became resistant to up absorbers with cell size and morphological alteration and that's not regulation of the cycle and depending kidneys inhibitor like p16 p21 and activation of nuclear factor like an fgpb it's also to create an array of factor called senescent associated secretory phenotype shortly that includes cytokine chemical and proteases growth factor and angiogenic factor those factors will lead to more degeneration of the disc and low back pain it's reported previously the presence of senescent cell in the general disc and we quantify in our first paper the difference between np and af with the in the general and non the general disc and we found an increase of those in the number of senescent cell by p16 staining and an increase also in that factor as an example here to cytokine i6 i8 and 2 proteases mmp3 and mmp3 it's important here to highlight that those degenerate discs are coming from symptomatic donor they go through vertebral vision or the disc is placed so then we decide because of this variability that came from the inter donor with age sex or way of life to look further within the same individual to this one degenerate and non-degenerate i try to find if there is any difference whether in number of senescent cell accumulation or size factor release and indeed it is and find an increase in both np and af in that factor that we can relate to more to disc degeneration than any other factor then we also tested different senolytics we show here the two most potential that are vananin which is a natural senolytic with known anti-inflammatory property and ergie 112 which is a methylene 3a inhibitor it's the synthetic senolytic and it's acting through the blocking the interaction between p53 and mdf2 and has not any documented anti-inflammatory so both senolytic reduce the number of senescent cell when we compare them to the entreated group we tried to look at what are the pathway possible pathway that or that are affected gene by the two compounds we did a qpcr with the preset place with 90 gene that are mainly uh uh cell cycle gene or senescent gene and we find out that the vananin is treating uh affecting 40 genes with 30 upregulated and 10 downregulated while ergie affected only 8g with 6 upregulated and 2 downregulated this brought us to look at the possible pathway affected with those gene variability and we found that ergie is affecting only one pathway which is the cell death and survival with touching uh affecting mdf2 and p21 as expected for vananin it was more broader effect with the affecting three possible network with the cell death and survival connective tissue development and function and cell cycle we found that now that vananin can act through p16 by blocking the cycle-independent kinase to drive the senescent cell to apoptosis while ergie will act through its p53 mdmt known pathway to drive the senescent cell on apoptosis and the proliferation of the remaining cell then we looked at the media of those cell cultures and we confirmed the effect anti-inflammatory effect of vananin but so as I think we are also able to see that ergie was able to reduce the SAS factor there is difference between the two compounds in the types and the effect of the on reducing SAS factor which we'll go later after on that then you try to validate this result in vitro in a next vivo organ culture we want to see if the drugs can reach and target the senescent cells in human IVD tissue culture it takes vivo so we isolate three days from the same donor and we try to inject them one with the control and two with ergie and vananin and we will culture them for 28 days by changing media everything for days we perform a name pre MRI and post MRI so MRI is the clinical tool to evaluate health data to validate to see the effect of the treatment we also collect the media and measure the SAS factor release to see the anti-inflammatory effect of the both compounds if it validates and we did also histology to confirm if the senescent cells are removed after the treatment first we looked at the histology and we find that both compounds specifically remove senescent cells with not really big effect on the proliferation just non-significant effect on the proliferation they also remove the SAS factor in the media and at the MRI we compare the pre-treatment disc to the post treatment so we found in the control here that the disc lose of the proteoglycan while ergie and vananin significantly increase this proteoglycan compound that we can identify here by a higher red intensity compared to the pre-treatment for the both of vananin and ergie so to resume this section when the disc is exposed to aging or environment mold and cell if they accumulate senescent cells that decrease cytokine chemokine that will degrade the matrix and lead to intervertebral disc degeneration and low back pain we aim with those senolytics to remove or reduce the number of those senescent cells and reduce the factor they release for a better health of the disc if we act late let's say for a degenerate disc we think that we will reduce the pain but earlier treatment will be able to prevent degeneration and this low back pain now we are working with to validate those results in vivo our model is the spark neural mice which have a progressive age dependence and the vertebral disc degeneration and back pain that starts at four months we looked at the senescent cell in spark neural mice and we compare it to a type and we see a accumulation of senescent cells in the degenerate IVD from spark neural and we test the two senolytics in the next vivo culture from nine or nine months old spark mice and we found that both senolytics reduce significantly IL-6 which is one of the main fat factors with those results we encourage to do the evaluation of the pain behavior in the animals we use for that the grip strength to measure the axial discomfort and the von Frey filament and acetone test to measure the radiating pain so we give this mice by oral gravage weekly starting at five months the two drugs and the wild type and spark were not treated with the senolytics so we see here in black the spark and in green the wild type and you see that there is more pain for the spark neural mice and we improve with a vanillin in blue and energy in it the grip force and reduce the discomfort the axial discomfort for those animals after two months of treatment we also reduce the pain the radiating pain by von frey for and the and the sensitivity to the cold with acetone test after two months of treatment also so to resume here we found that the two senolytic algae and vanillin first they both specifically tarred and killed senescent cell or in vitro and x vivo a vanillin through p16 and energy through p53 mdm two interactions to activate cycling-dependent kinase and direct those senescent cells to up of those they promote proliferation of the remaining cells and decrease that factor and pain mediator and protease protease which will induce an extracellular matrix tentative we validated also the specific compatibility and show an improvement in the behavioral sign of pain in vivo we are currently measuring the sas in vivo the effect of the two compounds and evaluating the pain in a female cohort so to see if there is any sex difference we also try to change the treatment whether as presented before so early treatment versus later treatment to see if the grafts can prevent the disk degeneration or it's treated for the older animal we also try now a combination treatment because we think that both compounds may target different cell population and the combination of the treatment will lead to better results I would like with that thank all the lab members that contribute to this work and our collaborator and the agent the funding agent and you for your thank you thanks haze that was really interesting so we have a couple of questions the first one is asking about a vanillin administration for patients and the specific question is what do you think would be the best way okay the question was about what would be the best way of administration of oh it's my connection of ovanillin for patients so that's a good question so we try with the animal model uh laurel gavage uh because we think that it will be maybe damaging the disk by needles when we try to do injection in the disk this will be uh uh to test now we show that with the oral gavage we have an improvement of pain behavior this will be an interesting way but uh i'll think that also ideally we if we could do a local injection or a local uh let's say diffusion of the compound that would be more perfect because it will be on site and it's a local uh i would say local will be better than systemic in this case but we're trying now systemic and it's showing a good result that makes sense uh the other question is also related to vanillin and are you aware of other groups that have published on vanillin senotherapeutic effects um is there any epidemiological data uh to suggest this relationship so the the thing on senolytics no but curcumin war there is some publication on curcumin and we did also publish on curcumin and which is the ovanillin is a metabolite of curcumin and they are shown senolytic effect uh for that but the thing is curcumin has a very low solubility and had some problems for treatment that's why we opt for vanillin that shows first better effect and lower toxicity to curcumin and yes there is if i'm gonna make sure yes there is some result on curcumin not vanillin but not on epidemiologic at that but you are like really at the early step uh the good thing is an FDA approved compound that makes things more easier after if you want to do testing like validate this as a drug and then a sort of related question to that is have you checked to see if there are decreases in senescent cells in other tissues from these kinds of treatments uh not for we just did for the disc but it should be a good we do on bone i think we have just the preliminary data that shows the decrease with those senolytic and in cartilage uh with uh congosat but just early in vitro study preliminary standard just but now we didn't really look at other tissue okay and then the the last well another question is what do you know about the mechanism of action for these commas the first one i think you said the rg drug is an ndm2 inhibitor do you know the mechanism is for vanillin for targeting senescent cells for now we know that it's acting by blocking those cycling depending on it but honestly with uh you know a preset uh place that we already decide to choose what are the genes that we want to look at which is more senescent and uh cell cycle gene and you have this network it looks to be a broader effect with different pathway that could be implicated but we didn't really look at that i will say that will be a good uh project to look at the mechanism to confirm the how those vanillin is acting personally perfect okay so so in the interest of time we're going to move on to the next speaker there are a couple of questions for you in the chat don't mind answering those and thank you for the the great presentation very interesting you're welcome thank you next up is uh five os borbolis uh is that right it's a video presentation and then first we'll join us for questions and it looks like we're queued up okay so he is at the uh biomedical research foundation the academy of athens in greece and the talk title is mrna decapping as a modulator of aging and development good evening everyone this is fever borbolis from athens and i will presenting recent work from our lab concerning the role of mrna decapping as a modulator of aging and development in eukaryotes all rna polymerase two transcribed RNAs are characterized by a methylated guanine cup structure that is added cotranscriptionally at the five prime end this five prime cup defines key aspects of an mrna's life cycle through its interactions with various cup binding proteins in the cytoplasm cup to mrna's interact with the translational machinery to produce the corresponding peptides although removal of the five prime cup a process term decapping is the first step in the major five prime to three prime mrna dk pathway decapped transcripts are not committed to degradation but can also be stored in ribonucleoprotein granules maintaining the potential to be recapped and returned to the translational pool such cycle rounds of decapping and recapping provide an extra level of post transcriptional and regulation of gene expression mrna decapping is performed by an enzyme comprised of two subunits term dcp1 and dcp2 this hollow enzyme interacts with many additional cofactors that regulate its activity forming multi protein decapping complexes our work focuses on the regulatory decapping subunit dcp1 which is essential for the function of the enzyme previous work from our lab with enumatode worm c elegans has revealed that depletion of the dcp1 worm ortholog dcp1 affects various aspects of its physiology resulting in short-lived animals that are also sensitive to stress produce less progeny and exhibit developmental defects neuronal dcp1 in particular has been shown to impact developmental decisions by regulating the expression of heterocronic genes in order to explore the requirement of dcp1 in each tissue for a normal lifespan we generated a series of transgenic lines that over expressed a dcp1 gfp fusion in different tissues of a dcp1 newton animal out of the four major worm tissues expression of dcp1 in the nervous system was enough to restore lifespan to wild type levels expression in the intestine improved lifespan to a smaller extent while expression in the epidermis or the musculature had a negligible effect on longevity the fact that over expression of this dcp1 gfp transient exclusively in the nervous system was able to fully rescue the short-lived newton phenotype suggest a strong positive regulation of lifespan even though dcp1 is missing from other tissues we therefore examined the impact of our expression all tissue specific dcp1 gfp fusions on the impact of otherwise wild type animals in agreement with our complementation analysis neural dcp of the transient resulted in a significant increase of both median and maximum lifespan intestinal over expression caused a smaller extension while epidermal or muscular over expression had no significant impact however both neuronal any intestinal over expression of dcp1 failed to extend the lifespan of dcp2 newton worms which lack any decupping activity indicating that the mechanism that mediates longevity depends on a marine decupping the control of organismal lifespan through the nervous system usually involves the function of neurosecreted molecules that regulate the function of longevity regulating pathways in distal cells and tissues the most prevalent among these pathways is that of insulin IGF like signaling which serves a conserved role in regulating longevity from worms to humans C elegans possesses a single insulin IGF-like receptor that integrates input from at least 40 insulin-like peptides to control the localization and activity of daf16 phoctotranscription factor under conditions of reduced insulin signaling daf16 translocates to the nucleus and regulates the transcription of longevity related genes we found that in the absence of daf16 overexpression of dcp1 fails to induce longevity moreover silencing of daf2 that reduces insulin IGF-like signaling and results in extreme longevity completely mask the effect of neuronal dcp1 overexpression which failed to have an additive effect and extend lifespan further collectively our results indicate that the cupping in the nervous system act through insulin IGF-like signaling to regulate daf16 activity and induce longevity in order to identify the signal that mediates the relationship between neuronal decupping and insulin signaling we assess the effect of dcp1 depletion and neuronal dcp1 overexpression on the mRNA levels of five insulin-like peptides with a well-established role in neuroendocrine lifespan control our quantitative PCR analysis revealed that four out of five insulin-like peptides are unaffected by dcp1 levels however the abundance of in-7 mRNA was almost doubled in young dcp1 animals and further increased in mid-daged worms parallel to age-matched wild types moreover in seven mRNA levels were significantly reduced in mid-daged worms that overexpressed neuronal dcp1 in comparison to wild type animals to clarify whether dcp1 affects in-7 abundance transcriptionally or post-transcriptionally we monitor the fluorescence of wild type and dcp1 animals current and transcriptional in-7 promoter gfb reporter in various ages in agreement with previous reports we detected fluorescence mainly in head neurons and some intestinal cells with advanced age resulting in increased intestinal fluorescence for both strains even so total fluorescence intensity of dcp1 worms was created at all examined ages arguing in favor of dcp1 regulating in-7 expression at the level of transcription this effect however is restricted to the intestine and does not apply to neurons that had no difference in fluorescence between backgrounds so far our data have outlined a model where dcp1 levels in the nervous system regulated in-7 insulin-like peptide transcription in the intestine strongly implying the involvement of active neural secretion we thereby measured the fluorescence of the same in-7 transcriptional reporter in mutant ang-31 worms where dense core vesicle mediated secretion of neuropeptides is impaired in this mutant background dcp1 depletion failed to induce the in-7 transcription revealing that the observed intertissue communication is mediated by a neuropeptide released by dense core vesicles previous work has shown that the in-7 is a DAF2 insulin receptor agonist that activates insulin signaling but is also a target of the downstream transcription factor DAF16 foxo that inhibits its transcription in intestinal cells this self-regulatory mechanism though does not apply to neurons where in-7 transcription is not affected by insulin signaling we thereby argued that increased neural secretion of in-7 could account for its increased transcription in the intestine of dcp1 newtons indeed we observed that fluorescence of in-7 promoter transcriptional reporter is not induced in in-7 dcp1w newtons where no functional in-7 peptide is produced collectively our data shows that neuronal dcp1 regulates in-7 transcription in the intestine by affecting the amount of neurosecreted in-7 peptide since we did not observe any induction of in-7 transcription in neurons if dcp1 regulates the abundance of neuronal in-7 that should occur at the level of mRNA stability we thus used in-7 newtons worms to express an in-7 transient exclusively in neurons and assessed in-7 mRNA levels which thereby correspond strictly to neuronal transcripts both young and mid-dates dcp1 newtons exhibited a two-fold increased in neuronal in-7 mRNA levels revealing that indeed dcp1 deficiency stabilizes neuronal in-7 transcripts due to its self-regulatory function in-7 is considered as a carrier of foxy to foxy signaling that coordinates the rate of aging across the organism consequently the deficit in in-7 induction during aging that we observed in neuronal dcp1 overexpressing animals could be the reason for their prolonged lifespan to support this theory post-developmental silencing of in-7 increased the lifespan of wild type worms but failed to further promote longevity of the already long-lived animals that overexpress dcp1 in their neurons it is thus safe to conclude that low levels of in-7 trigger longevity during neuronal dcp1 overexpression even the strict evolutionary conservation of both 5 prime 3 prime mRNA decay machinery and the mechanisms of lifespan determination we reasoned that the connection between neuronal decupping and aging and not be restricted to c elegans we therefore studied this relationship in the flighters of lamellano-gaster since nauticals of dcp1 that is the fly orthologue of t-cap1 our pupil lethal we use the uas gulf force system to express double-strand RNA and induce dcp1 silencing specifically in neurons the same approach was used to overexpress a dcp1 gfp transient exclusively in the nervous system of the fly in agreement with our c elegans data neuronal down of dcp1 caused a dramatic reduction of lifespan while its neuronal overexpression resulted in long-lived flies that exhibit a significant increase in both median and maximum lifespan the latter effect though only occurred when transient overexpression was initiated after the completion of development overexpression throughout development did not have an impact on lifespan suggesting that high levels of dcp1 in neurons are detrimental during development and counteract their longevity promoting effect during adulthood while studying the lifespan we observe that most flies with neuronal dcp1 deficiency exhibit a phenotype characterized by unexpanded wings and unexpanded thorax and the lack of cuticle tonic all these traits are affected by the execution of a posteclosion program that takes place during the late phases of the last ecstasy this process is orchestrated by the release of a neuropeptide term bursicon and its receptor rickets our preliminary results so that both bursicon and rickets mRNA levels are reduced by dcp1 knockdown in neurons suggesting that neuronal mRNA decupping affects bursicon signaling however further experiments are required in order to confirm this notion to examine whether the origins of wing malformation can be traced in early events of dermophogenesis we dissected wing imaginal discs from third distal larvae with neuronal dcp1 knockdown and examined cell and tissue morphology by visualizing effecting despite the neuronal nature of the knockdown most isolated discs exhibited widespread structural abnormalities associated with abnormal epithelial folding however by starting or stopping the knockdown after the completion of larval development we were able to isolate flies with normal wing discs but unexpanded wings advise versa consequently abnormalities in wing imaginal discs tissue pattern and aberrant adult wing morphology are two distinct results of neuronal dcp1 knockdown that occur independently at different developmental stages making clear that mRNA decupping neuronal cells can affect developmental effects in a cell non-autonomous manner collectively our data have uncovered an evolutionarily conserved role for the regulatory subunit of the mRNA decupping enzyme in the modulation of neuroendocrine signaling mechanisms that govern developmental processes and aging our model suggests that dcp1 controls the equilibrium between translation and storage or degradation of mRNA's encoding specific neuropeptides like insulin like peptides high levels of dcp1 promote decupping and storage or degradation by low levels fable translation and secretion of the corresponding neuropeptides such neuropeptides in turn act in distal cells to regulate lifespan and developmental events before concluding this presentation i would like to thank all the people that contributed to this work from the worm lab and the fly lab of the biomedical research foundation of the academy of Athens present and past members of our lab are collaborators from the university of Athens and of course all of you for your attention thank you very much great thank you fivas and there you are he's live all right uh so i'm speaking live but i do not trust my internet connection so i understand completely as mine's barely holding on um so if anyone has any questions please go ahead and put them in the chat um so so i'll start i mean i think you know this is first of all it's a really uh nice uh mechanistic story and i and i love the you know going from one model system to another and showing that it it seems to work the same way in worms and flies you know i think that then the next obvious question is you know is there evidence for any similar sort of mechanism of regulation in in mammals and do you think this might be conserved you know for for human aging well there's no evidence concerning aging but there is evidence that decapping can serve as a regulator of gene expression there are data from human cells that show that mRNA decapping maybe controls the expression of some immunity related genes so we believe that there is a good chance that the same mechanism could apply in aging of mammals and all organisms and that sort of leads to the other thing i was wondering about which is um do you think that this this regulation of the insulin like peptides and insulin signaling is something that has been selected for and this is asking you to speculate a little bit but i mean do you think that this is a regulated function of the the mRNA decapping and and and why might that be that that would be a mechanism that's evolved to regulate these insulin like peptides well i don't think that this specific to insulin like peptides i think that mRNA decapping has evolved as a mechanism to control a translation in general and there is this view that is developing that decapping can control gene expression through these cycles of decapping and recapping and there is a concept of homeostasis that is arising so i believe that it is a general mechanism of gene expression not specific to insulin like peptides but we have we have discovered some targets of regulation great thank you um okay well i will ask one more question because i'm not i'm not seeing any others in the chat i guess i'm wondering you know there are a variety of environmental interventions like caloric restriction that have been reported to extend lifespan and is there any evidence or have you thought about looking to see whether there are environmental interventions that regulate that could regulate lifespan through this mRNA decapping mechanism no we haven't really checked but that is very interesting that is very interesting and maybe we will check that okay great thank you and we are right on time so uh so really appreciate you giving that presentation and joining us in in person thank you okay so now we will come back to ann she's she's there no symposium of 2020 would be complete without dogs barking in the background and somebody's internet going out so thank you for for for saving us ann go ahead and load up your talk and just to remind everybody ann's talk title is late life restoration of mitochondrial function reverses cardiac dysfunction in in old mice hey um hello everyone hopefully this time the internet won't bow out on me and um again thank you my for the introduction and thank you the organizer for the opportunity to present my work which is based on this recently published e-life paper so i want to begin by acknowledging all the co-offers that participate in the study and contribute to this research in particular dr. Pindenburg binnerage which is my postdoc mentor and most of the work is a postdoctoral fellow in his lab and aging increases the risk of cardiovascular disease and in addition to increasing the risk of cardiovascular disease aging also results in deterioration in structure and function of the heart and these cardiac aging phenotypes are very similar between human and in mouse model which is what we use in this particular study and here i'm showing a few key cardiac aging phenotypes in mouse model so these are functional data data for mouse at different age groups from young four to six months old all the way to over a month old and one cardiac aging phenotype or one of the most important changes is the decline in diastolic function e-i-a-a represent diastolic function and this parameter decreased with age and for about 50 percent and systolic function on the other hand is relatively there's about 10 percent decline in ejection fraction over time over the cost of aging in this animal myocardial performance and other things we measure and this index actually increased with age and this increased index actually represent worsening of myocardial performance and structurally cardiac aging also result in increase in cardiac hypertrophy which can be measured by normalized heart rate and this index increase over the cost of aging myocardial dysfunction is a hallmark of aging and therefore targeted hydro oxidative stress to improve myocardial function is an attractive approach to try to intervene the process in this 2005 study the Rabinowitz lab generated a transgenic mouse model to overexpress catalyzed specifically in mitochondria of the mouse and what they shown is that by scavenging mitochondrial hydrogen peroxide in this mouse by targeting the catalyze in the mitochondria they are getting an 18 percent increase in lifespan in this animal and importantly when they target catalyzed in peroxism or in nuclear they are not seeing this improvement in lifespan which suggests the importance of targeting mitochondrial versus just overall loss and in that in addition to this lifespan extension MCAT mice also have attenuated cardiac aging phenotypes and the red line here shows the function of the MCAT mice compared to the black line which are the wild-type animal and this improvement in MCAT mice including input in DASOL reduced myocardial performance index which suggests improved myocardial performance and also reduce cardiac hypertrophy however all these study were done in transgenic animal meaning that their mouse have life long reduction in their mitochondrial loss and this can the development or prevent the development of this cardiac aging phenotype but from a translational point of view it's more practical if we can have an intervention that can be started at late life and can suppress mitochondrial loss and reverse mitochondrial dysfunction and reverse pre-existing cardiac aging phenotype which is the objective of this study is to determine if we can suppress loss only at late life and be able to resist cardiac aging and our hypothesis that if we give this late life to reduce mitochondrial loss and in particular essence 31 peptide or MCAT expression we'll be able to improve them as a function and reverse pre-existing cardiac aging phenotypes and I'm sorry to interrupt you cut out a little bit maybe if you shut off your video so that so that you have just a little bit less bandwidth usage so your picture I mean oh okay might help a little bit okay if not see if that's better okay so the intervention we choose is SS-31 peptide also named alamepratae so this is a tetrapeptide drugs developed by Dr. Hazel Seathall and Dr. Peter Schiller and this is the structure of SS-31 it's just a four amino acid peptide it has been shown to target to the inner membrane of the mitochondria by binding to cardiolipid which is a phospholipid that is exclusively present in the inner membrane of the mitochondria this peptide has been shown to reduce mitochondrial oxidative stress in various models and the major reason of why we choose this peptide is that this peptide shows similar protection in models of angiotensin induced cardiac dysfunction and tag induced heart failure that are in a very similar manner that with mitochondrial catalyzed mice transgenic mice so the graph in here is showing that for diastolic function which is impaired when we treat animal with angiotensin can be restored when we give animal SS-31 peptide and similarly or i am cat expression and this is the reason why we choose this peptide as our target and we first ask the question question whether late five treatment of SS-31 can reverse cardiac aging in mice and we do that by giving young and old C57 black six mile type mice with SS-31 through osmotic minimum subcutaneously and then we follow up on we follow on their cardiac function by echocardiography at baseline four weeks and eight weeks and we also assess their exercise performance at four and eight weeks by treadmill running so here's the result that we find for diastolic function which we measure as EAAA ratio this parameters reduced with age so compared to young animal which usually have EAAA ratio of about 1.5 this ratio decreased to about one in old animal similarly at baseline for the two different groups and only in the SS-31 treatment group we are seeing an increase in diastolic function an improvement in diastolic function and the other parameter myocardial performance index this index actually increased with age as i mentioned earlier compared to young animal which level would be around here old animal have increased MPI and only in the SS-31 treatment group we are seeing a reduction in the myocardial performance index so that's taking an improvement in their myocardial performance and old animal also developed cardiac hypertrophy which can be shown as disnormalized weight data in here the old animal have increased normalized heart rate compared to the young while in young animal SS-31 does not change cardiac hypertrophy in old animal SS-31 reduced cardiac hypertrophy and diastolic dysfunction is associated with exercise tolerance in human so we want to measure whether SS-31 treatment improve exercise performance and this is treadmill running time of these mouse after the eight-week treatment so in old animal they run significantly less time on the treadmill compared to young control and SS-31 significantly improve exercise performance in old animal because SS-31 target myocardial oxidative stress so we measure whether oxidative stress loss level is changed the heart after SS-31 treatment and we use mitral shocks to measure superoxide and these are the data summarized here and we show that SS-31 treatment significantly reduced myocardial superoxide level and similarly it also reduced myocardial hydrogen peroxide level which is measured by mitral PY1 indicator we then study whether SS-31 improve mitochondrial function and we did that by isolate cardiomyocyte from young animal control animal or old animal that are treated with eight weeks of SS-31 so in this cardiomyocyte we measure the OCR oxygen consumption by using seahorse mitral stress test protocol and the key findings is that old animal old cardiomyocyte actually have increased basal respiration compared to young and this increase is completely contribute by an increase in proton leak without actually in ATP production and so this age-related increase in basal respiration and proton leak are both attenuated by SS-31 treatment at the time of the study we don't know the molecular maximum that drive this normalization of age-related increase in proton leak but a follow-up study that should published today on eLife by the Rabinovich lab have shown that ANT is probably the target of SS-31 what they show is that similar to SS-31 ANT inhibitor can also suppress the age-related increase in proton leak in this cardiomyocyte and ANT actually can interact with SS-31 so this is attenuated SS-31 binding with ANT and this binding can be compete by three SS-31 and also this binding is inhibited when treat ANT and a different study from James Spool's lab also use cross-linked mass spectrometry to show that SS-31 can interact with ANT and they have more detail mechanism of how what interact with ANT and reduce proton leak in these two papers and in addition to these changes in cell level we also look at a tissue level how SS-31 affect oxidative stress so we perform proteomic analysis to look at as good a violation of myocardial protein and what we see is that this is not too full change of good a violation and the bull bar here are young versus old changes and all the most of the bull bar are on the right hand side of zero suggesting that this increased level of good a violation in all heart compared to young heart and if we look at old SS-31 treatment versus young control we can see this red bars here all shifts compared to the bull bar two zero axis here which suggests it attenuate please in good a violation in old heart we also measure another protein oxidative modification which is protein tabonylation and aging heart has increased level of protein good a violation and this is reduced by SS-31 treatment phosphorylation of malfilament protein is a key regulator of diastolic function and one of this malfilament protein is malcine binding protein C and we observe reduction in phosphorylation of serine as serine 282 at malcine binding protein C in old heart and this is reversed when we treat this animal with SS-31 and to determine if increase if reduced mitochondrial oxidative stress is sufficient to improve diastolic function or reverse cardiac aging we also express mitochondrial catalyze only in old age by using dinofsoce virus to administer this transition in 24 month old animal and what we see is that by overexpressing MCAT using the virus we can also improve diastolic function similar to what we observe with SS-31 treatment and we also try to combine the two interventions by giving MCAT animal or MCAT animal with SS-31 and when we treat SS-31 to wild type animal we similarly we can increase their diastolic function as we shown in our earlier data but when we look at the MCAT mice after SS-31 treatment there's no significant improvement in diastolic function which suggests that MCAT and SS-31 improve diastolic function in very simple in overlapping mechanism so when we combine the two we are not getting additive benefit in this case and so I want to end this presentation by showing you our proposed model based on our finding so what we show is that SS-31 can reduce mitochondrial oxidative stress and suppress proton leak in the mitochondria and the recent paper from Vrbinovich labs also identify ANTS target of SS-31 to suppress proton leak and this reduction of mitochondrial oxidative stress lead to downstream change in oxidative modification and lead to changes in phosphorylation of myofilum protein and results in improved relaxation or diastolic function of this mouse in mouse and similarly express catalyzed in mitochondria we can also get similar improvement in diastolic function and there's some overlapping mechanism action of SS-31 and mitochondrial catalyzed expression and I will end the presentation with this and we'll take any of your questions thank you great thanks Anne so we have a question from Alex Chen and he's asked he has specifically in the context of the MCAT mice but I think the same question applies to the SS-31 whether or not you looked at the effects in tissues other than heart like brain or or other tissues where you know mitochondrial activity is thought to be particularly important so other study have looked at that not myself but many other groups have looked at the effect of MCAT expression in other tissue and SS-31 also have been looked at in other tissue and for example SS-31 we have collaboration with state mastic groups and they show SS-31 can improve skeletal muscle function so attenuating mitochondrial loss other than the improvement that we see in cardiac muscle there's also improvement in skeletal muscle and in fact this intervention that target mitochondrial oxidative stress like SS-31 for example is in clinical trial for studies for disease that are heart related or in other organs like in eyes and in muscle so it's not specific just put it to the heart is actually have for therapeutic benefit in multiple organs and sort of related to that do you know if anyone has done a lifespan health span frailty experiment just looking instead of looking at one tissue at a time you know aging out mice on SS-31 and trying to characterize how well they're aging and how long they live right so I think for SS-31 because the way that we administer is by osmotic menopause we're limited to a very short period of treatment so what we use is zero four weeks menopause and we can only perform the treatment for eight weeks and we were trying to develop other regimen of SS-31 that can be different without doing the osmotic menopause and but I don't think we have yet there yet and so there are studies because this is a poem project at the within the research lab there are studies that they look at different uh health span parameter in SS-31 mice SS-31 treat mice this is limited to uh late type SS-31 treatment but not uh long term SS-31 treatment got it that makes sense uh and then we have a question uh does SS-31 treatment induce mitophagy um we haven't looked in our heart tissue uh so I don't know what this will do to mitophagy in particular I think we have for only for the heart we have not published the data but we have looked at uh autophagy marker but we don't see an increase in autophagy with SS-31 treatment in old heart okay and then uh the last question is sort of more generally related to um you know how this might tie into the the mixed results with antioxidant treatments and I mean your model right is that this is at least largely a ROS-driven mechanism and and this is sort of related to a question I had which is that you know there's also evidence that that low levels of ROS this sort of mitohormesis model can be beneficial so so you know what are you thinking about in terms of what does this mean with the varied data on antioxidant therapies and could it be the case that you know SS-31 is good in some context but might actually you know prevent some of the benefits if you believe the mitohormesis model and things like that you have any thoughts on that yeah this is a great question so uh it's um has been pretty well documented low level of ROS is important for signaling um but uh ROS signaling so it's not something that we want to completely wipe out all the ROS in uh the cell in the mitochondria um and in fact there are studies that shown for MCAT expression for example if we have MCAT expression at uh uh super high level it can actually be detrimental to uh some uh in in a model of mito induced cardiomyopathy and another model that is related to some bactericidal response so um we definitely don't want to reduce ROS to a very low level and which is why uh for in SS-31 we probably want to target OH to reduce age-related increase in ROS but not to suppress ROS at when it's not at a high level or excess level and which is the reason why this later life intervention is more uh translatable compared to uh some intervention that can act on especially because um I think in is it also an MCAT study so MCAT induced proteomics changes in Yang or in all animal in a very different way so in uh all animal is actually reversed the age-related change in proteome uh while in Yang animal it actually makes the proteomes look older so uh which is the basis of why we started this study at OH trying to see if we can improve by suppressing this excess ROS mitochondrial ROS only at OH. Okay perfect thanks Tan uh that was great and I want to thank all of the speakers for this session and for the entire symposium I'm sad to say we have reached the end uh so um so again thank you to all the speakers and and all of you who um stuck out through the whole thing and stayed with us um I just want to say sort of overall again you know how impressed I have been with the talks that we heard today and the the submissions that we got for the special issue I think you know as you've seen um this really reflected the diversity of of science in the field of aging from very basic research detailed mechanistic studies all the way through to to translational applications that are just on the cusp of really making it into the clinic and we had a good representation of model organisms yeast worms flies fish mice humans um uh so it's it's just been been fantastic to see uh how many how many great um papers we've had submitted and published in this special issue um I just want to thank in addition to the speakers Anya and Laura at Elife for uh running this thing and keeping us on track um I think it's gone particularly smoothly considering all of the challenges that we're all dealing with these days and I will just leave with uh encouraging you to please consider submitting your best papers to Elife I think um you know my experience now as a senior editor at Elife is that the the process works really well obviously it's not perfect and not everybody is going to get the outcome that they want but it's my feeling that it's it's a very fair process it's about as fair as we can make it and it's a collaborative review process that that I think has some benefits over the way a lot of other journals handle their papers and and I know that I speak on behalf of Jess and all the other senior editors when I say that we're committed to giving you a a fair review for your papers so please consider submitting your best papers to Elife um and I'll turn it over to Jess if she has any sort of final concluding remarks I just thanks Matt I want to also acknowledge Maria Guerrero who you guys don't know but she has been phenomenal she's the Elife person that's been behind the special issues she's been working with us um helping us get through these 160 papers so far and I also want to thank the speakers and the attendees for asking such great questions finally I just really want to thank Matt it's been amazing working with you you're so responsive he looks at the papers really quickly answers my emails amazingly fast and he knows his breadth of knowledge of aging is just really impressive so thank you so much Matt and thank you all