 So our next speaker is Michael Stout from the Oklahoma Center for year science. And is that better? Yes, that looks perfect. Also from the Oklahoma University Health Science Center. And the talk of talk title is estrogen receptor A is required for 17 A as ready all to modulate health parameters. So I would just remind everyone again to please pose any question for Michael in the chat function, we will take them afterwards. So please, Michael, go ahead. Thank you. Can you hear me? Okay. Yes, sounds perfect. Okay, well, thank you very much for the opportunity to speak here today. Again, my name is Michael Stout. I'm an assistant professor at the University of Oklahoma Sciences Center. And today I will talk about 17 officer dials effects in human cells and rodent models. But the overarching theme of our lab is really sex differences and metabolism and aging and what role sex hormones play. And so I know we're on a clock here. So if anything's not clear, please feel free to shoot me an email and put that here at the bottom. There we go. Okay, so as a quick reminder, about 80% of circulating beta estradiol is created in the granulosis cells of the ovary in females. And as everyone knows, this goes out and has major endocrine actions throughout the body. Conversely, males only produce about 20% of their circulating beta estradiol in the Certoli cells of the testes. It's really unclear what role these these this pool of estrogens has or plays in terms of an endocrine actions. However, it's exceedingly clear that in females, when you eliminate this 80% of circulating pool in diseases such as polycystic ovarian syndrome or in conditions such as menopause, this has the deleterious effects that lead to chronic conditions such as obesity, liver diseases, metabolic dysfunction, osteoporosis, etc. Again, it's really unclear if we were to eliminate this 20% of production by the male testes what role this has in chronic disease and aging. However, it is very well known that estrogens are playing a role in male metabolism and probably aging as well. And so if we were to block, this has been done now in humans, if you block aromatase pharmacologically, these men increase their adiposity. Or if you modulate ER alpha activity in male rodents, you get a modulation of gluconeogenesis and lipid metabolism, which appears to be regulated by FOXO1 in the liver. More recent data is showing that a nuclear or membrane ER alpha or genomic or non genomic actions of ER alpha appear to be different between the sexes. And so this seems to play a role in insulin secretion and insulin sensitivity. So it appears that the vast majority of estrogen actions in all these tissues are occurring in males through aromatization of androgens to beta estradiol, which has autocrine, paracrine, and incretin actions on these tissues. However, more recent data is now showing that males also have the ability to express steroidogenic acute regulatory protein, which is the rate limiting enzyme responsible for de novo steroid biosynthesis. And so it's really unclear at this point, what is going on with regards to local estrogen actions in this role in aging within these tissues and males. And so this is kind of how we wound up in the 17 officer dial field. I'm going to get this out of here. And so this is a naturally cranitantimer of beta estradiol. It has minimal binding affinity for ER alpha and ER beta, which has led many to believe that this has its own novel receptor. Although through this publication, I think we've shown that at least with regards to a lot of the stuff that we study, we believe this is through ER alpha and not a novel receptor. It's also currently prescribed as a hair loss therapy in Europe, and South America under the under the name of Pantostin. And it accounts for about 10% of Primeran, which is a hormone replacement therapy that has kind of fallen out of favor, but it's been prescribed for decades. It came to be known in the aging field due to its neuroprotective effects related to models of Alzheimer's and Parkinson's. And that's been studied for the past, we'll say 30 years. But more recently, there's a belief that this estrogen provided exogenously may have a role in systemic aging. And this first came about when the interventions testing program here in the US as part of the NIA was able to determine that when you provide this estrogen at two different doses, it extends median and maximal lifespan, but only in males. And so there's probably a story here as well as to why the females are not responsive, which we believe has to do with competition between endogenous beta estradiol and exogenous alpha estradiol. But we don't have time to discuss that today. So I'll just talk about the effects in males, but computer keeps freezing. There we go. Okay, so when we saw these longevity curves, we certainly wanted to understand if this is affecting hallmarks of aging in a tissue specific manner. And is this reversing or can it reverse obesity related comorbid conditions? And what this could tell us about systemic aging biology. And so we don't really have time to go over all these, we've published a series of papers showing that when you provide this estrogen to male rodents, you have a myriad of beneficial effects first starting with reductions in calorie intake. However, I must say that all of these benefits are not totally contingent upon reductions in food intake. If we use hyperphagic models, or we do K-Cal matching, a lot of these benefits are still present. And these include reductions in adiposity, particularly visceral or central adiposity, fatty liver disease, liver fibrosis, hepatocyte DNA damage, improvements in glucose homeostasis and insulin sensitivity, and a shifting of the polarity on monocytes and macrophages to more of a surveillance and repair type of phenotype. We've also started looking at B cells, and it seems there's a phenotype is there in there as well. However, these are all fairly descriptive. And we still at this time did not know what the signaling mechanism was, or if this is acting through a specific tissue, thereby having or leading to systemic benefits on aging biology. And so the first thing that we wanted to try to do in this in this paper that we were fortunate enough to get an ELIFE was determine whether or not 17-alpha estradiol and 17-beta estradiol elicit divergent effects or genomic effects with ER alpha. And so we took U2OS bone carcinoma cells, which are a human cell line that don't express any estrogen receptors. And then we transfected them with ER alpha. And then we hit them with 17 alpha at two different doses or 17 beta estradiol, and look to see where ER alpha is going to bind in genomic locations. And is what we find is that regardless of ligand or dose, it's going to the same places. And the magnitude with which it's binding is almost identical, regardless of dose or ligand. And when we look at specific binding motifs, is what we find is a lot of the common estrogen related response elements are coming up, including EREs, estrogen related receptor response elements, serotogenic factor one. And again, regardless of ligand or dose, they're essentially identical. So we repeated this and did the exact kind of experiment and looked at transcriptomics. And is what we found is that all of the treatment samples clustered together away from the vehicle, and the differentially expressed genes, including upward and downward are again, essentially identical, regardless of dose or ligand. And this is just the representation of the gene body and where ER alpha is binding. Again, these are essentially the same. And so the take home point from these first two panels are, or is that there is no difference, at least with regards to genomic activity through ER alpha, whether or not you have ER alpha or ER beta as your ligand. So we then went on to look at global ER alpha or estrogen receptor alpha knockout mice to see if this can ablate the effects of this estrogen. And I took out a lot of panels from the paper trying to make this easier to follow. But the take home point here is that pink, which is the knockout receiving the estrogen versus the light blue, which is the wild type receiving the estrogen, these curves diverge. And so there's certainly an ablative effect of removing ER alpha on this estrogen with regard to mass, fat mass and K cal intake. And so this tells us that ER alpha is at least regulating these three specific phenotypes that are listed by this estrogen. When we look at metabolic homeostasis, we see the same thing. Insulin goes down significantly in the wild type. So does a one C, but this is not seen in the knockouts. The same can be said for glucose tolerance, the wild types come down significantly, so much so that they mimic what's going on in the low fat diet control group, despite weighing about 15 grams more. So they're obese, but they're highly glucose tolerant and likely insulin sense. And so we use liver disease in a rodent model is kind of a read out of metabolic dysfunction because they have such a large liver. And is what we find is that, as I've shown before, the estrogen reduces fatty liver disease and fibrosis. But when we repeated this in this model, the wild types respond and the knockouts do not. And you see this both qualitatively through oreto and quantitatively through fatty acid and triglyceride quantification. When we look at liver fibrosis through tricrumpstani, and then we also have looked at Sirius red and hydroxyproline, you see that the wild types respond and the knockouts do not. Again, suggesting that the effects of 17 alpha estradiol are dependent upon ER alpha, which could tell us something about what ER alpha is is doing with regards to aging in males. And these are just fibrotic markers at the transcriptional level that somewhat mimic what we see from histology. And so given this very strong association between hepatic insulin sensitivity and metabolic regulation, and the emergence of fatty liver disease and fibrosis, we thought that the way to prove this was to determine whether or not this is affecting hepatic insulin sensitivity. And so the first thing that we did was just take these levers and look at genes that have been closely linked to a panic insulin sensitivity in rodents. And as what we find is that this estrogen reduces full of statin and hidden in IRS to significantly in the wild types, but not the knockouts. This also reverses some of the obesity related changes in gluconeogenic enzymes. Or these are at the transcript level, though. So then we thought as a direct measure, we should feed another set of mice, get them sick with high fat diet, and then provide the estrogen or just maintain them on the high fat diet and then inject them with insulin prior to dissection, so that we could test the effects of hepatic insulin sensitivity through looking at AKT and FOXO one. And as what we find is that the estrogen works or improves hepatic insulin sensitivity as measured by AKT phosphorylation and FOXO one phosphorylation, but only in the wild types and not the knockouts. So we thought, okay, the last way to prove this is to provide this estrogen acutely so we can circumvent the effects of reductions in food intake. So we just infuse this very acutely. And then we perform a hyperinsulin making glycemic clamp. And as what this is is an infusion of high insulin, and then that's that's clamped, it's consistent. And then you have a variable rate of glucose infusion to maintain your glycemia. So the higher amount of glucose that you have to infuse more insulin sensitive the whole animal is. And then you can look at rate of appearance, due to labeled or unlabeled glucose and rate of disappearance and tell you whether or not this is peripheral tissue versus liver. And as what we find is when we infuse this estrogen under hyperinsulin conditions, glucose infusion rates go up, indicating that the animal is more insulin sensitive. And this is due to a massive suppression of endogenous glucose production or gluconeogenesis. And so, but the rate of disappearance is unchanged. So in this top group of experiments here is what this is telling us is that indeed, hepatic insulin sensitivity is improved when we acutely provide this estrogen. But since there has been an established connection between the hypothalamus and the liver, in regulating systemic glucosomia stasis, we thought we should also infuse this into the brain at very minor doses to see what happens and to make sure we thought that this would not have an effect on hepatic gluconeogenesis. It turns out that when we infuse this into the brain, it phenocopies what we showed in A through E with increased glucose infusion rates, the suppression of hepatic glucose production and no change in the rate of peripheral disposal. So as what we learn from this is that this estrogen is probably multimodal in its actions. It's probably crossing the blood-brain barrier and affecting the hypothalamus. There is an established connection between AGRP neurons and the regulation of hepatic gluconeogenesis. And there's more emerging data suggesting the POMC neurons can do this as well. And so as what we're in the process of doing now is trying to figure out whether this is a direct action in the liver or indirect actions on the liver by actions in the hypothalamus. And so we're in the process of removing ear alpha throughout the hypothalamus or in specific neurons or in hepatocytes and cell cells. We're also trying to snip this crosstalk between the two to try to tease this out for one to tell us how 17 officers dial is working. But more importantly, tell us what this may or what role this may play in male aging. And so I think I'm probably slightly over my time here. So I want to mention Shavani Mann here first. She is a fantastic graduate student in the lab and she ran most of these studies. Here's the rest of my lab is Shini, Kaitlyn, Samim and Kennedy. The primary collaborators associated with this project were John, Sabu and Derek and Bill Freeman locally. And then certainly I would like to thank all of my funding sources for supporting this work. And at this point, I'll take any questions if you have any. Thank you so much. Thank you so much, Michael. I think you're perfectly on time. Really interesting. So maybe I can start when posing some questions. And so you're really targeting some of the sex-specific effects in aging, I guess, here, because you're only studying male mice, right? Yeah, I didn't mention that. So we have females in this study. The females, we have them in most studies. We didn't initially because we just wanted to see what the phenotype would be in males. Since then, we include females in everything. But and they're in this paper as well. But it's very complex and there's competition between endogenous alpha and endogenous, I'm sorry, exogenous alpha and endogenous beta estradiol. And in the context of OVX, we do see that this estrogen works. And so we don't know if that's because it's replacing some of what the beta estradiol is doing endogenously. Or if it's eliciting some other, that's what we think, or if it's eliciting some other effect that is replacing what the over with such as a micro RNA, because there's some belief that the ovaries are producing micro RNAs that are having systemic effects. And so we're in the process of teasing that out, whether that is so we're not just doing OVX, we're also doing VCD menopause induction in female mice and providing this estrogen to see if that if we can uncouple these kind of things, if that makes any sense. Right. So I mean, is it then possible to for the main mice to kind of catch up with the female mice on some of these phenotypes that you're studying? Yeah, that's a good question. Certainly they do. And in and so this is where the interventions testing program gets really interested in this because they when you provide this estrogen to males, their argument is that these males live longer than the females do. But there's, in my personal opinion, there's a lot of variables that play there. Yes, but the dosing has to be and the binding affinity and the activity of your alpha has to be equal to determine whether or not. You know what I mean? We have to dose ERL or 17 alpha accordingly to mimic the endogenous 17 beta effect to really make those comparisons. And so I don't know if that answers your question. I know I get tangential, but that's kind of the way we're thinking about it. Yeah, yeah, I totally understand it's complex. So we have some more questions here, one from Amanda Neo. Is there a role of GPER in the effects of 17 alpha estradiol? Great question. So yeah, GPR 30 or GEPR? Yes. So we're familiar with it. We tried, we've done a little bit. And so we looked at, so this is a membrane receptor that has been linked to estrogen signaling. And so we are aware of it. And we've taken cells that we know express it and we've hit them with this estrogen and we've done RNA Seq to try to figure out if we can get any readouts that would be indicative of GEPR activity. And we haven't found any, but we haven't, to be honest, we have not went super high in depth to try to figure this out because of what we've seen with ER alpha ablation. And but to answer you directly, we've tested a little bit. We don't have any evidence to suggest at this point that it is signaling through GEPR. Right. So maybe time for one last quick question. So this is from Dario. Great talk, Michael. Why don't males tune 17 alpha E2 to improve their metabolic aging? And what's the cost on the males and for doing this? So say that again. I'm sorry. So the why don't males tune the 17 alpha to improve their metabolic aging? You can't see the question. Why do they chew? I didn't. It's it's in the panelists and attendees. So okay, so let's do it like this. You can, you can read a comment and maybe you can type an answer. Trying to get to that one. It's in the chat function. So I think it's time to move on. So you can read the chat function. Yeah, yeah. Sorry, Dario. I just got it to pull up. So I will definitely shoot you a message real quick. Sorry I didn't get it. No problem. Thank you very much, Michael. Great talk. Thank you so much. I appreciate it. So let's move on to the next speaker. It's Yang Liu from the Department of Molecular and Integrative Physiology and Geriatric Center at the University of Michigan in the U.S. So your talk title is Sarah Tonning to a receptor signaling coordinates central metabolic processes to modulate aging in response to nutrient choice. And you share your screen. Yes, perfect. Thank you, Yang. Please go ahead. Okay, thanks Sarah for the introduction. First I want to thank Eli for hosting such excellent symposium. Again, my name is Yang. And I'm posted off from the Scott Pledge Lab at the University of Michigan. And the story I'm going to talk about today is about food choice, serotonin sickling, and aging in food flights. As many of you already know, doubt is arguably one of the most important determinants of aging. Early in 1935, MacKay and his colleagues for the first time found that the restriction of calories without malnutrition promotes lifespan in rats compared with their other liptoid fat siblings. Then it almost took 17 years until we realized that beyond the calories, dietary composition is sufficient to modulate aging thanks to the studies on esochloric diet and the nutritional geometrical framework. As a matter of fact, in recent two decades, increased evidence indicates many of aspects of diet influence aging in the house, including food perception, meal time, and many others. Today, I'm going to focus on how food choice influences aging. Food choice is natural. Animals take carbs and proteins to satisfy their energy demand all the time. On top of choosing specific nutrients, human also makes food choices based on taste and flavor. For example, here I show you our traditional Chinese hot pots always put that to divided wells, so people can choose from two different broths to make their own meal. In our lab, we have designed a neutral style paradigm, which is not inspired by Chinese hot pots, but the idea is similar. For food flies, sugar and yeast are two major macro nutrients. Well, yeast is the primary protein source. We separate sugar and yeast into different wells, as you can see here, so animals can construct their own meal. This is different from the conventional fixed styles where you have no choice but a chunk of nutrient mix. What we have found here is that food choice can dramatically reduce lifespan in both males and females as shown in dash lines, compared to their siblings that fit our fixed styles, and that has the same concentration of nutrients. We have validated these choice effects in several different white-type flies, including two white strains collected from the orchards in Pennsylvania and see the similar results. What effects are usually bigger in males, as shown here, the following parts of the presentation will be focusing on the male data. Now we know flies live shorter on a choice doubt. The first thing we ask is that do these flies live shorter because they choose a poor meal? So we measure the food consumption of flies living in these two different dietary environments. We observe flies eat the same amount of yeast, but about nine times more sugar when they fat on a choice doubt. From previous studies, we knew that high sugar doubt is probably detrimental to longevity, but is that why choice doubt reduce flies lifespan? To answer this question, we added to high sugar fixed doubts as controls. Although they have slightly different sugar to yeast ratio, we can see from the feeding they have blow that both diets approximate the nutritional composition that flies eat on the choice doubts. With that, we can test whether the choice lifespan effects are from the differences in sugar intake. Effects, we show that the effects of doubt with choice relative to the standard as white in doubt are much bigger than that from homogenous high sugar diets, which are shown as purple and white lines. The lifespan and the feeding data in this slide together in points that nutrient decisions significantly influence aging in a manner that is at least partially independent of sugar intake. So far, our observations suggest that animals respond differently to macronutrients depending on how they are presented and that effects of choice lifespan may be mediated by processes that are independent of macronutrient intake. Then what is the cause and the mechanisms of aging in the choice environment? All lab has been interested in neurotransmitters and neuron peptides that are required for nutrient perception as we hypothesized that neuronal segment is important modulator of aging. One of the screen we have been using is to put flies in the two choice feeding paradigm as shown here and see which mutants alter the food preferences. Now the data I show here is from our press publication also on your life back in 2016 where our press graduate student Jenny Rowe in our lab found flies will adjust the macronutrient preference based on internal needs. So in a choice essay fully flies always go for sugar after starvation flies will switch to yeast suggesting nutrient decisions are dynamical processes driven by internal states. Jenny also found a mutation in serotonin receptor 2A also called 582A abolish the switch. This data suggests that 582A means either internal energy demands or external nutrient sensing with those and we're interested in whether this molecule identified by the short-term essay can modulate aging in the long-term choice environment and indeed we found the lifespan differences between a fixed and choice environments are fully aggregated in the 582A mutant in the perfect way as shown in the blue lines. In addition to genetic mutants I also use other approaches to validate the involvement of 582A to convince myself as well as to convince you because 582A is highly expressed in the central neuron system. We use the pan-neuron driver ELVV-GEL4 to knock down 582A expression and we observe that this manipulation abrogates the choice effects using one of the online allies as shown in the center and significantly reduced the effects of the second as shown in the right. We also found an administration of Pyrrhon Perron of 582A antagonist during the adult stage is sufficient to reduce the choice effects. Together this data suggests that neuronal expression of 582A in adults mediates the doubt rate effects. Our previous feeding data has shown that 582A modulates protein drive up this dilution so we ask whether it might change nutrient consumption in fair flies. Here we show that 582A mutant exhibits actually same the nutrient preference compared to control flies in the choice environments. They both consumed significantly more sugars than yeast when given choice as shown here and in addition video tracking data also suggests those genotypes actually spend most of the time surprisingly on the east side as shown here. This indicates 582A does not influence health flies interact with the full environment when in a homeostatic state. Settling doesn't change the way fly interacts with the full environment. It does have significant influence on lifespan which make me think that 582A might alter the nutrient integration inside of the body and changes on the level might be sufficient to affect aging. To investigate this we use metabolomics to examine metabolic changes in flies. Here we started both control flies and 582A mutants in heads and bodies. This work is collaborated with the Daniel Poem's lab at the University of Washington. For this study we found a consistent upregulation of some TC intermediates and amino acids that induced by choice in white types which are fully aggregates in the mutants. For example alpha ketone glutrate and its precursor glutamate glutamine are both increased in the heads. We further investigate the direct role of biochemical reactions that produce the TSA metabolites and the modulation of aging in response to that way of choice. So we targeted snuck down expression of this enzyme. Glutamate dehydrogenase were GDH which converts glutamate to alpha ketone glutrate. Here we used RU486 dependent gene switch system to knock down GDH ubiquitously. On the left the controls where RU is not added and flies ashore left on the choice just as white type flies. On the right where the R&I instruction has been activated by RU the lifespan differences between two environments are aggregated. This data suggests a causal link between alpha ketone glutrate and the choice lifespan effect. Here I hope I can win you with this model of well-nutritional environments modulates metabolic states in peripheries through 5.2a in brain which further determines lifespan. I speculate that in the choice environment serotonin succulent turns the animals into energy burning mode which shortens their lifespan. With that I would like to acknowledge the people who have contributed to this project my mentor Scott collaborator Daniel and our contributors all the members in the pleasure lab and funding resources thanks you for your attention and I'll be taking your question at this time point. Thank you very much young really super fascinating that it's you would think that it's the sugar that's causing this difference but it's not really it's the partially yes. Yeah exactly so I saw that Daria posted a question it was actually the same that I had in mind so that's the choice of sugar or yeast changes a function of age in the flies. That's a great question we haven't do any age super second measure of feeding we do that but the observation is therefore I start to die at really early ages like two weeks so we don't know we need to look at early ages is what I'm saying but it's yeah it's necessary to look at multiple data points at this time. Right another question from Andre that was just speaking prior are micronutrients affected whether micronutrients affect food choice um we don't know about micronutrients I that's the text in the chat no do you mean it's micronutrients because there might a bad food choice but we absolutely know nothing about micronutrients at this time point. Okay another question from Daria could the choice I mean the reason why we see this effect then in the choice is it because it's some sort of stressful event or see costing costful for the flies or other things yeah that's an excellent question that is actually what we want to prove in the paper but it's highly speculate we actually have some evidence that flies could be stressful so there is a stress paradigm in flies that if flies are stressful they are going to consume more ethanol so we did that I say we expose flies to choice for fixed doubts and flies exposed to choice doubts they consume more ethanol which as indication they are stressful but we don't know for sure it just always hard to see that and we also have evidence flies have less triglycerides when they fail on choice and they leave shoulder in the stress resistant resistant experiments which is also consistent with this stress scenario but we just don't know for sure yeah that's what we want to approve at some time point right so is there any evidence for translational effects in other models or even in mammals for this type of there are lots of food choices experiments in mice but not regarding to Asian and we are curious to know that as well so we have another question from Kershid Wani do you know how serotonin acts in the peripheral tissues is the peripheral tissue the intestinal flies that's a very excellent question that's something we want to do in the next step so we actually know much clear in say like elegance and mice that's the gut serotonin regulates of feeding and the physiology and the metabolism we actually don't know much in flies and there's some expression of the 5-HT 5-HT receptors in the gut but nobody in the the expression level is not super high and we don't know any function evidence about those but that's what we want to know next right well I guess thank you very much Yang it was really really great talk thanks Sarah yes and with that yes I hand over to Dario to move on to introduce the next speaker thank you Sarah so our next speaker is Hughie Liang Zhang from the department of medicine and pathology university of Washington in the United States and the title of the talk is reduction of elevated proton leak rejuvenates mitochondria in the aged cardiomyocytes Hugh Liang thank you for presenting your work we're looking forward to your presentation and as a general reminder again please do write questions in the chat if you have them thank you yeah thank you for your introduction can you see my screen yes we can see you okay yeah great yeah thank you for the organizer to give me this opportunity to introduce my work today I'm going to tell you a story we published at the eLife Journal yeah this story is about reduction of elevated proton leak and the rejuvenates of mitochondria in the aged cardiomyocytes in the last century 1950s Dr. Dermot Ham Hamman already proposed out the free radical theory of aging and later on going to mitochondria free radical theory of aging but already 70 years past so far no clinical drugs can treat the cardiac aging either clinical treatment for our research we focus on SS1 peptide in the clinical name is called a lamin primary card this S31 is a small peptide has four amino acids that can incorporate to cardiotherapy which is abundant in the mitochondria inner membrane and then can promote to keep mitochondria curvature previously our lab has found we treat the aged mice with SS31 by osmotic mini pump we find eight-week treatment can reverse the diastolic function in the aged mice also eight-week treatment SS31 can reverse the hypertrophy with old mice however these observations are functional improvement the mechanism needs to be further studied in the mitochondria isolated from the heart we find this is young this trace is from the old mouse we find to keep the same mitochondria memory potential the old mitochondria need more oxygen consumption in other words this means the old mitochondria has but decrease the mitochondria proton coupling efficiency that they will hypothesis that whether SS31 can improve the proton coupling efficiency and then subsequently we do when it's the old mitochondria to test these hypothesis we isolated the cardiovascular and then do the seahorse mitochondria stress acid and evaluate the proton leak in the mitochondria we find old mitochondria has more proton leak and SS31 acute treatment two hours treatment on the isolated cardiovascular can reverse this increase the proton leak however the oxygen consumption measurement of the proton leak is a indirect method then we developed a direct method to evaluate the proton leak by a mitochondria targeted pH indicator the we over is pressed the CPYP in the red cardiovascular for three days then use pony to formula as the cutting mass then we exposure the mitochondria to whatever pH we can add it to the turn off solution as long as we lower the pH we can see in the old mitochondria when we lower the pH the fluorescent goes down that means the old mitochondria in the membrane are very like to resistance to the pH stress and in the young mitochondria we can see the young mitochondria resistant very well to the pH stress and SS31 treatment can make the old mitochondria goes back to the young level of resistance to the pH stress because the pH stress the fluorescent change dramatically at pH 5.3 we focus on this pH for the rest of the research we can see at a pH 5.3 the fluorescent goes down most dramatically and SS31 treatment can restore it cool to the young level we also test the dose effect of SS31 we found SS1 can affect you as a very low dose going to 100 nanomolar moreover we test the how fast of the SS1 can take its effect we found SS1 treatment can affect you at 7 to 10 minutes which are brought to you of its effect that is a phenomenon called a mitochondrial flash this is a transit increase of the mitochondria signal also indicated by CPYFP in a single mitochondria you can see here the arrow shows where the location of the fluorescent the mitochondria flash happened this we can see a transit increase signal and there are three mitochondria flashes in a single mitochondria research study shows that the protons goes into the mitochondria matrix through the inner membrane can trigger the mitochondria flashes then we hypothesis that whether the increased mitochondria proton leak can trigger more flashes in line with the increased mitochondria proton leak we see increased mitochondria flashes activity in the old cutting mass site at SS31 acute treatment can decrease this increase the mitochondria activity next we asked where is the proton leak site the protons in the mitochondria inner membrane goes back to the mitochondria matrix through the ATPase to generate ATP also the protons leak back to the mitochondria matrix through ANT1 a dinonucleotide translocator 1 this is a constitutive proton leak also the protons go back to the matrix through a coupling protein 2 UCP2 this is regulated proton leak so we use the inhibitors of this proton go back to matrix site and then to see which aid the major proton leak site we use ATPase inhibitor oglomacin A and ANT1 if you big test Bunkeric acid BKa all complex attractor-lucide CaT at UCP2 inhibitor GenP we see this is the data yeah ANT1 if better either BKa or CaT treatment can worse the proton leak and make the mitochondria inner membrane resistant to the proton graded stress but GenP, UCP inhibitor or ATPase inhibitor or MaCa ease of that does not restore the proton leak this did educate that the ANT1 is the major contributor of the proton leak is age mitochondria in line with the that the ANT1 inhibitors BKa or CaT decrease mitochondria flash activity next we asked how does acetyl-1 can prevent the ANT1 mid-aited proton leak whether acetyl-1 can bind directly to the ANT1 because acetyl-1 is a small peptide it's very hard to do the proton experiment we use biotin nullated acetyl-1 acetyl-1 binds to biotin and then biotin can bind to the strep avidin using beads we use this system to pull down the acetyl-1 binding proteins and then do west blot to test whether we can see ANT1 we found biotin acetyl-1 can pull down the ANT1 also acetyl-1 can compete with the biotin nullated acetyl-1 moreover, the ANT1 inhibitors BKa or CaT can partially prevent this binding this data suggests acetyl-1 can bind directly to the ANT1 to prevent the proton leak this data is supported by a recent published paper at PAES by our collaborator Dil Masnik and Jim Bruce we use the Yat Rectum method to test acetyl-1 can bind into the ANT1 the whole pocket of the channel moreover, because ANT1 is a component of the ATP system we wonder whether acetyl-1 binds to ANT1 can make the ATP system more stable what we did is prepare the mitochondria and then run the nitrile gel and do west blot of the ATP base we found old mitochondria has lower ATP synthesis and access to more treatment can make it to the young level this is our working model old mitochondria has increased the mitochondria proton leak and the proton leak is through the ANT1 acetyl-1 can bind directly to the ANT1 to prevent this proton leak and makes the mitochondria get less working load then to rejuvenate the mitochondria I would like to thank my mentor Peter Rabinovich and my colleagues also my collaborators Nathan Elder and David Masnik at 1-1 also our collaborator Hito Statham who is the inventor of acetyl-1 our acetyl-1 is provided by Stealth Biotherapeutic I'd also thank to the AHE Career Development Award to me and NRA graduate to my mentor thank you for your attention I'd like to take questions thank you very much Yuliang this is fantastic work we have a few questions from the audience so have you investigated SS-31 in diabetic cardiomyopathy models as I believe proton leak is increased in this condition due to an elevated fatty acid oxidation oh yes we did a touch the mitochondria right up rose level by the mitreous socks we find a mitreous sock signal is decreased that means the decrease of rose stress right of stress is as I think I can decrease it in the age of the mitochondria thank you so can you distinguish if the mitochondrial decline is the cause of the result of aging in cardiomyocyte oh this is a great question because in the aged heart what we observed the mitochondria mass not decreased but somehow a little bit increased but not reached a significant increase so we cannot see the decline may be based on the mass we cannot tell but for the function yes the function is decreased so I actually have a similar question so is increased proton leakage with aging necessarily pathogenic dysfunctional or could it be actually compensatory to some extent or this is completely erratical oh good yeah a great question yeah that is that is a theory of for accompanying to survival theory the proposal to increase the proton leak to make the less stress of the mitochondria then decrease the rose production yeah that is majority happened in the physiology condition what we think we observe is at a pathology condition at a pathology condition increase too much our coupling is a bad thing but in a physiology condition increase the coupling that is benefited to the mitochondria are you able to measure heterogeneity and you know across different cells during aging in in leakage probably yes right so you with your imaging method you should be able to to see how widespread or how concentrated in single fibers this phenomenon is during aging your question is about the population of the mitochondria proton leak well yeah actually it's a very big question so it's both about you know within cell how heterogeneous is the coupling with aging but also how many different fibers muscle fibers whether there is like a muscle fiber specific uncoupling basically that occurs during aging sorry this is a little bit is not directly connected with the the impact of SS 31 with the NT but it's a more general curiosity yeah that's a good question maybe in the future we can think out a method to evaluate yeah thank you for this question yeah all right thank you if there are no further questions let's thank again Yuilang for its excellent talk and we can move on to the next speaker that I will thank you or to Sarah yes thank you all for staying with us it's been super interesting so far so we have two more talks to go and we are even before schedule I think so that's great so I will introduce the next speaker Karen from UCSF University of California San Francisco in the United States so thank you for joining us I guess it's early morning for you and the title of your talk is small molecule cognitive enhancer reverses age related memory decline in mice and we can see your presentation so welcome and please go ahead Karen hi thank you so much Sarah I'm excited to present today so I wanted to tell you about a story that started as a collaboration between Susanna Rossi shown here and Peter Walter who wanted to investigate if interference with the integrated stress response could affect memory loss so I'll just give you a very brief background on what the integrated stress response or ISR is it is a universal intracellular stress pathway that responds to a number of different cellular stressors some of which are listed here that results in phosphorylation of a translation initiation factor EIF2 and this ultimately leads to decreased protein translation with a small subset of stress mediated proteins upregulated so I like to think of this system as our body's fire alarm system when there is an immediate threat the system reacts through phosphorylation of EIF2 and there's this shutdown of the system obviously this is really important if there's an immediate threat like a fire but the problem becomes if the system remains upregulated for too long and so what was previously shown was that if the system remains upregulated for too long it can result in some memory problems so the question became is there a way to briefly interfere with this system to try to mediate some of these long-term negative effects and prior to my time in the labs members of Peter Walter's lab discovered a drug the integrated stress response inhibitor or ISRIB that could briefly interfere with this system it acted downstream of phosphorylation of EIF2 and was able to briefly interrupt this stress system so we wanted to see could we use this inhibitor to mediate memory loss and the first system we looked at was models of traumatic brain injury and I'm not going to discuss these studies in detail today although I'm happy to answer questions on them either at the end or by email but the main takeaway from these studies is that we used a variety of mouse models and found that brief ISRIB treatment could reverse long-term trauma-induced cognitive and behavioral changes so we were able to administer ISRIB briefly to the mice and see long-term changes in both their memory loss and other behavioral paradigms so the next paradigm we decided to look at was healthier normal aging and it's been hypothesized by others that traumatic brain injury or TBI is a model of accelerated aging so if it truly is a model of accelerated aging we would expect similar mechanisms to underlie the deficits we see in both such as memory loss and we would expect that therapeutics such as ISRIB that was effective in reversing trauma-induced memory loss could also reverse age-induced memory loss so we decided to test this in a mouse model in which we administered ISRIB to young and old mice and we measured memory loss using the radial arm water maze I have a cartoon depiction of what the radial arm water maze looks like here and what you see is a large pool filled with water with eight arms in one of these eight arms there is a platform that is submerged just below the surface of the water it allows the animal to escape the water but is not visible to the animal over a period of several days we train the animal to locate this escape platform using visual cues placed throughout the room this is similar to how when you navigate your way home at night you use street signs or buildings in order to know where to turn in order to find your way home so it's commonly used as a measure of spatial learning and memory deficits so for this trial what we did is we trained the mice over a period of several days to find this escape platform during that training the mice received ISRIB injections this is the exact same paradigm we used in our trauma studies in which there was three injections of ISRIB and then we test memory deficits later and so we looked on day 10 to see if the animals could locate that escape platform and in order to quantify performance in this tool we count the number of incorrect turns the animal makes prior to finding the escape platform so every time the mouse turns into an arm that does not have the escape platform it is counted as an error so more errors is poorer performance in the tool so when we looked at young and old male mice we see that old male mice perform worse in the tool making more errors when compared with our young mice this was not new this has been shown previously but what was very exciting to us was that when we administered ISRIB to old mice we see that they perform at levels comparable to our young mice so the brief ISRIB administration is reversing this age-induced phenotype so I mentioned this was an old male mice we also looked in old female mice and we see an identical pattern where ISRIB administration reverses this age-induced deficit so we're able to see the potential for ISRIB to reverse age-induced memory decline we then wanted to look to see how long do the effects of ISRIB administration last so as you'll remember we were testing the radar water maze at day 10 so we decided to keep the mice alive and continue out later so starting on day 20 we introduced the mice to a different behavioral paradigm the delayed matching to place barns maze this is a top-down view of this behavioral paradigm it's a large table with 40 holes in one of the 40 holes is an escape tunnel that allows the mouse to get off of the table similar to the radial arm water maze the mouse uses clues throughout the room in order to navigate its way to the escape tunnel however unlike the radial arm water maze we change the location of this escape tunnel each day making it very difficult for the mice and it is a task that's known to be very difficult for age mice with them seeing little to no improvement and this is exactly what we saw our old mice shown in the light blue here show almost no improvement over our four testing days we're measuring the time it takes the animal to find the escape tunnel and you can see from day 20 to day 23 the mean average of the group remains about the same what was exciting to us is when we look at our mice that received ISRIB now they received ISRIB 20 days prior there were no additional injections administered during this behavioral paradigm we see that their performance is improving over the four days so that by the fourth day they are performing the tool at a significantly faster rate than their matched age counterparts so now we're really able to see the long lasting effects of this brief ISRIB treatment in our aged animals so next we wanted to look at how is ISRIB doing this and the first thing we wanted to look at is neuronal function and we decided to begin looking at this during ISRIB administration and there were a couple reasons for this the first is that this is when we first started to measure behavioral differences we wanted to look within the hippocampus which is one of the brain regions that is known to control learning and memory and we did some electrophysiology on these animals and this was done by Amber Nolan a former postdoc in Susanna's lab who has since left the lab and started her own lab at the University of Washington and I should say that Amber did a lot of work and a lot of recordings on these animals but I'm just going to show you the one where we measured age differences and ISRIB differences so we looked at the after hyperpolarization amplitude and what we're looking at here is following a series of spikes we look at the amplitude of after hyperpolarization zoomed in here what was known prior to this study is that young animals have a low after hyperpolarization whereas old animals have a larger after hyperpolarization and this can affect subsequent intrinsic excitability in the animals and what was exciting to us is what we found is that during ISRIB administration that old animals that received ISRIB this after hyperpolarization amplitude was reversed to levels equivalent to young mice so similar to what we saw in the behavioral paradigms we're seeing that ISRIB is able to reverse this age-induced phenotype to levels similar to our young mice so having seen these differences in neuronal function we next wanted to look at neuronal structure and this was done by a graduate student in the lab Alma Frias who I'll tell you a little bit more about later in the talk so Alma was looking at neuronal structure specifically she was looking at dendritic spines and again we're looking at the hippocampus in the same region that amber was doing recordings and we're able to use fluorescent mice that have excitatory neurons labeled and so we can identify these spines on dendritic length and so what we measured was a density so the number of spines we counted within a dendritic length and what is known is that with age we see a reduction in the number of spines um in the hippocampus and what Alma found is that when we give ISRIB to these mice we see an alleviation of this age-induced reduction in dendritic spines so unlike the behaviors and the electrophysiology we're not seeing a return to young levels but we are seeing an alleviation with ISRIB administration so we're able to identify the fact that ISRIB is able to change neuronal functional and morphological changes previously we've shown this in traumatic brain injury here we're showing it in normal or healthy aging so next we decided to ask does ISRIB administration have any effect on immune cells and initially we looked at microglia in the hippocampus as these are the main immune cells in the brain and we didn't see any differences between young and old mice and so excuse me we didn't see any differences between old mice plus or minus ISRIB so around this time a paper came out from a group down at Stanford that with age there's an infiltration of T cells into the brain so we decided to look at if we see this in the hippocampus and if ISRIB is able to affect this and so using QPCR analysis we looked at a T cell marker CD3 and what we did see is that with age there is a significant increase in this T cell marker in the hippocampus of our old mice and that when we administered ISRIB to these old mice we see that this age induced increase is reduced to levels comparable to our young mice and what was also striking to us about this is that this impact of ISRIB on T cell levels was not solely seen in the brain we also saw in the blood of these old mice where ISRIB administration reduced T cell levels so what could T cells be doing when they enter the brain of aged mice it's not exactly teased out in aging but in other models such as viral infection models it's been shown that when T cells infiltrate the brain they can influence cognitive function through production of interferon cytokines so inflammatory cytokines so we decided to look at these with age and we also decided to look to see if ISRIB could affect interferons and so I'm not going to show you the graphs today but I've listed some of the interferon response genes we looked at here and what we did see is an age effect where we saw increased interferon responses and these responses were reversed with ISRIB administration but what was equally exciting to us is that we saw these significant positive correlations between T cell markers interferon response genes and cognitive performance in the radio arm learning maze what does this mean this means that animals who have less T cell markers in the brain have less interferon response genes and perform better or make less errors prior to finding the escape platform whereas animals with higher T cell levels have higher interferon response genes and make more errors when locating the escape platform in the radio arm water maze so we're really starting to tease out and define this potential mechanism of T cells in the aging brain and the effect that ISRIB can have on these molecules okay so I've been able to show you that ISRIB administration affected long-term learning and memory loss we see changes in neuronal function we see changes in neuronal structure and we see changes in immune cell function but what if we look more molecularly inside the cell I mentioned before that we know that ISRIB acts downstream of phosphorylation of EIF2 so can we see the effects of ISR activation in the brain following ISRIB administration in order to do this we looked at ATF4 levels in the brain and as expected with age we see an increase in ATF4 levels and when we administered ISRIB to these old mice we see that this age induced increase in ATF4 levels is reversed to levels of young mice what was even more striking to us is that although ISRIB acts downstream of phosphorylation of EIF2 we wanted to see if there was a potential feedback loop and if ISRIB could be resetting this ISR activation and this is in fact what we saw when we looked at old mice plus or minus ISRIB we see that the old mice that received ISRIB had reduced phosphorylation of EIF2 suggesting that ISRIB is able to reset this ISR related this age-related ISR activation so in conclusion we were able to see that brief ISRIB treatment just three injections could rescue age-induced cognitive decline in mice we saw it in male and female mice and we saw it lasting for weeks after administration when we looked mechanistically at what ISRIB was doing we see that it improves neuronal functions specifically intrinsic neuronal excitability we saw that it increased enderexpine density it reversed age-induced changes in T cell and interferon responses in both of the brain as well as in the blood and we also saw a reversal or a resetting of ISR activation and this was the work that we did there's been a number of other groups that have looked at the ISR the integrated stress response as a main mediator of cognitive health and what is beginning to become an emerging picture is that regulation of the ISR could be important for mediating a number of different cognitive decline states and then just finally I wanted to share some exciting updates from the lab the patent rights of ISRIB are owned by Calico a company located in south San Francisco and they recently released that an ISRIB derivative is currently in phase one clinical trials and is expected to move into phase two clinical trials later in this year so this would be in this is in clinical trials for ALS patients but it is exciting to see the potential of manipulation of the integrated stress response as a therapeutic for regulating memory loss and I am no longer in the Rossi lab I have moved on to my next position but I did want to highlight some of the future directions as to where this study will be going I mentioned earlier in the talk that Alma Prius who's a graduate student in Susanna's lab did the spine density work well Alma is continuing to look at the spine dynamics and how ISRIB affects spine dynamics so when we look at spine density we're looking at a single snapshot of what is happening at this single time point and what Alma is doing is can she look at spine changes over time in a live animal and can she see how ISRIB affects the spine changes and her work is very exciting and I would say look for this coming out hopefully in the next year and another postdoc in Peter Walter's lab is looking into the cell specificity of ISRIB so we showed that ISRIB was able to affect neurons but we wanted to we're wondering is there a specific subset of neurons are there specific brain regions that ISRIB predominantly affects and this is what Morgan Boone in Peter Walter's lab is working on so I just wanted to show you all of the individuals involved in this study this these are the members of the Rossi lab and the Walter lab some of the authors aren't pictured here and then of course our funding sites so with that I can take any questions that you might have thank you so much Karen really really interesting we have some questions in the chat as we can start with one here how long lasting is the inhibition of ISR using ISRIB and could there be any long-term costs to inhibiting ISR using ISRIB yeah great question so we haven't looked to see how long lasting the down regulation of the phosphorylation of EIF2 is I can tell you that in the aging study we see that the brief ISRIB administration lasts for three we see cognitive changes asking for three weeks and one of our trauma models we see behavioral changes lasting for three months so we do think that there are long lasting changes with ISRIB administration as far as the cost of interfering with the ISR long-term I think this is a great question because obviously the ISR is a critical cellular pathway we wouldn't want to permanently shut it down and that is where some of the pharmaconetics of ISRIB are interesting where we've been able to find that the mechanism and I'm not going to dive into this too much because this really isn't my area of expertise but we found that ISRIB interferes with intermediate levels of ISR activation so if there's a very high ISR activation ISRIB is not able to inhibit it so it's really working in this intermediate state suggesting that hopefully we wouldn't see any deleterious effects and to date we haven't seen any in our mouse models okay and the follow-up question here then also are mice able to elicit ISR after being treated with ISRIB if they find themselves in a very stressful scenario in which the activation of ISR would be beneficial yeah so we haven't specifically tested that in our mouse model but yes we believe so and I think actually this brings up a good point from the trauma studies and one of the directions that we wanted to go is when we look at the trauma study as we do either a single or a multiple trauma model and then we administer ISRB later what we haven't looked at yet but I know they want to in the future is what if that mouse then receives another trauma after receiving ISRB is there any protection there or likewise if they received another insult how would ISR affect that and we just haven't looked at it yet okay thanks yes some more questions here ISRIB seems to elicit different responses in old mice there is heterogeneity in the responses among mice better and worse responders do better performers also have more young looking AHB I don't last this yeah so we do see a lot of heterogeneity in just old mice that haven't received ISRB there is a lot of heterogeneity in the old mice I don't have a lot of explanation for why that is as these mice are genetically identical they're bred together and they've lived in the same facility for the entirety of their 18 months of age but I think that it is just organizational differences that we see and I think we see that same heterogeneity reflected in the mice that receive ISRB we have not we do see the correlations between T cells and our neurons and cognitive performance we haven't looked at the electrophysiology or the spine dynamics in relationship to cognitive performance there I think that's what the AHB is in that question okay but yeah I mean I think similar to what we see in humans there's heterogeneity with aging and we see that mimicking our old mice as well yeah exactly one more questions are the cytokines kinds responses in old plus ISRB also changing in a young looking manner in the blood in other words is the immune phenotype in the brain organ specific or does it rather reflect systemic changes in immune profiles yeah I think that's a great question because one of the reasons we started to look at the T cells in the blood is could we identify a biomarker for an individual who would be at risk for cognitive decline in aging and we haven't looked at the cytokine profiles in the blood but I think that's a really great point we're still kind of looking to see if we can identify a true biomarker for this but so I don't know for the cytokines yet but hopefully soon and I think that's a great question so one more thing I was thinking about is this ISR system response responsible also for other aging or not aging but brain health effect so for example in burnout situations where you have a long chronic stressful situation is this system also in activation and could you then inhibit that with this ISRB functions I mean for stress conditions yes we usually do see ISR activation we haven't looked at it but I do think a system where we're seeing this kind of chronic low-grade activation of ISR yes I think ISRB could interfere with it and then could reverse some of the detrimental consequences that we see so it's not really aging specific it's more about the mechanisms yeah and that's where that review from Peter and Mauro really talks about a number of different degenerative states where they're seeing that ISR activation occurs and that using some sort of interference with the ISR pathway can alleviate in some way these different these different states so I do think we are moving towards a better understanding of maybe the ISR is this pathway that is common between a lot of different degenerative states and we've shown it in trauma and aging but a number of others have shown it and most recently it was shown in Alzheimer's disease as well great so I think that was a great ending for this discussion so thank you so much Karen super interesting thank you and move on to the last talk of today so let's see Gada are you there yeah hi hi welcome so our last speaker is Gada Alsali from the Kennedy Institute of Rheumatology University of Oxford in the UK so the title of your talk is Autophagy as a pathway to rejuvenate immune responses yes thank you Sarah for the introduction and I would like also to thank Eli for organizing this symposium and to give me this opportunity to present my work and as you mentioned this would be about autophagy and rejuvenate immune response so as you all know that one of the great transformation of our society is the increased number of people who are eight than more 60 years old and of course this great achievement of increased lifespan is affected by the increased number of people who suffer from one or several age related disease and this is open a new challenge in front of us to understand the mechanism of aging and it's the process and actually scientists do make a great effort to understand what is the mechanism of aging and they proposed to us nine candidates that contribute to aging and all together could determine the aging phenotype which we can generally classify in a three group the first one which is the molecular alternation and this considered as the primary cause of aging and this of course leads to the cylinder dysfunction and which finally does end in the tissue damage all this factor together lead to increase the risk of developing or inducing many age related disease and actually autophagy which is the main degradation and recycled pathway in the cells seem to be situated in the heart of aging mechanism it's linked to all this factor directly or indirectly we know that autophagy level is full with age and that's lead to increase the synthesis associated secretory phenotype several aesthetic kinds such as al1 beta tenef alpha it's also increased the damage of mitochondria damage and increase rose reduction of course this have also great impact on the damage on the other side when we overexpressed the gene of autophagy we can increase the life span in mice and the treatment with rapamycin or the color restriction also increase life span in different spices in autophagy dependent manner so we know that autophagy is reduced with age while the immune synthesis is increased with age so the immune synthesis is a theory of function declined with age in the immune system including the increase of inflammation or most likely we all we call it also inflammatory aging we have the increased number of macrophages neutrophils however we have a lick of their function in addition to that we have also decreased in naive tnp cells and also a decrease in memory of tnp cells for a new antigen so all this factor lead to a compromise immune response against infection and vaccination as we witness today with the pandemic of COVID-19 so in our lab we wanted to study and to understand the role between autophagy and immune synthesis so thus is this we inhabit first autophagy specifically in t cells as you can see here we inhabit we use etg7 which is gene for autophagy and actually Dan who was a PhD in our lab in his elive paper in 2015 showed that if we take these knockout mice in t cells specifically and infect them with a flu we have a normal response to a flu infection however a normal effectory response however we have a decrease response as you can see here in the memory response and these effects seem to be mimic what we find in aging as you can see here when we also infect mice with the flu we have a normal effectory response of cd8 t cells however we have a compromise of cd8 memory response so we can see that there might be a correlation between the phenotype aging and autophagy so we wanted or Dan wanted to induce autophagy using spermidine which is a natural polyamine it's synthesis by the eukary itself and we can also take it from the netto or blue cheese and other food so when we treat spermidine to these old mice we give it in water over all the experiments and we can find that actually when we give spermidine to these mice we increase the response of the effectory response and also specifically the memory response of the cells to the flu infection so I wondered what is the case then what is the role of autophagy during human during the vaccination in human cells so to assist this we try to get some sample from vaccinated trial and we could first get a different sample from hepatitis C trial where you can see we have several time points and then we measure autophagy in the cells using a fax essay this essay have been developed in our lab using different primary cells to validate it and then we apply this in the sample from the vaccine what was interesting as you can see here that autophagy was induced specifically in the hcv specific CD80 cells and but not in the non-specific cells and this effect was reduced in the end of the study so this might show that there is a role important or autophagy might be important in the response to a vaccination but as I mentioned before that the vaccination is most likely affected in the elderly I wanted to study and we want to study what is the rule of autophagy between and compare it between old and young so we get sample from RSV trial it's a respiratory synestical viruses which have sometimes a serious effect in the elderly and we compare we measure autophagy in the blood in the CDT cells in these vaccine and as you can see here that autophagy was reduced in the elderly compared to the young people but the most interesting things that actually autophagy was correlated with antler furonga medicines in the elderly and not in the young which means that autophagy might be important for the secretion of antler furonga and to study this we take CDT cells from mice where we inhibit autophagy it's of course knockout for autophagy in T cells and as you can appreciate these here after activation the cells for three days with CD3, CD28 we can see that the secretion of antler furonga is dramatically decreased in the superneton and also in the cells intracellular staining by facts moreover we also find that the periphery in which is important for the cytotexity of T cells and their function was also reduced in these cells we find the same data after we activated cells from a human sample from young people and we inhibit autophagy using two different inhibitors the hydrochloricine which inhibits the fusing of autophagy with lysosome and SPE which an inhibitor of ulk when it's in the initiation of autophagy and as you can see here antler furonga mass secretion also was reduced in the superneton and inside the cells and also periphery was reduced so I would we wanted to see if we could increase the autophagy by adding speramedin are we going to improve CDT cells function speramedin was reduced with age we measured this in different BPMCs from different people different age and we found that speramedin was reduced so when we added to the cells of T cells of course they were as isolated from elderly and then they activated for several days adding speramedin to them as you can appreciate here that antler furonga mass secretion was increased in the superneton and also intracellular by facts and the periphery was increased on the other hand when we inhabit autophagy speramedin was unable to induce and improve T cells response we we have actually recently identified how speramedin regulate autophagy and and Hanan who was also a PhD student in our lab identified the mechanism those speramedin have been used since several years but they are correct or the the mechanism of how speramedin induced autophagy was still unknown so Hanan showed that a speramedin induced the hypersination of ephabe which is a translational factor and this hypersination is amino acid exists for the moment only known to be exists in this specific protein ephabe and the rule of the hypersination is to facilitate the translational of a 3-proline motif of protein and one of these protein is T-phib. T-phib is a master regulator of autophagy and lesothome so we find that speramedin is important for the hypersination of ephabe that leads to induce ephib and T-phib is important for autophagy and autophagy was important for B cells response so I wanted to know if this mechanism also it's the same mechanism that speramedin induced autophagy in T cells so to do this I take a blood from BBMCs from Ordina and we inhabit as speramedin using the FMO this is inhibiting the entogenics as speramedin we inhabit the synthases inside the cells and as you can see here that we could decrease at the hypersination and also T-theb-label in the cells however when we supplement speramedin again in the speramedin we could increase the hypersination and the T-theb-expression and you can appreciate here the quantification of this with some blood on the other hand in the elderly when we take the blood we have already decreased of hypersination and a T-theb-label in their BBMCs and T cells so when we add speramedin to it we could enhance and induce T-theb-expression and the hypersination so we find that speramedin mode of action is via the hypersination of T-theb and the hypersination of alpha-v and inducing T-theb however is T-theb important for T-cells response to answer this question we use cells where they were knocked out for T-theb and we activate them for several days with CD3, CD28 and we look at the secretion of interferon gamma and periphery and as you can appreciate here we can find that the interferon gamma was reduced in the cells and the periphery and however when we supplement speramedin we were unable to re-secure the secretion of interferon gamma or the protection of periphery so in summary we find that the T-theb-expression was reduced with H and that's lead to increase of autophagy which lead to increase the immune synthesis in T-cells and that's have a great impact on their response to vaccination in the elderly so in this we cover a new drug targets that we might be used to enhance a vaccination efficacy in the future I would like to thank you for your listening and I'd like to thank all our group we seem to be all happy when we still meeting all again face to face hopefully this will come again I would like to thank specifically Isabel and Hanin who participate in this work and all our collaborators and I'm happy to have your question thank you Gada really really nice presentation and very and topical right now I guess with the pandemic so we have a question here from Dario is the change in autophagy levels between young and old mice cell specific or is it about measure because there could perhaps be differences in amounts of T cells between young and old mice yeah so actually we look at it separately and in the whole cells so even if you look at specifically B cells or specifically T cells we find that in mice and also in human we could find that autophagy with H is reduced so it's not a bulk it's really like if you look at the bulk you have this reduction but also if you look separately autophagy was reduced also oh great sorry I just want to mention that autophagy seem to be start to be reduced around 60 65 years old and also T-fib these protein the mass circulation of autophagy is also dramatically reduced after 65 so we could easily distinguish people aging just from how much they have T-fib in their blood and also this is dependent in T cells or B cells okay right and is that have you pulled the the cells from both men and women or have you looked at sex specific effects no this is was like a independent of sex whatever is yeah right there is one more question from Dario is autophagy involved or necessary for T cell receptor assembly uh you mean for the priming is that his question I think Dario exactly yeah so this is something we are trying actually to answer because we are studying also the the effect in rule of autophagy in CD4 and CD8 so in CD8 I remember that Dan was looking if the cells were defect in priming but this is was not the case because he used actually a chimera mouse where he mixed the knockout with the the white type and then he could distinguish that is not actually the lymphovenia because of course you will have lymphovenia when you have deficiency with autophagy was not the primary because of course in these T cells mice model when you use the CD4 you inhibit it in T and for CD4 and CD T cells however now we are working in a project in CD4 T cells and we are investigating if autophagy are important for the prime if it's really the priming or after after that very interesting okay one more question just came in here the T-fab also plays a role in aging could you speculate whether T-fabs role in immune response that you showed could be linked to its role in aging yes of course I think I think that T-fab might be used as a new bio marker for aging because T-fab have been also studied in different age related disease especially neurodegenerative disease of course we need more study to show if it's a bio marker but what we are working now we are trying actually to find a drug which enhance this expression on T-fab and I know that several actually company trying to use adenoviruses or like a drug specific to T-fab to to enhance and improve the age related disease so that would be interesting to follow up then to see how that goes yeah hopefully I mean we will get some data at the end of this year we are gonna we are doing actually I'm doing some of CRISPR screen to find what is the regulator of T-fab why T-fab is really reduced and it seemed to be like a translational in a translational level and on the other hand we are we are trying to find a drug to enhance this pathway very interesting so thank you very much got a really nice thank you thank you it was nice to meet you all so I think with that Dario we thank we think the symposium is has come to an end so well we'd like to thank everybody at eLife and all the speakers and the participants thank you Sarah thank you all for attending and then bringing up pretty interesting questions and for staying on the whole afternoon thank you bye bye bye thank you