 Okay, good afternoon everyone, and Robin, I have this on, good, okay. Welcome to our first inaugural international Steenbach Lectureship. So as you know for many years now, the biochemistry colloquiums flagship lectureship is in honor of Harry Steenbach, and this year we're adding for the first time an international version of the Steenbach Lectureship. So the Steenbach Lectureship, of course, is named after Harry Steenbach, who was a famous scientist and here at UW-Madison, in fact. Harry grew up in Wisconsin and spent most of his education here in Madison, and fittingly for today's speaker, the last place Harry was before returning to Madison to finish his PhD and really spend the rest of his career was in Germany, he was in Berlin. So Harry was known for, made many similar scientific discoveries and many contributions to the university, but none more noteworthy perhaps than his contributions to vitamin D fortification. So Harry was instrumental in getting a rat colony here at the university and discovered that it was the radiation not of the animals themselves, but the food they ate was sufficient to cure rickets, it was to, it led to the activation of vitamin D. And Harry used then $300 of his own money to patent his invention and was later offered in the 20s a million dollars from the Quaker Oats Company for the rights to that patent, which he turned down because he believed that the university should really be the beneficiary of his work. So with along with eight other Wisconsin alumni who each contributed $100, they started the Wisconsin Alumni Research Foundation, WARF, and in 27 WARF had its first licensing agreement to the Quaker Oats Company that uses technology to fortify their cereals with vitamin D. So I think it's pretty clear that Harry is someone worth honoring and I am excited to today to honor our speaker with a lectureship in his name. So Professor Thomas Langer received his Ph.D. in 1993 from the University of Munich and part through his work he moved during his Ph.D. and spent time at Memorial Sloan Kettering with friends over Cartel who did pioneering work in protein folding and is now the managing director of the Max Planck Institute for Biochemistry. So Thomas' early work had to do with protein folding and chaperone mediated protein folding. Then he moved back to Germany and joined the lab of Walter Neupert where he did his post doctor work and then received a full professorship at the University of Cologne in the year 2000. So Thomas' work deals with mitochondrial proteases. Everything about proteases, how they work biochemically, how they are regulated and all in his work is quite eclectic in dealing with proteases, things like the control of mitochondrial fission and fusion, the membrane dynamics, lipid trafficking, many different aspects of mitochondrial function. It's really put him at the forefront of really a burgeoning field of mitochondrial dynamics. And so I'm very excited today to introduce Thomas and he'll be giving of course two lectures tomorrow, same time, same place, 3.30. And before I introduce his first lectureship entitled mitochondrial proteases, membrane dynamics and disease, I'll remind you that right after the lecture today we'll have reception outside running till 5.30. So please join me in welcoming Professor Thomas Langer. So thank you very much Dave for this nice introduction and for bringing me here to Wisconsin. It's actually the first time that I'm here and I have to say I'm truly impressed already. I'm very much honored to give the first international Steenbork lectureship. It's really a pleasure for me to be here and I should say I really spend already a gorgeous day the whole day over talking with students and many faculty and I just can say it's really a great place and I'm really happy to be here and I'm looking forward to even more of these talks. But before that I will actually give you a first part of glimpse on our work on mitochondria and as Dave already said we are interested in mitochondrial proteases in this organelle. And this organelle is not only beautiful as shown here for primary hypochampal neurons where this mitochondria highlighted with GFP forming this showing that they form this reticulated and tubular network. These organelles are also very essential. And actually as I'm aware that I'm talking, many of you don't think of mitochondria each single day which is maybe a mistake but I thought I just remind all of you in the first slide that these organelles are really essential. So there is no proliferating cell without having mitochondria. And you will all immediately think that mitochondria energy producing organelles and of course all cells need ATP and you are absolutely right but this is presumably not the reason why mitochondria are really essential for those cells because there are cells just think of bakeries that can survive without respiration in mitochondria and this just shows you but they still need mitochondria and there are many other functions in mitochondria and this is just a list of those certainly not comprehensive list. And just highlighting here that what is now recognized as the essential function of mitochondria is this synthesis of iron and sulfur clusters which takes at least in part in mitochondria. So mitochondria are really essential for cell survival but there was really a boost in the last 10 years I think in the research in mitochondria because it was recognized that these organelles are not isolated organelles that as you may know are derived from bacterial ancestors but that these organelles are really deeply integrated in cell physiology and mitochondria are now much more sort of kind of signaling platforms in a cell which are integrated in major cellular signaling pathways and this interconnection of mitochondria with cell physiology really has boosted mitochondria research and in the interest in mitochondria as an essential cellular organelle over recent years. Now although or maybe even though mitochondria are essential organelles their biogenesis is extremely complicated and it is for the cell really a challenge to preserve mitochondrial activity which is essential for cell survival and this is in part because mitochondria have their own genome and although the coding capacity is limited this genome encodes a subset of essential respiratory chain subunits that have to assemble with nuclear encoded respiratory chain subunits into functional respiratory complexes and you can imagine that if there's any imbalance in this expression from these two different cellular genomes that there's a problem in the mitochondria kind of protein stress as you will hear later in more detail mitochondria highly dynamic they form this tubular network that I have shown you before and they're constantly rearranged but they must preserve their functional integrity during these processes just think of the proton gradient that is built up across in a membrane that must be maintained during this membrane rearrangement by fusion and fission and of course mitochondria are the side of respiration and a by-product of this respiration are so-called reactive oxygen species which are of course damaging or damaging agents that react with protein lipids in DNA in the nearest neighborhood and this is the side of production in mitochondria but and in this sense can also cause damage and oxidative stress but now it's also becoming more and more clear that this Ross rather than only acting as a damaging agent also has important signaling functions now but this just should highlight that it's really a challenge to preserve mitochondria integrity and function and in fact if you have a dysfunction of mitochondria this is associated with many diseases diseases caused by mutations in mitochondria genes but also secondarily affecting many of a neurodegenerative diseases in the pathology of these diseases and there's a decline of mitochondria activities during the aging process and there's a lot of debate about it what the contribution of this dysfunction of mitochondria really is directly to this aging process now this also just tell you that it's very important for a cell to preserve mitochondrial activities and function to specifically recognize mitochondrial damage and remove this damage from the cell just to ensure cell survival now therefore it's not really considering the importance of mitochondria maybe it's not really surprising that there's a really complex network of quality control mechanism in place that basically just fulfill this function and a key or key component of this quality control system are proteases that reside within mitochondria so these are just regular proteases that you know and mitochondria have a whole complement and have basically the ability to completely degrade proteins down to the amino acid level so the oligopeptidases ATP dependent proteases now originally proteases were recognized in mitochondria as simply as processing peptidizes that chop off mitochondrial targeting sequences of nuclear encoded proteins and therefore mediate the maturation and ensure the functional activity so this is the classical function but it became clear later that there are a whole complement of different proteases present in mitochondria and many of them are ATP dependent so they utilize ATP for degrading proteins and they were originally considered mainly as quality control proteases to do exactly what I just described you recognize damaged proteins and remove them degrade them to prevent the accumulation of possible deleterious effects but and this actually also the time when we started to get interested in these proteases but it turns out over the years that these proteases in addition or maybe even to say much more than having a quality control function they have important regulatory functions during mitochondrial biogenesis so there the proteases recognize specific proteins of regulatory with a regulatory function and preserve in this way both the biogenesis and the maintenance of mitochondrial activities and therefore it's now actually of broad interest in the field to understand what really the function of these proteases is not least because there are many many diseases associated with these proteases in mitochondria actually with any almost almost any of the proteases you have a disease has been associated with now in recent years now these regulatory functions really affect various aspects of mitochondrial biogenesis and function and this may be a bit busy slide here you see that all these function include the biogenesis of mitochondria the synthesis of proteins within mitochondria ribosome assembly mitochondria dynamics quality control like mitophagy and apoptosis and so on and so forth also the force for lipid metabolism and so on and so forth now my lab has a long-lasting interested interest in mitochondrial proteases and basically looking for substrates and proteases regulated by these enzymes in mitochondria we actually yeah as Dave pointed out touched upon many different aspects of mitochondrial biology and I would what I was thinking what I do now in these two talks today and tomorrow is basically introduce you to two of these processes that are regulated by proteases and describe a bit what we have done on these in this and these areas and one with today's lecture will be on the regulation of mitochondrial dynamics by proteases and tomorrow I will talk more about phospholipid metabolism in mitochondria and the trafficking of lipids within mitochondria and we look at these processes in general using yeast cell culture and mouse models and try to bridge actually our understanding from a more biochemical analysis to really more to study these processes in a more physiological context and today I will have a more physiological talk and tomorrow I will talk more about the biochemical analysis of these processes now mitochondrial dynamics I mentioned already mitochondria are highly dynamic organisms that constantly fuse and divide and this determines the topology of mitochondria in a cell can be very different in different cell types in different tissues you may know this from any textbook if you look at mitochondria they can look quite different you know in a this is a fibroblast you see again this reticulated network of mitochondria that is maintained by these fusion and fission events now these fusion and fission events have many functions to preserve are very important to preserve mitochondrial activity the fusion is called usually in which results in tubulation of mitochondria is considered as a pros survival mechanism it pushes mitochondrial activities preserves mitochondria integrity maybe also to preserve contact allow contact mixing between different mitochondria in a cell whereas the fission is associated with a mitochondrial quality control a fragmentation of the mitochondrial network for instance allows the orthophagic removal of damaged mitochondria you separate them from a tubular network and then can selectively remove damage from these organ damage parts of this organ and if it comes to the worst and the cell cannot fix the problem anymore mitochondria the outer membrane is ruptured and pro apoptotic proteins like cyrochrome C are released from the intermembrane space triggering cell death and if you think this is maybe the final quality control mechanism if you want now this fusion and fission therefore is very important for this maintenance of mitochondrial quality control now the reason why I'm actually highlighting this is because the fragmentation of mitochondria is observed under many also pathologic conditions and keep in mind fragmentation can be brought about by both either by inhibiting fusion in this way ongoing fission event will result in fragmentation or you can stimulate fission and this will override the fusion event and you will in both cases result in mitochondrial fragmentation so this is all a balance between but the fragmentation of then of the mitochondria network is really observed in many pathologic conditions and it is becoming and this actually also fostered a lot of research in mitochondria because people notice oh something happens with the mitochondria under these cells what I will tell you today or will try to convince you today that this is a very general stress response how mitochondria basically adapt to cellular stress under various conditions that is actually under control of proteases that reside within mitochondria and that affect the balance between fusion and fission now there's a whole set of proteases but the two proteases I'm focusing on are these two guys here and then in both proteases that reside in the mitochondria in a membrane one is termed by me one and the other one is all more one and I think I have another one no actually no so why me one is an ATP dependent protease an ATP dependent metalloprotease forms hexameric ring complexes and is a classical quality control enzyme and Oma one as you will see later is a stress activated metalloprotease also in a in the inner membrane now these two proteases actually control a central player of the mitochondrial fusion machinery and this is the OPA one now fusion and fission of mitochondria is mediated by dynamine like gtps is both acting on the outer membrane and at the inner membrane and this is a central player of the fusion machinery in the inner membrane and it's termed OPA one because mutation in this dynamine actually are the most frequent case of inherited blindness in human and this is how it was also discovered and these are rather complex molecule because there are actually eight different splice variants that are expressed in a tissue specific manner and I can we can come back to this later if you want what maybe the purpose of this is and even more they are not only splice variants but they also undergo proteolytic processing and for the sake of this talk is actually sufficient to memorize that this proteolytic processing is really essential to preserve mitochondrial morphology so and therefore it was this fostered a lot of interest to understand how this what what are really the proteases that are mediating this cleavage event and how are they regulated and there was a lot of they can tell you a discussion about it what the proteases are but I think this is a bit settled now because there are these two proteases that I mentioned already the OMA one and YMI one were shown to cleave OPA one at two distinct sides so there are specific sides that are close to each other that where these proteases cleave OPA one converting it into long forms into short one now this looks a bit complicated here but in fact it is not so complicated once you distinguish long and short forms and they are different sides so if you delete OMA one here then you basically abolish cleavage at side one and this form C and E disappear if you cleave the second protease which is YMI one then you selectively lose the cleavage product of cleavage at side two and if you combine and this is most important for what I'm going to say next you basically lose any processing so these experiments that were actually done by Ruchika Anand and Tim Wey in the lab actually demonstrated I think unambiguously that these are the two proteases that really cleave this dynamic like gtps that was by itself interesting to us but in particular it was interested that this now allowed us for the first time to really look what is the function of this processing it was understood that really you need this processing somehow to preserve morphology but it was really unclear how this is brought about and what the function of this processing is so therefore we looked at these cells and these were knockout cells that we derived from respective animals then we had really a surprise and I don't want to go into any showing you any data here just want to say so we looked at this protease there was no processing of OPA one and the mitochondria looked completely fine so there is mitochondrial fusion is ongoing the ultra structure of mitochondria they form this peculiar crystal was completely normal and also the apoptotic resistance so the resistance to release cytochrome C from these cells was completely normal so that was really a surprise almost a shock to us I have to say because we were also assuming that this is really important for the you know for the maintenance of mitochondrial morphology now further experiments then actually confirmed that really this long unprocessed form of OPA one is sufficient to mediate fusion and what OPA one processing is doing it limits the the accumulation of the fusion form and even more we think but here I think we are not absolute it's not the last word it's not said that the short form of OPA one is instead associated with the fission process so what the proteases do by cleaving at these specific sites they basically cleave long OPA one forms which is the fusion active form limit the accumulation of the fusion active form and at the same time accumulate a short version that is associated with the fission so they basically in both cases they drive the balance of fusion and fission in such a way that they stimulate the fragmentation of the mitochondrial network so this is basically the idea so the balance fusion and fission and the special thing is why do we need two proteases we think that these proteases response to respond to different input signals so the proteases are different differentially regulated the product is not so the we think that the short form of data generated by these proteases are functionally as far as we can tell now up to now completely equivalent the differences only come from differences in the cleavage specificity they differ by a few amino acids but they all convert both proteases convert long forms into short forms and by that shift the balance to mitochondrial fragmentation that's maybe the first important take-home message for today now to which signals do these proteases respond then now we see as i will show you in a minute OMA1 is a classical classical it's a typical stress activated peptidase and responds to different types of stress whereas YMI1 seems to be regulated and this is by far less clear by different metabolic use and the principle is therefore by differential expression of these two proteases in different tissues by differential expression of these two different eight splice variants which differ in their content of the proteolytic sites you can basically adjust any level of long versus short form in any tissue that you want to have so it's basically a perfect system to trim and optimize this or adapt this balance between fusion and fission to any to any physiological condition condition you may think of so therefore i think it's a very cute system to really regulate this balance between fusion and fission now i mentioned OMA1 is a stress activated peptidase this is basically just the same when you activate then this OMA1 you basically have this shift this to my to my to the fusion fission and you have a fragmented mitochondrial network now OMA1 is a stress activated peptidase and this is just a panel of different conditions that Philippe Lampier actually at that time a master student in the lab did during this thesis where he basically treated isle as in this case these are maths with different stress conditions and i admit these are extreme stress conditions for instance depolarization of mitochondria mitochondria will die actually when when they are depolarized but in in a cell you can do it it's also a reversible event i should say but then you basically trigger the conversion over time of the long forms into these short forms and this is mediated by this OMA1 peptidase and this you can actually do under various conditions so you can do it you know under oxidative stress conditions heat stress conditions you can inhibit activities of respiratory chain complexes under hypoxic conditions under all these conditions these peptidase is activated and cleaves these long forms of OPA1 limits fusion and shifts the balance of fusion and fission to mitochondrial fragmentation okay so this seems to be a very general stress responsive pathway now how this is regulated one aspect that was actually we were surprised when we started to look at the peptidase what happens to the peptidase itself during stress conditions and this is shown in this experiment that Michael Baker did in the lab so where he basically expressed a tagged variant but it doesn't matter it's the same for the endogenous protein and to look at it at that time we didn't have a good antibody for it and what he observed is that he did when he applied stress in this case is depolarization he did not only see this conversion of the long forms into the short forms but he also saw this turnover of the OMA1 protein itself so it turns out this is autocatalytic because he didn't see it in sales expressing only the proteolytically inactive variant indicating that there's autocatalytic processing of not only processing of OPA1 but also autocatalytic degradation of OMA1 under these conditions so active stress conditions like for instance depolarization of mitochondria activate we think somehow OMA1 peptidase and we would love to understand how I'll come to that in a minute this has two consequences is induce the processing of OPA1 shifting the balance as described but at the same time it initiates its own degradation and we think this makes a lot of sense although it looks weird at the same because it ensures the reversibility of the stress response otherwise anything that activates a protease irreversibly will basically there would be no way back until the lifetime of the protease but in this case because the protease basically limits its own lifetime you basically ensure the reversibility of the stress response and indeed you can show in vitro that if you alleviate the stress after some time the tubular network recovers which is only possible because the protease is gone again so therefore it's by we consider this as a kind of inbuilt timer in this regulatory in this stress response now we are of course interested to understand how this is activated best on a structural level but I was talking with some people already it's really difficult to get it for these proteins so we don't have it but we made a very simple observation actually and that is we looked at our second model system that actually also express an OMA1 variant but much to our surprise we didn't see any turnover of OMA1 and there is also no stress induced cleavage of the OPA1 homolog MGM1 in yeast so this pathway is this stress pathway does not seem to occur in yeast so therefore we thought we can exploit that to maybe learn a little bit more about how the stress response pathway is regulated and we expressed in yeast cells lacking the yeast OMA1 the mouse OMA1 and basically when we then treated those cells or stressed those cells when then we basically saw autocatalytic turnover of this mouse protein so the mammalian proteins have the intrinsic property to degrade themselves that the the yeast proteins don't have so there's apparently a difference between the yeast and the mammalian proteins now of course we looked at the sequence and what you becomes very apparent is a conserved protein family metallopeptidase family and we noticed then that of course the mouse one also the human one they have n and c terminal extensions when we compare them to the yeast one so the yeast seem to have the really the core peptidase domain but maybe the the mammalian proteins attained additional domains that make them them responsive to mitochondrial stress and we therefore started to mutate these areas in the mouse proteins to really learn more about it and I don't want to say much that's a very busy slide here but just maybe focus on that there is a patch here of hydrophobic amino acid and charged amino acid that somehow attracted our attention and it turns out when we mutate this positive charges which are in this n terminal region on the other side of the membrane we basically can obtain a mutant that is still active it still cleaves OPA1 we still see this OPA1 forms but is barely responding to stress like depolarization or oxidative stress so this seem to us so this actually indicated to us that this region is very important for the sensing of the stress so we have a positive cluster in the amino terminal end that basically I should show put this figure here because you can show it best that basically in this part which we think somehow senses the stress and allows activates this peptidase under stress conditions upon depolarization and basically allows the increased processing of this OPA1 but this is about what we know and we would very much like to understand what really is the signal that is sense whether it's really only the only the membrane potential or whether there are other things okay good so this is basically what I've told you so far this OMA1 is a stress activated peptidase it cleaves L OPA1 which is active in fusion and in this case inhibits fusion and it also in this case accumulates S OPA1 which we think I still put a question mark here is activated is activating fission and in that case OMA1 activation results in mitochondrial fragmentation we have an internal sensor domain that is important for this response and after this response OMA1 degrades itself limiting basically ensuring the reversibility of this response only if you have persistent mitochondrial stress so if you have persistent stress then this basically results in persistent mitochondrial fragmentation and we think this fosters or renders the cells to prone to cell death so we have this fragmentation as an initial response and if it is persistent it results in cell death now this is all done in vitro and cultured cells in vitro and of course we are very much interested to understand or basically prove the relevance of the stress activated pathway also under in vivo conditions and therefore we moved into mouse and this is actually the second part what I wanted to tell you today our work where we looked at the role of this stress activated pathway mediated by this stress activated OMA1 peptidase in vivo and to do that we generated two mouse bottles for the OPA1 processing peptidase and this was done by Tim Wei who did most of the work that I'm going to tell you now actually almost exclusively and Michael Baker who generated the OMA1 conditional knockout mouse for OMA1 now a knockout mouse for OMA1 has been described by Carlos Lópezotín groups in Spain and they observed that these mice are viable so the complete knockout a conventional knockout of these mice and they observed the diet induced obesity in these mice and impaired thermogenesis but otherwise these mice are fine in contrast when we deleted the second OPA1 processing peptidase these processing these mice were embryo this caused an embryonic lethal phenotype so we used therefore we generated conditional knockouts and we used Cree different Cree driver lines and in this case and this is not really all the work I should really highlight this of Tim he used a Cree line that is specific for cardiomyocytes and this will become later more important later during my talk and when we looked at these mice the loss of these YME1 in specifically in cardiomyocytes limited the lifespan of these mice they all died you know with about one year this is just to show that there is a selective loss in cardiomyocytes this was associated with body weight loss and so on and so forth in these mice but of course we wanted to look how does it affect the heart function and therefore teamed up with the group of Boria Ibanez in in Spain who is who are cardiologists and a really specialist for doing the heart function and they did actually Tim went there also they did this heart analysis and basically what you see here the echocardiograph of these mice I was really happy to see that because even me being not a heart specialist could immediately say see that there's a phenotype I should say this is now 18 weeks so rather early in the mice and the next is a progressive phenotype now when the mice many of the mice are dying already at 40 weeks you see that there's a more severe phenotype so the heart function is severely impaired upon loss of YME1 in in mitochondria specifically in cardiomyocytes now these mice and I don't want to go into much detail show all characteristics of a dilated cardiomyopathy so these mice show dilated cardiomyopathy they have a reduced volume that is ejected from the left ventricle they show signs of fibrosis and we see the deaths of cardiomyocytes and this death is a necrotic cell death so various characteristics of dilated cardiomyopathy including a typical shift in metabolism that is observed or has been observed in other mouse models for a mitochondrial dysfunction and this is a shift in metabolism from the utilization of fatty acids to the glucose utilization and this we could basically show by doing some pet CTs where we basically see that in the YME1 knockout mice there's a significantly increase in the glucose uptake in the heart and we also see reduced level of total cardiocyanitines in these animals so this all illustrates and other experiments I don't show you that there's a shift from the utilization of lipids to glucose so we have an increased glucose uptake in the mice and as I said this is a really typical sign of cardiomyopathies that are associated with a dysfunction of mitochondria now what is the reason for that of course we look that I look at a mitochondrial proteins everyone I'm sure most of you will immediately think of course we'll knock out a mitochondrial protein ATP production is impaired and the muscle doesn't work and we have a heart failure of course we also saw that but it's clearly not the case we looked here at the enzymatic activities of the respiratory chain complexes of oxygen consumption in the mice and there was no phenotype whatsoever if anything we see a slightly improved activity of some respiratory chain complexes that we still try to understand but the message here is now there is no impairment of respiratory activities in these mice no reduced ATP production so it's the failing heart in this mouse model is definitely not caused by an energy crisis in these muscles so what can be the reason I mean we started to work on this because we wanted to understand the function of YME1 as an OPA1 processing peptidase of course we looked at mitochondrial morphology and did this via EM careful EM analysis the first thing that you see here the ultra structure of mitochondria is intact so they have normal crystal so that seems to all seems to seems to be okay but what Tim noticed when he carefully evaluated all these electron micrographs that there's a significant shift down shift of the size of mitochondria indicative of mitochondrial fragmentation in the heart and this is exactly what we would have expected because we have shown before in cells or many as actually several labs I should say have shown this before in in cells that if you take away YME1 you basically induce mitochondrial fragmentation you induce mitochondrial fragmentation and it seems that the loss of YME1 is causing us imposing a stress on mitochondria that activates the OMA1 peptidase and by that shifts the balance of fusion and fission so we see a mitochondrial fragmentation and this we could also recapitulate in cardiomyocytes you see an aberrant mitochondrial morphology in this primary cardiomyocytes and we did this because we wanted to look more biochemically if you wish at least directly look at the proteins in these cells and this we could to assess does it affect mitochondrial activities mitochondrial OPA1 processing in mitochondria and for sure it does and this is exactly the result that mirrors what I've shown you before in mouse embryonic fibroblasts we delete YME1 in these three animals here we lose the form D which is basically generated by cleavage of OPA1 generating at S2 and we see some accumulation of form C and D which is less pronounced but we do see it which is which are actually the forms that are generated by OMA1 just here this is another pro another substrate of YME1 which is also accumulating just as a control here so we see here exactly the same like a mouse embryonic fibroblast loss of this YME1 form as expected but also accumulation of these other forms that are generated by OMA1 now this led us to hypothesize and we have done this first in cells before we did in the mouse for time reasons that the loss of YME1 may impose a kind of stress on mitochondria that activates this stress-induced peptidase OMA1 and shifting the balance down to this C and E this is now again in maths you see a more C and E in these cells now these these mitochondria are fragmented in maths and seemingly also in cardiomyocytes in vivo but in maths we did what we did is we looked at the double knockouts and when we basically take this away we take OMA1 from these cells away we prevent the formation of these short forms and accumulate the long forms and this resulted in tubulation of mitochondria so from these cellular studies we would think why the cells fragment in the absence of YME1 because we activate the stress-induced processing by OMA1 of OPA1 by OMA1 so can we test this also in vivo here we basically generated double knockout maths and could restore mitochondrial morphology can we do this also in vivo so for that we basically or Tim took the heroic task to cross three alleles in one mouse which you can do but it takes time therefore the cell culture work was definitely faster so what he did is he crossed not only the conditional knockout or the floxtile allele of YME1 with cardiomyocytes specific creeline but also introduced the floxtile allele of OMA1 in this creeline so he ended up with a double knockout cell that specifically lost YME1 and OMA1 specifically in cardiomyocytes only so and now what is the phenotype of these mice now first of all western blot of these mice shows that there's really cardiomyocytes isolated from these mice shows that there's the expected processing defect we do not see any processing of OPA1 in these mice which is nice also in vivo YME1 and OMA1 are the only processing peptidases for OPA1 we see the accumulation of the YME1 substrate as expected what about the heart and what about mitochondrial morphology we do see a significant suppression of mitochondrial fragmentation in the heart so this is the same analysis as I've shown you before we just did EM and then Tim carefully quantified the basically the diameter or the mean surface of these mitochondria and looked at plotted their distribution here and as you can see here when you look at the yellow versus the red one the deletion of OMA1 in this YME1 knockout shifted the size of the mitochondria clearly to the larger side I should say there are not there's not restoring the the size of mitochondria and wild type cells so this is not the case but we have a significant shift to the larger size which is what we expected based on our cell culture work because we prevent stress-induced processing by OMA1 so we have an increased in size of the mitochondria how what about heart function the heart is completely normal under in these animals this is just again a cardiography echocardiography of these mice and you see there is no significant difference moreover without showing you we have no signs of necrosis fibrosis the heart the pumping rate of the heart is completely normal so by deleting OMA1 we can completely suppress this dilated cardiomyopathy now this basically told us just to wrap up this part that the loss of YME1 basically activates OMA1 this results in stress-induced processing of OMA1 mitochondrial fragmentation which then results in chronic heart failure which is associated with this metabolic shift from lipid metabolism to glucose metabolism so this basically shows first of all that the loss of YME1 triggers the stress-induced processing of OMA1 and highlights the physiological relevance of this stress pathway because it causes actually heart failure in this model identifies that OMA1 is a critical regulator of cardiomyoside survival because if we inhibit OMA1 and take it away cardiomyosides live longer in this model and therefore the heart is fine it also shows however and there are some people who like it others don't that the long form of OPA1 is actually seems to be sufficient for the survival in the in cardiomyosides seems to be sufficient for the survival of these animals so we maybe don't need this processing at all we are still breeding these mice we're still doing a lifespan on these mice so i want to see how long they really live in a year from now maybe i can tell you but at the moment it seems that they are really fine now this seems to this is basically we are played with this and then we basically see now this seems to be kind of a story we could make sense out of it but there was one observation that really puzzled us a loss and this is shown here so Tim i mean he was really an excellent postdoc now he's now his own group he basically crossed these YME floxed YME1 mice not only with a CRE-deleter strain that specifically disrupted in cardiomyosides but he also used another CRE driver line which basically deleted both in the skeletal muscle and in the heart and i should say this is broadly used in the mitochondrial field to understand what how muscle is affected in the biospecific intervention now he did it i actually ask him why do we do the cardiomyoside specific one if you have the other one he told me that this is better to do so and if i saw that three years later this is one of these experiments i said it to someone today you know this is three year experiment basically to get this curve so i have to spend a bit time on it anyway it's just to to highlight so this is again the the lifespan curve for this cardiomyoside specific one and the lifespan curve of the other mice that lack it in cardiomyosides but in addition in skeletal muscle is completely fine and here we did a full lifespan curve so deleting YME1 in the in the skeletal muscle rescues or suppresses any effect that we see of a deletion of YME1 in the heart this was completely unexpected and puzzled us a lot as you can imagine this is just again the western blood test to make convince ourselves that this is really the right mice we are looking at so we have a specific deletion in the heart and in the muscle now when we looked at the heart heart function in these animals was indeed fine expected it after seeing the lifespan so there's everything everything fine no sign of cardiomyopathy or anything even more surprising when we look in the cardiomyosides then we do see mitochondrial fragmentation so there is the phenotype in the cardiomyosides seems in terms of mitochondrial morphology seems to be identical so we have mitochondrial fragmentation in these mice but still the mice are fine because they lack YME1 in the skeletal muscle really weird so we were thinking what so this is basically just a so we have basically this mitochondrial fragmentation which causes chronic heart failure but the ablation of YME1 in the skeletal muscle renders these mice healthy so how what can this be now we saw a lot one key issue seems to be this metabolic switch that is associated with the failing heart from glucose to fatty acid metabolism and we were actually thinking about so what is special about this skeletal muscle now many people of you may be more familiar than this than i am but of course the skeletal muscle is an insulin signaling tissue not the heart but the skeletal muscle so we were thinking starting to think could this be that there's a endocrine effect perhaps via insulin signaling that the deletion of YME1 in the skeletal muscle actually affects insulin secretion or insulin signaling and in this way affects metabolism in cardiomyosides restoring or suppressing uh yeah suppressing cardiomyopathy in this mouse mode now we therefore did some glucose tolerance test and indeed it turned out that these mice lacking YME1 both in the heart and in the muscle show our glucose intolerant and we see a reduced level of fast in fasted animals reduced insulin in insulin level so this is all consistent with this hypothesis that that basically maybe insulin signaling affects the metabolism in the heart i should i just added this here in this gtt's here show are also done in double knockout mice we also generated double knockout mice lacking both YME1 and OMA1 both in skeletal muscle and cardiomyosides and this completely suppressed the phenotype because so this glucose tolerance test shows that this in this systemic glucose intolerance in this HMYKO mouse is also caused by stress induced OPA1 processing in the skeletal muscle in this case but that's just a side thing so the hypothesis is basically that by deletion of YME1 we basically impair insulin signaling we render the cell or we generate a systemic glucose intolerance changing the metabolism in the cardiomyosides and suppressing cardio or the cardiomyopathy in this YME1 specific mouse model now can we prove this in another way now another way to induce glucose intolerance is of course hi-fat diet never in my life I thought that we are doing these experiments in my lab but here we did and so we put these mice on a hi-fat diet to basically induce a systemic look in this mice I should say these are now the cardiomyoside specific mice so these are the mice that show heart failure so we put these mice on a hi-fat diet and you may think this is completely weird I would join you in that but and then tested what effect does this has on the functioning of the heart in this mouse model so first basically this is more or less a controlled experiment we see systemic glucose intolerance in these mice regardless whether YME1 is there or not so these mice you feed them with hi-fat diet they gain weight become fat show this systemic glucose intolerance as you would expect it became more interesting when we basically looked at the glucose uptake in cardiomyosides because the hi-fat diet indeed planted the glucose uptake in these cardiomyosides and actually also restored the acyl carnitine levels in these mice so this indicated to us that we indeed could shift the metabolism in these in these cardiomyosides now what about heart function now it turns out that these hi-fat diet completely restored heart function of these mice as long as we measured them huh so we have a new therapy here and this is just a joke hi-fat diet is good for against heart failure and I was always in the lab I was saying I will write some grant applications for that I know some companies who should give me a million but I'm these mice suffer of any deleterious effects of a hi-fat diet I'm just saying that and sooner or later they will die of I mean we had to stop the diet of course but they will die of obesity obviously so I'm not saying that because but in this time window actually it suppresses the failing heart in the in this mouse model actually I don't show you that but it even seems we are just doing that it can even reverse the onset after if you start the hi-fat diet after onset of the heart failure we seem to be able to reserve it but that's still very preliminary okay and these are just some data to convince you that they really restore this heart function there is basically we have no signs of fibrosis in these mice necrotic cell disease is gone suppressed and also the and also the left ventricular injection volume of the heart is normal when we treat this mice with a hi-fat diet now what does it leave us with this is just to wrap up what I just now told you is the the loss of volume one causes the stress activated pathway it activates OMA1 triggering mitochondrial fragmentation and resulting in a heart failure and this can be this heart failing heart can be which occurs around one age the mid-age mice can actually be suppressed in two ways genetically if we delete OMA1 and restore mitochondrial morphology this prevents necrotic cells as we believe because it stabilizes mitochondria OMA1 is a pro apoptotic protein in this in this context and basically prevents necrotic cells and suppresses heart failure however we can also suppress this heart failure by metabolic intervention that limits glucose uptake in the heart because this dilated cardiomyopathy caused by YMI1 deletion is associated with a metabolic change in cardiomyocytes we see an increased glucose uptake in a decreased fatty acid oxidation and if we interfere with this metabolism either by hi-fat diet or by deleting YMI1 the same gene in another tissue and impairing insulin signaling we can basically affect this metabolism and suppress heart failure independent of mitochondrial fragmentation this of course suggested this altered metabolism is somehow downstream effect of mitochondrial fragmentation but we don't understand it that is something we were very much interested but we don't know really how this what is behind this error which seems to error which seems so simple now this is also I think a good example coming back to the first part of my talk that the stress-induced mitochondrial fragmentation is indeed physiological relevant and actually OMA1 may be an interesting target because in the heart when we activate OMA1 in this case by deletion of YMI1 mitochondrial stress activates OMA1 we call this causes heart failure and we can suppress it by OMA1 I also showed you at least one piece of data that when we delete activate OMA1 by deletion of YMI1 in the skeletal muscle this also impairs insulin signaling so also there we have a consequence of a stress-induced mitochondrial fragmentation by OMA1 and we did another study that I don't have time to look into where we looked actually in a mouse model where we specifically induce a dysfunction of mitochondria in the forebrain of the mouse by deleting another mitochondrial protein I will say a bit more about it tomorrow prohibitins which is a membrane scaffold this triggers neurodegeneration and in this case when we delete OMA1 and prevent stress-induced processing we be significantly delay neuron loss we don't suppress it but we delay neuron loss also highlighting that also in neurons this is a pathway that is really important for cell survival okay now with this this is basically the summary what I've told you today I hope I have introduced you to an important function of mitochondrial peptidases in particular of OMA1 in the in the regulation of mitochondrial dynamics the balance between fusion and fission OMA1 and YMI1 are both OPA1 processing peptidases which ensure this balance of long and short form balancing fusion and fission OMA1 is a stress activated peptidase that degrades the fusion active form of OPA1 long OPA1 limits fusion and also forms the short one which we think is associated with fission in that way it balances or it shifts the balance between fusion and fission to the mitochondrial fragmentation to the fission side and mitochondrial fragmentation whereas fusion is considered a beer cell survival mechanism mitochondrial fragmentation allows quality control like autophagy to occur but we think in a persistent stress this also renders the cells more susceptible to cell death and this is what we look in this genetic model like OMA1 deletion which is of course a persistent stress that we impose on mitochondria and therefore we prevent the cell death by ablating of OMA1 but I don't want to but I do think that under physiological conditions OMA1 may actually be a quality control mechanism that is important here for autophagy now with this I really I'm at my end I really want to acknowledge the people in my lab I really have had the pleasure over the years to have worked together with really many exciting and really absolutely outstanding co-workers in the lab now this story that I told you was the mouse story was largely based or was largely the work of a really outstanding postdoc in the lab is Tim Wei he is now his own group in Paris and he really did all this heart-specific characterization of the consequences of OMA1 deletion now his work he also worked together with Ruchika before on the you know in the cell culture with the cell culture work to understand the role of OPA1 processing Michael looked together with Philip at the activation of OMA1 on his work I just briefly touched upon you she looked at OMA1 in the brain and we have really great collaborations in particular for this talk the I have to really highlight the collaboration of Boya Ibanez that's one of the examples that you can do all the certain things that frankly I never thought that I look at an echocardiography in my lab but we did and I think it was a lot of fun so they really helped us a lot and did many of the experiments as I showed and Pierre did the Pierre Restor in Paris helped us with the oxygen measurements in the heart and with Elena we are looking at all the mouse models together since many years thank you very much so in principle the idea is that it's basically the question whether you fragment them stress induced or not that's the idea so if the stress is so strong that you basically in hip depolarize them as a most extreme one that you activate this peptidase then you induce mitochondrial fragmentation and then basically if the stress persists they remain fragmented and basically they are removed either by autophagy and if then the stress persists even further then you induce apoptosis huh so you have to think of this as a kind of a gradual step you basically elicit this response you have fragmentation and then this if the stress persists the the cell tries constantly to refuse but if the stress is persistent and OMA1 remains activated you shift the balance constantly to the fragmented side and if kinetically if you basically stay longer on the fragmented side you allow the autophagy machinery to to basically being activated and if worse comes to worst you trigger cell death huh so it's I think at the beginning you have the activation of this pathway and then there is a kinetic argument to it that you basically if you have a persistent then you basically don't have it yeah yes I should really emphasize there are different types of fission in the sense of the physiological output so mitochondria fuse and divide all the time this is not necessarily mediated by activation of OMA1 so OMA1 is under stress conditions I should really say that but you have normal fusion and fission events ongoing all the time in a cell and under these conditions you don't it's basically not deleterious so after fission event I mean there are experiments done by Uri and Shiri and and he basically looked kinetically so each fission event is immediately followed by a fusion event so the the time that the mitochondria is fragmented is different but this may be very different in different cell types and in different tissues but in principle you have this ongoing all the time this is a pathway that is specifically activated in under stress conditions for instance we think that YMA1 is a pathway where you basically shift this balance more which may be more important under different various metabolic use so this is what I meant there are different input signals that basically affect this rheostat and allow the adaptation to the different conditions well so let's say these are metallopeptidases and you can inhibit them with any metallopeptidase inhibitor if you think of a treatment it's not so helpful first of all because there are many metallopeptidases and the main problem is to to bring it there actually we're trying to set up an essay to do a small molecule screen to look for inhibitors in particular of OMA1 of the OMA1 peptidase but they are not way level at the moment and it's not so trivial to reach the side there ah sorry not that I'm aware of so the question where they are really yeah this time yeah so we have no evidence for that yeah I mean that's the only thing we know it's always better to answer like this so there is no there's actually no evidence we have that they're really in him in case of YMA1 it's a bit more complicated because it's an ATP dependent protease and there I think there are we have some other assembly partners that affect the ATPase but in terms of the OMA1 peptidase which is really a classical metallopeptidase we have no evidence for a physiological inhibitor yeah yeah this is a very important question because one one aspect of all what I've showed you in the second part in the mouse book also relies on the assumption that we are looking here at the same substrate under all conditions and I have clearly to say we don't know there are several arguments why I would argue we look at the same substrate which is a correlative most importantly that this is the only substrate that we know where the both peptidases act we actively look at other substrate of OMA1 there are one or two candidates let's put it like this they are all stress-regulated proteins so in the literature there's maybe one or two described where I'm not so convinced about yet but this is absolutely out there and ongoing work is actually addressing that really not a I mean there's one substrate this is established I think but this doesn't have anything to do with this pulse but it's consistent with this being a stress-responsive peptidase can you speak up a bit sorry in vitro you mean this we haven't really looked at I must say I must admit this we haven't really looked at in vitro I mean of course in vitro you have your culture conditions where you impose the cell anyway under certain conditions you have to start somewhere I'm not sure whether this is most informative under those conditions but we haven't really looked whether they use then utilize in the presence of glucose maybe more lipids this we haven't we haven't really looked I think there was some yes this is possible this has been done by Kasmiara's lab years ago and you but they're basically they deleted 10 amino acids you have to delete more because there seems not to be a cleavage specificity and that would be the best in our model now to re-express this in this mice there are two caveats and I should say this has been done in a kidney disease where they're basically rescued you know a renal failure or suppressed renal failure by re-expressing this delta as OPPA1 so this has been done in this physiological context here we have not the critical thing why we haven't done it or even started it is that the expression level of OPPA1 is very critical so you need to adjust it very much but otherwise you have difficulties to distinguish between that but there are these variants and that will be in terms in particular what we said before in terms of different substrates something very valuable absolutely no unfortunately not you know there are so many things now yeah yeah no we didn't go in this direction because you know we are not really not yeah that's simply okay so in the downstream the downstream effect should be the same and there is no evidence that there's really a difference whether mitochondrial fragmentation is induced by increased fission or decreased fusion however there are quite different signaling cascades that impinge on these two pathways so there are pka dependent pathways that are basically acting on fission or the chunk dependent pathways act on fission so they act on the fission machinery at the outer surface whereas this is a pathway that is basically which impinges on fusion that based on the inner at the inner membrane so what we think is that there are simply many different signaling cascades that impinge on this fusion and fission machinery making it responsible to different input signals and you have to always coordinate both inner and outer membrane that makes it very complicated in the case of mitochondria because we have these two membranes there but from the product so to say of the change in the morphology that doesn't seem to be a difference so that seems to be the same okay a little bit of time now so let Thomas go please join us for a reception