Darren Baker – Cell Senescence, Brain Aging, and Senotherapeutics for Neurodegenerative Disorders

Introduction welcome to brain ponderings I'm your host Mark Matson my guest today is uh Darren Baker he's professor of biochemistry and molecular biology and pediatrics at male clinic in Rochester Minnesota he's uh expert on regulation of cell cycle and the process called cellin Essence which is what I'll talk with him about today he's done a lot of pioneering work uh somewhat on mechanism molecular mechanism of sin Essence but he's got really interested in brain aging neurodegener disorders and has some exciting data suggesting cellin Essence does occur with brain aging and Alzheimer's disease and may play a role in the pathogenesis and so he'll talk about that as well as about about uh emerging therapeutic approaches based on targeting these senescent cells and removing them or otherwise affecting them so welcome D thank you very much for having me Mark and you are you from Who is Darren Baker Minnesota originally I am originally from Minnesota my hometown is about three and a half hours away from Rochester Minnesota so I knew Rochester Minnesota as or at least what I knew of Rochester was the Mayo Clinic the clinic part of what is being offered here because I was actually a patient here when I was in second grade I was really quite sick when I was a kid so when I was sick my parents had options about where they could take me for treatment and of course they were going to take me to Mayo Clinic instead of my local hospital that was there and so I was a patient in St Mary's Hospital for quite a long time when I was in second grade so I was a I was again quite a sick kid I had some kidney issues that were then essentially alleviated so now I've been really healthy since then and so uh I knew Rochester and Mayo Clinic for that perspective but it wasn't until I was graduating from my undergraduate which was at the University of Minnesota that I was walking around and there was a career fair and there was a big booth that was talking about Mayo Clinic research and I was thinking well I kind of want to do some research versus what I had worked on in my undergraduate was things about looking at herpetology studies looking at chorus frogs and salamanders was what I had worked on in my undergraduate and thinking that if I wanted to go to medical school what better place could I get for having research experience would be than Mayo Clinic so that was essentially my entry into why I became interested in coming here in the first place okay and and so but you didn't Darrens path to Mayo come directly to Mayo right you you went did you go to Europe uh for your PhD so so my path is a little bit confusing if you look at it so my path I did my undergraduate the University of Minnesota I came to Mayo as a research Tech originally with the intention of being here getting research experience and going to medical school during the time that I was doing my research which we'll talk about I did all of the parts about medical school of doing the MCAT doing the admissions process getting into medical school um and I deferred my matriculation for a year before I was going to start and that fall when I would have been starting medical school which was at the Medical College Wisconsin in Milwaukee was when my first son was being born and so I kind of made a judgment call at that point in time of should I go to medical school and do this thing with a A young developing family or do I really enjoy what I was doing from the research side and I chose to really stay in the research side and so then kind of have changed trajectory from that point in time at the time Mayo I was able to to enroll in the employee master's program from Mayo Clinic so my master's degree is from Mayo in the context of tumor biology which will talk about where tumor biology is relevant for senescent cell biology and how we got into it that's what did first for my Master's Degree and my PhD work is from the Netherlands uh radboud University in Nan is where my PhD was so the the work for phds in the Netherlands is based much more on your scientific contributions to the field meaning Publications more so than having a particular thesis project that you propose you go through and you end up hopefully with a paper at the end of it at least that's typically what our graduate school here of mayo is in the Netherlands it's much more of a comprehens iive you have to have kind of a collection of stories around a topic that you can then apply for and so because of my primary Mentor being originally from the Netherlands having still associations with radbot University in fact my mentor from the UN from the Netherlands side was my primary mentor's Mentor so like the scientific grandfather if you will uh and so that's that's kind of the way that path had gone oh very interesting and and early Aneuploidization on then you got interested in cancer M and of course uh cancer cells divide out of control correct and they're generally resistant to process called apoptosis which is a way that the body naturally removes cells that may become cancerous so what can you talk a little bit about that work because that that really LED you to inessence that you're studying cell cycle regulation exactly and so what I started in my research career in the laboratory of yan vanerson that Yan was very interested in a concept about aneuploidization meaning an abnormal complement of chromosomes a a German person named Theodore bovy about 120 plus years ago now made a recognition of looking at human cancer cells when he was looking at them he saw that they were very abnormal for the number of chromosomes that were in those canc answers you know typically you and I are walking around with 46 chromosomes having just one extra copy of one chromosome for example in Down syndrome most of those patients have one extra copy of chromosome 21 that's the only abnormality that those people have from a cellular basis but it's very stable meaning that all of the cells of the body have the same thing but there's pretty significant ramifications for the overall Fitness of those individuals by having just that one extra copy what theor Bouy had noticed when he was looking at human cancers was not that it was plus minus one or two chromosomes they were crazy spreads of 6070 chromosomes so very unusual and he made a postulation at the time of maybe chromosome instability where you have these random gains and losses of chromosomes is sufficient to promote the early stages of tumor Genesis where you maybe have a chromosome gain that has at the time it wasn't known about this but having a lot of tumor promoting effects an oncogenic activity you maybe had a chromosome loss that had a lot of tumor suppressive activity or the various protein imbalances that come along with that that those would be sufficient to kind of stimulate the process of tumor Genesis so what Yan was trying to do at the time was we were trying to convince cells to go into these abnormal segregation of their chromosomes and or at least having aneuploidy and the way that we thought we would do that is by really manipulating a particular point in the cell cycle you know about the cell cycle and kind of the idea about cancer was that we were looking specifically in mitosis if you've looked and seen live cell Imaging videos of cells as they're going through mitosis you have this very beautiful condensation of chromosomes they all align in the metaphase plate and there's a very synchronous beautiful division into two identical daughter cells lovely lovely cellular process that doesn't just happen by chance or accident there's a whole bunch of proteins that play a role in making sure that this is maintained in a very high adelus manner something that's called the spindle assembly checkpoint or the mitotic checkpoint make sure that all of your chromosomes have the proper tension and will then segregate into two identical daughter cells there's probably about 30 or so proteins that play a role in that process and so what we started doing I'm a mouse biologist I run also the transgenic and knockout core facility of the Mayo Clinic so any mouse that you want to make we can definitely do that but what we were doing was we were then manipulating different players that were part of the surveillance Machinery to try to convince cells to missegregate thinking that then we are going to test the hypothesis that chromosome segregation alone is sufficient to promote tumorogenesis okay we made probably about 15 different models that were in that space most of them if you really stimulate or challenge them with a carcinogen especially they they do demonstrate a clear predisposition to cancer suggesting that in situations where now you're maybe having oncogenic mutations or tumor suppressor loss the addition now chromosome instability on top of that is really stimulatory for cancer there was one model that was very unusual in that it didn't really have a very strong spontaneous cancer phenotype we did Drive cancer if we treated with a carcinogen but these mice were dying really really rapidly and so instead of like throwing that Mouse in the bin because it wasn't a very good cancer model and we were tumor biologist I became very interested in why is this mouse so sick and so that's why kind of we got into where this mouse has pathology we were trying to at the time in the early 2000s when we started this work we were then noticing it was not uncommon in high-profile journals to see DNA mutation Mouse models or mitochondrial mutation Mouse models or tiir deficient Mouse models things that are very uh prematurely aged having pathology in the pages of science nature and cell and so looking at those they talked about these mice having Pro progeroid or premature aging syndromes where maybe they had hair graying or hair loss or all kinds of other phenotype and so by reading those papers we started then looking a little bit more at our mice again and saying well you know they don't have hair graying or hair loss so much but all of my mice have cataracts by two months of age I have elderly grandparents cataract surgery is something that may Clinic does with a high degree of regularity around here uh so I know that that's very much an age related condition and so we started then really examining this mouse model to say well does it have a premature aging syndromes and we found a number of different tissues that develop these pathologies but when we tried to jam it underneath the known pillars of the time about why a mouse would have a premature aging phenotype because of maybe DNA damage M DNA damage that's unresolved or short tiir or excessive mitochondrial damage things like that it never fit under underneath any of those pillars which is why we really kept digging into what is going on with this mouse and we found in that in certain tissues there was a really high acquisition of ccent cells which we characterized at the time of being primarily p16 expressing but then also betag gaco sidas positive a couple different measures that we'll talk about in the context of senescent Cell Biology that those mice seem to have some of those features early on Darren could you Ccent cells could you could you describe the phenotype or what a ccent cell distinguishes ccent cell from a normal cell we're still working on that question even though it's it's something that has been known for quite a long time the the concept about Cellar inessence so the Theodor bovy idea about aneuploidization driving tumor was 120 year old hypothesis that we were kind of testing the idea about sence that's much more recent that was in the 1960s was when that was about the idea about noticing that cells in culture at least human fiber blasts up until that point in time the IDE the prevailing thought was that cells were Immortal that if you brought human cells into culture you can continuously proliferate them pretty much forever which was somewhat unexpected that's a thing that you might expect in the context of tumors or cancer cells but normal cells you maybe not necessarily expect that so it was really Len haick that was the person that had done a lot of the work in the laboratory of Paul Morehead in the 1960s to really demonstrate that human fiber blasts by now having the right type of media by passaging over time that you would see that there is a phase where they will proliferate but then after a certain number of Divisions which seems to be really well programmed it goes into a state where they're no longer capable of division those cells the apoptosis that you talked about the cell death process don't seem to be activated incent cells in fact there's a lot of Pathways that seemingly are on in encouraging those cells to stay in a survival state which is where some of the Therapeutics are coming into to now try to convince these cells that have turned on these Pathways to be alive to now go into a death process but they become in a state where they're very metabolically active they're still secret and what we've found since that point in time is they start secreting a variety of factors they really change their behavior completely so the concept about senescent cells and when we're talking about your brain ponderings podcast related to neurons neurons by definition are an irreversibly growth arrested cell which is one of the things that people talk about about syence but I don't really think that that's alone sufficient to really call a cell ccent what I think about syence is I more think about the behavior changes that are now taking place in those cells they're starting to do things that they haven't historically done maybe becoming very nonfunctional in relevance to what they've been doing historically as well and part of that then is the acquisition of a variety of different features some of them might be a very pro-inflammatory state where they're secreting a number of molecules that can influence other cells in the environment the concept about sence is you know largely if you look at much of our work it's essentially sence equals bad is kind of the simple simple answer but in essence we have this process for a variety of purposes and in a short stage kind of what we Define more in the acute stage there seems to be a very important role of syence in terms of activation of immune responses having benefits for wound healing responses having other things that kind of play a very transient role for benefit of having these cells that maybe are now having the secretion of these factors one of the places even even in in normal development of different organs right Senescence there's evidence that sence is is kind of a normal correct correct and I was just going to get to that in terms of the in developmental process I think we're still it was surprising to find sence there because in development you're having a lot of cells that are dividing very rapidly and now why would you trigger this irreversible or this long-term stable growth arrested state but one of the conditions that I like to talk about uh because I think it's a a good example is that there seems to be senescent cells that will accumulate at the tips of the digits that developing within animals when they do that they now start to secrete a variety of factors things like the Matrix metall proteinases that are destroying extracellular Matrix such that then you get actual separation of the digits of the hand so it plays a very important programming effect of that if you don't have the ability to engage s essence you see that this doesn't happen with the same type of regularity versus if ccent cells are here they secrete the factors that are necessary for then having the separation of what was the developing digit just as one of the examples for where developmental syence seems to be playing a beneficial role there's there's a certain kind of Labrador Retriever dog Cell Senescence Chesapeake Bay Retriever and they have they swim really good they have webbed feet I'm wondering if they have like a mutation in a Metallo proteas gene or something or if there's a roll or a bypass I'm not sure because there's all kinds of animals at sence markers in the feet of those dogs with development y possibly okay but anyway so cence it's important for normal development uh can suppress tumors in a way right that from a cell intrinsic effect healing some other things correct from a cell intrinsic effect and this is where we think about inessence maybe that's occurring in the context of that bubble one hypomorphic Mouse model is that their cells are becoming ccent for a particular reason so this mutant Mouse model that we made it was having chromosome missegregation but we had other models that were having chromosome missegregation that had no syence effect so it was something really unique about that particular Gene and that protein about why are those cells now becoming sensitive to really becoming senent versus maybe others that we mutated did not have that same effect from a cell intrinsic mechanism you can think of syence being really a beneficial anti cancer response because sence like what we talked about for the cells in culture if you now stop that cell from dividing any longer that's a really potent intrinsic anti-cancer mechanism that cell will not be able to make now two cells or four cells or a it it's stopped at that point in time it would be better if the cell would die entirely so that then it's not not hanging around but maybe there's some benefit of having that senescent cell that it will maybe trigger immune response or something to then say hey something's not quite right in the system here maybe we want to activate some sort of response to this and so that's maybe where the the pathway was somewhat activated or induced to become ccent and we see that with this particular model where we became very interested and so in the context of why did we continue in this work because it was very easy to make mice sick and in fact at the time when we were you know first shopping this story we had frequently the comment of you can mutate any one of hundreds of mouse genes and you can make a mouse sick so what what is the importance of this which is which is a valid point what we were doing at the time was we knew that that mouse was missegregate purely prone to cancer development if we now add chromosome instability on top of these mice that have a predisposition to cancer is it much like what we see in our carcinogen treated experiments where we see a synergistic effect and we get more cancers okay so that was the simple idea of what we were doing because Yan doesn't like to do things in small scale we threw a bunch of different tumor suppressors at this mouse at the same time many of them p-53 knockout mice p-53 is an extremely important human tumor suppressor Gene mutated in lots of human cancers p-53 knockout mice very tumor prone p16 knockout those animals also are tumor prone not to the same degree as what we see for p-53 but p16 is also a very commonly mutated tumor suppressor Gene in human cancer p21 is part of the p-53 pathway p-53 engage cell cycle arrest largely through activation of p21 those mice also don't really have a really strong tumor phenotype but we started asking that model as well as p19 which is Upstream of modulating of p-53 so essentially we have the p53 pathway based on p19 p53 and p21 and the p16 pathway which both of them will inhibit different cell cycle regulators and so the idea is if you now are knocking out these important Regulators you don't have a stop of the cell cycle when it should be stopping and you get transition through and you can then proliferate very happly okay so that was essentially the idea what we found was actually quite remarkable is that this mutant Mouse model that we had that had a premature aging phenotype it was it was quite severe we could make it even worse so if we knocked out the p-53 pathway essentially p19 or p-53 we made the mice even sicker than what they were originally those mice also seemingly had a predisposition to tumors as well which fit along the line of what we had anticipated related to the tumor development it was a tumor study was what we were doing interestingly when we were looking at the p16 knocked out animals if we looked at them for their tumor phenotype it was a Synergy where now we were getting cancers in those mice that didn't have P6 but they looked really healthy so it was this very uh kind of dichotomous effect where these mice it was a damned if you do or damned if you don't scenario that if you had the ability to express p16 you had a very strong premature aging phenotype if you didn't have the ability to express p16 you had a strong cancer phenotype but they looked very healthy so you were dying one way or the other premature aging disease or you're dying of the cancer disease which led us to then the idea about this this particular regulator that was the only one of the group that I told you about that kind of had this differential effect suggesting that the p16 expressing cells were playing a very important uh protection against cancer effect but by their presence were actively promoting degenerative disease which led to then essentially our next tool in the mouse model world of then saying if these p16 expressing cells are so important for driving pathology but yet so important for suppressing cancer can we do something about those cells can we maybe manipulate that cell type so that it does so it still keeps the ability to engage the arrest mechanism but then we don't let it reside longterm within the organism to really promote OTE the degenerative process yeah and so this has been a Ink Attack Model System really valuable tool and kind of so you develop mice where you can selectively um eliminate cells that are expressing p16 correct so in my career so we go back to the career at the time so I had just received my PhD from the University from radboud University in Nan I was thinking very seriously about leaving Mayo Clinic at the time and going to the laboratory of Judy camping an absolute Giant in the field of sell inessence related research interested even though not she herself is not a giant but her work is you know very influential within the field that she was at the buck Institute looking still in the context about syence and cancer and she's done a lot of work related to why senescent cells have impact on other cells in the environment just you know an amazing individual that we unfortunately lost earlier this year um but so at the time and I've always been very transparent with mayo when I've been thinking about doing something different and at the time was just when the Cod Center on Aging was just starting at Mayo Clinic that was uh started by Jim Kirkland was kind of our first director of the of the Aging Center and at the time I was still having a bunch of cancer related work but there was a a desire to have a program project like grants that would be able to hit a number of different things within the Aging Center that do we have the right type of ability to do something about senescent cells or not and we thought because we had this bubber one mutant Mouse model that had the premature aging phenotype that we show that p16 expressing cells were important for driving pathology but also preventing tumor Genesis we thought we had the tool TR test can we do anything about that and that was where we developed this ink attack model system system the ink attack model system the ink comes from p16 ink4a that's the ink part of the machine the second part is apoptosis through targeted activation of caspace is what attack stands for that had been previously shown with different promotors to be able to actively eliminate cells not via the typical way of having DNA chain incorporation through like hsvtk mediated cell death because in the context of siness we knew molecules or we thought molecules that were really important that we used for selection of embryonic stem cells in culture where you have a a lot of Rapid division DNA is replicating so if you do something that then will stop that replication you can promote cell death ccent cells are by definition really not cycling so there's not theoretically much DNA incorporation Judy's group has now suggested that the cell death mechanism be a hsvtk can happen through a mitochondrial mediated cell death but we didn't really think about that as a potential at the time so we were looking for a molecule that we could do a selective induction of apoptosis which was this attack trans Gene the attack transene has a modified fkbp protein with C base 8 that requires a artificial drug to be added for its dimerization and subsequent activation of caspase8 that drug ap20187 which I'll just call AP I could fill you and I with as much AP as has been produced and it will have no effect because we don't have the transing so it's it's not it requires the genetic modification for there to be a benefit and so what we had done and this was again a a crazy experiment because we made transgenic founder mice that had the Inc attack trans if you remember from your genetics approaches when you make classic transgenic Mouse models every transgenic founder line is different than every other transgenic founder line so line one line two line they're all different because they have different incorporation sites different number of trines and so forth we tested in Vivo the ability of the Inc attack trans to have benefit on physiological function in the bubble one hypomorphic Mouse model so bubble one the mutant Mouse model that I've told you about itself is sterile so we cannot interbreed these animals it was a one in4 shot of getting mice that had the right mutation to promote premature aging then you had half of those that had the attack Trine and half that did not so then you're at one and eight of mice that have this four we tested I think 11 different transgenic founder lines was what we had tested at the time and what we found is that there were ultimately in the context of the bubble one hypomorph there were two transgenic founder lines that functioned so you can look at this in one of two ways we had nine failures if you want to look at it from the pessimistic point of view where the trans Gene was not Incorporated in the right spot or it wasn't behaving in a p60 dependent manner or we were maybe having killing in cells that we didn't anticipate having killing in cells so a lot of weird stuff can happen when you're in the context of making transgenic mice but two of the founder lines look like they behaved quite well in the context of the bubble one mutant Mouse model where we saw a similar benefit for what we had done with the genetic deletion of p16 by now removing p16 expressing cells with this drug started early enough in life before pathology had come on we're able to largely repeat the benefits that we had seen with The p16 Knockout animals where we had attenuation of disease primarily of cataract development skeletal muscle degeneration and atopos tissue retention by now eliminating siness and cells and did you look at lifespan extension good question question so in that in that experiment when we looked at the hypomorph we had not a strong Improvement of overall lifespan but the mice were not dying of cancer unlike The p16 Knockout animals we had no tumors that were developing the phenotype that we think was largely killing this progeroid model because I told you they di prematurely without a lot of cancer relates to a cardiac arhythmia effect that the cardiac arhythmia effect doesn't look like it's a senescent cell dependent context so in the context where we're now removing the p16 expressing cells we really don't have a strong Improvement of the cardiac function of that particular model interesting that that's really interesting is it is anybody gonna Heart Function pursue what's going on in the heart we've done a little bit of work uh it's it's interesting because this mouse model uh even though we published the first report I guess in 2004 so 20 plus years ago I still work with the mouse model it's still upstairs breeding or still finding out various things about it uh but in terms of the cardiac phenotype we haven't done a whole lot of work in that space specifically for the bubble one mutant at this point in time we had an idea at a point in time that it looked like there was a mislocalization of conon 43 that it was part of the retent ability to then have a synchronous signal that then lead the Gap Junctions between the cardiac cells correct and that connection 43 seems to be dependent on its phosphorilation that doesn't occur when bubber one is at low levels the gene that we mutated bub one is a kinas it's never been really defined of what are its particular substrates but now science is getting much more improved in our techniques and technology versus where we were playing at the at the time for what were various kyes activities and so forth so it might be an interesting thing to re-explore now that our TOS are getting Brain Function better you know I know we we're Limited in time so I don't want to take it but in context of the brain let's move to the brain and sure just follow asites which are the most prominent type of G cell equal in number to neurons they're connected by Gap Junctions so it would be interesting to look at those but can you get to the brain so sure so so so in the context of the brain I'll I'll tell a couple things so related to the bubble one mutant Mouse model this prematurely aged Mouse we have found now with collaborators from our Florida our maroc Clinic Florida group that there seems to be a a strong induction of sence in endothelial cells that occur within the bloodb brain barrier they cause disruption such that these might have a lot of leakiness into their brains if you remove their senescent cells you see that there's again a large benefit suggesting that the presence of senescent cells in the endothelium can promote dysfunction where now proteins that shouldn't be getting into the brain are getting there and vice versa and we've also been then looking in the context of normative aging so this Inc attct model we had the comment that it was very easy to make mice sick what relevance does this have for a normal Mouse we did do an overall lifespan experiment on normally otherwise wild type mice of doing the same treatment Paradigm of treating prior to disease onset and what we could find there is that many diseases associated with age that occur within a laboratory Mouse can be prevented with senescent cell elimination there's also an improvement of median lifespan in those animals I think we didn't have enough to really make a judgment call on the maximum lifespan extension we just didn't we didn't do it quite right but I also told you that we started with two transgenic lines one of the two lines if we were looking at male wild type mice for whatever reason those mice had the insertion somewhere really negatively that in the absence of treatment they were dying after about 15 months of age something we didn't see in our hypermorphic mice because they never get to 15 months of age they're dead by 12 months of age and so that line has some significant impact that we can't that we can't prevent there was some improvement of lifespan with those models but then we've now been basically eliminated down to one founder line from the 11 where we started from so that's the inot attct model that everyone is using is this one particular founder line that we ended up doing the invivo testing of at the time so I was going through my career I was doing my postto making this Inc attack model seemingly was turning out and I was intending to for a variety of familial reasons was trying to maybe stay at Mayo Clinic so again being very transparent my mentor was still here at Mayo and we made a a very simple delineation of saying I guess my lab and what I'll focus on is what I call neck up biology and he'll work on neck down biology okay so that was my entrance into brain ponderings was I was taking the neck up because I was also very interested in the Cataract phenotype that we had in these mice unfortunately funding agencies are not so interested in the molecular mechanism behind cataracts which I have a really good understanding for um maybe we'll be able to re revisit that at some point in time but that was why I got into neurodegenerative research was really trying to find something of a tool because we had this really rapidly aging Mouse model I wanted to use tools that were also rapid disease models of neuropathology to now see are their ccent cells relevant for these particular diseases so my lab I came at it very naively I think and I think I'm still very naive in this space because we were looking at Alzheimer's disease I've talked with Ron Peterson who's here at Mayo many different times and I was looking at ad pathology and essentially separating it into the two main pillars of whether it's an amalo based model model or a towel based model of disease instead of putting them together and so we had done a couple different experiments where we had bred the ink attack model onto some more amalo based models of disease or to a tow based model of disease and we'll talk primarily about the to based model of disease for the remainder of this because that that's where we have some more information because to this point in our hands we don't see a very strong induction of inessence in the amalo alone models in our hands we don't have the right ones and that's the problem with the amalo model with with any of the models so we're doing for Mouse model neurodegenerative research is that they can behave somewhat routinely repeatedly in some places and maybe not so repeatedly in other places but we didn't have when we got tissue from other places that had done more amid alone models we didn't see strong upregulation of sessen related changes at the time related to p16 up regulation factors that are D in this sence Associated secretory phenotype the pro-inflammatory signal occurring from sent cells so we focus very much on this tow dependent model of disease the mouse that we use is the ps19 strain that has a p301s mutation developed by Virginia Lee uh and first published I guess in 2007 I was somewhat impatient based on you know starting a lab and wed to have phenotypes so those mice get pathology really very aggressively and quickly and so we did an experiment where we cross the Inc attct model onto the ps19 mice treating them prior to disease onset so this is not relevant for people that are coming into the clinic here because you're not coming in prior to disease you're coming in with some sort of ailment that then you're wanting to have some treatment what we found in that model is that it's very important the timing of treatment which is not surprising right you know in the context of neurodegenerative disease much like cancer so if you make still the same analogy is it better to treat a cancer when it's a stage one or a stage four well your outcomes are probably much better at stage one than it is at stage four same with neurogen disease that how much of an ability does the brain have intrinsically to repair these processes once the SCAR or damage has occurred seems to be not terribly awesome but if you can kind of change trajectory that's where we've had some benefit so what we were doing was we bred this Inc attct model onto the ps19 strain treating mice to remove their senescent cells prior to disease onset just to see whether we had an impact or an improvement overall in their pathology what we found is that we did and so then okay why now we need to figure out what is actually happening we saw that we could really negatively influenced so there was a ccent cell induction what we were trying to then figure out was what is the origin of the ccent state in this ps19 strain naively we utilized sa beta GLA todas staining sessen Associated bet galact bet galac totis something that was first published about 30 years ago from Judy campes lab of a notice that sessen cells changed their Lal activity to be very active at a particular point in time we coupled that because of work that we had done in the context of normative aging we had done beta galactia staining followed by electron microscopy to look for where there were the inclusions of the beta gal crystals to now look at cell morphology to give us a of a hint of what cells are now having the beta galactosidase expression because in the space our tools are still not awesome for ccent cell research and if you were to ask me today what is the best marker I can't give that to you because I think it's very dependent on very different context so in the brains of the SP ps19 mice what we found is that there seemed to be just a lot of background neuronal beta gal activity that was change we found that too yeah so so maybe I should Cell Types be asking you questions coming up here no no no we found we we followed you right y but what we found so then but that was not changed with our treatment so that background that was happening within neurons didn't really change with our Inc attack mediate elimination strategy we found that there were other cell types primarily cells that look like asites and look like microa that had the inclusion of these excal crystals that we then were doing our treatment Paradigm so like where you maybe have a drug or something we have this genetic model where we can remove p16 expressing cells and we saw that there were fewer of these microa that had the excal crystals and fewer astrocytes that had excal crystals both of them in the model the ratio that we had for numbers overall we had probably about three times as many astrocytes that had the xcal inclusions as microa but I don't I can't tell you which one gets which or if there is any relationship between those two so that's and there there are a lot more as sites than there are micr and so maybe it's just a numbers thing that because there are so many asites that's why there's three-fold more but what we've been doing since that point in time so in in work that's currently unpublished is now we're trying to convince the brains of the sps9 mice in a cell type specific manner to be better or worse in a sessen Cell context meaning we're trying to make more asites ccent in the ps19 mice or less asites ccent in the ps19 case interestingly the model doesn't care about how many ccent astrocytes it has the pathology is consistent so if there's many more doesn't care less doesn't care pathology is identical microa on the other hand that it cares about so if we push the dial on one side of making more ccent microa theology is worse if we prevent senescent cell expression specifically in microa of the ps19 animals there's an improvement now it's been known for a long time that there's what are called activated microa Activated Microa associated with the pathology in Alzheimer's and these it's also well known that these activated microa produce a lot of pro-inflammatory cyto kindes that are thought to contribute so what what distinguishes that phenotype from you know a cell that has a microG that has sence markers um great great question and we thought we would be very very smooth in answering that question of doing something in vitro of having microa that were resting activated microa and then we'd convince microa become ccent we just bend diagram that and then say what was unique about the one versus the other and so forth in vitro we were extremely efficient at making reactive microG that we had no problem doing but anything that we tried to do to convince them to not be so stressed out didn't really work so we had no node of the the resting state and when we were trying to then stimulate them to go into a senescent process they were dying was the consequence so in vitro we couldn't really do that we resorted recently to just doing a microG isolation from the ps19 mice to then pull out the microa from the brain and we found the three populations we would have anticipated so we wasted a ton of time and energy in an invitro approach that we then were able to answer with some invivo because technique and technology has improved such that we can do single cell profiling of the microa that are coming from these animals to then look for these distinct States what we found and what I think is somewhat in agreement with others but there's different tools different Mouse models are probably behaving somewhat differently what we found is that the ccent population in the ps19 mice was underneath the overarching umbrella of the disease Associated microm so there are many different subcategories of disease associated microglia in this ps19 mice there seems to be one particular group by single cell profiling that looks to be much more ccent or having markers that look like different po inflammatory signaling molecules but some of those are going to be in agreement with a lot of the disease Associated microglia or the activated state of microa as well which in the inessence associated secretory phenotype area of the sasp there's many of those phenotypes that look like they're just part of the activated state of microglia so it's kind of this new transition state where they've gone into the disease Associated microglia something that maybe you can explain to me is that it looks like maybe within our within that population if we do pseudotime analysis it looks like the ccent state for the disease Associated microa has first gone through something where it tried to proliferate or there was a proliferation signal that then maybe there's a blockage or something that goes Ary in those cells to then trigger them to go into a inessence state or maybe the the induction of inessence itself or the induction of proliferation specifically of these activated disease Associated microglia stimulates them to transition into this different state something we're still kind of exploring at this point um off the top of my head I can't you Disease Associated Microa know think of what what to do next with that particular question but um and and work from others not us has some has done some interesting studies where they've done co-culturing of neurons with neurop fillary Tangles so our model theoretically is having disease in the neuron the neurons we don't have real strong evidence that they're becoming ccent we have cells in the micro environment that are becoming ccent so why what is that relationship we're still working on that in terms of what are the poent potential cues coming from neurons that are stimulating this I do have an idea okay now now that that stimulates them we did Neurofibrillary Tangles all this work beginning in the 9s early 90s showing that in in neurons and Alzheimer's disease that affected in Alzheimer's disease and we used P postmortem patient tissue and then we also exposed cultured neurons to oxidative stress amalo beta peptide that um two things one there's a lot of membrane lipid peroxidation occurring and release of a of a toxic alahh it's a little bit of the phospholipid that's cleaved off by free radical attack and it can coal modify proteins on lysine histadine and cysteine residues and affect their function so one experiment I would do is just you can buy four hydroxy nonl and just dump it into microa cultures and then another thing we found was there's a accumulation of longchain ceramide and you know so these are a couple things that i' say might have a effect on microa that pushes them maybe towards a inessence phenotype that's just two things off the top of my head and so work from not us but from Ria Gracia spillantini lab has shown an idea about the idea of having neurons that have neurofibrillary Tangles being co-cultured with microG that the those neurons have the eat me signal such that the microa will go and they'll phagocytose the neuron the problem is is that those particular neurons don't seemingly become destroyed within the microa and the microa then changes its Behavior to then say holy cow now I have a problem of I can't do what I was supposed to do and looks like when it goes into that state starts to secrete many of the factors that we think about of the ccent state the cell the then loses its ability to phagocytose any more neurons so it kind of becomes stuck and now activates that signal which would be basically in agreement with our model where we have the neurons with neur Tangles the microa they maybe engulf them become aescent if we then go in and eliminate them then you remove this negative signal that's that's occurring but then you know it's it's at least conceptually the idea of what we're kind of working with right now huh that's really intriguing and and Senotherapeutics so so you made these mice where you can selectively remove cells with a ccent phenotype and you see in in Norm this normal aging and then in pathologies associated with multiple organ systems including the brain uh one accumulation of ccent cells and two if you remove them beneficial effects you focusing on the brain and your mentor focusing on everything yep down okay now there's a big effort and even companies uh being developed around uh developing chemicals drugs if you will that uh can remove senent cells or I guess maybe otherwise modified and these are referred to variously as senolytics or Seno Therapeutics can to finish up can you talk a little bit about that because there's even even clinical trials going on in in certain disease conditions correct so in the context of the company development we were involved with a company that formed that's now known as Unity biotechnology that still is in existence in California is where they're at but the company when it started some of the scientific co-founders were my mentor Yan venders Judy campy was another of the founders and daong zoo is the third one and what we were doing at the time when the Company formed I would not advise as a as a great way to start a company when we didn't really have a drug nor did we have a disease we had an idea about that syence was bad for you largely based on some of our strategies where we had removed ccent cells so since that point in time we had shown that that it was safe and effective to remove ccent cells from mice that they weren't killing over dying many people here the first time that we started doing our treatment in the ink attack model system was that they anticipated that we were just going to have all of our mice turning up dead because maybe the transing was on in the neurons or you know something where there could have been a lot of negative effect but we don't see that there's not a real long-term negative impact with our genetic strategies of eliminating siness and cells which gave Credence to the idea of well let's now look for agents that can do this Jim Kirkland was one of the people that was pioneering in this space related to looking for what is really different about a senescent cell that might be a space that can be intervened with and what they were looking at was something called ccent cell anti-apoptotic Pathways or these scap Pathways where ccent cells have a high up regulation of many different Pro Pathways that are actively keeping the ccent cell alive we used to think about sessen cell biology at least I used to think about Cent cell biology of the analogy that they would exist in a valley and it was be very challenging to convince them to behave differently than what they were that they were very stable and convincing them to move out of that or to die after that point in time was going to be very challenging because all of these Pathways that were on what seems to be repeatedly now is that cesson cells are much more teetering on the Cliff of Destruction more so than that they're very stable because many different players that we have that can now manipulate some of these certain Pathways seemingly are sufficient on their own to to promote death because there's so many things that are apparently going on within that cell what my lab has been focusing on primarily is molecules that are involved in inhibiting bcl2 factors of anti-apoptotic pathway the negative consequence is that there are certain cell types within our body that are reliant on the same pathway for their survival so there are of Target negative I say of Target but it's completely On Target it's doing what is designed to do in a syence cell but there are normal cells that have the same pathway required for their survival so that that's an an effect that is not desirable but it's very much on target for what the molecule is doing things like nid is the molecule that we use very frequently there's other molecules a combination of dasatinib and ctin which you yourself have published on related to how there seems to be a benefit for various Mouse models of pathology with the satin and ctin treatment in mice and that was based off of Jim Kirkland stuff related to Pathways that look like we being able to be targeted by molecules that we already had on the Shelf such that we could get them into a and have already been used in humans in fact theatin is used for many of these molecules so your idea about C Therapeutics is I use again I like analogies that I use the analogy about C Therapeutics that senescent cells are a tree that of all kind kinds of different branches of different inflammatory signaling or whatever is going on within the tree with a senolytic approach with our genetic approach we're cutting the tree off at the base that we don't want to have anything that's going on from that ccent cell whatever the pathways different are theomorphic strategies those are cell those are abilities to try to take off particular branches that if you think that this is the branch that's really driving pathology let's only take away that and leave the rest of the tree intact maybe a bonsai type effect maybe if you're thinking of it that way that then there's still healthiness after you've taken away the disease tissue something like that so that's the analogy I think about about senolytics versus xenomorphic largely much of our work is focused on the senolytic strategy because of our genetics our genetic approaches that we've been using are taking the tree off at the base so that's where we're coming with our senolytic strategies in the context of companies so Unity was one many others are playing in the space now so Unity since that point in time has now developed molecules so we have now a way to eliminate senent cells deatin and ctin is one but there's also proprietary molecules that are being used in different cases as well as different disease conditions the first clinical trial that was done from Unity was related to osteoarthritis was what it was and in that particular case unfortunately the the clinical trial did not yield positive result and so that had a pretty significant impact on the company and kind of change what they were working on now what they're working on is is really quite exciting research on diabetic macular edema where this is a condition that diabetics get within their eye they've developed molecules that are injections into the eye such that it's a local administration because they think there's endothelial cells inessence that's occurring within these particular patients when that happens there's a lot of reorganization of the endothelial network within the eye leading to a lot of problems such that if you do this treatment you kill off the senescent cells there's not very many of them but by killing off those endothelial cells there seems to be a replenishment with normal cells such that those patients the first clinical trials have been published that if you look at DME patients that are treated with vehicle over the period of a year so a one-time administration of the cenotic drug patients that receive the vehicle you have glasses on I have glasses on so we've done Vision tests what diabetic macular edema patients typically have is a loss of two lines of text over a year meaning that they have to move up two lines on the chart to see the the the letters these patients that have received the senolytic it's stable so they do not lose their Vision over two years so pretty exciting work related to a specific patient population with a specific agent with a very targeted delivery and that and that's Treatment one that's one point even in the animal studies right uh the the the treatment doesn't have to be continuous it it can be you know intermittent or one last thing well much of our work so we can go over it's fine so much of my work we've been we've been more working in the space of we want to see biological effect so I I'm sure we're overdosing our mice we haven't done the intermittent kind of strategies that would be relevant for the clinic our patients our mice upstairs are very compliant with their treatment regimens you know we don't see all target toxicity with the molecules that are administering so we do pretty much consistent treatment to to really change the trajectory as much as we possibly can what we found and what you know related to some of the unpublished work because it's not terribly beautiful is that in the context of our degeneration model there is a point of no return where if we're treating mice that are too diseased they don't care about whether the senescent cells are removed or not because the pathology is already severe enough that it it has no ability to restart there's maybe ideas that you can think about by now coupling ideas of cleaning the soil if there's contamination of senescent cells to then maybe have a reactivation of maybe various factors like yamanaka factors if you're thinking about doing reprogramming to now try to stimulate those cells that are remaining that are the healthy cells to now have ability to divide or replenish or something at that point in time so there's all kinds of things that you can think about that could be really fun to do is that we don't have the type of money that would be required to do all of these experiments and that's why we haven't done a lot of the intermittent type of treatment but with that with that uh clinical trial for immunity that shows clearly that if you're on target for the disease causing ideology and you can prevent that there's pretty significant impact in the context of AD pathology there is a relatively well-known clinical trial going on right now with the satin and kettin trying to also really change TR of disease course in people with mild cognitive impairment for example of these molecules are relatively safe they don't seem to have really they seem to be well tolerated and that's not work that I'm directly involved in but obviously following the literature in that space so it'll be interesting to see because those molecules the deatin and kettin or other even much of our senolytics are molecules that can have on target senescent cell killing ability regardless of where they're occurring even our strategy our genetic approach is also if it's developing ccent cells in the liver we're killing the ccent cells in the liver as well as in the brain and with the interorgan communication that's occurring about how sessen cells can be having really distal impact you're changing phenotype in many different tissues such that then maybe overall you have a dampening of the signature of sence by removing the ccent cells that might have a benefit for the patient and that's ultimately what we're looking for is improvements for the patient we'll have to figure out mechanistically why we see those improvements in the end if we end up having improvements but those are some of the some of the caveats with our current approaches for intervening with the senescent cell phenotype yeah one thing you've mentioned several times is uh you know in the animal models and and it's the way it is in as far as clinical trials in Alzheimer's disease the current kind of con consensus is that by the time someone is so symptomatic and diagnosed with probable Alzheimer's disease there's so much pathology that it it may not be possible actually to completely halt the process or probably definitely not reverse it so the idea is to have early diagnosis and and Ron Peterson your colleague up there has been kind of a Pioneer in in establishing criteria for mild cognitive impairment and so a lot of the clinical trials now are kind of going to that stage now I've been interested as you may know for a long time in how can we reduce someone's risk yeah and epidemiologically the only well there's kind of three things that seem to stick out and are supported by studies in animal models for example um physical exercise regular exercise not overeating being uh and keeping your mind intellectually challenged MH um you've done some work actually we did study long time ago where we had evidence that daily caloric restriction and intermittent fasting can be beneficial in an Alzheimer's model if we started fairly early actually before there's our Landing pathology in this 3x TGA model and you followed up and you also done um you did a calorie restriction study too right we we did yeah so in the context of ccent cells anytime that I give a talk then people say well what should what should I do now what what can I do today diet exercise you already hit on two of the three related to things that we can modify that we know healthy living has an has an impact on that we see that there are fewer sent cells that are accumulating the chloric Restriction study that we that we did just recently it the con the concept behind it was not exactly a caloric restriction study the concept behind it was actually a hibernation uh idea was what we were trying to do so with colleagues from the Netherlands what they have seen is that in hibernating species many of them mammals bears and other sorts of things possums uh ground squirrels that if you look at their brains as they're entering into the state of torpor or this their state of hibernation they have a very high upregulation of phosphorilation of toel okay so their neurons become very hyper phosphorated with to when they're going into the spot of Slumber when they're coming out of this torper state that goes away so it's a very reversible phosphorilation state of towel which is unlike what we see within pathology so with ad pathology it phosphates and it stays phosphorated so there's probably different kinases and phosphatases that are involved in this process and so we were then thinking was that if we can convince laboratory mice to go into hibernation which this collaborator had done it was through a pretty significant caloric restriction Paradigm we can then induce the phosphorilation of of too and now when they're coming out of it maybe we can have a prevention of that and we're doing that in a disease model so they have the point mutation of to and is there now a particular kinas or phosphatase that is activated as they're coming out of Slumber that then we can potentially utilize as a new approach for intervening with to Dynamics in pathology was kind of the concept of the study that's intriguing because it turns out to is hyp this microtubular Associated protein is hyper phosphorated during early brain development right when there's a lot you know the axons dendroides well it's mainly axons uh where TOA is but anyway they're rapidly growing synapses are forming so it's interesting that in in states of kind of normal plasticity healthy plasticity tow can be hyper phosphorated but it's also hyper phosphorated in the neurop fibrillary tangles yeah and so what we had done then was we were trying to convince these mice to go into hibernation but their hibernation protocol is on a daily cycle so they go into Slumber for about 21 hours and then they're awake for 3 hours and then they go back into Slumber and the way that we're doing that is by in by influencing when they can eat their calories is the idea so what we had done was we had done this in our tow Mouse to then see whether or not there was a change of now could we change the pathology what we found is that there was a clear impact on their behavior so there there were behavioral improvements in these mice that was not just because they were more active wanting to eat when during the time that it was actually something that we saw by mazes and other things for the mice that they were behaving as if their cognition was improved but if we looked at the overall neuropathology of these mice there was not really significant changes it was kind of a situation where we could improve phenotype and behavior Without Really changing underlying biology but part of that looked like there maybe was a bit of stimulation of some adult neurogenesis maybe that was going on in the model but it was a system that we were working with a shared mdphd student that she then came here for a period of time and then return back to the Netherlands to finish her work but that was where the concept was very solid but then overall the results were a little bit uh hard to interpret I think at theend it's interesting though that these you know so bats right bats also have this state of torp or right they go and bats live a lot longer than mice even though you know some species of bats are same size as a mouse Y and there's some thinking that this going cycling in between torper and non- torper well and you also think about the energy expenditure of bats of how that they have to go out at night then they're hunting bugs that they have to have good echo location that their ears need to be completely functioning otherwise they're also not going to find their prey that they're needing to eat and the maintenance of that system is very robust if you look at those animals being able to live two three four decades even in the wild relative to not having mice living that long in the wild I'm pretty sure yeah okay so um what what's on what's going on now I mean you you've done a lot you know you've been really a leader in this area and I'm kind of want to know what's going on now that you can that you can mention without yeah without uh getting myself in trouble so so to me I think something that is still surprising to me is how common the sence node is in promoting dysfunction in different pathologies including within the brain because you look at different disease models whether it's a Parkinson's model or an ALS model or an ad model or a to model or an AP model you have different cell types and you've published this too that in your model you seemingly find a ccent cell context but you're not finding microG and asites that look like they're becoming ccent Li the prec or lead endite cells look like they're the ones that are becoming ccent but yet that node of involvement really promotes dysfunction such that if you're intervening in it you can have many improvements I'm still shocked that that is the case regardless of what is the tool or the model so I don't think that you're wrong and that I'm right or that I'm wrong and you're I think that the tools are kind of suggestive that it's going through this particular path in your condition and it looks like it's actively promoting dysfunction so that that's you know to me still somewhat surprising so lots of work is being done in the space still related to how relevant are these cells in the context of normative brain aging normative Cognitive Behavior I've kind of steered myself a little bit away from that uh area largely because I don't want to be competing very actively with others here at Mayo so there's a number of different researchers that are working in the normal spaces of cognitive aging to see whether or not sessen cells are there and largely it looks like they are as well and maybe it's different cell types but it also kind of has hints of micral involvement of sence as well within my program what we try to really do is develop new tools new approaches new technique where we can really identify and promote dysfunction within particular cell types I mentioned driving senescent pathology in particular cell types to then say how critical is that one cell for driving the pathology or not if we can just intervene alone in that sample is that sufficient because then that might be our next generation of approaches for targeting intervention where we're now not hitting all of the other sessen cells about what might be a beneficial State let's only go after the ones that are really responsible for the pathology or if we're actively promoting apoptotic death of those senescent cells alone does that have disease modifying effects or not so those are some of the things that we're working on within my laboratory right now and I guess it's a to be determined or stay tuned kind of approach that's very exciting goal and I look forward to seeing how that turns out uh one thing I want to mention to you might be useful you may already know about it uh before I retired a neurologist under me demetros copanas developed methods to all cells secrete these small vesicles they're called exosomes or extracellular vesicles and using an antibody that's fairly specific for uh protein on on neurons uh he pulled out of the blood extracellular vesicles that we think are coming from neurons and then he molecularly interrogated them it would be interesting if you could find some you know it's called that syence Associated secretory phenotype these ccent cells are undoubtedly pumping a lot a lot of these vesicles some of them even from the brain but from anywhere may get into the blood and it would be interesting to identify like a a blood-based biomarker of sin Essence yep absolutely we we've been looking a little bit in that space and you're right there are a number of these extracellular vesicles or exomes or all kinds of different things that are secreted that senent cells seemingly do what we haven't had a really good tool for is looking at that on a cell type specific manner so then identifying which ones are coming specifically from a microa for example that might be sent and so forth so tools are getting better all of our approaches are getting better what we used to think was you know Cutting Edge about microarray technology nobody does microarrays anymore you know it's single cell RNA SE can then you can easily identify where these populations occur which we would have not had the right tools for doing that a decade ago and so it's just moving so fast that you kind of have to keep up with what is changing to really understand and identify what some of these factors are with our toel model of disease what we were trying to do was really characterize because the strain is good but there is also a variability of penetrance of the phenotype much like happens in many Mouse models where we can have some six-month old mice that are very diseased and others that are really not diseased even at 11 months of age and so why is that so we were looking for tow depositions in the retina of these mice was what we were trying to do to then stratify those that are now having aggregation of TOA within the retina to now say that those are probably the most diseased let's intervene on those animals relative to those that are maybe more mild to kind of stratify populations so a different strategy that is also not terribly invasive because you could do the Imaging with just not with just having the animals being asleep and not needing to do anything else but blood-based markers markers of inessence in the blood a lot of that work is being done very actively right now and including by you know a lot of people from the Nia are are doing a lot of that work yeah very good uh Darren it was a pleasure to see you again it's been a long time you were came to ni I don't remember when that was but that that was when I was uh very seriously thinking about leaving Mayo Clinic for the ni in 2016 I think that was so that's right yeah and uh Mayo Clinic's a great place as you know I'm from Rochester I was I was born in St Mary's Hospital which is the main hospital for them same place I was treated when I was a kid yeah um yeah so it's great to see you and I appreciate you taking the time it's always great to talk to you Mark and if you need a part two I'm always happy to come back too okay we may do that all right sounds great have a good one talk to you soon bye