 תודה רבה ניסים. ניסים נכון, ובאיינתן, אני חייבה שאני עשיתי על פורטי, ואני לא נשמע שם, אבל לא הרבה. אז אני usually give this talk in front of molecular biologists, biochemists, sometimes cancer specialists, but never before mathematicians or philosophers ולכן לפני סיסטון ביולוגיה ובאינפורמטציה, אז אני חושב שאולי אני מבחינסים טובים. אז כשאתה יכול לבין, אני מבחינס קצת קצת. עוד אוטיזם, כשאנחנו already עוד פשוטים, אני חושב שזה קצת קצת קצת קצת קצת קצת. אז אני חושב שזה קצת קצת קצת קצת, ואני עוד פשוט, אני עוד פשוט, שזה פשוט שייתה, שאנחנו לא נתפליש עד. אז אנחנו נראה איך זה ידעה. אז כשזה מה ניסים הולדה, כשאנחנו נשארים, אני נשאר את זה 4 דקה כך, To try to find out which is the protein, this was part of my post-do, which is the protein that binds here to this very unusual structure that was discovered in 1975, more than 40 years ago, that was called the cap. It was called the cap because it was the first nucleotide at the 5-prime end of the mRNA, so it's a cap for the mRNA, very unusual. It's a 7-metal G that is linked to the rest of the messenger RNA that is going this way via a 5-prime to 5-prime triphosphate bridge, which is very unusual. Again, all along the messenger RNA, the bases are connected via the ribals in a 3-prime to 5-prime phosphodiester bond. So, of course, this was the first question that came up. How does it work? And it asked to usually everything works through protein, what's the protein? So I purified this protein. This doesn't work. So I did this experiment that turned out to be, you see it was published here in 1978, so exactly 40 years ago, and it turned out to be the best experiment I ever... It works. You have to press it hard, huh? So this is the best experiment that I did in my life, and this is cross-linking a labeled cap to a mixture of protein in the cell, just a cell mixture, and see what cross-links specifically. And I got a very good advice from an excellent chemist, Charlie Cantor, without him, I wouldn't have been able to do it. It's using sodium cyanobuara highlight to stabilize the bond, the shift bond. And basically what we found, I found is that there is a protein here, we call it later EIFOE. Is that cross-link specifically to the cap? Because here you see the cross-linking, you have the cap analog, just a seven-metal GDP, and you see zero cross-linking. So of course the next step was to purify it, and then later it was called EIFOE, standing for eukaryotic initiation factor 4E. So there are many initiation factors that are involved in recruiting the ribosome to the messenger RNA. There are 13 of them, many of them are multi-subunits, so there are at least 40 different polypeptides associated, maybe not as complex as transcription, but still complex, sufficiently complex to make life difficult. But this one behaves in a very clear way, it recognizes the cap structure, but it's not by itself, it's in a complex. And the complex is rather stable complex, consists of EIFOG, which is a scaffolding protein, and which binds the EIFOE, the cap-binding protein, and another protein EIFOE, which is a helicase, which unwinds the secondary structure of the messenger RNA in the 5-prime-mount translated region. That's very important in order to enable the ribosome to move and get to that initiation column. So this is a simplified model because there are many other proteins involved assisting in this. There is an EIFOB, EIFO-Rage, et cetera. But this is the core mechanism of all this process. Once you have the unwinding, then the ribosome can bind and then continue with the translation, basically what we have is an ordered process here is the messenger RNA. This is here the AUG, where you start a translation making the protein. Sometimes this is very long, it could be about 3,000 nucleotides. And you have this interaction of the EIFOF or the EIFOE. This recruits now another factor, which is EIF3, a multi-subunit. This recruits now the ribosome, the small subunit with other factors. Only one of them is shown here, a ternary complex which brings the MET-TRNA. And all this big complex that assembled here at the end of the messenger RNA has to now scan or traverse the 5-prime-UTR to reach here the AUG and then the 60S joins the large subunit and then you start to make the polypeptide, adding amino acid to make the polypeptide. So now this is a very important process in the cell as to be tightly regulated and there are many mechanisms. One of the major mechanisms we found are proteins which are called 4EBPs, 4E binding proteins, small molecular weight proteins that binds to EIFOE prevents the assembly of the complex and then you in a bit translation. And I'm not telling to you today about this mechanism and the consequences, but it's very important in cancer because it turns out that this is a reversible complex and as you can see here these 4EBPs can dissociate when you apply hormones, ghost factor and mitogen and then of course the 4E can function again as part of the complex and the key to all this regulation is that this 4EBPs is via the mTOR pathway through this PI3 kinase and all these components that are involved in cancer. So you see here downstream of this mTOR complex there is a 4EBPs which are first correlated here and released from the EIFOE. So I won't tell you about this pathway today. I want to show you another pathway in which EIFOE is involved and can be activated and this is the last pathway. Again, very important in cancer but I won't tell you the cancer part that we are doing. I tell you about the neurodevelopmental and the disease, the fragile X syndrome and I tell you about depression. So the EIFOE as I told you is a part complex with EIFOG. So it's part of a complex with EIFOG and it can be phosphorylated on one side, 0 to 9. And there is a very specific enzyme, the only enzyme that we know that can phosphorylate, only kinase, phosphorylate EIFOE is called mink. There are mink 1 and mink 2 first discovered by Tony Anto 20 years ago and mink gets a signal from the arc pathways. We see here arc make rough rust. And once the mink binds to the carboxyterminal part of EIFOG and phosphorylate the EIFOE. So we wanted to study this in cancer we make a knocking mouse so you have a serine to alanine mutation it cannot be phosphorylated so very clearly turns out that in cancer these mice are resistant to cancer and we have all ideas how it works and all this but again I won't tell you today about cancer I'll tell you about the Fragile X syndrome and I'll tell you why we got into that. So first of all once you have the phosphorylation of EIFOE you have a subset of mRNAs that became better translated and the way to do it is what you do is a polyzone profile actually you separate the number of ribosomes on the messenger RNA on the sucrose gradient so when the messenger RNA is translated well it has many ribosomes on it because the initiation rate is fast so you can have 5 up to 10 maybe more depends on the length of the messenger RNA and then in the sucrose gradient you have them at the bottom a messenger RNA that doesn't is a poor translator we'll have 1 or 2 or maybe 3 we'll have it close to the top of the gradient you separate them so now you can do different treatments and see how you change the position of the polyzone for different mRNAs depending now on the condition so we looked one condition here is comparing wild type fibroblast to fibroblast in which the serine 209 was mutated so instead of serine you have an alanine you cannot get phosphorylate and then we came up with a list so I don't give you this traditional IPA ingenuity pathway analysis and all the rest because we didn't have enough targets to do the statistic so basically I have a list here very short list it's a cut off of 1.5 as you see and just when you look by eye you don't need all the bioinformatics et cetera and you start to see the poor inflammatory you know aggressive behavior for cancer et cetera so you see all the CCL1 CCL2, CCL7, CCL9 the inhibitor of NFKB you see MMP9 you see MMP3 for some reason here is MMP3 so this led us some experimenting cancer but also led us to experiment on Fragile X syndrome because of MMP9 what happens in Fragile X syndrome many proteins change because the proteins that is can you imagine what is a Fragile X syndrome? that's what I'm going to tell you so yeah, so better instead of giving the introduction by words maybe I'll show you what it is this is the Fragile X syndrome so it's X linked it's encoding one protein which is called FMR Fragile Mental Retardation protein and what you can see here this is the Fragile site it breaks, you see it here and then what you have is I'll show you in a minute in this patient in males you don't make the protein I'll show you why so Fragile X syndrome is the most common form of inherited intellectual disability boys are 30 to 60% called diagnosed with autism but there are many many other syndromes you can recognize them if you once learned what is Fragile X you recognize them you know, the long ear long face the way they behave like autistic and repetitive behavior and this is what happens so this is the gene as I told you encoded by the Fragile X site so there are always repeats in humans in males there are no repeats 145% repeats of this triplet CGG there is no disease and this isn't the first in turn which has a promoter activity so you make the messenger RNA you make the Fragile X FMRP protein between 45 and 200 repeats you have some disease not all the penetrance is not 100% but sometimes you have disease when you have 200 then you have the the critical disease and this accumulates generation by generation yeah, yeah, it's from generation to generation and then you have expansion generation between parents and children right, exactly so here what happens once you have this many repeats this C come hyper-metallated once you are hyper-metallated you don't have a promoter activity so you don't have a messenger RNA you don't get now the protein, the FMRP FMRP is a translation inhibitor right so many people did different assay especially the clip etc they found out a subset of mRNAs but there are too many about 4 of them 4% of the mRNAs are substrate to FMRP so you don't know which one are the important one of course it's always a problem it's not just specific to this this one but we wanted to concentrate on MMP9 because we saw the MMP9 in our list of mRNAs that are affected by the serine phosphorylation so what we know we know that MMP9 proteins levels are elevated in fragile X and I have to say that MMP9 plays a major role now in the function of the synapses in the brain and we don't know exactly the mechanism but you need them in order to get a synaptic plasticity and learning in memory what was shown also before we started the experiment some inhibitors of MMP9 and 2 they are not specific so you see 2 is a target some others they rescue the fragile X behavior in mouse so you can make a mouse look out of the fragile X of the FMRP and then you can get as you see in a minute the very very very similar symptoms to what we see in humans so this is the symptoms so this is the fragile X FMR1- so in females it contains 1 X chromosome in the humans it doesn't have so most of the experiments are done in males not humans in males most of the experiments are done in males and these are the things we are looking we are looking at impaired preference for social novelty which is a classical markers of this patient and that's also for autistic children in general hyperactivity or geogenic seizures this is the electrophysiological measurement which is called long term depression so it measures synaptic activity so you give this drug DHPG and then you see a depression and the potential so you see in wild type you see a depression and goes up almost to normal when you see it in the knockout this knockout you see this exaggeration it's exaggerated it goes lower and never reaches back so this is a classical now again electrophysiological measurement of this disease so here are just some other things that we can measure as I mentioned already the repetitive behavior repetitive behavior social interaction or geogenic seizure, hyperactivity these LTDs that I just explained we can also look at the glintic spine abnormalities that always correlates with synaptic activity and learning and memory and it's part of the synaptic transmission so basically this is what I described before in the world where we do the polyzone fractionation and we got so just to show it to you I should have put it before so this is the polyzone we separate them and we can hybridize them to see DNA and that's how we got the list that I showed you and this is the list and again the MMP9 is the one that we picked up to study the fragile X so how do we show now that it's really important that it's associated this MMP9 reduction and the phenotype are associated with the EIF-4E phosphorylation so first we did genetic manipulation so in the genetic manipulation what we did is we took the knockout mouse for the FMR we crossed it to our EIF-4E which is the knock in the serine 209 to alanine and we crossed it also to heads of the mink 1 and 2 just the kinase that phosphorylates and as I said this is the only kinase phosphorylated serine 209 what we expect now is the lower amount of phosphorylated EIF-4E and because the amounts are lower then you expect less MMP9 you have less MMP9 if MMP9 is really important as really advertised we should rescue the phenotype and that's as you can see we published in 2014 we showed the rescue of most of the phenotype actually all and I'll show you just one of them and this is the exaggerated LTD, the long term depression and basically what you can see here in this case we didn't use a genetic genetic test we used a drug so this is an acute recovery and rescue so this is a sarcosporamide we developed with lily for cancer it's toxic but still we can use it here and this will reduce inhibit the mink 1 and 2 and reduce the phosphorylation of EIF-4E so you see here the reduction in the phosphorylation of EIF-4E and it's more than 70% and here is the test of the long term depression as I told you when you use the knockout mouse, the black one you see that the exaggerated LTD goes very low and doesn't recover this is the wild type the white one and here is the one when you are the sarcosporamide you see you completely rescue it you know it goes behaves exactly like the white one so now the problem is we use drugs that could never be used for this children with the fragile ex we have to come up with something better and we of course everybody thought about something and I had a poster he came up with the ideal drug and you will see why I say ideal because it indeed seems ideal and this is metformin so before going into that what cells is it working and what cells is it working in the neurons which neurons the pyramidal neurons for example they pockampus also in the PFC SH RNA for mink SH RNA for mink would be much more specific no it is that SH RNA for mink but it is the mink but it is a mink no car so it is very specific but we use for other thing but when we published I told you 2014 we published sometime came up a paper that actually showed it even in a clearer way than we did they asked a simpler question and the question was there is always to find this the question was let's take a knockout for the MMP9 so we remove it and to see what it goes to the fragile ex knockout if it can reverse the symptom and that's exactly what they did we thought it's an excellent complimentary story we sent together to sell the papers so the reviewers want us to do their experiment and she was asked to do our experiment so there was no use so we tried to convince this is etel etel and we tried to convince them that maybe we should do but they had a grant they had to submit anyway so they submitted this and we submitted somewhere else so as I told you Arkady Kortursky came up with this bright idea to use it for me this was two years ago took us a long time until it got published published in nature medicine in 2017 why do I say ideal metformin is an FDA approved drug used to treat diabetes type 2 type 2 diabetes 100 million people take it every day around the world this is a drug with the least side effects that you can imagine so you the worst you can get is diarrhea of course if you have a kidney, a damaged kidney then you have a problem you're not allowed to use it you get the ketosis et cetera so that's why this is the first line drug for diabetes and most importantly it's cheap, extremely cheap for a very long time so why did we use metformin because metformin suppresses mRNA translation via inhibition of arc and mTOR we saw that it will go through the mTOR but it actually worked through the arc as I told you arc is upstream of the mink which is upstream of the phosphorylation why metformin that's the big question we don't know we know we don't know actually most people don't know I know that don't have a list of things that metformin says right, right, right yeah, yeah, yeah so in answer to your question look at this paper so you have one, two, three, four yeah, yeah, don't believe everything so we have five papers but we could by ourselves because you don't believe everything you do the experiment yourself and mink inhibits arc one in our hands so again you can challenge even the people who did it in the lab but then you ask several people so at the end you run out of challenges so I accept now that mink that metformin inhibits arc so here what we do is we injected metformin if I find it again metformin 200mg per cure that's about a little bit more than you give to patient with diabetes and over 10 days so here you can see the schedule and then at 11 days we look at booming we look at social days a test after 12 days this is what I showed and this is the there's a very classical experiment to people study behavior so you have here two tests one is the social choice test one is the social novelty test and the first one basically what you do is you see here on the left there are three chambers and here you don't see anything here there is a stranger and the mouse that we testing always is social so most of the time not always will go here and sniff this mouse and sometimes it will go also to the other one goes around this it doesn't see anything and doesn't spend much time you see how fast it went out so this is the second test here and as you can see here here you have two mice on the left is a familiar mouse so you cohabituated your mouse with this one he already knows it, he's a friend here is a stranger he always likes to go more to a stranger so he will spend more time with them with the stranger rather with the familiar mouse so of course you can measure it very easily and here are the results so this is our mouse so it can be either a wild type or a knockout of fmr this stranger one, stranger two and you see now with the wild type as I told you it will go more to the stranger to the ones that he doesn't recognize rather the familiar when you look now at the black which is the knockout is no difference so no social preference for the stranger now this is the critical one you admit forming the way I described to you it corrected so now you do other test like grooming so this is the classical test of repetitive behavior so you groom yourself there are different stages of grooming you can learn from the old stages something and basically you can see that now the knockout mouse comes itself twice twice the time as the wild type but when you admit forming you correct it also the number of grooming bars is corrected something that very interesting that is corrected and still this we don't understand but tells us a lot the fact that it's corrected tells us something about the specificity and this is the macro-orchidism which is enlarged testis and again it's amazing that this is the same phenotype in males and also in the mouse and you can see here although it's not as strong as before you can see even here some correction of the size or the weight of the testis so I told you about the dlitix spine so here you can see again what you can see by microscopy and basically you can see the spine density in the knockout is increased and most of them are immature spines and again corrected by metformin and here you can see the spines that are increased so you see a lot of פילופודל which is immature and the mature one are less and again corrected by the metformin the LTD yeah I mean the effects are very clear but I have one problem I was told that metformin only works in the liver only the liver has the transporter for metformin right yeah that's another question whether the effect of metformin is systemic or cell-autonomous I am not sure that metformin even can go through the brain barrier yeah yeah and you find the metformin inside the brain so if you cell-autonomous you do IP is it the cell-autonomous or systemic effect is that still a question because what could be happening metformin reduces insulin level in the blood so therefore you get less insignia we thought about this and we have some experiment that shows that it's not it's not the case but I cannot hold it out completely so it's a very good point but as you know I put the emphasis on the correction of the disease not the mechanism so so the mechanism we are starting now we for example we know that there is an increase in the insulin receptor on the cells increase increase of insulin that's Eric what no insulin receptor so you get more more carapactin yeah so that's that shows that it's cell-autonomous so if we I'm almost convinced but 80% maybe it's a feedback effect they see less insulin dilavate the insulin etc. so these are all important questions but let me go through the data and basically the LTD is also corrected like I showed with the circus I don't have to show this again and so let's so here you do all this electrophysiology which I don't to myself look at spontaneous miniature excitatory post synaptic currents and you have always to do it if you want to really convince the people that know something about synaptic plasticity so again you can see the excitatory signal is much much much higher in the knockout and you correct it again with the metformin also where you correct the basic biochemical phenomena which is the increase of translation again this is inhibitor the fmr so it's inhibitor you increase it now you increase the translation there's the increase you reduce it now with metformin seizure you reduce and what I want to show here you see a lot of panel but the message of all this is everything that we actually predicted in terms of the signal now of the phosphorylation is exactly what we predicted so for example as you can see here this is the mech the phosphomech you see is increased now in the knocking you see the phosphorylation is increased you see the phosphoric is increased and what's most important the mmp9 is increased in the model and when you look now all the one the last one and the blueish one which is metformin all of them are corrected this can be done as you say both cells both in the hippocampus also in the prefrontal cortex so two major two major areas of it's not good so two major areas now of the brain to do now is the intellectual ability and the other syndrome so basically once you presented it we presented at golden conference they are sitting all the clinicians there is no good drug to fragile X and what's easier than to take metformin and give it to the patient so it's a very low hanging food and that's what exactly they did so Randy Hagerman she's not on this paper but Randy Hagerman she did and gave to her patients in Davis California and actually the slide I showed here shows you that you can get a similar phenotype in drosophila the phenotype is not similar, exactly similar to the mouse but the title is insulin signaling misregulation under lies circadian and cognitive deficits in drosophila for a gel X model and this are the now the clinical trials that will be done now a lot of patients now take metformin as off label right? so that's the kind of problem for the clinical trials because how could they get into this in the control group or placebo etc but still they found enough patients and they started it and this is the paper that came out almost together with us showing this metformin as a targeted treatment for a gel X syndrome as you can see again from Davis California from Niagara she didn't have many in this study like seven but in all of them there were improvement and then there is this anecdotal things that I get emails from people in Spain or whatever they are in the society the fragile X society so the father tells me this is big improvement of course it doesn't say anything scientifically but it still looks like if metformin is really helping him helping these patients this will be the from the beginning the ideal and as you know many people take it to extend life so they can live even longer than the average person so these are the different grants I should mention here Israeli he's a very rich developer he had a son yes he is dead but he has a son that had fragile X he was very interested he's giving a lot of money now the family for brain research and these are the people that did this work I still have time because I want to talk about depression so I mentioned Arkady Kudoski Christos Gokas is also one of the originator these are other postdocs that work on autism and fragile X and this is Jean-Claude Lacaille he has many many machines for doing the LTPLTD and we use them a lot so the role of here for reforestation depression so why did we do it some people noticed that the mice are depressed but maybe the students themselves were depressed they think that the mice are depressed there are many reasons to be depressed these days but there is another more scientific reason why we used it so first everybody knows about major depressive disorder so it's not this kind of depression that you have for a few days so persistent low mood irritability persistent diminished interest in all activity changes body weight appetite, sleep disturbances psychomotor acceleration and the lifetime this number is really alarming because the lifetime prevalence of 17% in the general population higher irritability the only drugs that are used today are not the only but the major are the serotonin reuptake inhibitor are commonly used but they are good only in 30 to 40% of the patient so this is not good so how do we get now from the EIFRE to depression and we get there because of the reduction in elk both you see in the pre prefrontal cortex and also in the apocampus there are other things that are going down but the one that of course is related to us is the elk so we wanted to determine whether the elk phosphorylation downstream of mob kinase, controlled depression and the anxiety barrier we wanted to determine the activity of the dorsal raffus or a tenorgic mpfc circuit so that's in the dorsal raffin most there are most of the cells that are producing the serotonin and maybe I don't get to this at the end but we'll see we saw a lot of changes in the dorsal raffin and we wanted to investigate the molecular mechanism underlying behavioral and surface alteration induced by deficit in the EIFRE phosphorylation this I think I took it from you you write a review on the EIF right? so I didn't give you credit here anyway so the next time actually it's my postdoc so he didn't give you credit so here are the tests so you start with a very simple test of course you need always everybody that knows about this behavior you need a lot of control because you can get this kind of things because of many many anxiety many other things so one of them is the first swim test the other one is tail suspension test as you imagine the first swim test if you put a mouse now that is depressed he won't try much so he will stay immobile the same is with the tail suspension test you see here is suspended by the tail and again if you have a depressed mouse he will stay immobile and what you see very clearly is there is a difference here when you take the mink wild type and knockout and you can see this time immobile you see here in the knockout you see there are much more immobile that here on the bottom both male and female are more in the wild type the same is true with the EIF-4E so the EIF-4E the wild type and the knockout and again you can see that the knockout or the knocking in this case it's the knocking there are much more immobile so in this case they will be counted as depressed the same is true also with the tail suspension test and you see the difference maybe in this time I will go a little faster so you have also basically just to mention that you have also other tests you cannot rely on two tests you have the novelty feeding so basically you put food food here in the middle and the mouse which is not depressed of course will go to the food the mouse that is depressed here you see stays in the corner again there could be other reason but this is just one test then you have the open field test it's exactly the same the depressed one will stay in the center they are afraid to go outside it's like also anxiety and it's true for the depressed one so here what you do is again to try to correct it like we did with the Fragile X so we use the circle's parameter I told you remember it's the mink so you have less EF4E for correlation and you see very clearly here that you have less 4E for correlation and you see that you correct now again so basically you see that the EF4E for for correlation is reduced and when you ask the amount of EF4E it's not reduced just the force for EF4E and here you can see with the circle a sporamide you correct so this is 20 mx per keg this time immobile you change it from 70 seconds to about 140 seconds okay again we go to the electrophysiology we change it and you can see very clearly the changes that you make here this is the EF4E the wild type and you can see here sorry with 50 micromolar here we didn't make a change but with 100 micromolar we could correct again so one question maybe in your physiology plot you have baseline and you have sedimentary yeah so I didn't explain because I was rushing sorry about that thank you for this so basically what you look here is now the signal in the electrophysiology so one lesson is not too harsh so you see with the EF4E here is the baseline you have here a serotonin you see now that you have an increase right when you do now with the knocking you see that you see that the increase here you can see at the blips at this you see that it's much less you say serotonin do you mean you are injecting serotonin or what does it mean how do you do the serotonin you inject it right so you inject the serotonin ICV ICV inside the prefrontal point exactly so here you have all this connection you know with the dorsal raffa here this is the region this is the prefrontal context and you see the projection so many experiment we did on this and we could show the really the importance of this connection for the depression and I want to go that's why I was rushing with this one because I want to go to the molecular explanation like we are right with the MMP MMP9 4 minutes so in 4 minutes I'll be able to explain you here what is now the target it's not MMP9 now so we look now through the list right and the ones that looks the most promising is this inhibitor of NF kappa B so the official name is nuclear factor of K like polypatterge in B cells inhibitor alpha which is called IKK alpha and the expert here I think in the room I kappa B alpha no I kappa B alpha but so he was sitting the expert in the first row so why why did we pick up because it's very important place a major role in inflammation and there are this idea in the field although no accept not completely accepted especially not by the not by the psychiatrists because when we are working with the psychiatrists we tell them okay you can inhibit the inflammation it's not sure it's not this etc but we thought that this could be a very nice clue to do experiment and you see inflammation in many papers in major depression disorder and again not everybody believes it but you see it in many days so this is a protein that is on the outer membrane in the microglia and goes up in inflammation and you see very and MDD you see in different regions in the brain you see here it's increases in the amount and you see the for elevated poor inflammatory cytokines IL-6 and TNF-alpha so as I told you what we saw and this is what we saw in the list of course we validated and basically you can see the inhibitor now and the knock in is much less and again you can see it also by the profiling that it translate less than acting for example so here is the inhibitor and you can see in the wild type it sediments at position 6, 7, 8 of the polyzone in the sucrose gradient in the knock in you see its position 2, 3 and 4 so the rate of translation initiation is lower which fits with the connection to the ear for ease that works for in the translation initiation so you can see very nicely that this is controlled by the mink 1 and 2 so here for so again here you can see the absorbance of this polyzone profile here you can see active on the polyzone profile you see that there is no difference here between the wild type and the knock in, the ear for in knock in and here you can see the inhibitor of NF kappa B and you can see a big change so you see in the wild type here it sediments in the every every fraction in the knock in it goes to the light fraction so we can show also increased increased TNF alpha expression in the prefrontal cortex of the ear for in knock in you can see here again the TNF alpha so what you see here some other marker this marker is is a marker of the microglia so again increased this is another marker here so again increased so the idea can we correct it now by reducing the amount of TNF alpha the depression so we use the dominant negative that has been described before and basically this treatment of 12 days and they use 10 milligram per ml and so this is the the fourth swim test and the novelty surplus feeding that I showed you before and you see very clearly that this is again the time immobile that present the depression this is increased in the knock in you are the dominant negative again this is ICV you do the dominant negative it's going down the other test the novelty surplus feeding the same thing so this is the model so this the model is based on many other many data in this your data actually so basically the NF kappa B alpha inhibitor is a target of the ear for E and when it's not phosphorylated you make less of it so under normal condition the NF kappa B when there is no inflammation is inhibited by the inhibitor that doesn't let it go to the nucleus you don't get the TNF alpha and this is the last one before the last before doing the acknowledgement here what you have depressive state so basically you make less now of the inhibitor of the NF kappa B NF kappa B goes into the nucleus you make the poor inflammatory cytokines and depression so I want to finish with something that's the most difficult was the most difficult to do from all the experiment that I explained and this is how to get lymphocyte from patients with depression so you go all this bureaucracy you have to fill this and that and etc so we ended up only with 24 samples that we measure and we see something interesting you wanted to put in the paper but it's only 24 I told him don't put in the paper and basically what you see is that in female here in the major depression disorder you see less of force for EIFOE than in the wild so this if this turns out to be if we can get more samples etc it will be I think a major major breakthrough because we have all the work with the mouse and these are the people that did the work so again these are psychiatrists Gabriela Gobi all the group Gustavo Torecki and these are the people that did the work in the lab so basically this is again going for molecules to disease going from basic science to understanding disease and maybe treating disease and killing people thank you again we have time for one or two questions but before that I want to ask you for education so the inflammation is mediated by the microphase microglia microglia is the one that they secrete the TNF alpha what do you mean microglia? in the brain okay but you are testing the mRNA level where in which cells so we testing from so we test from all kinds of first of all we test the amount of protein no no you check the mRNA level with the inhibitor the mRNA level I don't remember which one oh yeah yeah yeah yeah so we test when you looked at the air for E so we test the hippocampus so but this is mediated by the by the hematopoietic cells no by microphase the inflammation so the microglia are the ones that are the microphases in the brain yeah they are microphages he is checking the mRNA level in which in all in all cells wherever you look you see a difference but the ones that secrete the TNF alpha and all these are to be the microglia לרמידל cells etc okay yeah so repurposing drugs I mean this can be extremely powerful and getting tools to develop more specific compounds and I mean having compounds are well tolerated is a great way of getting patients and so on the question though is as many of you know with diabetes the compensatory changes very rapidly eliminate the benefit depletion so I guess the question is in mice you can certainly use many erg specific inhibitors do those do those have benefit so the biggest problem with this erg specific one are the side effects right so that's why in cancer why didn't they become the you know the the major treatment but in mice you can do the experiment there are in clinical trials right now there are mac inhibitors in clinical trials I know but many of them are in this but so that's why you started with repurposing and you have one of the safest drugs in the world right so of course you will use that instead of this but again people try I don't know for some for one of the reason is this is out of pattern you cannot make much money so people try make the derivatives now of the drug of metformin for obesity so I don't know maybe this can be used but the erg for sure you cannot compare them to metformin there one more question good question so in your fragile eggs Have you tried rapamycin just to rule out that the poor pathway is also important and sort of a side question from this discussion why would an urchinivit that have side effects metformin not since it inhibits metformin okay so there was a paper with metformin actually published and it had the opposite effect you must admit I didn't read it very carefully why should it have the opposite effect but again metformin has many targets because it acts M-Tor and downstream of M-Tor there are about 40 different targets so metformin was tried and he's asking about the safety situation the metformin actually doesn't have side effects it's actually beneficial in general when you inhibit air you inhibit signaling everywhere in the body so everybody should take what about rapamycin rapamycin in this one in fragile eggs so that's why I said there was a paper but it made fragile eggs even more severe didn't correct so it's very interesting in terms of our data anyway we have to continue with the program you can ask