 Thank you very much for the introduction and also thank you very much for inviting me it's a pleasure to be here I think it's the first time that I'm in a meeting where I don't know anyone so and for that yeah I didn't know but I'm learning to know people more and more and well so for those that still stay till the end I quickly want to say where Loewe is so Loewe is a university town close to the capital of Belgium to Brussels Brussels has not really a big university so the biggest university of Belgium is in Loewe I'm not from Ghent like is indicated on my name tag Ghent is somewhere here so it's closer to the seaside and so now we are sitting somewhere here this is France and so you should know Loewe at least that's what I hope you should learn to know Loewe from the historical things there's a lot to see in Loewe so whenever you're in Belgium just give me a call or an email so I will guide you around it's a very old university it's one of the oldest university on the continent and if you don't know Loewe from yeah from the research or from the students you should know Loewe from the beer Stella Artois is from Loewe and the combination beer and students is apparently a perfect combination okay so that's the introduction what I want to show you today is what aging is doing to our population so especially the western population is becoming older and older it's also increasing the number of people is increasing and what you see here in the red dashed line is the number or the percentage of people over 60 that we will have over time and you see that more than 20 percent of the population in 2050 will be over 60 so older people also means that there is much more neurodegeneration and neurodegeneration will come with the cost and so what is this what is shown here is a simulation of what it will cost to the society if we all become older and if we all start to suffer from neurodegenerative disorders there is no cure for any of these disorders and one of the reasons that there is no cure is because of an insufficient understanding of the pathogenic mechanism of the etiology and the pathogenesis of these diseases and that's why I think we need to do a lot of research on these diseases this is a slide showing what happens in a number of these neurodegenerative disorders they are different in the sense that they affect different regions of the brain they're also different in the aggregates that are formed in these different regions and today I would like to focus on one of these neurodegenerative disorders it's called ALS amyotrophic lateral sclerosis and that's the disease that we are studying in the lab what is ALS? ALS is a motor neuron disease it affects selectively motor neurons and we have two types of motor neurons we have the motor neurons that are in the motor cortex are the higher motor neurons and these higher motor neurons they connect with the lower motor neurons and the lower motor neurons are in the brainstem and in the ventral horn of the spinal cord these lower motor neurons they connect the ones that are in the brainstem they connect to the facial muscles and the muscles that you need for swallowing and the lower motor neurons they go to your arms and to your feet so legs and feet and so yeah that are the ones you need for voluntary movements but also the ones that you have here you need them to talk so what happens if these motor neurons start to degenerate you cannot move around anymore and you cannot talk anymore so which means you're in some kind of a locked in state so you cannot communicate anymore with your environment the consequences of this dramatic disease well it's a very dramatic disease because you die from the disease usually two to five years after the detection of the first symptoms it's a rare disease having said that at this moment there are four to five million people running around on the world that will die from ALS so it's yeah it's lethal so the incidence just to give you another number the incidence is the same as the incidence of multiple sclerosis which is a disease that is much better known there are also many more MS patients running around but that's because they don't die from the disease so it's an incidence between two to four per hundred thousand per year so so what are the first symptoms first symptoms are benign so spasticity and hyperreflexia atrophy of skeletal muscles and then as I already said loss of speech and also people get paralyzed and there is no cure so it's a dramatic disease without a cure what is also what is what is also important to know is that in 10% of cases it is an inherited disease so in 10% of cases there are more family members that suffer have suffered from the same disease which means in 90% of diseases we have no clue what the cause is so that's an open question so in these 10% of familial patients we know in almost 80% of cases what the underlying genetic cause is and I've here indicated the most important ones I will come back to these genetic causes in a minute what I also would like to point out is that what the pathology of these diseases I told you in the beginning that every disease has typical aggregates every neurodegenerative disease has typical aggregates in the case of ALS 95% of patients have mislocalized tdb-43 which aggregates so it's mislocalized in the cytoplasm usually normally tdb-43 is a nuclear protein but in the disease cases tdb-43 is mislocalized in the cytoplasm and in the other cases it's SOD1 or FOS so okay so what is our goal is to understand the initial steps of the disease and the major question we would like to answer is how do these mutations that we know how do they result in the pathology and one of the things that I've just indicated is that mislocalization of that protein called tdb-43 we hope sorry yeah it's an RNA DNA binding protein so it has a role in RNA transport in well DNA damage rescue against DNA damage so it's quite well known what it's doing it's less well known sorry well it's unstructured yes so it has low complexity domains and I will come back to that later during my talk that because that's important so we hope that by studying the underlying mechanism that we can find also therapeutic targets what I also would like to indicate that is that there is a lot of variability in a family for instance with the same mutation and the example I've taken now is mutation in another gene superoxide dismutase 1 it's the enzyme that we all need to get rid of our free radicals to down-regulate oxidative stress that's a cause a genetic cause that is already known for a long time more than 20 years and so we have in Belgium patients with the mutation in SOD1 and you can see that the disease duration varies from two years to 20 years so patients belonging to the same family have a difference in disease duration that is so significant and so dramatic and there again what we want to know is why is that so which factors which modifiers are responsible for that major difference and again if we know these modifiers we can also try to develop therapeutic targets against these modifier modifiers by the way there are exceptions on the rule that patients die after two to four years and you all know one person Stephen Hawking has ALS but he has a typical form of ALS he has already disease for 30 years so he stabilized in a far advanced stage nobody knows why but nobody knows the mutation what no no it has a sporadic form but that doesn't matter so there are also in the sporadic forms there is also a lot of variation the sequence was done yeah yeah well he has not a genetic cause of ALS like in 90% of cases Stephen Hawking also illustrates another aspect of the disease cognitively these patients are still okay so only the motor system fails only the motor only the motor neurons die sorry so that's just to illustrate that there is a lot of variation in the disease I want to come back to one of the causes one of the genetic causes of ALS and it's a very special one we have already heard about repeats in in in genes when there was a talk about fragile x there is also a gene causing ALS that also contains repeats in a non-coding sequences also in a non-coding sequence it's c9 or 72 why is it called c9 or 72 it's located on chromosome 9 it's an open reading frame it's it's it's called number 72 so nobody really knows what the gene product of this open reading frame what it actually is the repeat is a hexa nucleotide repeat four G's two C's so GGGCC normally well I hope that we all have two to eight maximum eight of these repeats in patients you have hundreds and several thousands of this repeat and it's located like I said it's located in a non-coding region it was the well disease of its mutation happens yeah when when when you have such a large expansion of that hexa nucleotide repeat you will get a disease the only thing you don't know is when but already before we started they haven't been checked before they had developed disease yeah yeah yeah yeah you can't do that now because now we know what the cause of the disease is now indeed we have pre-symptomatic carriers we have families where we know that there are patients that have the repeat and that will get sooner or later the disease it's important because now we can even look much earlier what is going wrong in these patients well we should not call them patients at that moment they are not yet sick so the big discussion in the field now and well I think most of people working in ALS are shifting to yeah the hexa nucleotide repeats in C9 or 72 is how do these hexa nucleotide repeats how do they cause ALS and there are different possibilities I will come back to that in a minute but there is another thing I have to tell you and that is that these repeats are translated they are translated in a non-ATG mediated fashion so there is no ATG in the in this repeat in this intro but despite that they are translated and not only the sense RNAs formed also the anti-sense RNAs formed and so because it's a hexa nucleotide repeat and because there is no ATG they can be translated in every reading frame so theoretically that means that there are six potential dipeptide repeat proteins but because there is one in the sense the GP is translated both from the sense and from the anti-sense trend so we have five different dipeptide repeat proteins that can be translated from this repeat in a non-ATG mediated fashion do you know which is the polymerase no that doesn't not much known about that so no it's it's well there is now they are now investigating how it actually works well in the beginning there was even a discussion does this happen in the nucleus or in the cytoplasm it's a lot of investigation yeah yeah yeah indeed yeah yeah and so is it cap dependent or cap independent they know mechanisms now they know but I you know it's still not published okay yeah well yeah yeah yeah but I mean that's not well that's not what we are doing actually so but indeed it is important to know how this works because it's also happening in other repeat diseases also in Huntington there is also repeats and they are also translated and the the translation seems to be upregulated under stress so cells under stress they increase this non-ATG mediated RAM translation is it called okay large amounts are mean well that's that well nobody well you can detect it in the CSF and in the blood you can also find them back in pathological material it's not really known how much of these proteins you have the concentrations well very it's also there's also not a very good correlation between the place for instance where they find these aggregates and where you have the motor neuron that so there is a some kind of a discrepancy there so there is still there are still a lot of open questions but yeah these repeats were only discovered six years ago five six years ago so it's relatively recent so what's the tissue specificity of this it's it's present in every tissue well you mean the DPR production yeah it's mainly in neuronal cells that you can find them but also in astrocytes so it's but not in as far as I know not outside of the cns but a lot of things that basically are appearing yes at the onset or associated with ALS right that's happening late in this happened well that's of course a problem you can only well you can see it in the CSF and in the blood already at disease onset and that's why these people that have the repeat but that are not yet sick why they are so interesting but the fact is that most of them are still not sick I mean but in like let's say five 10 15 years some of them will develop the disease and well we are following them now already so we will see when for instance these DPRs show up which was previously only possible in patients when they were already sick or when you have to look well postmortem material yeah that will always be the same it's at the end of life so that's that doesn't make much of a difference but in for biomarker research you can now go much earlier to detect what shows up okay so we focused on the DPRs that's why I introduced them so extensively and before I go on and focusing on these DPRs I would like to give a little bit of a broader picture in the sense that it's still not yet known whether it is due to loss of function or due to gain of function that these repeats cause ALS although there are a lot of arguments that it's not just loss of function I'm not saying that loss of function couldn't play a role but it doesn't seem to play a major role so the expression of C9 or 72 for instance seems to be quite similar in patients and in controls and then there are two gain of function mechanisms proposed there are RNA foci seen in the nucleus and these RNA foci they contain these repeats these hexanucleotide repeats and they also bind RNA binding proteins and so maybe by doing that they deplete the nucleus from some essential RNA binding protein so that could be a gain of function and then the other gain of function is what I just described is that run translation the production of these DPRs that you usually don't have and that can become toxic what have we done when these repeats were discovered and when DPR translation was suggested as being important we have made constructs where we have introduced an ATG in front of a sequence but and we used the wobble positions to create a coding sequence that doesn't contain the repeats and these constructs also they only express one of the DPRs because that's a problem when you just express for instance these repeat RNAs in cells yeah you don't know what actually is translated from them so you will have a mixture of all of them so what we have now is constructs with just one of the DPRs expressed and these constructs don't form RNA aggregates for instance okay so we use these constructs to find out which mechanisms are responsible for DPR induced motor neuron that and the story I would like to share with you or the first story I would like to share with you is the role of nucleosytoplasmic transport that we discovered a few years ago this was done both in yeast and in flies and so in yeast you have an inducible system so you can turn on the expression of the different DPRs and so then you have a dilution test so you dilute and you see how many of the yeast colonies survive when you turn on when you turn on the expression of the arginine containing the GR and the PR then you can see well for instance for the PR here that there are no yeast cells growing and also for GR you can see that there that the number of yeast that is growing the number of colonies is lower while the other two that are tested here don't have any effect on survival we did similar things in the fly eye and so here is a control eye it looks like a control eye but in fact it expresses one of the DPRs PA and it doesn't show any defect while when we express again an arginine containing DPR you can see that the eye looks a bit sick it's not only the eye that is affected when we just express because in that case we only express the DPRs in the fly eye we can also express the DPRs in the motor neurons or in the whole body and what you see then is that the ones that again express the arginine containing DPRs that they have a shorter survival whether these are females or males it doesn't make a difference and now that we have this system where we have clearly cell that that is induced by certain types of DPRs we can use that system to look for modifiers why why is the arginine contained that's a good question if you replace it with lysine will do the same thing no it's the arginine is important so but again i will try to give an answer i don't i'm not saying i know the answer but we have some hints what what could be before question this repeats that for all patients are only having this in the family it's only for the ones that have the hexanucleotide repeats in c9 or 72 all patients have this if you have the repeat you have the DPR if you have this disease do you have this DPR no no no no no because yeah yeah yeah yeah so again so only 10 percent is familial and from the 10 percent of this familial 60 percent have the repeats that i just yeah the rest have or other mutations in other genes or mutations in genes that we don't know but the majority and that's important to know well in the end it's always tdb43 that is found and that is independent of whether it's genetic or sporadic that's very important to to mention indeed so the the pathogenic end result is always the same independent of you have whether you have the repeat or whether you have no genetic cause or whatsoever so that's that's indeed interesting observation and to be honest we don't really know why that is but and i will come back to that also in the end so now we have two models yeast and fly and we can use these models to screen from modifiers so for instance for the fly we just have uh as iRNA expressing flies for the yeast you have deficiency strains and you just mate them you just cross them and then you can see whether this improves the condition of the yeast or whether it improves the eye of the fly whether it has no effect at all or whether it makes it worse so that's a classical modifier screen that you can do in these organisms and they have the advantage that all these lines are available so it's relatively easy to do these screens and so this is just an illustration what it means to be better or worse so this is the control condition where we just express the arginine containing dpr here is a modifier that makes it worse here is a modifier that makes it better the same for the fly eye this is what we normally see with some uh pigmented places in the eye here the pigmentation is is much uh more pronounced so this modifier makes it worse here you can see that it almost looks like the healthy eye so this is a modifier that makes it better okay so which modifiers did we find and that's uh illustrated here the modifiers that had the biggest effect had to do with or the nuclear pore or the nucleosytoplasmic transport and so these are a number of enhancers that all have to do with nucleosytoplasmic transport here are a number of suppressors that have to do also with nucleosytoplasmic transport and also in the yeast screen these modifiers were over represented so the the genes that have a role in nucleosytoplasmic transport were over represented in the modifiers both in the enhancers and in the suppressors which makes it more complex of course and so if i summarize a lot of work in one slide so this is this is a representation of all the enhancers and all the suppressors that we have found both in flies and yeast so and in red it's it are the enhancers and that are mainly the the constituents of the nucleosytoplasmic transport mechanism that are in the cytoplasm while the suppressors are mainly localized in the nuclear pore but don't ask me how they influence toxicity that's something we are investigating now it's well it plays a role the only thing we don't know is which role i mean that's more complicated to find out than we expected that i don't know by heart yeah it's in the i don't know what i should i should check which one it is so i don't know all the i'm i'm still learning how yeah the same name too many port yeah and and these are to make it even more complicated these are the names of the fly genes yeah so i'm not sure whether it's the same m tor no i don't think so so these are the fly names so i can check which what it is in basically you're getting anything that is affecting the nuclear pore yeah and it and it's doing it in both directions it would be nice if it was always preventing toxicity but that's not the case and that's we are not the only one by the way that found this so other groups have similar problems so to say but it's always clear and because there are two other groups that came up with similar data it's always clear that it's uh it has to do with nucleosytoplasmic transport so that's uh that's the the interesting thing let's say so did you look to see if there is any direction here directionality here that's that's something we are investigating now so we are now because the the enhancer versus suppressor could be explained by who helps to go to one way yeah what well the first thing we thought was maybe the dprs are just binding to uh the nuclear pore it doesn't seem to be that simple i mean that's that's not no that's uh i mean that had been too easy i suppose so could it be that this particular it's actually the rna that is no in this case in yeah okay yeah no no no it's bound to a particular problem that's moving around because it's so rich in in the all options are open so yeah it's uh because of course yeah there it is uh it's rna both as proteins that have to pulse uh no it's yeah not have to pulse the pore yeah yeah yeah so it's but it is translated so it means that no no you don't know but no you have no idea at this point no whether the effect present the rna or is the effect on the problem i know i know but it's impossible to believe you that the rna is stuck in the in the pore so how is it not translated yeah but no but it could be it could be other rna it could be it would be other rna that is it doesn't need to be the repeat rna that's what you i think are referring to it wasn't there a question okay my point was the very one what the hexanucleotide repeat was recorded so the repeat is no no no in this case the repeat is not there so in the uh models that we have there is clearly no effect of repeat rna but of course in the patients yeah we don't know so it's and of course it could also just be other rna that's that's the point i just wanted to make okay so that's uh nucleoside or plasma transport we made some nice movies that's uh these days uh well what's what we think is happening and so you have to think about the uh orange dots as being tdp43 when something is going wrong with nucleoside or plasma transport it could very well be that they mislocalize in the cytoplasm so that in one way or another but again this is an oversimplified model because we have no clue how it exactly works if i then just go to what happens with tdp so normally tdp is localized in the nucleus what we see happening over time is that tdp43 gets mislocalized not only in the patients that have the hexanucleotide repeat so this happens in all the patients so do you so if you would express this in a mammalian cell uh-huh do you get also this mislocalization through cell division in other words um i'm i'm trying to um figure out whether we see we don't see mislocalization of tdp43 in um yeah in simplified cell system yeah so we also have and i will come back to that in later we have for instance now IPSE lines that we can differentiate into all kinds of cells we don't see in these cell systems we don't see the same mislocalization of what you see in the patients but of course these patients when they become sick they're 50 60 70 years old in the flies we also don't see mislocalization uh yeah well we don't know that's uh that's the honest answer that's uh no yeah fuzz is also miss i could i could more or less uh tell the same story uh with fuzz instead of tdp43 but fuzz is much more rare that's only in two to three percent of patients in which cell did you do that the mislocalization uh the mislocalization is in the patients so the mislocalization is in post mortem material but of course you start from what you see in patients so it's mislocalized so the question is what's the explanation for that the explanation of that could be that there is a problem with nucleoside or plasmid transport that's the point i just want to make with these movies the patient you're integrating after many years this is not the same as in your culture of course not i mean that that's the that's exactly the point i want to make i mean so it might not be anything to do with you try but no but it could be there is it's well and it modifies it can modify the nucleoside or plasmid transport is dpr yes any yeast no no no you can generate cell line from the patient and yeah yeah uh all the nuclear poor proteins that were uh yeah that's that's indeed what what people are now doing uh that is whole genome CRISPR-Cas9 screens on um yeah on ipsc lines or on uh motor neurons that are differentiated from and there is well there was i think yesterday or the day before yesterday was there was a paper in nature genetics where they have done um a dpr screen it's it's a group from stanford where they have done a dpr screen on uh cell lines and they found again um that the nucleoside or plasmid transport was over represented in the hits they had together i must be honest together with for instance er stress was also there uh dna damage related proteins were there so it was not only uh nucleoside or plasmid transport but and of course the next question is what in a neuronal system because this was not yet in a neuronal system any idea what's special in what are neurons so what particular they have very long axons and uh yeah that that's and they are also they have also another way of signaling so in the glutamatergic signaling they don't use nmda receptors they are more dependent on ampereceptors um and there are well less calcium binding proteins i mean there's there is a lot of things that are different in motor neurons in comparison to other neurons whether that's all related to the disease that's of course another question is it not that these aggregates are actually the cause of the neurological symptoms no no no and that's of course that's that's also an argument i sometimes use uh when i when when i have to answer the question uh how do you explain the fact that aggregates don't correlate the aggregates of the dprs don't correlate with uh yeah the cells that die in the disease maybe it's just a way of the cell to get rid of something that is very dangerous it's possible yeah this could be that due to the long axon that uh the protein needs to travel a long time in order to get to the nucleus uh yeah because yeah because yeah uh to get translated they'll get translated but then they have to yeah yes and that's indeed a good point because one of the roles of tdp43 is to bring RNA to the neuromuscular junction so in that sense but then of course then it has to go back and to go back to the nucleus to get the next RNA so that axonal transport is important uh well that that seems to be uh and i will come back to that if i still have time this particular transport i would be engaging with what is the mechanism the mechanism of what who is this transport what are proteins which move it uh the the normal the normal yeah yeah the normal motor proteins dinectin uh kinesins this the normal the normal and i will i will come back to that uh in the end of my talk hopefully the dna damage proteins you spoke about yeah are they known yeah yeah they are known but not by me have they something to do with uh slippery or uh with the repeats no no it's i think as far as i remember it's a single strand dna breaks and double travel i mean but it's i can i can try to nothing to do with the repeats no no no no no no most like no no no it's just well apparently um there are sorry how do you generate the repeats in which case then with uh what you showed yeah that's all overexpression that's also something you have to keep in mind everything i showed until now is overexpression of a construct with the repeat no okay i mean what you you showed in some cases there are repeats hex and yeah so so is it known what is the genesis how the mechanistic genesis uh how how this how these how these are yeah no the question now of uh uh some some the machinery yeah yeah yeah replication yeah yeah no that's not it's not even known it's not even known whether the length is the same in every tissue so it could even be that for instance in neurons you have much longer repeats than in other tissue that's what people are doing for the moment yeah yeah it's trying to figure out and it's also not that the next generation always has longer repeats than the previous ones but there is variation there is clearly variation but the last word has not been said i mean this is this is relatively recent like i said so it's it's work in progress okay um so yeah to end this part of nuclear cytoplasm transport it seems to be a key part way and that's confirmed now again with uh also the whole genome CRISPR-Cas9 screen that i just mentioned and that is published i think the day before yesterday um and uh yeah what we are investigating for the moment is what the exact mechanism is so that's still an open question but while we were doing these experiments we purified the DPRs um and we had pure protein pure DPR protein and when we looked at it um in in a test tube then we noticed that these um yeah that these test tubes could become opaque for instance when we cooled them down when we just put them on ice then we could see that something like this happened so normally this solution is transparent if you cool it down or if you add a molecular crowded it gets opaque and so what you then see under the microscope is that droplets are formed so that was not known uh until we observed that just by accident so these are droplets of the arginine containing DPR so when we just have arginine containing DPRs in the right conditions then we can just see that they form these kinds of droplets that they face separate that's actually uh what happens and so that's actually the same as what happens uh yeah oil and vinegar uh so you have face separation so there is no membrane around it so they these proteins seem to cluster together and what is now a very popular view in in the field and that brings me back to the formation of the aggregates is that you have these physiological granules which are these droplets that are not only formed by DPRs but also for instance TDP 43 which is also a protein with a low complexity with low complexity domains but also fuss for instance that they form these physiological aggregates sometimes in combination with RNA but I will come back to that in a minute and that then over years this can result in pathological aggregates because this process is reversible so going from soluble to physiological granules is a reversible process similar with here if we just uh yeah well uh warm this solution then these droplets disappear and we can do that a hundred times there are no aggregates formed in our test tube but maybe in the cell under certain conditions you can have the formation of these physiological granules these droplets and then over time they can form uh pathological aggregates um yeah I already told you but it's a characteristic only again of the arginine containing DPRs so when there is no arginine in the DPR you don't see it yeah here is what you see under they're not the only arginine containing even their APS what or arginine contained it's common for all DPRs no it's only for the arginine containing DPRs so you need to have it's PR or GR so and you also need to have the right buffer conditions because the problem of course is you have a lot of positive charges so you need to have counter ions so and in the case well you have to have the right buffer so it works best with the phosphate buffer so that's what you see here for instance in potassium chloride you don't see them and that's why also a lot of people missed this formation of these droplets and what is also important to indicate is that RNA can also um yeah can also be a counter ion so the negative charges of RNA and so when you for instance what you see here is increasing concentrations of RNA added to a solution of the arginine containing DPRs so the more the higher the concentration of RNA the more turbidity we see the more droplet formation we see okay we can also do frappe so we can bleach fluorescence recovery after bleaching so we bleach and then you can see that the fluorescence if we label the arginine containing DPRs fluorescently you can see that the fluorescence come back so it's it's a dynamic process and here you can see it over time well we have young and old droplets it doesn't make much of a difference so it's not that we have aggregate formation in our test tubes because these are test tube experiments so then what we did was we did masspec on the proteins that associated with these arginine containing DPRs so a little bit to our surprise when we centrifuge a solution with arginine containing DPRs we found that there were a lot of proteins bound to these DPRs and then if we did masspec the most prominent pathway that popped up was was where the stress granules and stress granules are also structures that are formed by phase separation so there are also no membranes around the stress granules and so then we started to look at stress granules so this is a cell line where the ggpb1 is labeled is fluorescently labeled and then if you put these cells under stress then you can see that droplets are formed in the cell and you can even see if you focus here in this region of the cell you can see that they also start to form bigger droplets that they fuse a little bit the same as the droplets that I've just shown you in the test tube but the difference is that these are stress granules in a cell where also RNA is included in these stress granules while what I've shown before no no no no yeah this is a cell I mean you have to label them with which what what taking the stress granule yeah well it's it's a similar process the formation of a stress granule is the same thing as what you see when the olive oil is separating from the vinegar so that that's it's a it's a similar process of course I mean here is also RNA involved which is not the case when you are looking at your cell at the dressing room yes yeah yeah yeah and I think yeah g3bp1 is indeed a protein that binds to RNA to prevent it from being translated because indeed these stress granules yeah stress granules is a way of the cell to keep RNA shielded and untranslated for a while while the cell is taking care of surviving so it's up-regulated proteins that that keep it alive and the other RNA that at that moment is less important is just stalled away in the stress granules this is a combination of proteins and RNA so this is a this this what you see here is a blob of proteins and RNA combined and it's a very small so this is the cell so this is very small it's very small granules but you can see them with fluorescent microscope yeah yeah this is this is fluorescent microscope yeah something like that I mean large well they can they can have you can have them in different sizes but they are subcellar so what is this response to what what is stress yeah stress can be everything so this in this case it's yeah well what you see here is arsenide that's a very popular way of putting cells on the stress but it can also be oxidative stress it can also be heat I mean whatever you can do to a cell yeah that that puts them under stress will lead to the formation of these stress granules and once the stress is gone these stress granules disassemble so then the cell just well starts to use starts to use the RNA again I mean then then that's at least well what what should happen it's probably to preserve ATP in part because mRNA translation is the major consumer of Facebook as well yeah yeah so but in in a way it's just to protect protect the cell from stress it's a it's a stress response and the point I want to make is that yeah you can do it by arsenide so by addition of arsenide but you can also do it you can induce these stress granules also by adding the arginine containing dprs so that's also apparently a stress it also induces stress granules and what is also indicated here is that in case in the case that you induce the stress by these dprs you have more tdp-43 in the stress granule if you compare it to stretch granules that are induced by arsenide so that's the my second conclusion so the arginine containing residues they are well the arginine containing dprs they undergo liquid liquid phase separations these arginine containing dprs can also induce the stress granules and it seems to be but that's a well more general conclusion that numerous pathways not only nucleocytoplasty transport but also stress granule formation are influenced by these arginine containing dprs and the patients are those actually liquid droplets or these are yeah well you should well what yeah yeah these are aggregates yeah yeah yeah yeah the whole thing is what we believe is that what we are looking now in in culture is something that happens years before the formation of the aggregates and that's actually what is also illustrated in this summarizing figure that is that this process so the phase separation and the formation of stress granules but there are a lot of other structures in the cell that that are formed in a similar way that that is a reversible process and that then over years this can become irreversible that gels can be formed and that after certain years also these gels can form microscopic aggregates that's the idea i mean not so easy to prove that that's the same happens in patients because well what you see is the entries out you should well be able to go back years before the patient dies which is a bit difficult and the aggregates in the patients are they're intracellular or intracellular intracellular yeah yeah they're all intracellular so cytoplasmic yeah i'm just confused with the liquid liquid where the aggregates are liquid no the aggregates are not liquid so the aggregates is but that's that's the so what you have first you have soluble protein then you have the liquid liquid phase separation which gives you these droplets or these stress granules then that can lead to gels that's that's the next step where where there is maybe a core that is a bit more stable and that that can then over time generate these aggregates so it's a stepwise process and the first steps of the process are reversible the last step once you have an aggregate it's not reversible anymore but when you do the experiment in mice you say you're less in the models you see right away you don't you don't see the the liquid yeah no you don't see no no no well so in human if we look at the mice yeah but there there is not a good mouse model for foreseen yeah there is one there's a good one for for ALS but that's based on salt one on SOD1 of which people think that that's the there is there is one there is one good model but it's not it's not available to the community yet so once that it becomes available we can say they saw aggregates right away in the patients no in the mouse in the mouse there are yeah there is one model where they indeed see aggregates but it's yeah but before seeing the liquid that's what you should check in this in this mouse model but that has as far as I know not yet been done yeah that's indeed an interesting experiment can you tell me about the gel stuff yeah the stage I hope would you know about it yeah well it's it's just I mean a less how do you distinguish it from the stress granular yeah well in in in in just in test tube you can you can see that it gelifies that it's that it's more yeah in cells it's not possible to to see that difference you see either the stress granular yeah yeah yeah the gel the gel thing is is more something that comes from a test tube a test tube experiment where they can indeed see and some people well that's a big debate in that field they say that there is a stable core in these gel like structures which then is even another argument for saying there is already yeah it's an intermediate step to yeah to to the formation of the aggregates so that's yeah okay um maybe also an astrocyte but that we don't know yet that should be checked yeah so um yeah so what is what is uh part of these uh stress granules yeah yeah stress granules happen everywhere yeah yeah but but the formation of the aggregates the the transformation of these of these reversible structures to irreversible aggregates that seems to be something unique for motor neurons maybe because most cells are reproducible they're reproducible neurons are not so they're very they they live for many years yeah well for your entire life but muscle cells also don't need to be used muscle cells regenerate yeah muscle cells regenerate yeah yeah but motor neurons don't regenerate so you're born with a number of motor neurons and in the best case you die with the same number and less patience yeah they they die when they have lost a large proportion of them so that's uh so they live well if you're lucky 90 hundred years which is makes them well all neurons do that neurons that depends when you take the very long neurons uh to these merciatic but you know well the neurons uh that's the big yeah that's a big discussion yeah yeah well whether there is indeed yeah yeah but for motor neurons there is no regeneration that that's yeah for also because it's it's very unlikely that even when you have a new a stem cell that is transformed into a motor neural we'll find its target because well i want to come back to that in a minute i'm not sure whether i will get there but the cell body of the motor neuron that is innervating um yeah your foot is just at the end of your back so it's it's one it's an axon of one meter in some people well or less or more yeah so it's it's it's well the first signs are on the leg period yeah yeah yeah yeah so well yeah you have two forms uh but the most common form is where the legs and the arms are first uh yeah involved in the disease okay so so this is uh yeah now what i want to come to axonal transport so i think the introduction was already given um so what we have is uh ipsc so induced pluripotent stem cells that are generated from fibroblasts from als patients so that the big difference is that these cells are from patients are human and they have the same mutations as the patient and they have no overexpression that's i think very important to mention so you have the mutations in the same genetic context without any overexpression and what is very interesting is that you can differentiate these cells into all kind of cells including motor neurons and that's what you see here uh in the in this picture so and you can keep them some people can you can keep them for 150 days well we can keep them for 60 70 days and then they start to detach so you can keep them for a very long time they form very long axons and what you can do in these cells is you can measure axonal transport and this is a chymograph what you see here uh so these are cells so this is the cell body these are the neurons and this cell is loaded with a dye that is going to the mitochondria the functional mitochondria and what you see here then is this is the length of a neuron and so we have taken a picture every second and so uh when we put all these pictures all these lines next to each other you you get what they call a chymograph and so um vertical line is a is a signal mitochondria that has not been moving so all the lines that go from here to there or that go from there to there are moving mitochondria and if you know for instance if did if this is the cell body and this is the end of the neuride so then is this uh entrograde transport and this is retrograde transport and so you can see this is um the control a control line this is a line of a patient with a mutation that is causing uh ALS but it could also have been a patient with uh with a repeat so these patients show exactly the same over time in culture they don't die by the way that's one of our major frustrations we can keep these cells for uh yeah months and they don't die but what they start to show is a defect in axonal transport in this case of mitochondria and so this is what what i just explained it's over time so uh at week two yeah mitochondria are transported along the axons very important because you need energy for instance at the neuromuscular junction so you can see that there are a lot of stationary mitochondria as well as so there are mitochondria that don't move so that that stay always at the same place but the majority of mitochondria are moving in both directions so uh it's not so clear why but anyway so they sorry yeah yeah yeah yeah there are motors for both directions and that and that's what they do especially in controls so here you can see that it is developing over time so the uh after week two of differentiation the transport is still okay after three weeks it's lower and then it's going down and if we waited longer it would even go uh more it will even become lower it's not only mitochondria by the way we have been looking at other cargos this is just an example of ER vesicles but it could have been lysosomes it could have been RNA which we can also visualize it all shows the same phenotype over time uh you get less axonal transport yeah but these these cells don't form uh well that normally they should form a neuromuscular junction uh because these are lower motor neurons so they don't form and that's also what we see they don't really form synapses they are looking around for muscles I suppose and I will show you a picture where we have indeed combined motor neurons with muscles that's what we are doing now so we're trying to make yeah motor neurons muscles in a culture dish uh yes not the ones I just showed you that I've just shown you but we have also IPSCs from patients with repeats of which we have shown that they contain DPRs also in the medium by the way and they also show the same axonal transport defect so but yeah that seems to be a general and just to show you how movies look uh yeah okay it's running so this is um yeah a culture it's it's it's a fast patient but it could have been a c9 patient as well so this is the cell body and if you look very good you can see some movement here so this is um yeah a situation where you don't have much transport anymore the controls they look like this but I will tell you immediately what is this so I hope you can see that there is much more transport now so you can if you you can see here for instance this is a very nice one it starts again you can see it comes from here and it's going all the way yeah that's what I'm gonna tell you now um we can rescue this um by uh incubating these cultures overnight with an H6 inhibitor and it's a misnominers so it's doing everything except deacetylating histones so they have classified it as a histone deacetylase but it is deacetylating cytoplasmic substrates and one of the substrates are the microtubules and I will I will show it in a minute so uh so the difference between this and this is just an overnight treatment with an H6 inhibitor by the way we have other systems and other diseases where we also see axonal transport defects if I ever see a situation where there is less axonal transport I always tell my people add an H6 inhibitor and then you get surprising results I mean it's uh it always rescues um but and this is by the way also how it looks in a control so this the situation uh this is the patient this is control whatever the mutation what is the pressure used sorry the enhanced the H6 inhibitor what it is doing uh yeah I will first show the the the cartoon so what the what you have is for to get normal transport you need to have tracks you need to have the microtubules and they need to be acetylated um if they are acetylated the motor proteins are well connected to these microtubules if for one reason or another the microtubules get deacetylated and uh the the the protein the enzyme that is doing that is HDAC6 histone deacetylase 6 but it should be called tubulin deacetylase uh instead um so then you get a situation like this and then there is less uh axonal transport because the motor proteins detach and so by uh taking these HDAC6 inhibitors we reverse this situation into that situation and then I go one slide back to prove that that it's indeed true so this is acetylated tubulin um in in patients this is this is a patient so this is the patient this is by the way the isogenic control so with CRISPR-Cas9 we have corrected the mutation that's also an advantage of these iPSCs you can just have the same line same genetic background in just one mutation corrected and then these are treated overnight with two different HDAC6 inhibitors and you can see that the intensity of the acetylated tubulin is dramatically uh increased and by the way you can also see that there is a slight decrease in the muted in comparison to the isogenic control and that's also something we see in a lot of situations that the the acetylation of tubulin is going down in disease conditions but you can use it in for for patients that's a good question um there are well we are negotiating to to start clinical trials not in ALS because that's that's still uh well we are not able to to cure ALS but we are also using the same strategies in peripheral neuropathies and just to give you one illustration of which peripheral neuropathies we are thinking about so you have chemotherapy induced neuropathies and there you can uh yeah there you have uh similar things as here axonal transport is is going less well and also acetylation of tubulin is going down so there you could say okay we give Vincristin for instance together with an HDAC6 inhibitor to a patient in order to prevent the neuropathy from uh happening okay it seems that I have to speed up a little bit but I'm almost there I just wanted to show you a disconclusion but where I also show what you see here is a two compartment system so here are the motor neurons cultured in one side of the dish then you have mini grooves and what you see here are the axons that are crossing that groove and they make contact with what you see here in is it pink well in pink these are muscle cells and so we this is this is very recent so we are now also trying to measure axonal transport in this system and we are also trying to figure out whether we have or whether we can get neuromuscular junctions and I think the conclusions I've already stated them and just to uh well make sure that you understand what we are talking about so it's this these are these very long axons of up to one meter the motor neurons are in your spinal cord and then the muscles that you have are in your arms or in your legs and this is the problem that we hope to solve by using HX6 inhibitors just to wrap up the nucleosytoplasmic transport effects could be responsible for the mislocalization this is a two hit model like it is proposed in literature not sure whether it's completely true the second hit could be an aberrant liquid liquid phase separation and then this could lead to a number of things including for instance also axonal transport defects I also want to say something about whole genome sequencing I will be very short because that was actually what was asked so apart from the four mutations that I've been focusing on today there are many more genetic causes already known for the disease and the most recent ones because this is a function of the time the most recent ones are the result of exome and whole genome sequencing and that is for the moment a project going on in the ALS field where patients and that's why they call it project mind patients are giving their DNA and then well we have to give money or the public has to give money you can buy the sequencing of one chromosome or you can buy a half a DNA profile of a patient and so they want to go up to 15 000 ALS patients and 7 500 controls and just one example that it works there will be because this is a slight under embargo there will be this will be the cover of Neuron in two weeks where they have found mutations in KIF5A as a kinazine protein so which is a motor protein which is mutated and what is well illustrated so here you have the Axon shipping company that is transporting all these containers along the microtubules and so apparently when you have a mutation it doesn't work that well I mean it's a visual way it's made by geneticist by the way but I like it so I think it's a nice and here has to come Neuron so it's it's not yet finished okay I'm almost done if you give me can I have three more minutes three more minutes just to well to wrap up everything what is known in the field but you have so many kids so many motors yes yeah yeah not all are mutated yeah no no no yeah that we don't know yeah but we don't know how in some cases it will be genetics in other cases we don't know I would like to end with a movie of three minutes it's not it will not last more than three minutes Nature Neuroscience Review asked us to summarize 20 years of research in ALS it was not easy but I like the end result and I want to share it with you amniotrophic lateral sclerosis shortened to ALS is a neurodegenerative disease which usually begins in adulthood and advances rapidly it's characterized by the deterioration and loss of both upper and lower motor neurons as the motor neurons stop sending signals the muscles weaken leading to paralysis when the muscles in the diaphragm are paralyzed this can be fatal there is no known cure for ALS for most people the cause of their ALS is unknown but some people inherit the disease from their parents by studying the genes that are altered in these patients scientists have pinpointed processes in the neurons that might be causing ALS it's too early to know which of these altered processes are a result of the disease and which could be a cause but it's clear that they affect many different aspects of the motor neuron function let's start in the cell body here proteins that are not transported into the nucleus build up in the cytoplasm errors in the systems that build up and break down proteins also cause other misfolded proteins to accumulate the different proteins can then aggregate and can become toxic to cells in several ways for example they can damage mitochondria the cells power generators mitochondrial damage can lead to oxidative stress which can trigger breaks in the DNA of the cell ALS seems to affect DNA repair processes as well when DNA breaks are poorly repaired it ultimately contributes to the death of the neuron the cells transport machinery can also be damaged for example ALS effective neurons often have problems transporting RNA proteins and vesicles both in the cytoplasm and along the neurons these vesicles contain important cell signaling molecules called neurotransmitters if these can't be moved along the axon or released the neuron can't send messages to its target cells damage to the cytoskeleton can also cause the axon to retract if this happens the axon can no longer connect to the muscle nearby and can no longer signal the muscle to contract other cell types can also be involved in ALS oligodendrocytes which electrically insulate the axon and provide support to the motor neuron don't work in people with ALS reduced uptake of neurotransmitters by astrocytes can lead to over activation of the receptors at the synapse and death of the neuron finally astrocytes and microglia can produce factors that protect or damage motor neurons neuronal death occurs when excessive damaging factors are produced further research is needed to find out which of these many processes caused the motor neurontogeneration seen in ALS patients targeting new drug therapies at those processes might lead to a treatment for this incurable disease that's it perhaps not surprising question from me why do you're looking for money sequence subjects affected and controls but why do you need to re-sequence control I think they also rely on public databases but they want to yeah well they want to increase the number I think that's also why they focus much more on on patients than on on control and yeah yeah yeah there are there are I have a question do you know where the peptide containing our gene are located in the cells um in the cytoplasm not in the AR could be because AR stress is is is something that yeah yeah yeah yeah could be I don't exclude it yeah yeah yeah yeah yeah yeah that's that's just that's exactly well the paper that's the point that the paper that was published the day before yesterday Nature Genetics want to make yeah you should it's interesting I mean I had to I had to write a commentary yesterday evening so I know everything about it maybe you said that I missed it yeah how is the transcription of the repeats is it that's a polycystronic a very long long long messenger which you should maybe detect or is it cleaved right away and then the translation how is that yeah well but it's some there are also people that say that it stops in in the in the middle of the of the repeat yeah that the that the transcription process well that that you have also messengers that are could be yeah but not not all these pieces of them are named get translation right yeah but but I mean these these RNAs are not polyadenylated are also not kept they are not so they are they are they are excised from the introns from the they are they are translated as part of the mRNA starting from the middle that's the whole idea that he doesn't yeah well I'm not so sure about that the beginning the start from the middle and that's why it was so controversial at the beginning because there was no any precedent but it also happens in the reverse in the reverse way in the anti-set when you do you do a reverse so you have also a mRNA that we translated you have a transcript yeah coming from the others but that's not polyadenylated it has also polyadenylated it is the expert on that then then I should sit together next to him this evening yeah I can learn more from you than than yeah but it's not polyadenylated won't go out of the no-closet where you'll get stuck in the day okay that's good to good to know yeah since this is completely multifactorial yeah that's what we think so everybody is right everybody is right in the field right that's the problem yeah that's good that's how you keep friends yeah okay if we go now to try to deal with patience it's a first yeah um where where where you think one we should admire the day it's a very good question well the the the field at this moment is focusing especially for the genetic forms on antisense oligos so because it's gain of function in that's at least what what we think it is for salt 1 tdp fuss and c9 orph so if you can prevent the mutant form from being formed so if you can prevent the translation of the mutant containing transcript then you don't have the mutant protein and that should well help so antisense oligos is the thing people are investing a lot of money in for the moment but that's only helpful for a limited number of patients let's go through the antisense right yeah i will have to do this how the whole life of the individual um these antisense oligos are relatively stable but but but you have to yes but you have to re-inject intratically regularly you're not going to do that well it depends what kind of this issue an sma for instance is spinal muscular atrophy the the therapy that is there is also injection of antisense oligos no i realize this right but yeah it's very expensive first of all and it's indeed an intratical injection it's not yeah it's not something you're looking for you would like to find a way to maybe cross the blood barrier or yeah yeah yeah indeed or well the way that the easiest way to to stop people from getting familial al s is prenatal screening that that's the i mean that's the most obvious thing to do and that happens already really i mean it's 100% penetrance yeah close to not 100% but it's close to 100% so it justifies prenatal yeah and it's a terrible disease as well so if you can it's the same with well peripheral neuropathies the the the inherited ones also there you have prenatal screening already as a routine kind of thing although you don't die from these diseases so that's uh it's a it's more an ethical thing than anything else but but it happens i mean pretty sure that it happens if we eliminated organs what i mean he would not go through this screen what does he think about it this famous al s patient the actually two of them one but he's not genetic he's not genetic so for him it doesn't it doesn't help so it only it's only helps for for for 10% well maximally for 10% of patients 90% yeah are not helped so we have to continue doing research even if we can solve all the genetic forms yeah and so for the for the other forms we hope that we can find a pathway like for instance axonal transport that is a common mechanism also seen in sporadic al s patients that we can symptomatically treat these patients and that it could help them to survive longer sure but for example you describe the acetylation actually but then you also have a kindness yeah yeah yeah yeah yeah yeah it's a well you don't have to convince me that it's a complex disease yeah you're speaking about detailed therapy but what do you think about therapy right Chloe putton or total putton stomach cells is that not well the problem there is right because i think for brain for for Parkinson's and several other yeah yeah but the problem is that you have to restore so you have to restore functionality yeah so what what you if you if you replace well for instance this motor neurons dying so the axon so yeah first of all what we think is happening that the first problems happen here so you get denervation then you get axonal retraction and in the end to motor neurons die so what are you gonna do you're gonna transplant stem cells here and how will they find their targets one meter further away that's their job they should find because in heart if i yeah but what's it's a distance informed it's the way they inject and and then the sense yeah but how what distance do they have to cross i think it's it's i mean this this is one this is one meter yeah well stem yeah it has been tried in china so i mean and not in the best possible way i suppose but anyway there was at a certain moment it was a big hype in the al s field so people thought that they and not for the simple reason to replace the motor neurons but to replace the environment the toxic environment because well astrocytes and microglia also seem to play a role so if you can replace the astrocytes maybe then you will have a benefit in the end everything that they have tried failed so it didn't have much i'm not excluding that it will help but for the moment it's not the way people are well going forward let's take it that way because that's a radical yeah yeah but here i mean an axon well we we are so happy that we can bridge and the the culture has we can bridge maybe well it's it's a few hundred micrometers 800 micrometers it is i think and that is already a challenge so how can you get it one meter i mean hopefully i will yeah hopefully i wouldn't uh yeah you know uh i don't know you're on a maronic translation is mostly concentrated at the scene out all right so the question here i don't know about motor on my own yeah yeah yes yes yeah indeed yes yeah yeah absolutely no no there is there is clearly translation i well there is clearly translation here at the at the sign us for the simple reason if something has to be made here and the message has always to come from from the nucleus that will take too much time so if the if there is something needed here to react very fast to whatever situation local translation is the only way to do that and there there is RNA there so that's uh and there is RNA transport as well thank you very much