Prof. Dr. Rubén Fernández Busnadiego (Universitätsmedizin Göttingen) spricht im Rahmen der öffentlichen Ringvorlesung “Herz und Hirn gemeinsam im Fokus” an der Universität Göttingen. Die Veranstaltung fand am 16. Januar 2023 in der Aula am Wilhelmsplatz statt. Es moderierte Prof. Dr. Prof. Dr. Silvio Rizzoli (Universitätsmedizin Göttingen).
Erkrankungen des Herzens und des Gehirns gehören zu den häufigsten Todesursachen weltweit. Manche Erkrankungen betreffen sogar beide Organe. Obgleich Herz und Gehirn auf den ersten Blick sehr unterschiedlich erscheinen, weisen ihre elektrisch erregbaren Hauptzellen viele Gemeinsamkeiten auf. Beide verwenden ähnliche Funktionseinheiten, die der Erregbarkeit der Zellen dienen und über die sie physiologische Leistungen als Teil aktiver Netzwerke erbringen. Fehlfunktionen dieser Nanometer-kleinen Einheiten führen oft zu Erkrankungen.
Ziel des Göttinger Exzellenzclusters „Multiscale Bioimaging: von molekularen Maschinen zu Netzwerken erregbarer Zellen“ (MBExC) ist, diese Funktionseinheiten von Herz- und Nervenzellen zu verstehen, um daraus neue Diagnostik- und Therapieansätze entwickeln und gesellschaftlich relevante Fragen in der Herz- und Hirnforschung beantworten zu können.
Im Rahmen der Ringvorlesung gewähren MBExC-Wissenschaftler*innen am Beispiel spannender Forschungsergebnisse umfassende Einblicke in den einzigartigen Forschungsansatz des Clusters. Sie stellen innovative Technologien vor, die ihren Ursprung oft in Göttinger Pionierarbeiten haben und am MBExC weiterentwickelt werden, und zeigen auf, wie genau man heutzutage in das Gewebe und die Zelle „hineinschauen“ kann. All diese Technologien liefern uns einzigartige Einblicke in unser Herz und unser Gehirn.
Weitere Informationen https://www.uni-goettingen.de/de/680128.html
#unigöttingen #herz #gehirn
Okay good evening everybody and thank you for coming uh for tonight’s lecture my name is syia roli from the University medicine gingan and I’m going to introduce our wonderful speaker for tonight um Professor Ruben Fernandez Busan Diego who um has been hired in 2020 201920 has been hired to our campus
To introduce a technology to this campus which is called cryo electron microscopy and this as he will no doubt show us it’s a it’s an excellent I would say very very important technology highlighted by Noble priz with in 2020 I believe was it right 2020 17 already oh
My God how time flies I’m getting older losing much of my hair um so that’s just how life is now Ruben um originally is from Spain and he was he did his PhD here in in uh in Germany um with a group of ladan lukic one of the first people
In Germany and in the world overall to use cryo electr microscopy to look at the brain to look at copses and then he graduated there and went across the Atlantic to the group of Petro de camili Petro is one of the biggest sinaps and brain people in the world still alive so
To say he’s one of the very very fundamental people um in this field that made a lot of discoveries much of how we understand the brain comes from petro and and Ruben had a couple of really seminal fundamental papers there I mean you your papers on on the synaptosomes
On purified synapses from the brain are still cited like there’s no tomorrow and um you know they they they made an Epoch I mean those those really were seminar works and then um instead of staying in the US Ruben decided to come back to Germany come back to munan um in the
Institute of wolf wolf K bowm there the Pioneer in Germany for the for for this technology which I meant I I mentioned cryoem and there he had a very successful group leader position for a few years and then finally um join our campus to strengthen us to bring this
Technology and really allow us to see things that there’s nothing else out there that can show them to you so he will show us very small objects invisible to most other techniques but coming to life in in his hands so you know I don’t want to explain how this
Works but it’s probably the most exciting technology that we have today so I look forward to the talk thanks a [Applause] lot so um yeah can you hear me yes I think so thanks so much uh Sylvia for this uh very nice introduction thanks uh everyone uh for coming here tonight um
And the lecture is going to be in English if you have questions uh in German I think I can manage uh so um yeah today uh I I want to uh share with you uh how developments in electron microscopy technology are allowing us to see uh fundamental or to make
Fundamental new discoveries into the physiology of uh our bodies and also uh understanding disease mechanisms better um so well Sylvia already went through this but I just want to tell you a little bit more about my story so um I cannot really uh Point very well in this
Geometry but um I was uh raised in in Spain where I studied physics and then I went to all these different Labs uh around the world uh where I was introduced to uh biological research and also um electron microscopy technology and in 2019 I joined the university uh Medical
School here in gingan and um a couple of years ago in 2021 with the occasion of the inauguration of our lab uh we made a little advertising video uh and I want to share uh this with you today because I think it gives you an idea of the
Machines we are using of our technology and the kind of work we do so uh I’m afraid the music might be a little bit on the loud side but let’s see how this Goes The getting gen multiscale bioimaging Excellence cluster aims to decipher the molecular organization of functional units that are relevant to disease in excitable cells such as neurons and cardiomyocytes cry electron microscopy is Central to this Vision as it Bridges the scales between the Nano and the the microw world from molecules to cells and
Even tissues at the University of gutan we have set up a state-of theart cryoem platform which includes equipment for sample preparation cryolite correlative microscopy cry Focus time be milling and a high-end cryo transmission electron microscope using these instruments we can image micro molecules at very high resolution both isolated in their
Purified form but also within their native cellular environment we are grateful to everyone who made this possible and of course we’re excited to start revealing the structural mechanisms that allow excitable cells to work and to investigate the structural causes of their dysfunction in disease all right so that’s us uh and
Maybe you know looking at this video and from the stereotypical view one has of scientist one can think this is mainly how scientists look like you know with this very Sleek uh Hightech world you know working on on very complicated scientific challenges um but so there is
Some truth to that but there is actually a lot of that as well you know just being bombarded with lots of task tasks which you know sometimes have something to do with science and and other times not that much and actually you know when I came here to getting Gan my first task
Was to work on a construction site uh to put together the facility uh that that you have just seen and that was not a trivial task because these microscopes are on one hand very expensive so we had to raise a substantial amount of money to be able to purchase them first and
Then also uh the these microscopes are very sensitive to the environmental conditions so we had to plan the the construction of our uh facility very carefully and uh this was uh we started this process in 2019 and everything was going okay uh but uh in 2020 a year
Later something got in our way and maybe you can guess what that was uh This was um probably you recognize what this is this is a SARS scov 2 virion and I’m sure all of you uh have also stories of how this guy impacted in in your life and how the pandemic
Changed the plans you may have had um so I think this was a a pretty traumatic event for the whole world and and I feel now that the the worst is over um we we’re just trying to move on and and kind of forget what happened but in my
Opinion our duty as scientist and and maybe also as a society is to learn as much as we can from this traumatic event so that we are better prepared for the future uh so the topic today is not Public Health uh so I want to focus on
Lessons that I think are important and that I think we learned or we should have learned from the pandemic and I want to say that the first of these lessons is that fundamental research is important and it it has a direct impact in our daily lives often in ways that we
Cannot anticipate and this was recognized last year by the Nobel Prize in medicine to Cataline Caro and uh Drew Weisman for research they did some uh three decades ago on RNA modification so back then this experience ments were seen as kind of obscure research with unclear impact but it turns out that
This uh RNA modifications that they pioneered Ena the development of uh mRNA vaccines which saved uh millions of lives during the pandemic so I don’t know if you remember how these vaccines uh work how what what is it that they do but uh basically um I mean you see this
Varion it has all these uh spikes these are actually actually called the spike proteins um and what the vaccines do is make our cells produce such spikes in in isolation so the spikes alone do not lead to an infection but they train our immune system to recognize the virus
Once we’re exposed to it um so you may be wondering why uh I’m telling you all of this and may you think you you just walked into the wrong lecture but uh you know just uh bear with me for a second I’m telling you this to to make the next
Point uh I wanted to to convey today that within fundamental research uh I think the kind of work uh we’re doing in structural biology um has also been quite important during the pandemic um so that in what what uh we are doing in in structural biology is to understand
Uh the structure of proteins and how they are arranged and distributed uh within their cellular and and Native environment and um I think uh also you know the the the the research that took place also in the early days of the pandemic has shown the importance of this type of research
So what you see here on the left is an uh magnified view of of one of these spikes uh and if you look at the date at the bottom of the reference you can see this this all this work was done in 2020 so months and even weeks after the
Genomic sequence of the virus was available many Labs worldwide were already determining structures that uh in the end were actually very important to establish the development of vaccines and and all kinds of Therapeutics oops um right so um if if we look at uh uh at
The the structure of this Spike we uh one of the first thing uh one of the first things that scientists found is that it has um an important confirmational change and it can go from this close confirmation that you see on the left to an open confirmation and
Maybe you see that this little domain here is just going a little bit upwards and this little movement in this protein is actually uh of very high importance uh in physiology because it turns this Spike into a key and this key can now fit into the lock of human cells and
This was also uh visualized a few months later this this is the lock this pink part is a A2 receptor of human cells that can be open with the Kei the virus has so a few months later in the pandemic this variants started to emerge that were more infectious than the
Ancestral strains and if we look for example at the Omicron Spike we can understand why these variants were more infectious you can see these these red balls are uh the points where uh mutations have happened and you can see that in the Omicron Spike the mutations are accumulating this little piece here
Which is the receptor binding domain which is exactly that part the spike uses as a key to enter our cells so um we can see the key inside the lock today with actually with atomic detail with atomic precision and we can understand how these mutations make the key fit
Even better in the Lo and therefore we can understand why these variants are more infectious than uh their predecessors and using structural biology uh we not only can see what you know what the virus is doing but we can also understand how we can start to
Fight it so again also in the very early days of the pandemic um the first structures of um the spikes the spike protein uh being bound by uh neutralizing anti bodies uh were determined again with uh a very high level of detail and uh not everything
Was done on the spike but also on other parts of the virus for example the polymerase which allows the replication of the virus and uh several groups and I want to highlight in particular this work here which was done in gingan by the group of Patrick ker also within
Mbxc so several groups were able to determine structures of this polymerase in complex with antiviral substances and these structures inform us of of how these uh drugs work how they uh impair the replication of the virus and perhaps what’s more important they inform us on how we can design
Better drugs that you know uh function more efficiently and with um Lower Side Effects so in in mbxc um you know we we uh uh believe that that uh research and in particular microscopy must have a multiscale dimension so it is very important to understand biological phenomena at Atomic detail as as shown
Here but it’s also very important to see these proteins in their native context to understand their distribution and to understand their behavior um so if we want to zoom out a little bit from the single protein and and and look at their complete Arrangement within a virion
Then uh we need to use uh different techniques and and these uh Technologies allow us to to analyze samples that are more pleomorphic so when we look at the spikes they are all of them they are more or less the same but if we look at
The varans we see that they are very heterogeneous they all of them have a different shape all of them have a different number a different distribution of spikes um but also these kind of samples uh we have been uh or or groups worldwide have been uh able to IM
Image in quite a bit of detail and actually if we move further in the scales we can also see how these viruses um actually infect cells and how they replicate inside them um so um the reason I’m telling you all this is that all these uh images that I
Have been showing you uh so far from the isolated uh Spike proteins to the virion to this uh in ecle cells and the replication of the V of of the viruses inside the cells all this uh data has been recorded using the the same technology Tri electron microscopy
Different uh different forms of Tri electron microscopy and this is um the main topic of the lecture today and uh I believe this is also as Sylvia mentioned a very important technology in this mission of mbxc of Bridging the scales of going from the atomic detail to to
Really the molecular to the molecular details within cells and even within tissues so um as I said this technology is called cryo elron microscopy or electron cryomicroscopy and it allows Imaging of biological samples in close to Native conditions at high resolution and in three dimensions so what I want
To do today is basically just go across the three words that make the the name of this technology so I want to explain uh to you why we are using electrons what does cryo mean and how we do this microscopy so the first topic um I think
It’s it’s relatively simple so why do we use electrons because we want to achieve very high resolution so um if we look at this uh all formula uh from Ab we can see that the resolution here D is directly proportional to the wavelength length of the radiation that we are
Using in in our microscopy if we are using visible light uh we uh the wavelength we are using is of a few hundreds of nanometers um so if we want to increase the resolution one of the things we can do is just to increase uh to to reduce the wavelength um so when
We are working with electrons the voltage uh that we use in our microscopes you can see that the wavelength of the radiation is uh several orders of magnitude lower than uh visible light and therefore uh we can reach much higher resolution there are also of course other advantages of
Electrons and disadvantages um but I think this is uh perhaps the most um important thing to discuss and in the next slide I’m going to show you a movie of how actually different techniques come together uh and this is going to be we’re going to start with the uh
Magnetic resonance imaging of the whole brain and this is a actually the brain of a group member uh Felix boand who also made this movie so um and now we’re going to zoom in progressively and as we zoom in we’re going to see that at some point the pixels do not contain enough
Resolution anymore so that technique was very good to look at the whole organ but if we want to see for example individual neurons we need to go to a different technology and this is what we seeing here uh using live microscopy using live microscopy we can see individual neurons
And we can also go inside neurons and look at and see you know their subcellular organization but again we’re going to face the same the same problem the more we zoom in at some point we’re going to hit this resolution limit of the technology and at some point the pixels are just not
Going to contain enough information so we need to change technology again and now we’re already jumping to cry electron microscopy in this case cry electron tomography where we can see already the organel for example here’s a mitochondrian here’s a nuclear envelope already in a much higher degree of
Detail but maybe we want to zoom in further and want to see we want to study for example these macro molecules those were individual ribosomes and for that we change technique again and now we’re going to look at uh single particle cry electron microscopy where we can resolve
The structures of protein complexes um into too close to Atomic resolution so um I’m going to tell you in the next slides how you know what technology we need to use to to obtain such structures but first maybe uh we take a look at the microscopes themselves and um this is roughly to
Scale like a maybe simple light microscope to compared to one of our um uh current electron microscopes and at first sight uh they look very different but actually uh conceptually they are not that different so both of them uh have a light source uh here uh this one
Is producing photons this one is producing electrons then uh they have lenses and uh that that Focus the radiation into the specimen and if here is the specimen in the electron microscopy column then there are some more lenses and at the very end there is a detector so conceptually they’re
Actually quite similar but of course technologically they are very different um so uh sources of photons and electrons are very different this is is uh this is not a light bulb for example this is what we use to generate electrons in our microscopes a field emission gun the lenses are also very
Different we don’t use uh glass like in Optical microscopes but magnets that can deflect electrons because they are of course charge particles the detectors are of course also different it’s different to detect photons and electrons but in my opinion maybe the the main technological difference is uh this this this property of electrons
That they can they need to propagate in vacuum so we need a very high vacuum within the electron microscope to allow the propagation of electrons because if we would have just the electrons going uh within the air there would be too much scattering and you know we wouldn’t
Have sufficient electrons uh arriving to our sample and to our detector so we need to keep everything inside the microscope at very high vacuum and this includes as well the sample and this poses a technological challenge that I want to discuss now in with with the next part of the name of
This technology so uh we have cry electron microscopy we talked about electrons how that uh we use electrons to go to high resolution now uh I’m going to discuss the cryo part which uh enables to look enables us to look at these cells in close to Native
Conditions um so so for that I want to uh maybe bring to your attention this movie which you may or may not have seen uh this was like a big thing when when I was kind of young in the 90s or so um and and in this movie uh Arnold goes to
Mars uh and somehow he starts to mess around with the wrong people and uh he gets into this mountain and you know things happen and somehow he gets expelled uh from the mountain from this uh uh interior of Mars and he gets exposed to the surface of Mars and in
Mars there is an atmosphere of course it’s not vacuum but let’s think of it as something that is pretty close to vacuum and as you can see Arnold is not very happy on the surface of Mars so um Arnold is not very happy uh in something that is quite similar to vacuum and
That’s doesn’t apply only to Arnold it applies to actually any biological sample and the reason is that um biological samples including humans are mainly made of liquid water and if we put liquid water inside High vacuum water will sublimate and samples will be destroyed so we have this problem that we have
Samples that are mainly made of liquid water but we cannot put liquid water inside an electron microscope so you know how how can we then analyze um such as specimens by electron microscopy so what can we do with the liquid water of our samples and historically the first answer to this
Question was let’s just get rid of the water um so for example uh so this this slide shows that the procedure that is used to or one of the procedures that is most commonly used to get rid of the water of cells uh here we have cells in
Culture and we’re going to apply a chemical fixative and what this fixative does is to build a scaffold in the cell that tries to hold things more or less in place once the cells are fixed we will dehydrate them so we will remove all the water from the cells and substitute this
Water by a plastic resin this plastic is then hardened sliced mechanically using a knife and this results in sections that can be placed on an electron microscopy grid last ly these sections are contrasted using heavy metal stains and in this uh contrasted State they can be loaded into an electron microscope and
Analyze so even though you know we are um introducing quite a bit of distortions into the sample using this this technology this uh this this method of sample preparation has been incredibly useful over the last decades and and has shown us most of the things we know actually about the organization
Of the cells um so so this preparation has been incredibly useful in revealing the alra structure of the cell its General organization but it also has its limitations of course and if we want to look at individual macromolecules we cannot do it this way so this gentleman
Here uh Jax du uh who got the Nobel Prize uh in in chemistry a few years ago for develop techniques of of doing something else with the water of of the samples he used to say um maybe he still says this that if you want to study macromolecules within a cell that you
Have dehydrated where you have removed the water this is something similar to studying the distribution of fish in an aquarium after removing the water so you know what happens to the fish if if you do that so similar things happen to proteins inside a cell when the water is
Removed um they also kind of fall down and stick to whatever is standing around so this is these are meant to be uh proteins that are happily floating in the cell here we have some perhaps some cytoskeleton and when the water is removed these proteins will tend to
Stick to each other or to this kind of scaffolds and actually when we look at the hydrated cells this is what we see but we are not looking at the native organization of of these macromolecules so dehydration leads to to aggregation and this is this is a problematic
Artifact so if we want to understand the native organ molecular organization of cells we need to study them in a fully hydrated state but as we learned uh liquid water is not compatible with high vacuum so we need to make this water solid we need to freeze
It now we Face the next problem if we freeze something in a conventional way let’s say we take ourselves and we put them in the freezer um this is also problematic so if we freeze samples slowly that will lead to the formation of ice crystals and ice crystals will
Have several detremental effects on our samples when so crystals will typically start to form outside of the cell because that’s the more diluted environment and they will start taking up the liquid water um this will have a otic effect on the cells that will try to balance the the uh concentration of
Water in and outside so they will start pumping out water they will start to dehydrate themselves and therefore the concentration of all the solutes that we have inside the cell is going to automatically increase this has a detrimental effects and Toxic effect to the cell and when ice crystals get even
Bigger if we keep the cells in the freezer for longer uh these crystals will start uh exerting actually mechanical damage on the cell that might be um you know just too much to repair um so we need to avoid the formation of ice crystals and
The way to do this is to freeze the samples faster than the crystals can form and if we manage to do this and for that we need to apply cooling rates of around 100,000 degrees per second we will achieve so-called vitrification so we will have a form of ice in the cell
Which is not crystalline but it’s amorphous it’s a glass-like uh form of ice in which the molecular organization uh of the solid is very similar to the molecular organization of the liquid and this uh allows the preservation of samples in very close to Native conditions um and this allows
Structural examination at very high resolution and by doing so we also avoid these procedures that that we were using uh that have been used in In classical electron microscopy where samples are chemically fixed dehydrated and then stained with heavy metals so uh this is uh a very high
Cooling rate and the the goal here is to get basically to liquid nitrogen temperature this is about minus 200° uh however uh um Mr uh duoset found that actually with a pretty simple device it’s possible to achieve these kind of cooling rates on very thin uh
Specimens so um this is the the device uh he uh he came up with what you see here is some these are some tweezers and the tweezers uh are holding an em grid here at the very bottom and and these Twisters are mounted on a piston that will shoot the specimen into this
Reservoir that contains typically Li liquid ethane that has been cooled down by by liquid nitrogen at about as I say minus 200° um if we put our example here for example some viruses um this uh solution of of viruses or other proteins can be vitrified can achieve this state of
Solid amorphous water and immediately this was recognized as a very important finding with uh uh quite quite high impact in the scientific community so uh vitrification is key to all forms of cry electron microscopy uh but there are some people that you know like vitrification too much and take it too
Far um and and you know some people claim that you can actually vitrify yourself uh when when you’re sick and then you know just uh stain one of these tanks for 200 years and at some point technology uh will be good enough to cure your uh incurable disease and bring
You back to life and let you live another 100 years um so you know don’t don’t spend your money on this because um as I said for vitrification we need to achieve this 100,000 uh degrees per second and this we can achieve with a with a simple device on very very thin
Specimens on these suspensions of macromolecules but we cannot do so on on thicker samples so it’s already difficult to vitrify single cells it’s much moreal to vitrify tissues and it’s essentially impossible at least with current technology to vitrify organ and uh much more a whole human body so you
Know uh save your money for for better Technologies so that was the cryo part of of cryo electron microscopy and now um I want to tell you a little bit about the microscopy itself on and how we managed to image these specimens in three dimensions using microscopes that actually can only produce flat
Two-dimensional images and uh so so there are two main types of specimens that we uh study in cryoem one are purified proteins so these proteins can be expressed and and purified and we can have millions of copies of the same Macro Molecule um and to study this kind of specimens we use
The so-called single particle analysis and there are other specimens of which we cannot have two of the same so as we saw before there are not two uh expan that are identical to each other and there of course not two cells that are identical to each other and to study
That kind of specimens we use a different form of cry electron microscopy that is known as cry electron tomography um in single particle analysis um so when we work with purified proteins this is basically uh what we have to do so this schematic is showing the same thing as in the video
We have a solution with our purified proteins we will apply it on an em grid which is held by these Twisters similar to the duoset device now we blot the sample we remove the excess liquid and we shoot it very quickly into this cryogen and by doing so we achiev this
Very thin layer of um that that you can see here in the bottom you we have this very thin layer of amoros siiz and our proteins of Interest are suspended within the layer and in principle because the proteins were freely floating in the in solution they will be in this layer adopting
Random orientations so we will have a slack of ice that you can see here with millions of copies of the same Macro Molecule in different orientations and then we will take pictures of it from here this is these are our electrons that are Imaging this specimen and
Because each of them is in a different orientation we will get different views of this of very similar objects then computationally we can try to figure out where do these views correspond to in a three-dimensional structure and we can reconstruct this this structure um in in
With a lot of detail and to illustrate this with a practical example I want to uh tell you a little bit about the proteome uh and this is the work of uh AR of the group of Aris sakata also at the University Medical Center Gan the
Proteome is the main machine in the cell for uh targeted degradation of protein it’s highly conserved and it basically consists of two modules this uh regulatory particle here is recognizing substrates is recognizing what needs to be degraded so this is kind of this recycling station just sorting out the
Trash and um handing over the trash to the to the next module of the machine there is this atpa that uses ATP energy to unfold proteins and feed the proteins in into the last unit of the machine which is this uh Pro prootic core that is basically chopping the proteins into
Little pieces so that they can then be recycled and Aries work or ar is very interested in understanding how this machine is built because only then we can really understand how it works and only then we can understand how it doesn’t work in certain diseases so uh her group reveal one of
The first uh high resolution structures of the proteome which you can see here already uh some years ago you can see that resolution is local and that the parts that are more solid more stable of the protein can achieve higher resolution than the more flexible Parts but another important uh character
Characteristic of cryoem is that when we freeze these machines they are actually carrying out their work so um they are performing it their native function and like many other machines like a car or anything anything else they need to move to carry out this function uh when we
Freeze them we have snapshots of individual particles in different uh stages of this uh confirmational cycle that they need to undergo to carry out this function and um AR’s work has also revealed the most important stages of this confirmational landscape of the proteome so the proteome has to undergo
These major confirmational changes to recognize the substrates unfold them and then chop them into little pieces so this is basically what single particle analysis does we can uh obtain a very detailed blueprint of these machines we can understand how they move but we do not see how they work in their
Native environment within cells and to do that we resort to cry electron tomography when we have specimens that are unique we don’t have millions of copies of the same cell because there is each cell is unique we need to image them in a different way um and for that
So this is our specimen and what we will do is we take pictures of it from different directions by tilting the specimen within the microscope and then we end up with the series of pictures that we can reconstruct into um into a three-dimensional object and now um
Moving to the topic of that that we are working on in in my laboratory we mainly use cry electron tomography to study uh some of the effects of aging so this was Arnold in the ‘ 90s and this is him few decades later um and of course aging has very
Profound effects on our organisms and now we understand that it is actually the major risk factor for many diseases and this includes neurodegenerative diseases uh which as you know are uh affect mainly uh our last Decades of life and this is a big problem especially in in our societies uh where
Where uh the average age of the population is increasing so it is estimated that nowadays uh around 7 million people in Europe uh suffer from Alzheimer’s or related dementias Alzheimer’s is the most uh prevalent of these diseases and this uh um prevalence of the disease is of course expected to
Increase as the population ages and this is a big drama for the 7 million people and their families and also poses a huge burden on a huge financial burden on our Health Care Systems one important aspect of Alzheimer’s and other neurodegenerative diseases like Parkinson’s and others is protein aggregation so we just saw
Before how proteins need to achieve their native structure that we can study with cryoem and this native structure is necessary so that they can carry out their function in many of these diseases proteins fail to achieve this native structure they misfold and when when they misfold they Aggregate and they
Form this these uh mesoscopic structures that pathologist can observe in the brain of patients that died of these diseases and um the driver of aggregation in each of these diseases is a different protein but somehow they all end up going down the same Cascade and
What I think is amazing is that we know this for more than a 100 years so these are uh original drawings from allo Alzheimer more than 100 years ago so we know that um aggregation is is a main uh character characteristics characteristic of Alzheimer’s and these diseases but we
Still do not understand the role that protein aggregation plays in disease so we do not know to what extent these Aggregates are the causal agent of the disease and we do not understand whether these are really the main therapeutic Target that we should be addressing and
This is one of the questions that we want to investigate in in my laboratory and to do that we want to image these Aggregates within their native cellular environment within the neurons in which they form and within the neurons they may eventually contribute to kill and we want to understand the native structure
Of these Aggregates and maybe more importantly how these Aggregates interact with their cellular environment because we think that if we understand these interactions we will understand better the contribution of this Aggregates to disease and to do so we use cryon tomography and how we do that
Is shown in this movie so here we have a living human cell that contains one of these Aggregates that we mark fluorescently this is a polyglutamine aggregate found for example in Huntington’s disease and others we have a living cell now we’re going to freeze it we’re going to vitrify it at liquid
Nitrogen temperature and from this point on we’re going to keep it at this temperature at all times the first thing we need to do is we need to thin down the cell specifically at the location of this Aggregate and for that we are going
To use a machine like this one this is a cryo Focus I beam scanning electron microscope what you’re seeing here is the loading of the samples in the shuttle that contains two em gits and on these grids we have vitrified cells containing Aggregates we can ignore this
Step it’s kind of a a technical thing but now we’re going to start using the Electron Beam of this microscope to perform correlative microscopy so we want to find back the cell we identified first in the line microscope that contain that green aggregate we want to find it on this Ian grid after
Vitrification and it turns out to be this one which is has been uh Frozen on the on the substrate of the EM grid and we know uh from the live microscopy that our aggregate our aggregate is somewhere there and now we’re going to start using the ion beam to remove material from the
Cell basically everything that’s above and everything that’s below this aggregate is going to be removed by the ion beam and and every the only thing that remains from the cell is this very thin slice which is about 100 nanometers thin and as you can see it remains
Suspended by the parts of the cell that have not been uh touched by the ion beam in this machine we only do this step of sample preparation and then we transfer the samples to the next microscope to the transmission electron microscope where we do the high resolution imaging
And this is shown here um all these transfers between microscopes and everything else all the Imaging is done at liquid nitrogen temperature to get these threedimensional images we take pictures of the specimen from different directions by tilting the stage of the sample and then we can computationally back project these images to reconstruct
The three-dimensional volume of the aggregate here uh I’m going to show you one example of of one of these tomograms uh this is um this polyglutamine aggregate within a human cell and and we’re going to see the the raw data here we’re going up and down in Optical
Slices and now we’re going to see the computational analysis of this data where we can um separate the different elements we can see these uh blue fibrils of the Aggregate and different uh cellular components in other colors today I want to tell you a little bit more about a disease that is called
Amyotrophic Latos sclerosis and a related uh condition known as frontal temporal dementia this is the third most common neurodegenerative disease and affects mainly motor neurons so this is not always uh linked to to dementia to cognitive problems but it’s linked to um impaired muscle function and it’s a really devastating disease because life
Expectancy is only a few years typically after diagnosis although there are cases in which patients manage to survive for much longer but this is really exceptional most most uh ALS patients also suffer protein aggregation in their brains most cases are sporadic and in those cases there is aggregation of a protein called
Tdp43 but uh there are also familial cases and most of those patients suffer a mutation in this Gene called C or cti2 and the main uh driver of aggregation here are proteins called polyga so these are just repeats of glycine alanine residues up to hundreds or thousands of times
So I want to show you some work we’ve been doing with these kind of Aggregates within neurons we can use this workflow I mentioned before to take this kind of tomograms so now again we’re going up and down in this uh in this volume of the tomogram we can see the Aggregate
And now you’ll see the the result of a computational analysis separating the main macromolecules found in this volume so you see the aggregate in red it forms kind of planner twisted ribbons um you see also some other macro molecules like ribosomes here in in yellow and here in
Purple is the trick chaperonin and maybe you could see that those macro molecules are mainly found in the periphery of the aggregate but um what was really dramatic in this case was the accumulation of these green macro molecules within the aggregate volume and directly from their structure we can
Tell that these green macromolecules are proteosomes uh and we can do so because the sample has been Pres oberved in these close to Native conditions where we can recognize mcro molecules directly from their structure so we found that Within These Aggregates there was a concentration of proteomes that was 30 times higher than
In controlled neurons and this was very interesting because proteosome dysfunction has been implicated in many diseases including many neurodegenerative diseases so I showed you before how this uh single particle cryoem work re from from Aries group uh reveal this confirmational dynamics of the proteome and this work was done in
Vitro with purified proteins but now we can try to find all these different confirmations also within the neurons so what we can do with this type of data is uh we have here these individual proteomes and we can extract them from their cellular environment and we can
Align them and average them like we do with purified proteins and then we can also try to separate all these different uh confirmational uh States the result of those such analysis in these Aggregates is is shown here we have these four main classes of of proteins and we could assign their functional or
Confirmational States we could see that these green uh groups of of proteins corresponded to the ground state of the protone where it’s not engaging in substrate degradation this other group of of proteon corresponded to the so-called S2 State and finally this group corresponded to the so-called S4
State and this is what we found most interesting because in vitro AR’s work showed that this S4 state is a very transient confirmation and we can only observe this confirmation if the proteosome is stuck with nucleotide analoges that cannot be hydrolized efficiently however in our samples in in
These Aggregates within the neurons we found that almost a quarter of the particles were adopting this confirmation that is supposed to be very transient suggesting that perhaps the proteome is kind of getting stuck at this particular time of its confirmational cycle and what we can do with the tomograms what I told you
Before is we find individual particles we extract them we figure out their confirmation we know now the confirmational the functional state of each individual particle and now we can put them back we can put them back into their cellular volume and we can see if these different states follow any
Particular patterns one of the things we measured was the distance between each proteome and the closest Aggregate and this is shown here in this graph you see four dotted lines and these are these four uh confirmations that I showed you before and this San line is this S4
Class and if we look at proteomes that are within 15 nanometers from an aggregate like this ones here that are essentially touching the aggregate we can see that about 40% of them are in the particular confirmation and if we move away from the aggregate by only 30
Nanometers so this is less than the length of one individual proteome we can already see that the fraction of particles in this confirmation has been reduced to half indicating that it is really this direct interaction between the proteome and the aggregate which is stabilizing this uh particular confirmation indicating again that the
Proteome is getting stuck because of this interaction with the aggregate which we later proof using uh functional assays that uh I will not describe here um but I think what was important is that also our collaborators could corroborate this finding these findings in patient issue so here you can see um
That a patient that died suffering this mutation if we look at the proteosomes within this brain most of them colocalize actually with disaggregate so this is a phenomenon that is actually happening in the brains of of these patients and this may uh hopefully have some therapeutic relevance as well because preclinical
Experiments have shown that if we try to counteract the effect of these Aggregates if we pharmacologically activate the proteosome we see an improvement in cells or mice that carry these Aggregates so um I mentioned that there are familial and sporadic forms of the disease I told you about the familial
Cases and now very briefly about some uh work we have done on this uh tdp43 aggregation that is characteristic of sporadic uh cases this is one tomogram uh recorded in a neuron that has one of these tdp43 Aggregates um now again we are going to see the mcro
Molecules in red is the aggregate the aggregate itself is very different from polyga now it doesn’t form this ribbons anymore it’s more of an amorphous uh um uh structure but but we see that there is also a a profound accumulation of proteomes that are shown here in blue within the aggregate within
The aggregate volume and again we could show that these proteon were in pair were functionally stuck by this interaction with the aggregate so we could show that both in for Aggregates that are characteristic of familial cases but also for sporadic cases of ALS and frontal temporal dementia we have
Different proteins that are aggregating these Aggregates have a different structure but somehow they mess around with the neuron in a similar way they attract proteomes they recruit them and they get them stuck in there the effect is that this fundamental Machinery of the cell that is taking care of of the
Trash of recycling is not correctly functioning and what we have in the end in in these neurons is an accumulation of trash which of course has many detrimental effects and can contribute to tox toxicity and to the neuro degeneration that we observed in these diseases so um with this um I think I
Would like to finish these are the take-home messages uh that hopefully I could explain to you that vitrification this very fast freezing preserves bi biological samples in close to Native conditions if we are working with repetitive structures such as purified proteins we can image them by single
Particle cryoem at Atomic or close to to Atomic resolution and and I told you a little bit about the proteosome work in that context if we are looking with more complexes we are working with more complex specimens such as cells uh we can image them by cry electron
Tomography um and with this uh so I I Illustrated this with this example of the the protein aggregates related to amyotrophic lateral sclerosis on frontal temporal dementia how they recruit and stall proteomes um so hopefully I can show you how we’re trying to do what we call structural cell biology using these
Techniques so how we can start to understand basic fundamental cellular phenomena and also um how these phenomena um are altered in pathological conditions directly from a structural perspective directly in C2 within cells and um globally i al hope I I could also uh convince you how cry electron microscopy
Is emerging as a powerful tool in biomedical research addressing problems from uh viral infection to neuro degeneration with this I’d like to finish uh thanking my group here’s a picture of uh my group and arys together um most of the work I’ve shown here was
Done by Chang when he was a poog in the lab now he has his own lab in China uh these are uh our funding sources our collaborators to which we are also very thankful and uh of course I’m very thankful to you for your attention thank you very much [Applause]
And uh I’m sure we are all eager to ask questions so yes we have a question that’s okay okay you want to do the running around okay that’s perfect this is this is super fascinating I’m working for hofa Russian Basel and and have some insights into
Some of the diseases in this area I’m just wondering when you see at the sheer number of aggregated proteins in there when you think about proteosomal activation principles right you know at a certain point in this aggregation uh accumulation there is a point of no return right likely where
Even protal activation may not really change C of disease anymore have you got any ideas about you know the quantification and how much of you know this aggregation will then in a certain stage of a disease where still an intervention may be feasible yeah so so
Uh there’s probably a point of no return in terms of aggregation but we don’t know if there is a point of no return in terms of proteosomal um stalling so our idea idea is that activating the proteosome may or may not help aggregation per se but it
Will help help the cell to keep its recycling Machinery active so other substrates Beyond this aggregating protein uh other substrates that need to be recycled will be able to be recycled within the cell um so this is this wouldn’t be a therapy that will cure the disease I think this is not something
That you know anyone is thinking you know for the moment for any of these neurogenerative diseases the hope is that you know one can prolong basic Quality of Life by helping the neurons cope better with this misfolded Aggregates yeah I mean prolongation let’s say yeah other questions I actually have one and this
Is really a sneaky question did you uh get some some help from Alex a chisik on these wonderful movies that you shown or who made them yeah no I mean these movies are coming from different sources um but the one where for example we explain the workflow of tomography this
We did together with thermop Fisher so they were making you know a very nice promo movie for their microscopes and we just sneaked in our Aggregates in there um and yeah could collaborate with them wonderful nicely no because we actually have um specialist in and that did some
Movies for some other some other people very very similar to your to your ther ficial movie you know astoundingly similar so that’s why I asked yeah okay thanks a lot okay other points yes um I wanted to ask you about if you have studied different stages um of
Those Aggregates in the cells and if so um I’m curious about where do you get those samples from are they human samples so um different stages um we have not directly addressed because it’s not easy to temporarily control the aggregation of proteins uh we have looked kind of at early stages of
Aggregation and late stages of aggregation and we have not seen um qualitative differences so of course Aggregates get bigger the bigger they are the more for example proteomes they sequester but we can see that phenomenon already with the smaller ones um the samples you have we I have shown today
Um are not human so these are uh culture primary neurons uh that are expressing aggregating proteins but actually now one of the main drivers of our activity is bringing this to human systems that might be more direct directly relevant to you know what might be happening in
The brains of patients and for example we’re doing a lot of this work now with alphascan Aggregates which are found in Parkinson’s disease with and and you know in collaboration with our colleagues in the clinic we can obtain these Aggregates directly from patients either from patients that passed away
Already with the disease from their brains but also from leaving patients because we can obtain Aggregates actually from the cerebros spinal fluid of leaving patients and the hope there is that by um examining um Aggregates of patients at different stages of disease maybe even within one patient during disease progression maybe
We can also understand in with our system how this disease is progressing and how the Aggregates are maybe interacting with this with the cellar environment in different ways so this is definitely you know something that we are pushing uh for for the present and future work yeah thank you thank you
I mean that sounds great but in CSF you have a lot of different things I mean so how do you recognize that those are ccle particles I mean what we are using the CSF for is to seed aggregation in neurons seed aggregation so so we look at intracellular Aggregates that have
Been triggered by exogenous Aggregates obtained from this this great technique I mean there’s a couple of diagnosis for Parkinson that have been made with this um um last year FDA approved in the US so yeah very cool very cool um have there been studies on mice or other
Animal models where you can do use gene therapy or something to introduce the uh to rescue kind of the a good copy of uh functional protome right so so I mean this for example this pharmacological activation of the protome uh this has been tried by our collaborator in in in Munich using
Mouse mod models that you know uh Express this aggregating protein and just by feeding the mice a drug that is known to activate the proteome the mice basically get better so so you know these are kind of first steps into uh therapeutic workflows but this
Is kind of ongoing yeah but not not yet in gene therapy or something that and creates a no I mean uh I think gene therapy is probably something people I mean one would like to avoid if there are simpler Alternatives uh so if one can use a pharmacological approach this
I think is often the first thing to try um yeah so this is I I wouldn’t say this is a direct candidate for gene therapy or I wouldn’t know now you know how to use it um but yeah definitely in other for in other diseases this might be a very important approach
Yeah okay then I think some of our audience members indicate that it’s time to wrap up so yeah thank you very much again Ruben so thank you very much give him a hand and yes last word speaker um we are a little bit late um next medic
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