Interview: Alzheimer's in a petri dish

How would you introduce your research and what led you to it?
DB: In the lab, we study and model diseases using stem cells and their differentiation into neural cell types. I started doing this at Masaryk University Medical School after I returned from the United States around the time when the world was beginning to create so-called cerebral or brain organoids. We started to do it too and started to produce our "mini-brains" in a test tube. Following this, we were approached by Professor (Jiri) Damborsky's group from the Loschmidt Laboratories, saying that it would be useful for them if we started to model Alzheimer's disease using brain organoids. So it was also a bit of a coincidence that we started working on this disease.

So what exactly were you doing in America and how did you follow it up in the Czech Republic?
DB: During my PhD studies at Masaryk University, I was studying the mechanisms behind a stem cell becoming a neuron. During a postdoctoral fellowship in America, we conducted preclinical research in which we wanted to create neural stem cells that could be used in the clinic, for example, for patients with spinal cord injury or amyotrophic lateral sclerosis. In the Czech Republic, when I returned, I wanted to focus on in vitro disease modelling, which is a big part of stem cell research.

What do we know about Alzheimer's disease, what don't we know, and what about the disease are you most interested in?
DB: What is known are the pathological features of the disease, which are clusters of several proteins that patients deposit in their brain tissue. Why they are deposited there, however, is unknown. It may be a consequence of fat metabolism, cellular aging, and partly an immune response. Or pathogens, infections with certain viruses...

...or a combination of all of these?
DB: Or a combination of everything... Plus, Alzheimer's is not one disease. Some patients have a genetic predisposition for it. The so-called familial form can be seen in people in their 40s. But most people get the so-called sporadic form, which only manifests itself later, after 65-70 years of age.

Both the familial form and the predominant sporadic form, however, manifest themselves gradually without one noticing. Is the aim of today's research, then, to trace the disease to the earliest possible germs?
DB: Yes, at least that is the goal of our research. For years, therapy has focused on reducing protein clumps in the brain, but all clinical trials to date have failed in this regard. Moreover, it has been found that even if these clumps can be removed, it doesn't help patients. The possibility is that such help comes too late, because if Alzheimer's disease begins to form twenty years before a person experiences the first memory impairment, then intervention should have been taken twenty years ago. Another possibility is that the protein clumps themselves are not the cause of the disease, but just the result of something else. It's like having a bruise on your thigh that you're trying to nurse instead of moving away from the table that's pushing into your leg while you sit...

You're just basing your research on the assumption that these protein clumps are not the cause of Alzheimer's?
DB: We approached the research with the assumption that we don't know what is cause and what is effect. That's why we created a model - brain organoids - that we use in the lab to simulate the onset and development of Alzheimer's disease. We also assumed that in order to observe signs of the disease in the organoids, we would have to let them age. But it turns out that some of the signs are already present early in the development of the organoids, leading us to believe that the cause of the disease lies elsewhere.

What research potential does the possibility of creating a virtually miniature brain in a dish open up?
TV: Huge. First of all, we can work with human cells, even directly with the cells of Alzheimer's patients, because working with mice is not the most appropriate in this case. The very organoids created from these human cells will allow us to follow the very beginnings and development of the disease. Another advantage is that we can differentiate the organoids spontaneously. In principle, they are self-assembling, which gives us a multitude of cell types in which the disease is free to manifest itself without our interfering in the process. It is thanks to this method that we have been able to describe that the organoids formed from the cells of Alzheimer's patients develop differently.
DB: It is important to mention that we are working with a familial form of Alzheimer's disease, that is, cells from people who have a genetic predisposition to get it. They have a mutation in a gene that is known to affect the disease. In them, we focused on detecting protein clusters and several other features that characterise Alzheimer's disease. At the same time, we tried "torturing" our "mini-mammals" with potential therapeutics and seeing how they responded.

What have you traced?
DB: Differences in how organoids develop. The ones from healthy controls develop, in simple terms, as they should. That is, they have the basis of neurons, the eye, the basis of immune cells and other structures. However, organoids from cells from Alzheimer's patients do not develop in a standard way, which we believe is due to the fact that they do not organise themselves correctly at the beginning of the process and do not establish the developmental environment, a certain reservoir of cells from which these structures are formed. This leads to the formation of a large number of neurons that age rapidly and then there is nothing to rebuild the new neurons from, which causes defects in the tissues and probably an increase in the amount of amyloid beta protein.

The early signs of Alzheimer's disease can be detected by looking into the eye, from the retina, where the protein clumps can be seen. What does this have to do with your finding that organoids from the cells of Alzheimer's patients do not, for example, give rise to ocular structures?
DB: We plan to find out. My colleague Dr. Tomáš Bárta is an expert in the development of so-called retinal organoids and we want to start collaborating with him in this direction. We would like to combine the brain organoid with the organoid of the eye and create a so-called assembloid on which we could study the development of the optic nerve, which is really speculated to be a diagnostic tool for Alzheimer's disease. But the fact that our organoids do not form eyes is probably due to the fact that they skip the developmental stage in which cells are supposed to "line up" into the structures needed to form an eye, and do not form the aforementioned reservoir from which new cells can arise. Instead, the organoids prefer to form adult neurons. This is consistent with recent studies confirming that new neurons are virtually not formed in the hippocampus of Alzheimer's patients.

I'm going to ask this in layman's terms, but wasn't it clear that the stem cells of Alzheimer's patients would develop defectively when they are sick?
DB: It wasn't. It's really not clear why neurons in Alzheimer's patients start to degenerate. One theory is that microglia, which are the immune cells of the brain, may start to damage neurons for some reason and be the trigger for their degeneration. However, we show in our models that the beginnings of Alzheimer's disease pathology form even without microglia.

Listening to you, I got the impression that your research has challenged some of the existing findings and at the same time opened more questions than it has answered...
DB: At the very least, it suggests that the familial form of Alzheimer's disease is very likely related to the development of new neurons. But in the sporadic form of the disease, which is not genetically caused, the cause will be somewhere else. We're still planning to investigate that.

It has already been mentioned that you have tried therapeutics on the minimozyce you grew. What did you find? Could such tests contribute to drug development in the future?
TV: I think so. At least in the form of some basic testing that experimental substances work as they should. If we are able to create the symptoms of Alzheimer's disease in the form of protein clumps and changes in neurons, if we see that we can also correct them, that's useful. We've even done a study with colleagues in the Loschmidt labs to test the drug tramiprosate and see how it affects organoids. The changes we observed were related specifically to fat storage and cholesterol stores.

But then again, we're talking about a bruise that's already formed...
TV: From what we observe on our organoids, we think that the cause of the disease is really very much at the beginning. However, if in the future we come up with some candidate molecules that might work, we can test them on our organoids and look at the very beginning, which logically is always missed in Alzheimer's patients at the moment.
DB: But who knows where the therapy will eventually go. Because if we could reduce the amount of protein clumps early enough, we could extend patients' disease-free lives by ten to twenty years, and maybe that would be enough. So maybe we wouldn't have to deal with the root cause. Organoids could be used for that, too.
TV: Today, thanks to the work with organoids, we see that often what we would expect based on the available knowledge does not happen and we have to look in another direction...

Which brings me to a more philosophical question: as a scientist, do you work with the idea of an organism that functions like a machine, in which every cog has its meaning, and the slightest misalignment of a detail causes a defect in the whole machine, or do you allow for the possibility that some things just happen randomly in this system?
(both smile)
TV: I don't think anything just happens...
DB: The first idea is more like ours. We want to look all the way back to where it all starts, and we need to keep our eyes open in the sense that if there are any theories about where and how Alzheimer's disease arises, our model shows that this may not be the case. And then we need to look at another piece of the puzzle.

What impact do you think your study will have on the scientific community and the direction Alzheimer's research is going?
DB: At the very least, it will make a contribution that at least the familial form of Alzheimer's really starts sometime in development and that it is more related to that than to immunity or aging, which play a secondary role. In terms of drug discovery, the model we have described may again be of benefit, which can be used for further testing, perhaps by culturing the organoids from the start with some drugs.

What do you want to focus on next in your research?
DB: Definitely on early development, on what's going on and changing in it, so that we can tell what happens when, for example, there's a change in cell signalling or when some cells misfold so that they don't build up their reservoir. This is more of a theoretical research. But we would also like to set up some models or platforms to test substances, not only with organoids, but maybe with astrocytes that we are trying to create.

You're basing your research on sequencing individual cells, which has never been done before in organoids modeling Alzheimer's disease...
DB: That's right. And even this data analysis of ours beautifully demonstrates that the error is somewhere in the development. It shows the individual cells of the organoid, where we can see that the organoid from healthy cells has everything it should have - progenitors, neurons, the base of the eye and other structures... The one from the Alzheimer's patients has a different development, and its neurons are older because they're probably not renewing themselves because of the lack of storage. That's what our analysis suggested.
TV: I would also add that we grew a really large number of organoids for this study and all our conclusions are supported by the results from all the lines that were generated. We're not trying to make some wow conclusions, but ones that will be supported by the data. And that's what we describe in the publication in Cell Reports.

 

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