Neuro-degenerative diseases

On-demand webinar

webinar-image

Summary:

The progressive, age related degeneration of the structure and function of proteins and cells in the nervous system can lead to issues involving memory and motor function loss. Join us to gain insights into recent advances in this field of neuroscience.

Neurodegenerative

Speakers:

Dr. Caghan Kizil, DZNE, Germany
Zebrafish as a model for Alzheimer's disease

Moderator

Dr. Alfonso Martin-Pena, University of Florida, United States

Video Transcript

00:00 - 00:15: Hi, my name is Sian Constantine and I am the Strategic Marketing Manager for Neuroscience
00:15 - 00:21: at Abcam. Welcome to the fifth session of Spotlight on Neuroscience. We have two wonderful
00:21 - 00:26: talks for you today, followed by a panel discussion led by Dr. Alfonso Martín Piña.
00:27 - 00:32: Abcam is approved as a provider of continuing education programs in clinical laboratory
00:32 - 00:38: sciences by the ASCLS PACE program, and PACE credits are available for this event.
00:38 - 00:42: At the end of this session, a link to request credit will be sent in the chat box.
00:43 - 00:49: Attendees are automatically muted. Please submit any questions in the Q&A box at the bottom of
00:49 - 00:54: your screen. These will be addressed during the dedicated Q&A session after each talk.
00:55 - 00:59: If we don’t get to your question, please don’t worry, we will get a response to you after the
00:59 - 01:08: event. So, now I’d like to talk to you a bit about why Abcam is hosting this session on
01:08 - 01:17: neurodegenerative diseases and what our interest is. So, Abcam is focused on providing research
01:17 - 01:23: tools in neuroscience, and our current strategic pillars sit around neurodevelopment, neurobiological
01:23 - 01:31: processes and neurological diseases. So, we are aiming to provide research tools that enable you
01:31 - 01:34: to advance the scientific needle in your laboratories with your research.
01:35 - 01:41: So, if you are working in any of these spaces, obviously in particular the neurodegenerative
01:41 - 01:45: neurological disease space, we’re very interested to hear from you about
01:46 - 01:51: what research tools would be helpful for your research and how we can improve our
01:52 - 01:56: catalogs to support you better to achieve your research goals faster.
01:57 - 02:04: So, if you have any insights or any questions for us, please do use the neuroscience@abcam.com email.
02:06 - 02:14: So, within neuroscience, we have multiple ranges of product types that we provide for research.
02:15 - 02:21: We’re very well known for the antibody area, but a lot less known for ELISA kits,
02:22 - 02:26: proteins and peptides, although it’s probably not a surprise that we do offer those as an
02:26 - 02:34: antibody company. What’s maybe a little bit less known is that we also provide multiplex
02:35 - 02:41: microRNA and immunoassays, cellular biochemical assays, and then also cell lines and lysates.
02:42 - 02:47: If there is anything we don’t provide at the moment within these areas,
02:47 - 02:52: so perhaps a particular target of interest for your research, and it’s not particularly something
02:52 - 02:57: you’d like to wait for us to bring into the catalog, there is also the customization services
02:57 - 03:03: available. There’s a lot of examples, in particular, an example I can call to mind
03:03 - 03:09: that’s on our website is the collaboration we did with the MJ Fox, I’m sorry, the Michael J.
03:09 - 03:16: Fox Foundation, the MJFF, where we worked with them to create research tools for Parkinson’s
03:16 - 03:23: that were much needed in space. So, we have a track record of delivering on this, and so,
03:23 - 03:30: please do reach out to us if there is any area you think that we should be developing in.
03:32 - 03:38: So, within neurodegenerative diseases, we have a lot of different products to
03:38 - 03:44: support Alzheimer’s, and this slide just gives you a flavor of the different products we have
03:44 - 03:49: specifically for Alzheimer’s across all of those product categories.
03:51 - 03:57: What I’d like to draw your attention to specifically is some of the new tau pathology
03:58 - 04:05: samples that we have coming out. So, we’ve had tau as a target in our portfolio for a while,
04:05 - 04:12: including several phospho tau’s, but in November, available for purchase on our website will be
04:12 - 04:24: the phospho tau 217, both as BSA-AZ-free and as a normal antibody product. And what’s particularly
04:25 - 04:29: advantageous about these is they’re rabbit monoclonals, they’re not polyclonal antibodies.
04:30 - 04:35: And so, we’re really excited about having those on the market to enable your research.
04:36 - 04:39: Into this hot biomarker for Alzheimer’s.
04:42 - 04:47: So, we’ve talked a lot about Alzheimer’s and Parkinson’s through the Michael J. Fox Foundation.
04:48 - 04:55: We do support, within neurodegenerative diseases, multiple other diseases. We’re not just focused
04:55 - 05:01: on Alzheimer’s and Parkinson’s. And we, you know, we’re not opposed to doing work in the rare
05:01 - 05:07: disease space either. So, here, you can see three examples of rare diseases that are
05:07 - 05:15: neurodegenerative that we have a clear portfolio of products to support research in. So, do please
05:15 - 05:20: get in touch if there’s any areas that you think we should be investing in to support you with.
05:23 - 05:28: So, without further ado, I’d now like to introduce the moderator for today’s event,
05:28 - 05:35: Dr. Alfonso Martín Piña. Alfonso is a neurogeneticist interested in the synaptic
05:35 - 05:41: mechanisms of cognition. To pursue this long-term goal, his work focuses on two main areas.
05:41 - 05:46: The molecular and cellular processes that regulate the synaptic physiology that support adaptive
05:46 - 05:52: behaviors in healthy individuals. And the erosion of these processes in neurodegenerative disorders
05:52 - 05:56: that lead to synapse loss, movement deficiencies, and memory impairments.
05:57 - 06:02: His research focuses on the pathophysiological mechanisms of Alzheimer’s disease
06:02 - 06:07: and related dementias, including frontotemporal lobe degeneration,
06:08 - 06:15: amyotrophic lateral sclerosis, and Parkinson’s disease. Thank you. I’ll now hand the mic to Alfonso.
06:18 - 06:25: Thank you. Hello, everyone, and welcome to today’s session on neurodegenerative diseases.
06:26 - 06:32: We have today two fantastic speakers. So, before we begin, I want to thank the organizers for
06:32 - 06:39: putting this together and our speakers for sharing their work with us. I don’t want to delay
06:39 - 06:48: anymore, and I’m going to introduce our speakers today. First, we have Dr. Kagan Kizil. He’s an
06:48 - 06:52: associate professor of neuroscience in the German Center for Neurodegenerative Diseases
06:53 - 06:59: within the Helmholtz Association of German Research Centers in Germany. And he’s also a
06:59 - 07:05: visiting professor at the Taube Institute for Research on Alzheimer’s Disease and the aging
07:05 - 07:12: brain at Columbia University in the city of New York. He’s also the founder CEO of NeuronD. I
07:12 - 07:18: believe this was the first spin-off from the German Center for Neurodegenerative Diseases.
07:18 - 07:24: And his research focuses on stem cells and their therapeutic use in Alzheimer’s disease,
07:24 - 07:30: animal models of disease, notably the thera-fish models for Alzheimer’s, 3D culture model of human
07:30 - 07:35: neural stem cell plasticity, industry-scale high-throughput screening approaches,
07:35 - 07:40: and single-cell transcriptomics. So, please, Kagan, take the floor.
07:41 - 07:46: First of all, thank you very much for the nice introduction, and I’d like to also thank
07:47 - 07:53: Abcam and all the organizers for putting together this meeting and inviting me.
07:55 - 07:58: You can see my screen properly, right? So, everything is fine?
07:58 - 07:59: Yes, we can.
07:59 - 08:06: Okay, and that sounds great. So, actually, I chose today’s title as
08:06 - 08:13: thera-fish as a model for Alzheimer’s disease. Some people may think, why do we need another
08:13 - 08:20: model in Alzheimer’s disease while the models we already have, the animal models, are not so
08:20 - 08:25: sufficient to understand the disease? This is one thinking, but the other thinking is, well,
08:25 - 08:31: we couldn’t understand the disease so far. That’s why we need new models. I clearly belong to the
08:32 - 08:38: second one, and today I’m going to talk about why thera-fish may help us
08:38 - 08:46: to investigate certain aspects of Alzheimer’s disease and maybe tell us a few things that we
08:46 - 08:57: may learn. Actually, this is a classical slide I start with. This is from more than a century ago,
08:57 - 09:06: 1907, one of the drawings from Alois Alzheimer’s paper on where he saw two major pathological
09:06 - 09:14: hallmarks of Alzheimer’s, the plaques, formation of the amyloid plaques, and the
09:14 - 09:21: tangles that form these neuronal malformations. At that time, we didn’t know what they were,
09:21 - 09:27: but now we know that these are the two pathological hallmarks. But still, after one
09:27 - 09:34: century, we still don’t have an efficient drug for this disease, and we still understand.
09:35 - 09:42: In this hundred years, the histology, molecular biology, and genetics have helped us to look at the
09:42 - 09:49: different aspects of this disease. For instance, the functional imaging led us to investigate the
09:49 - 09:55: brains at earlier stages to understand how the changes start. Different biomarkers we identified,
09:55 - 10:01: and those biomarkers may define different stages of the disease. Genetic association studies
10:02 - 10:08: found the genetic basis of the mutations or changes in particular genes that increase
10:08 - 10:13: the likelihood of getting Alzheimer’s, and we know many of them, APOE, TREM2,
10:14 - 10:22: SOX1, and we now know a little bit more about the environment and its effect, the lifestyle,
10:22 - 10:28: and its effect on Alzheimer’s disease. And the drug development pipelines and the failures there
10:28 - 10:34: also told us how we should amend our way of looking into Alzheimer’s disease.
10:35 - 10:40: So, what all these things told us is Alzheimer’s is not only one disease, it’s a combination
10:40 - 10:46: of maybe many malfunctions in different cell types, and there are particularly a lot of important
10:46 - 10:53: cell types partaking in this disease pathology. Classically, Alzheimer’s was
10:55 - 10:59: characterized as a neuronal disease, a neurodegenerative disease, but there was a
10:59 - 11:04: neuroscientific view where we wanted to rescue the death of neurons by focusing only on the neurons,
11:04 - 11:11: which didn’t work. So, we now know that in the central nervous system, just as abundant as the
11:11 - 11:18: neurons, and sometimes more, are the glial cells, and these glial cells come in different flavors,
11:19 - 11:24: the astrocytes, microglia, and the oligodendrocytes. The microglia represent the immune cells in the
11:24 - 11:32: brain, and we know many genetic associations like TREM2, APOE, are affecting these cells so that
11:32 - 11:37: they contribute to the disease pathology. While the oligodendrocytes are myelinating proteins, they
11:37 - 11:45: regulate a lot of a plethora of functions in the brain’s disease progression, therefore their role
11:45 - 11:54: is important. But today I’d like to focus more on the astrocytes, which are a glial cell type,
11:54 - 11:59: which have different functions. They regulate the ionic environment in the brain, they regulate the
11:59 - 12:06: synaptic connections, they have immunomodulatory functions, but also they are the cells that
12:06 - 12:11: generate new neurons in our brains. Our neural stem cells, or progenitors, are of glial character,
12:11 - 12:18: they’re of astroglial character. So, neurogenesis has been an overlooked aspect in Alzheimer’s,
12:18 - 12:26: and because our brain is really poor in forming new neurons. However, there is also a big
12:26 - 12:30: discussion whether the human brain has neurogenesis, but there’s strong evidence that we have
12:30 - 12:36: neurogenesis in our brains, although localized to some certain smaller regions compared to some
12:36 - 12:45: other vertebrates. And recently, especially Lawrence Martin’s lab found striking evidence that
12:46 - 12:54: as Alzheimer’s disease patients develop in their disease, they reduce their proliferation
12:54 - 12:58: and neurogenic capacity in their brains; there’s less neurons produced.
12:58 - 13:07: So, this also brought a question whether the reduction in neurogenesis, or producing new
13:07 - 13:12: neurons, the reduction in producing new neurons in our brain, could be one of the factors that
13:12 - 13:18: leads to the disease, or can we target this aspect and maybe we can revert the disease?
13:19 - 13:22: For the different reviews published, and we also published one,
13:23 - 13:27: but we approached this from a zebrafish perspective. I’m going to tell you why.
13:29 - 13:35: Here, this is a very simple sketch, but I think it’s very powerful, and I generally start
13:36 - 13:44: my talks with that. So, zebrafish is a teleost, it’s one of the lower, or more earlier vertebrates.
13:44 - 13:51: It’s a necrotic vertebrate, and it has tremendous tissue regenerative ability and neurogenic
13:51 - 13:57: ability. Basically, whatever tissue you can think of in a vertebrate, it regenerates if it has it.
13:58 - 14:04: And also, the amphibians, salamanders, axolotls, they’re highly regenerative. But as we go higher
14:04 - 14:10: up in the phylogeny, as we come to the mammals, our regenerative ability declines in all tissues,
14:10 - 14:15: but especially in the central nervous system, quite dramatically. So the main question is,
14:16 - 14:21: we lose neurons in Alzheimer’s disease, and can we reform these neurons? Can we
14:21 - 14:26: restore the functional connectivity, and can we restore the resilience of the brain?
14:27 - 14:32: By understanding the molecular programs, how zebrafish can regenerate its central nervous
14:32 - 14:38: system, and maybe harnessing this information for humans for potential therapies. The basic idea
14:39 - 14:46: in neurodegenerative conditions is the pathology is generally forming by proteopathies;
14:46 - 14:51: different protein aggregates are forming in the cell, and they are not cleared well,
14:51 - 14:58: and they lead to cell death. But in Alzheimer’s disease, there’s also reduced neurogenic ability.
14:58 - 15:03: So normally, what you would expect from a regenerative system is that the stem cells,
15:04 - 15:09: our neurogenic progenitors would increase their activity and start making more neurons,
15:09 - 15:15: maybe those neurons on demand, replacing the lost ones. This is an ideal condition,
15:15 - 15:19: but it doesn’t happen in our brain. Rather, we reduce our neurogenic ability,
15:20 - 15:26: and at the same time, we increase our proliferation of the glial cells, the astroglia,
15:27 - 15:32: and they form scar-like tissues at the lesion sites where, for instance, amyloid plaques are
15:32 - 15:39: forming. So this cycle goes on, and it exacerbates as the disease progresses.
15:39 - 15:44: So our two questions are, can we induce the neurostem cell plasticity in human brains?
15:44 - 15:52: But this is not enough. Can we also coax these proliferating glial cells or progenitors into a
15:52 - 16:01: functional neurogenic outcome, which asks the question whether Alzheimer’s can be tackled by
16:01 - 16:07: enhancing the neurogenic activity? Zebrafish is an excellent model for that because it regenerates
16:07 - 16:11: everything. But this is a schematic view of the fish brain, and many different labs,
16:11 - 16:16: including our lab, contributed to the understanding of the adult fish brain and
16:16 - 16:22: the neurogenic response there. This is an adult brain. In red, you see the neuroprogenitor stem
16:22 - 16:27: cell regions, which are distributed all along the so-called lexus. In general, zebrafish brain is
16:27 - 16:33: more neurogenic spatially compared to the other vertebrate brains. And in blue, you see
16:34 - 16:40: the neurogenic niches where the produced neurons migrate and integrate at these regions.
16:41 - 16:45: So we and others have contributed to the understanding of the cell types.
16:46 - 16:50: We are particularly interested in the telencephalon, which is the forebrain
16:50 - 16:59: autolog or analog of the human brain. And there, this is a zebrafish brain, a cross-section of
16:59 - 17:05: the telencephalon. The ventricle is, it develops a little bit differently; that’s why the ventricle
17:05 - 17:12: is all over. And the ventricle is delineated by cells, like green here, which are the astroglial
17:12 - 17:17: cells. In this case, they are a progenitor form of radial glial cells. These cells are the ones
17:17 - 17:23: that we are interested in; they do the job for neurogenesis during development and also after
17:23 - 17:27: any lesion or in Alzheimer’s disease. So we want to understand the molecular programs there,
17:27 - 17:32: and then try to apply them to human systems to increase neurogenic ability.
17:33 - 17:39: So this is basically what you can think of, and there are probably many more steps from the
17:39 - 17:45: initial activation of the neural stem cells to functional neurogenic input into the circuitry.
17:46 - 17:50: So you have to proliferate your progenitors, so you have to activate them, proliferate them,
17:50 - 17:55: they go into the neuroblast stage, they differentiate into, they specify, they differentiate into
17:55 - 18:01: different subtypes. These subtypes, neuronal types, should mature on their way to their targets,
18:01 - 18:07: and they should also survive in this condition. They should integrate and functionally restore
18:07 - 18:16: the tissue behavior. Zebrafish can do this all, but humans have many different roadblocks
18:16 - 18:22: at this step. So we want to understand from fish how molecularly we can engineer our neural stem
18:22 - 18:28: cells and neurons to do all these steps functionally. For Alzheimer’s disease, we
18:28 - 18:34: wanted to generate a model where we could particularly recapitulate some pathological
18:34 - 18:42: aspects of AD in the zebrafish brain, and then simply go ahead and see whether zebrafish can
18:42 - 18:47: regenerate functionally and nicely and generate more neurons. This is one of the first cross
18:47 - 18:52: sections of the fish brain where we are using a collaboration with Yixin Zhang from Teodresen.
18:52 - 18:59: The human amyloid beta-42 monomers, we modified a little bit so that it’s endocytosed and it
18:59 - 19:05: penetrates the tissue. This is the ventricle. We inject here, and it’s taken up by the whole brain,
19:05 - 19:10: and these small green dots are the amyloid aggregates after a few days. And when you
19:10 - 19:15: look with the electron micrograph, these are proper beta-sheet structures that are
19:16 - 19:20: localized into early endosomes, which represent the earlier phases of the
19:20 - 19:27: intercellular amyloid toxicity. But we also later found that amyloid beta-42 kills the neurons.
19:27 - 19:37: It exerts a specific toxicity. And here, alplatin is a marker for immune cells,
19:37 - 19:42: and these immune cells get activated. You see these marking the neurons. And also looking at
19:42 - 19:49: the synapses, we see particularly in specific regions reduction in the neuronal connectivity.
19:49 - 19:56: This also leads to behavioral outputs that the fish that is injected with amyloid beta-42
19:57 - 20:03: fails in the learning and behavior tests when compared to the controls. But the striking part,
20:04 - 20:13: thinking that these phenotypes maybe are reminiscent of the human symptoms, that was
20:13 - 20:18: good for us in the beginning. But the real catch point was when we looked at the neural stem cells
20:18 - 20:25: and progenitors, which are marked in red here, and the green is a PCNA staining for a proliferating
20:25 - 20:30: marker. They increase their proliferation, although under a high amyloid load. And this
20:30 - 20:35: leads to extended neurogenesis. There’s increased neurogenesis. And these neurons
20:35 - 20:42: do survive and integrate into the circuitry. We expanded or improved our model. Now we have more
20:42 - 20:48: advanced forms of amyloid degradation, more to the fibrils and later forms. And still,
20:48 - 20:54: the idea holds true. Neurons die, but zebrafish tries to replace them by increasing the
20:54 - 21:00: neurogenic output. And we also found out several molecular programs that control it. Here, this is
21:00 - 21:06: a transgenic line that marks the astroglial cells. And in the pinkish, you see the proliferating
21:06 - 21:13: cells. Amyloid beta-42 increases proliferation. And we also found that IL4, a new neuroglial
21:13 - 21:21: crosstalk mechanism is key and is probably sitting at the top hierarchy of the regulation.
21:21 - 21:27: And IL4 itself is also positively regulating. We also found different neurotransmitter
21:28 - 21:32: control mechanisms, which positively or negatively regulate, for instance,
21:32 - 21:38: here the serotonergic signal is negatively regulating the proliferation. So there’s a
21:38 - 21:44: tight control of the neurogenic activity and the molecular programs there. And to get at that,
21:44 - 21:49: we first started with normal RNA sequencing. But when the single-cell sequencing approach
21:49 - 21:57: kicked in, we also adapted this to the zebrafish brain in different models. In our Alzheimer’s
21:58 - 22:03: fish model, we did single-cell transcriptomics to look at different cell types and understand
22:03 - 22:08: the heterogeneity in the nerve stem cells, their molecular programs, and their probably
22:08 - 22:15: differential response to different forms of amyloid. But also, we wanted to see how cell
22:15 - 22:21: types interact with each other to generate this regulatory condition. And we used this
22:21 - 22:28: system for different collaborations. How we started was we did single-cell sequencing.
22:28 - 22:35: We clustered the cells into different gross categories. But then we wanted to see the
22:35 - 22:41: ligands and receptors expressed in every cell. And these are the list of all the receptors and
22:41 - 22:48: ligands we could reliably find in the databases. And now we matched these points to each other
22:48 - 22:55: and then came up with an algorithm that led us to a complex picture which tells us that
22:55 - 22:59: different cell types may interact with the other cell types through ligand-receptor
22:59 - 23:04: interactions. And these interactions may change during the course of the disease.
23:06 - 23:12: When we functionally analyzed this and also compared it to the literature, we really found
23:12 - 23:19: that the predicted in silico interactions may match well with the known regulations. For
23:19 - 23:23: instance, a certain cell type through FGF signaling is regulated against another cell type,
23:23 - 23:29: which was experimentally verified but also present in our system. We did with this analysis
23:29 - 23:36: also, we’re just finding many different analyses, mostly the transcription factor codes or the
23:36 - 23:41: lineage progression of different cells, as well as the molecular programs which are
23:42 - 23:50: present in different cell types. And we found, as a case study, we found one significant regulator
23:50 - 23:55: of neurogenesis in zebrafish brain and Alzheimer’s disease, the BDNF-NGF signaling.
23:56 - 24:01: What we did, we knew that serotonin was reducing the proliferation of neural stem cells,
24:01 - 24:06: but we didn’t know how, because the neural stem cells didn’t have the receptor for serotonin.
24:06 - 24:12: It had to be a sequential mechanism. Therefore, what we did, we picked the cells that express
24:12 - 24:18: the serotonergic receptor and did a differential gene expression there and looked at the ligands
24:18 - 24:23: that are differentially expressed. And serotonin and interleukin-4 are oppositely regulating
24:23 - 24:28: proliferation. Therefore, if there is a proper and real mechanism that regulates neural stem
24:28 - 24:35: cells through this serotonergic signaling, these ligands should be differentially expressed
24:35 - 24:41: and reciprocally. And we found three of them, and BDNF, the brain-derived neurotrophic factor,
24:41 - 24:48: was the highest fold change. And BDNF is expressed in the neuronal clusters,
24:48 - 24:54: and its two canal neuroreceptors, NTRK2 and NGFR, are also expressed in different cell clusters.
24:54 - 25:01: NGFR is expressed in the neural stem cells, astroglia, and NTRK is expressed in the glia.
25:02 - 25:07: And when we did our interaction analysis, we really found that serotonergic signaling is
25:07 - 25:12: reducing, generally, the BDNF signaling pathway, while amyloid or interleukin-4
25:12 - 25:18: enhances the cellular interactions that lead to this contact with this mechanism.
25:18 - 25:23: And we also functionally and spatially distinguished the role of BDNF in different
25:23 - 25:29: populations, because here you see the stem cells expressing its ventricular region.
25:30 - 25:34: Stem cells do not express NTRK2, but neurons just next to that express that,
25:34 - 25:39: and NGFR is expressed specifically in neural stem cells. And when BDNF is injected into the brain,
25:39 - 25:46: it activates both signaling, but the downstream effectors are compartmentalized very clearly.
25:47 - 25:51: Phospho-AKT signaling is in the neurons, and NF-kappa-B signaling is in the stem cells.
25:51 - 25:57: So basically, normally, single-cell sequencing gives us a cellular resolution and heterogeneity
25:57 - 26:05: that we can pinpoint the sequential functions. And after our analysis, we found that serotonergic
26:05 - 26:13: signaling is, through BDNF and its receptor NGFR in the neural stem cells, is forming a
26:13 - 26:19: balanced mechanism, where if you block serotonergic signaling, you have more secretion of BDNF from
26:19 - 26:24: the periventricular neurons that are targets of serotonergic signaling, and they increase
26:25 - 26:29: the proliferation of the stem cells. Or the other way around, when you increase serotonergic
26:29 - 26:37: signaling, you reduce BDNF and reduce the proliferation. We also functionally dissected
26:37 - 26:47: different cell types, different heterogeneous, different clusters of subtypes of the astroglia.
26:47 - 26:52: NGF signaling is one. IL4 signaling, I told you one, but we recently identified another one,
26:53 - 26:58: which is aryl hydrocarbon receptor signaling through tryptophan metabolism leading to kynurenic
26:58 - 27:03: acid production. When kynurenic acid is produced by the periventricular neurons, it also generates
27:03 - 27:10: a balancing mechanism, but through repression. When we have more kynurenic acid, or the agonist for the
27:10 - 27:17: receptor for it, we suppress the proliferation. But when we have a blocker of kynurenic acid or
27:17 - 27:22: tryptophan metabolism, we increase the proliferation. So single cell sequencing and
27:22 - 27:28: looking at the neurogenic response and the cell types pertaining to that led us in zebrafish
27:28 - 27:33: to find different subtypes, and we are still scratching the surface. But one important
27:34 - 27:40: finding was majority was going through the tryptophan metabolism. So either conversion
27:40 - 27:46: to serotonin, which leads to BDNF signaling, or the kynurenic acid branch, which leads to
27:46 - 27:52: AHR signaling. And we temporarily found that some pathways should be blocked and some pathways
27:52 - 27:59: should be activated in order to produce a neurogenic response. So this is all good.
27:59 - 28:07: Zebrafish is telling us how vertebrate astroglial cells are leading into a neurogenic route
28:07 - 28:14: when there is amyloid toxicity. But we need to test, of course, these findings in an amyloid
28:14 - 28:20: model or in vivo models, in order to see whether this finding can be translated there.
28:20 - 28:26: That’s why with our academic collaborations in the Leibniz Institute IPF, in Dresden with
28:26 - 28:34: Karsten Werner and Uwe Ferdenberg, we developed a 3D hydrogel matrix where we embedded the primary
28:34 - 28:39: or epistriate human neural stem cells, which you see right here. These are the astroglial cells,
28:39 - 28:48: which lead to the formation of the neural neurons and a neurogenic response in these 3D hydrogels,
28:48 - 28:55: which you can see the human neurons in cyan and their interactions. Now we established a startup
28:55 - 29:04: to use this in an automated manner to go to the drug screening platform. So this 3D culture
29:04 - 29:11: was really nice for us for the neurogenic aspects because it generates from single
29:12 - 29:19: neural stem cells that are detached from each other, connected functional neural networks
29:19 - 29:26: that go through the neurodevelopmental paths that we know, like SOX2, NDR, and then
29:26 - 29:32: the neurogenic proneural factors and then the development of the neurons into functional networks.
29:32 - 29:36: But when we used amyloid toxicity here, which you can talk about in details further, and this
29:36 - 29:41: is a published work, the details can be found there, we lose this development, plus we lose
29:41 - 29:47: the neurogenic activity, plus the neurons, of course, don’t make proper connections.
29:47 - 29:53: We do see functional or morphological outcomes, like the dystrophic neurons or
29:54 - 29:59: disruption of the extracellular matrix deposition in these cultures. We see
29:59 - 30:06: de novo tauopathies. Just with the amyloid toxicity in healthy cells, we do see
30:08 - 30:15: tau tangles and loss of intracellular axonal tracts, which was very nice for us. But
30:16 - 30:23: also the SOX2-GFAP positive cells, which represent the neurogenic progenitors, are significantly
30:23 - 30:29: reduced, and neurogenic outcomes significantly reduced. We also did molecular studies, and the
30:29 - 30:35: gene expression patterns of different neuronal cortical subtype neurons are significantly reduced,
30:35 - 30:40: and these include the ones that are first affected in our brain in the intrarenal cortex.
30:41 - 30:47: So this model, by using this model, we tested some of our findings, for instance, the interleukin
30:47 - 30:52: and the tryptophan metabolism, and we found very comparable and parallel findings. When interleukin
30:52 - 30:59: 4 is applied to the system in the case of the amyloid toxicity, interleukin 4, through STAT
30:59 - 31:04: signaling, can block CAT2, the enzyme that produces the kynurenic acid. And we found the
31:04 - 31:10: kynurenic acid is exactly doing the same, is suppressing the neural stem cell proliferation
31:10 - 31:15: in humans, and also suppressing the neurogenic outcome. And we blocked this pathway through
31:16 - 31:23: the understanding from zebrafish. We can get back the neural stem cell response
31:23 - 31:28: and the neurogenic response to a certain degree, and the molecular, this is not simply
31:29 - 31:34: correlative, but this is also through restoring the molecular programs that in the end lead to
31:34 - 31:41: this neurogenesis. And we also went then to the post-mortem human brain tissue, and this is a
31:41 - 31:47: comparison of the healthy and AD human brains in the hippocampus and temporal lobe. The CAT2,
31:47 - 31:53: the enzyme that produces this toxic kynurenic acid that is suppressing the neurogenesis,
31:53 - 31:58: is highly upregulated in Alzheimer’s disease. So basically, these zebrafish findings can
31:58 - 32:06: functionally tell us the targets that we may address in humans, but also will help us to
32:06 - 32:12: understand how neurogenesis could be put into the game. And finally, with our collaborators
32:12 - 32:19: in Columbia University, and they have Alzheimer’s disease cohorts, and we have been checking the
32:19 - 32:24: bulk RNA sequencing patterns between different Alzheimer’s disease cohorts of the humans
32:24 - 32:31: and zebrafish, and we saw a good correlation. There are many pathways that are similarly affected,
32:31 - 32:35: and there are many pathways that are oppositely affected, which are both interesting in terms of
32:36 - 32:42: an academic point of view. And for instance, some of the pathways that we saw
32:44 - 32:52: oppositely regulated were the JAK-STAT pathway, or the apoptosis and cytokine receptor interactions,
32:52 - 32:57: and the biological process evolved around regeneration in immune system processes,
32:57 - 33:04: and others. And for many of those, we already found that zebrafish is using these programs to
33:04 - 33:11: generate a neurogenic outcome in Alzheimer’s-like conditions. Therefore, it seems that zebrafish can
33:11 - 33:17: really tell us how human brains maybe fail in regeneration of the neurons, and maybe
33:17 - 33:26: neural regeneration-aiming therapy options could be helped by zebrafish, and what we understand
33:26 - 33:32: from there. So this is a summary of my talk today. I believe, and we believe, zebrafish
33:32 - 33:40: recapitulates certain aspects of the pathological AD, and in these conditions, it can tell us about
33:40 - 33:45: how we can go to the neuro-regenerative path in Alzheimer’s disease by using the vertebrate
33:45 - 33:51: astroglial progenitors, and single-cell transcriptomics approach and the comparison
33:52 - 33:58: really revealed the cellular heterogeneity, which we need to understand because astroglia is not
33:58 - 34:05: one cell type. As immune system cells, microglia is not just one cell type. And human AD cohorts
34:05 - 34:13: and comparison of the omics approaches to zebrafish showed some correlation in terms of
34:13 - 34:20: gene expression changes, but I think it’s a promising future approach for further studies.
34:21 - 34:28: This is my final slide. I’d like to thank all present and past members of our laboratory.
34:29 - 34:35: Especially here, I highlighted the work of Pravesh Bhattarai, who generated the amyloid
34:35 - 34:41: toxicity model in zebrafish, and Mehmet Ilyas Çaşacak, who established the single-cell sequencing
34:41 - 34:48: and all these beautiful comparisons. I’d like to thank our collaborators and our funding sources.
34:49 - 34:54: And I’d like to thank Abcam for organizing and inviting us. Some of the images and the
34:54 - 35:00: antibody stainings we did with Abcam antibodies and reagents, which we have a nice collaboration,
35:00 - 35:04: and we’d like to continue doing that. Thank you.
35:04 - 35:16: Thank you so much. That was a great talk. We have several questions.
35:18 - 35:25: We’re going to go ahead and try to answer those. So, we have a question in the chat
35:26 - 35:33: that is asking, this is Emily Jan, she’s apologizing for the nice question, but
35:33 - 35:39: she had never worked with zebrafish. She’s asking, what is the administration route
35:39 - 35:43: to test drug candidates, and how relevant it is regarding translation to humans?
35:45 - 35:50: That’s a beautiful question, and the zebrafish field, I didn’t have time to go through that,
35:50 - 35:56: is becoming a good drug development tool as well. Not only in neurodegenerative diseases,
35:56 - 36:03: but also in oncology and other toxicology. There are many ways that some drugs or toxic
36:03 - 36:08: chemicals can be administered. You can just put it into the fish water and see how they are exposed,
36:08 - 36:15: or you can inject it into the IP in different ways. And people also test how these drugs or
36:15 - 36:22: compounds are metabolized and pass the blood-brain barrier or different membranes. There are many
36:22 - 36:29: different ways. In our case, we inject it into the cerebroventricular fluid, and just to not
36:29 - 36:36: to have it systemically, but we have transgenic animals that also generate toxic protein aggregates
36:36 - 36:42: systemically around the body. There are many different ways. So, whatever is present in mouse
36:42 - 36:46: and other animal models is being developed in zebrafish and probably is there.
36:49 - 36:57: All right. We have a few more questions in the Q&A. So, some anonymous attendees are asking,
36:57 - 37:03: is the amyloid pathology and the cell death associated with it a specific response or a
37:03 - 37:09: generic protein toxicity response? Did you check any other amyloid peptides?
37:10 - 37:17: We did. We did check different amyloid forms, which are known to be soluble and forming
37:17 - 37:24: aggregates but are not toxic, like 38, 40, and others. Plus, we tried other types of proteins
37:24 - 37:31: that aggregate but may not be toxic. So, this work and these controls have been performed,
37:31 - 37:39: and amyloid beta-42 is specifically forming certain beta-plated structures and forming
37:39 - 37:47: a specific toxicity. So, this we also tried in our 3D cultures, also in mouse cell cultures,
37:47 - 37:55: and in vivo, and this is specific to amyloid beta-42. So, that probably answered it. The
37:55 - 37:59: next question is, have you checked the tau pathology in your fish model?
38:01 - 38:08: Yes, we checked. There’s no de novo tau pathologies with the injection acute amyloid toxicity model
38:08 - 38:16: we have. We also generated a tau transgenic animal, which chronically expresses different
38:16 - 38:23: versions of the human tau mutations. We see all the way through the phosphorylation. We do see
38:23 - 38:29: hyperphosphorylation of tau, as we see in cell cultures or human brains, but zebrafish doesn’t
38:29 - 38:37: form tangles. We think that this could be a real mechanism that prevents tau pathology in fish,
38:37 - 38:43: but it could also be a technical issue. So, we are now trying to find out ways to increase,
38:43 - 38:47: to generate tau pathology in fish brain, whether that would lead to a second
38:48 - 38:53: complementation of the AD model that we have in common. That’s it.
38:54 - 39:00: That’s great. That is so interesting. You were mentioning at the beginning about
39:00 - 39:10: resilience, and I think a question that reminds there is whether the animals are developing
39:10 - 39:16: the pathology, but they are resilient to suffer it, or they’re just fighting the
39:17 - 39:23: disease. So, they are regenerating, and they are overcoming the issue. Whether or not it’s the same
39:23 - 39:28: or it’s related, it’s still fascinating. That’s right. That’s right. I think any
39:28 - 39:32: model that particularly reductionist looks at a certain aspect is valuable,
39:32 - 39:37: but I think we should agree that all models are good, and all models are bad in this case.
39:37 - 39:41: So, we can’t really have a good animal model so far.
39:42 - 39:45: Yeah. We have a couple more questions.
39:47 - 39:54: How do you rate the power of your single-cell sequencing? Do you need to sequence more cells?
39:55 - 39:59: Yes, I think we need to sequence more. The more we sequence, the more cell types and
39:59 - 40:07: heterogeneity, different programs we get, and this was a study we performed a few years ago. Now,
40:07 - 40:12: even the technology is more developed than we are already sequencing. We have already sequenced
40:14 - 40:19: much more. So, I think we started with sequencing a few thousand cells. Now,
40:19 - 40:24: hundreds of thousands of cells can be sequenced, and we did that. Yes, that’s it. That’s true.
40:26 - 40:31: All right. So, someone is asking to clarify and say, sorry, I may have misunderstood,
40:31 - 40:34: but did you say there were some transcriptomic changes that are
40:34 - 40:40: opposite in zebrafish model and human AD? Doesn’t that make the model flawed?
40:42 - 40:44: Does it make the model what, sorry? Wrong.
40:44 - 40:46: Flawed. Yeah.
40:46 - 40:54: Okay. No. So, I think it makes the model more interesting because these programs,
40:55 - 40:59: some of them are pertaining to the neurogenic programs, neuronal survival programs,
41:00 - 41:07: and other types of programs that might go wrong in humans, and zebrafish may be doing it the right
41:07 - 41:15: way. So, I think rather than disproving a model, it actually makes it more interesting. And some
41:15 - 41:23: of the programs or the changes that we see parallel in humans and zebrafish are mostly
41:23 - 41:30: pertaining to the pathological aspects, like what amyloid does, the synaptic connections,
41:30 - 41:37: and so on. So, probably we need to really look into detail, and I agree. So, zebrafish may not
41:37 - 41:45: be the, probably won’t be the excellent model for AD, and there will be some differences,
41:45 - 41:50: but we are trying to find what can be worked in parallel in zebrafish, but what cannot be,
41:50 - 42:03: which is also a very interesting scientific understanding, I guess.

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