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Parkin Maintains Robust Pacemaking in Human iPSC-derived A9 Dopaminergic Neurons

July 07, 2023
In this episode, we interviewed Prof. Jian Feng about his most recent paper covering the role of parkin mutations in human A9 dopaminergic neurons and role they play in locomotor activities of early-onset PD.
Journal CME is available until July 20, 2024 Read the article.

[00:00:00] Dr. Tiago Outeiro: Hello and welcome to the MDS Podcast, the podcast channel of the International Parkinson and Movement Disorder Society. I am Tiago Outeiro, professor at University of Medical Center, Gottingen in Germany. And today I have the pleasure of interviewing professor Jian Feng from state University of New York at Buffalo in the US.

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And we will talk about a recent study he co-authored together with Dr. Baorong Zhang from the College of Medicine in Zhejiang, University in China. Their recent study was entitled, Parkin Maintained Robust Pacemaking in Human Induced Pluripotent Stem Cell-Derived A9 Dopaminergic Neurons.

So although familial forms of Parkinson's disease account for a small percentage of Parkinson's cases, learning about the normal function of the various proteins linked to these forms of Parkinson's is extremely important. And [00:01:00] therefore we thought this study really adds to our understanding of the function of parkin.

So welcome, professor Feng. It's a pleasure hosting you here in the podcast. Let's dive right into it, and can you please tell us briefly about the study that you just recently published?

[00:01:16] Prof. Jian Feng: Sure. Thank you for the opportunity. It's a great honor to introduce you to this study that we recently have published in Movement Disorders. So basically, this study was to try to establish whether the mutational parkin is causal and necessary for some phenotypes in the IPS derived A9 dopaminergic neurons from human subjects.

So from many decades of painstaking work, people have realized that you have certain forms of Parkinson's disease that can be caused by mutation of a single gene. So one of the very clear example is parkin. So we have been studying this gene for the past 23 years.

Basically, the field has known there's a lot of [00:02:00] evidence that might have parkin mutation. You have Parkinson's disease in human right, but you cannot really establish the causal relationship. Say, is the mutation really unnecessary and sufficient? Is the phenotype caused entirely by parkin mutation? Not by anything else in the genetic background. So this can be established in a dish in the IPS derived of these A9 dopaminergic neurons. So we have developed a method to differentiate the human induced pluripotent stem cells into the A9 dopaminergic neurons, which are specific laws in Parkinson's disease.

We know that these neurons. Are very unique in that they're actually pacemakers, they're sort of like pacemakers in the heart. Basically their job is to constantly release dopamine. And when there is a need to start movement the release becomes even more. You have these sort of tonal firing, which is supposed to be constant.

And then you have these facing firing, which is supposed to [00:03:00] be related to the initiation of movement. The exact nature is not quite clear. But what we know for sure is that these functions are very critical for the movement for the function of these A9 dopaminergic neurons. 

[00:03:12] Dr. Tiago Outeiro: Let me just interject here to ask you, so we hear a lot about IPS cells at the moment, and we hear about people differentiating them into dopaminergic neurons. But here you are actually a lot more specific. So you differentiated IPS cells into A9 dopaminergic neurons.

So what are the challenges in generating such specific populations of neurons in culture?

[00:03:35] Prof. Jian Feng: So this really has been built on probably at least two decades of work. So this is like the third generation of differentiation in protocol. In the beginning, people do not really understand too much about the development of these neurons. 

Through studies are contributed by many, many labs in the field, we now actually know that these neurons are very unique and they have a special tangential trajectory. So [00:04:00] utilizing this information, we're able to generate the particular subtypes of these midbrain dopaminergic neuron as some of the listeners may know there are many types of dopaminergic neurons in the brain. 

Even in the mid brain, there are at least three types. The A8, A9, and A10. They project to a very different area. They control very different functions. The type that is lost in Parkinson's is mostly the A9. So these neurons, they reside in the substantia nigra and then they project very long axon to the caudate putamen.

So that's the challenge and we were able to develop a way to differentiate human-induced pluripotent stem cells, which is the IPS cells into the A9 dopaminergic neurons.

[00:04:49] Dr. Tiago Outeiro: Yeah, I was really excited about this because usually in the studies we don't hear people going this far and generating such specific populations of neurons. So this is really interesting. [00:05:00] And in your study you used two different methods to generate isogenic lines. So can you explain why this was important? And how challenging were the procedures you used to generate the isogenic lines?

[00:05:14] Prof. Jian Feng: Yeah, so this actually has been a multi-year effort. So we actually started when there was no CRISPR, there was only talent. So it has been a long journey for us. So first we have to repair the exon 3 mutation in a homozygously mutated subject. So this is a patient who have two copies of her parkin gene mutated.

So she lost the exon three of both her copies of the parkin gene. So in order to say that the loss is actually causing her phenotypes, we have to repair it. And we have to repair both copies, right? So at that time there was only TALENs. So this is a older generation of genomic modification technique where you [00:06:00] can do homologous recombination to repair any genetic defects by introducing a double stranded cut in the DNA and that will increase the recombination rate dramatically.

So we tried that and we spent a lot of effort. And then we finally are able to generate the repair. So while we are doing that the CRISPR technology came online. It was such a relief. It's much easier to introduce mutation, particularly if you bonded it to the point mutation. So then we say that to convince our colleagues we wanted to do the reverse way.

By introducing a point mutation of parkin that is found in Parkinson's disease patient. It was the A2E point mutation. Changing into glutamic acid. So we want to introduce this mutation to a normal IPSL. This is IPSL derived from a normal subject.

We are able to use CRISPR to precisely introduce mutation to two copies [00:07:00] of the parkin gene. So it's a homozygous introduction of mutation. Now we have basically two pairs. These are the isogenic pair. Have the exact, the same background. One pair is on the background of a patient, the other is on the background of a normal subject.

So with this, we are now in the position to answer is parkin mutation causal, right? If you introduce parkin mutation, or you can do cause a PD phenotype even on a normal background. So this is what we try to do, and the flip side is that if you have a PD patient, That has parkin mutation when you repair it, would that cause the phenotype to go away? So that's precisely what this paper has found.

[00:07:44] Dr. Tiago Outeiro: Yeah. But I, think it's really important to use multiple approaches also to avoid the unspecific effects that we know the different methods have. So I, really enjoyed that you were that careful and you took advantage of different technologies that are of course evolving and now enable us to do things [00:08:00] even more specifically.

And, now about the role of parkin in pacemaking, I find this really interesting. So is this something you were expecting? I mean, how did you come up to the decision to look at this particular aspect and also how do you think parkin is having such an effect?

[00:08:17] Prof. Jian Feng: Okay, so it was totally unexpected. When we started the project we basically know that there's some phenotype that we have already published. We want to see whether we can confirm these phenotype. These phenotype were obviously confirmed, but because they're a sort of confirmatory study, we don't feel that it will be competitive for us to try to publish them again.

As you know, it's very hard to publish confirmation results. So but at that time the other technology that we have developed came online, which is the method to differentiate IPS cells into A9 dopaminergic neuron. So then we say, yeah, now we have a very strong readout. So this readout is that these neurons are [00:09:00] pacemakers.

We know through decades of work in the rodents, in animals, all sorts of animals actually that the pacemaking of parkin is very important. And then I say to people in my lab, let's say, can we find out whether there's any difference in their pacemaking when we have this two pairs, four lines of iPSC derived A9 dopaminergic neurons.

And to our surprise, there's a huge difference. So if you look at the parkin deficient A9 dopaminergic neuron, the frequency of their action potential, of their pacemaking action potential is much lower then if you do not have mutation, now we have established this both ways.

So first you look at the patient. The patient has a fairly slow autonomous pacemaking action potential. And it's just like what they found in the rodent. It's really amazing that even the frequency is very similar. And then you repair the exon 3 mutation. Suddenly, the pacemaking frequency becomes much higher.

And [00:10:00] it's as if the neuron becomes more healthy and then we'll say, is it real? So then we have to do it the other way. We look at the control neuron. Then the control neuron has a very robust pacemaking. And then we look at the mutant that are introduced in the control, the A82E mutation. Just change two letters, one letter in each chromosome.

So this is out of 6 billion base pair. We just changed two letters, right? And then the pacemaking frequency is significantly reduced. So that was really shocking in a way. And we also saw that the absolute value is different. So the normal cells they have a higher pacemaking action potential, whereas the parkin cases, even when the parkin mutation is repaired, the pacemaking frequency is lower.

We do not know what to make of this. It could be due to the genetic background. So this patient is a result of a consanguineous marriage. She has a lot of homozygosity, so maybe the [00:11:00] homozygosity in her genome is contributing to the slower pacemaking. So I think there's still a lot of things to be studied.

We don't exactly know how parkin is controlling pacemaking because pacemaking itself is very complex. It is likely that many channels are involved in maintaining pacemaking because it's such a critical function, right? You can imagine. So there are many, many channels that are involved.

We first need to understand better what is going on in pacemaking, particularly in the human A9 dopaminergic neuron. Prior to this, there's no material to even just study that you can work on the mouse or the rat A9 dopaminergic neuron, but even there, we don't really know too much about it. So I think that it opens up new possibilities for the field to examine in more mechanistic detail, what are responsible for pacemaking in the nigral dopaminergic neurons and how parkin interact with these components to control robust pacemaking.

[00:11:59] Dr. Tiago Outeiro: Yeah, no, this [00:12:00] is fascinating. I'm really curious to see what you'll find out about how parkin is involved in controlling this pacemaking activity. And so this was looking in this A9 neuron. So if you were to look in different neuronal types or even in glial cells, would you expect to find other rules of parkin in these other cell types that go beyond this particular effect, in dopaminergic neurons?

[00:12:25] Prof. Jian Feng: Yeah, sure. We actually have published a body of literature to show that parkin has a variety of very critical function in the membrane dopaminergic neuron beyond pacemaking. So we first published a paper showing that parkin is actually controlling the utilization of dopamine in several ways. One is that, dopamine metabolism produce a lot of reactive oxygen species, part of which is done through the ML mediated, so degradation of dopamine.

These are done by the monoamine oxidase. So basically you have monoamine oxidase that catalyze the oxidation of dopamine, so they can be [00:13:00] degraded. So in this process, you produce a lot of free radicals. Parkin is actually suppressing the transcription of monoamine oxidase. So that normally you have a very low amount of MAO in these dopaminergic neurons so that it will not cause problem. When you have parkin mutation this check is gone.

So then the MAO transcript level is much, much higher. And then you produce a lot of free radical just because these cells have to metabolize dopamine. So this is only one aspect. And we also find that parkin controls the precision of dopaminergic transmission. So we have basically published several papers in this field using some older technology.

And we have already reexamined this in the A9 dopaminergic neuron and we can confirm that, as I said, the confirmatory experiments. I'm not quite sure how hard it is for us to publish it, but we do have confirmation on that. We intend to try to [00:14:00] expand beyond that.

As for the glials, we don't have the expertise to look into that. So that can be a very important area done by people with expertise in that side.

[00:14:10] Dr. Tiago Outeiro: Yeah. No, I think it'll be really nice to see your other data and then see what people do in other cell types. And so now from the perspective of the translation. So how do you see these findings helping us decide or define possible therapeutic strategies for patients with parkin mutations?

And do you see that this could have a broader application into idiopathic forms of Parkinson's as well?

[00:14:39] Prof. Jian Feng: So there are two components to this question. If we only focus on parkin first, I see that if you, for example, treat parkin cases as if it's a monogenic disease, like for example, Huntington's disease, right? It's rare, but it's caused by mutation on one gene. Then you conceivably already have a solution, which is to repair the [00:15:00] mutation.

There are a lot of ways to actually deliver the gene. Some people have been using AAV and a lot of biotechnology companies are trying those on other diseases. I imagine that if there's effort to develop a therapy for the parkin cases, it can be very effective because we know that their problem is actually caused by mutation of the single gene.

So it's causal, right? You repair the mutation. It is very likely that it can help them a great deal. So the second part of the question is whether the knowledge on parkin can inform Parkinson's disease in general, the idiopathic form. I don't really know for sure.

So my current thinking is that because Parkinson's mostly occurs when people are already beyond their reproductive age. So there's really no selective pressure to generate Parkinson's disease. It's not like, for example, sickle cell anemia, right? So there's a possibility that you can have many, many ways [00:16:00] to generate the idiopathic Parkinson's.

Reflect the intrinsic vulnerability of these neurons. If that's the case, then it'll be hard to translate the funding from the monogenic mutations into the idiopathic, that remains to be seen. We do not know. It's just a speculation. If there is a strong mechanism, so if the A9 dopaminergic neuron can only fail in so many ways, then I think there is at least some value in the study of these monogenic form. Many people believe in that. We believe in that as well. So I think that to say the least we can try. For example, let's say if we overexpress parkin in the idiopathic cases, whether it will help, that is a very useful approach. Because parkin does many, many things that is very good for the dopaminergic neuron.

We said that it controls reactive oxygen species. We also have another paper showing that it stabilize microtubules. It's very important for maintaining the long [00:17:00] processes of these neurons. These neurons are very special in other senses that they have massive, excellent authorization that relies on micro tubular to support the structural integrity and microtubule based trafficking.

So, If you just use a single for example, a small molecule drug or even use a protein to deliver into the neuron, it may not do the whole thing, but parkin it already does everything that the neuron needs. So that could be a potential pathway to examine whether it is protective.

[00:17:31] Dr. Tiago Outeiro: Great. Yeah, I think there's hope and of course this new knowledge will be informative in the future. And as you and others explore things further, I'm sure we will learn more and hopefully we'll be in a better position to use this new knowledge to improve any possible therapies for, patient use.

So Professor Feng, thank you so much. This was really nice that you could talk to us about your study. I appreciate that and we look forward to having you in the [00:18:00] podcast to talk more about your future studies. So thank you.

[00:18:03] Prof. Jian Feng: Thank you very much, Tiago. It's a great pleasure. Thank you.

[00:18:06] Dr. Tiago Outeiro: Great, thank you. So we've just interviewed Professor Feng about the recent publication in Movement Disorders on the role of parkin in pacemaking in iPS neurons.

Thank you all for listening and join us in our upcoming podcasts. [00:19:00] 

Special thank you to:

Dr. Jian Feng
Department of Physiology and Biophysics
School of Medicine & Biomedical Sciences
State University of New York at Buffalo

Tiago Outeiro, PhD 

Director of the Department of Experimental Neurodegeneration 

University Medical Center Goettingen, Germany

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