1st of october 2021
Parkinsonian mice with intestinal inflammation
A day in the life of patient researchers
Part 1. The visit
On Friday 17 September 2021, we were guests of the Utrecht Institute of Pharmaceutical Sciences, Division of Pharmacology at Utrecht University on behalf of the Dutch Parkinson’s Association (Parkinson Vereniging). We were guests with a unique mix of patient researchers and representatives of the patient representatives of the Parkinson’s Advisory Council (PK-PAR). We wanted to know everything about inhibiting intestinal inflammation in Parkinsonian mice. This research received a grant of 40,000 euros from the Dutch Parkinson’s Association. We were treated to a delicious lunch, insightful lectures, and a tour of the lab.
Stop, not so fast.. Patient researchers and representatives of the PK-PAR? What are those?
Haha, yes good question. Very briefly: Representatives of the PK-PAR advise, among other things, about which research should be subsidized and patient researchers represent the patient perspective within the Parkinson’s research project itself: thinking about the design, organization and implementation of the research and the translation from mouse to human! We – Leny Rietdijk, Joost van den Brakel, and myself – will be involved as patient researchers throughout the entire study. We will inform Monique Bosman and Theo Zuidema – who were there on behalf of the PK-PAR – during the project, including via this blog.
OK, tell me more
I’d love to. We were welcomed by professor Dr. Aletta Kraneveld about whom I blogged earlier. We met three young Parkinson’s researchers from her group: Anastasia Markidi, Joshua Elford and assistant professor Paula Perez Pardo. Joshua will work as a PhD student on the awarded project and Paula will supervise him. Paula previously showed – as part of her PhD research – that in the intestines of People with Parkinson’s pro-inflammatory bacteria are more abundant than in healthy controls.
What are ‘pro-inflammatory bacteria’?
These are bacteria that trigger a response from our immune system. These pro-inflammatory bacteria have so-called lipopolysaccharides (LPS) on their cell walls. Our immune cells possess a protein on their cell membrane that can bind specifically to LPS like a key on a lock. This protein is called Toll-like receptor 4 (TLR4). When LPS binds to TLR4, the immune cell receives the signal to secrete substances that cause inflammation. The intention of that inflammation is to clear up the bacteria, but as you know, too much of a good thing is often no longer good. If too many immune cells get to work, it causes local inflammation in the gut.
So what does that have to do with Parkinson’s?
The hypothesis is that local inflammation in the gut can lead to a leaky gut. Then the intestinal wall no longer protects you against toxins. They can get into the bloodstream that way. This exposure, in turn, can lead to a disrupted blood-brain barrier and chronic neuroinflammatory processes in the brain itself, leading to neurodegeneration – the loss of dopamine neurons – and .. well .. Parkinson’s. If you want, you can read more about that here.
Neuro-inflammatory processes.. I got it I think: that’s inflammation in your brain cells? Why don’t you just say that?
Neuroinflammation sounds a lot nicer, don’t you think?
Haha, yes, that’s right. But what about that mouse with intestinal inflammation?
In summary, it is Joshua and Paula’s hypothesis that the activation of TLR4 in people with Parkinson’s is out of control. Therefore, they want to investigate a drug that inhibits the activation of TLR4 in a mouse model for Parkinson’s disease. The researchers expect that the use of this drug will slow the progression of neurodegeneration in the mice. If the TLR4 inhibitor is able to show effect in this Parkinson’s model, this opens up possibilities to investigate this compound for the development of a drug for humans.
OK, but how can you prove that this works in a mouse?
They are going to measure 6 things: The motor skills (by making the mice run on a kind of treadmill), the spatial memory (cognition), they are going to make 3D recordings of the brain, they are going to see how the dopaminergic neurons and the microglia – cells that keep neurons healthy – are doing, they look at how things are in the gut (is there a leaky gut, for example) and the degree of activation of the immune system is monitored. Ethically, you need to get as much data as possible from the mouse experiments. After all, you’re sacrificing the mice.
Yes, that is definitely an ethical dilemma…
How can the researchers determine whether that TLR4 inhibitor works? What is needed for that?
Basically by comparing it to a mouse that doesn’t have Parkinson’s. Of the mice that get Parkinson’s by administering the pesticide rotenone, some are given the TLR4 inhibitor – in different doses. Another part is not given the TLR4 inhibitor. Then there is a group of healthy mice that receive only the TLR4 inhibitor and then another group of healthy and untreated mice. By comparing the results, you can draw conclusions about the effect of the TLR4 inhibitor.
And if it works, what’s the next step from mouse to human?
The next step is to perform this experiment on a cell culture of human cells. If you want to know more about this, you can also study the Anastasia’s research setup.
Anastasia?
Yes, we came to Utrecht because of our role in the TLR4 project. But that’s not the only Parkinson’s research being done in Utrecht. Anastasia also conducts fundamental research to better understand the cellular processes that play a role in the development of Parkinson’s. If you want to know more about that, you can open the accordion. Then you will also learn about the use of pluripotent stem cells (with the potential to become whatever cell type you want) in Parkinson’s research.
Anastasia Markidi is researching the role of the enteric nervous system – the nervous system in our digestive system – in Parkinson’s disease.
Do we have a nervous system in our digestive system?
For sure. In the wall of our gastrointestinal tract are all kinds of neurons, surrounded by so-called enteric glial cells. Glia comes from Greek and means glue. Glial cells are the cells that take care of our neurons. Anastasia looks at how the immunometabolism of these glial cells changes in Parkinson’s disease.
Immunometabolism?
Yup. Immunometabolism links two historically distinct disciplines: immunology and metabolism:
- Immunology is about our defense mechanisms against foreign organisms and/or substances;
- Metabolism is about our energy management. About converting food into the necessary energy to keep the processes in your cells running, converting food into building blocks for necessary proteins, fats, carbohydrates, DNA, etc., and about removing the waste products that remain after combustion and conversion.
Ok, but what then is immunometabolism?
Immunometabolism ((Robb, 2019) looks at changes in the metabolism of, for example, neurons or as a result of changes in our immune response. If there are a relatively large number of pro-inflammatory bacteria living in your gastrointestinal tract, this activates your immune cells. They are then, as it were, ‘overactive’. The illustration below shows how glial cells change due to this kind of low-grade inflammatory processes (immunology). It gives them a different energy balance (metabolism). They break down more glycogen into glucose and so more energy is released. Researchers call this a ‘metabolic switch’.
Can you turn it on and off, that switch?
Not consciously, but there are triggers that cause the switch to flip. Glycogen is an easily mobilized storage form of glucose. It is a very large, branched polymer of all glucose molecules together that can be broken down to yield glucose molecules when energy is needed. The moment the energy is so low that that conversion is necessary, this process occurs automatically. And when there is enough energy again, glucose is converted into glycogen. But with glial cells in an environment of low-grade inflammation, it may well be that that switch is always ‘on’.
You write ‘could it be’, so you’re not sure?
That’s right. Anastasia will investigate this hypothesis by simulating an enteric nervous system in a petri dish. She will use so-called pluripotent stem cells from healthy people and people with Parkinson’s disease. These stem cells will be differentiated into enteric neurons and enteric glial cells: a healthy mix and a Parkinsonian mix. Then Anastasia will feed that enteric nervous system products from bacteria and also a solution from the stool of people with Parkinson’s. Anastasia is curious about what happens next. Do the neurons die from exposure to the products of pro-inflammatory bacteria? Will alpha-synuclein – a protein that clumps together in a subgroup of people with Parkinson’s – clot in the neurons? Or is the metabolic switch flipping?
As befits scientific research, the questions are still ahead of the answers. The great thing is: Every answer helps to better understand the cellular processes that play a role in the development of Parkinson’s.
And what kind of input did you give yourself?
Good question. After all, the collaboration should yield something for both parties. We gave a presentation about the working group patient researchers of the Dutch Parkinson’s Foundation. We visualized how many studies we participate in and which subjects are involved by means of a data visualization. We also asked questions about the content, relevance, impact, and efficiency of the research. We did that during the meeting and also afterwards, for the people who had not been able to give their input during the meeting.
Are you curious about the type of questions we ask? Then you can open the accordion. The answers will follow in a next blog, so be patient 🙂
Validity: are we measuring the right things and are we measuring things right?
- What is the primary outcome measure or endpoint? In other words, at what degree of difference between the results of the intervention versus the control group is it concluded that the drug is considered an appropriate intervention?
- In the presentations, accumulated alpha-synuclein is considered pathological. In science, an alternative hypothesis makes the rounds. When alpha-synuclein accumulates, the ‘soluble’ and functional protein disappears. That protein has a function that it can no longer perform. And the hypothesis is that it is this loss of function that is pathological. Could we also measure soluble alpha-synuclein in this mouse model to test this? To illustrate the question about measuring free-flowing alpha-synuclein (the functional protein), we refer to http://regaintherapeutics.com/index.html. This startup is based on “rethinking the mechanisms leading to neurodegeneration and completely redesigning the therapeutic approaches to replace and regain lost protein function rather than just focusing on protein aggregation”.
Relevance
There are several clinical studies aimed at reducing inflammatory processes in Parkinson’s, for example with drugs that inhibit the so-called NLRP3 inflammasome. How could TLR4 inhibition complement such studies?
Applicability and impact: From mouse to human
- If the activation of immune cells is inhibited, don’t the mice run the risk of getting sick from other pathogens? And what about people in the future?
- There are many studies on inflammation. How should we understand the value of this sub-study for people with Parkinson’s disease? Is there a good chance that if the mouse study gives a positive answer about the TLR4 inhibitor, this will also be the case for people with Parkinson’s in the future? So that a translation from mouse to human can be made? In many studies, this translation is not possible. Can we point to reasons for this and what can we learn from it?
- Could the many lines of research in immunology lead to a classification of pathological subtypes of the immune system in Parkinson’s disease? Is that plausible?
Cross-pollinating and accelerating innovation
- (How) do immune system researchers work together? Are there collaborations of research groups in the world? If not, wouldn’t it be more fruitful if these connections were formed?
- When will the drug’s name – the TLR4 inhibitor – be revealed? At what stage of the investigation? And why hasn’t that already happened? For patients themselves, it seems important that as many eyes as possible are focused on interesting leads?
- Are you already a member of the Dutch Parkinson’s Scientists?
In fact, these are questions that are completely in line with the open science program of Utrecht University. Patient researchers at the Dutch Parkinsons Foundation also believe that working according to the values of open science is important. We think that this will allow us to achieve our common higher goal faster: eradicating Parkinson’s from the world.
‘Open Science’ stands for the transition to a new, more open and participatory way of conducting, publishing and evaluating scholarly research. Central to this concept is the goal of increasing cooperation and transparency in all research stages. This is achieved, among other ways, by sharing research data, publications, tools and results as early and open as possible. Open Science leads to more robust scientific results, to more efficient research and (faster) access to scientific results for everyone. This results in turn in greater societal and economic impact | National Platform Open Science
Part 2. The afterthoughts
Time passed quickly. There was no time left to evaluate and tick off mutual expectations for the sequel. But time itself doesn’t care. Time is patient and infinite.
So after the working visit, we did a tour of the patient researchers and members of the PK-PAR to ask for tips and tops. Then it turned out that we not only have to close the gap between mice and humans, but also between humans themselves. We don’t know each other that well yet and it turned out that unfortunately, it had been difficult for some of us to follow the presentations. This was due to a combination of Parkinson’s, the technique, the set-up, the background knowledge, and the dust that had settled on listening skills for (biomedical) English.
A tip from one of the participants was: “If you have to communicate in a language that is not your mother tongue, in a room with bad acoustics, then you should at least be able to see each other”.
The content matters, the form matters, and the context matters. And especially the coordination between the three. Nice learning point!
Furthermore, the Parkinson’s researchers and the patient researchers independently answered a number of questions about the expectations for the follow-up. By expressing expectations in advance, you increase the chance that the collaboration will pay off. That you really know how to bridge the gap between the two worlds in a way that adds value for both parties. In a next meeting – which has yet to be planned – we will compare expectations and discuss the questions we have.
It has already been an instructive journey, from mouse, to Parkinson’s researcher, to fellow Parky and back again. I’m looking forward to the sequel!
Marina, Monique, Leny, Joost, Theo