Modulating the peripheral immune response: LRRK2 and GBA1 mutations

In this study, the authors explore how two of the most important genetic forms for Parkinson's disease influence immune response to bacterial simulation highlighting a potential link between genetics, environment, and inflammation in Parkinson's. Today, I am very happy that I'm joined by one of the corresponding authors of the paper, Rebecca Wallings, researcher from the Indiana University at Indianapolis.

Becky, welcome to the podcast.

Dr. Rebecca Wallings: Thank you so much for having me, Michele.

Dr. Michele Matarazzo: So I'd like to start from the big pictures. This is a complex topic. [00:01:00] I think we are just starting to understand a little bit more about this things and about the interplay between neurodegeneration, inflammation, immune response, these kind of things. And there's been a growing interest in the role of immune system in Parkinson's disease over the past years.

Now your study really is in between an intersection between genetics and immunology. Can you tell us what motivated you to explore how LRRK2 and GBA1 mutation affect immune response? And particularly in the context of exposure to an external agent, a bacteria in this case?

Dr. Rebecca Wallings: Yeah, absolutely. I'll start off with why LRRK2 and GBA, so for listeners who might not be familiar with these two genetic risk factors associated with Parkinson's disease. We decided to look at LRRK2 and GBA, 'cause LRRK2 is the most common familial mutations associated with Parkinson's disease and GBA is most common genetic risk factor associated with Parkinson's.

And the reason that we wanted to look at these two [00:02:00] genetic perturbations in parallel is there's a lot of functional convergence between the two proteins that they encode. And they seem to converge highly in the immune system, both LRRK2 and the protein that GBA encodes, GCase. A highly expressed in the immune system, myeloid cells in particular.

And they also have a highly functional role in lysosomal biology, and we've known as a field that both LRRK2 and GBA modulate lysosomal function and their mutations can perturb lysosomal function in neurons, but also in the context of immune cells both inside and outside of the brain.

As a lab, this paper in particular was conducted whilst I was still a postdoc with Malú Tansey. I've since started my own lab and now she's my colleague which is very exciting. We were very much interested in the role of the peripheral immune system in Parkinson's disease as it's been becoming more and more apparent over the last, I would say, two decades, [00:03:00] that not only is neuroinflammation causative in Parkinson's disease in the sense that it is not just simply a byproduct of neuronal death and neurogeneration, but it's actually contributing to the disease.

But this inflammation is not, confined to this central nervous system. We see systemic inflammation in patients in our preclinical models. And prior data that we'd accumulated before this paper, in collaboration with the University of Sydney in Nicolas Dzamko's lab, we'd found that patient PBMCs harboring either these LRRK2 or these GBA1 mutations had these differential responses to stimulations with regards to their immune responses and their lysosomal biology.

But in that preliminary data we were looking at maybe a less physiological immune response because we were essentially taking these PBMCs from patients, plating them, and treating them with various different cytokines. Which is a great way to obviously stimulate and activate the immune system.

But we wanted to transition to some [00:04:00] more arguably physiologically relevant immune stimulants in the sense that we wanted real world bacteria, real world viruses, and understand how do patient immune cells respond to these. Because we believe that Parkinson's has a gene by environment interaction with regards to the development of the risks.

So we want you to understand how do these genetic mutations affect the immune response to real world environmental pathogens that people would encounter on a day-to-day. So why pseudomonas aeruginosa in particular? For those that might not know what aeruginosa is, it's opportunistic bacteria that is very commonly found in soil and in water.

It's also a big source of the infections that people will get in hospitals as well. There is not necessarily epidemiological data linking Parkinson's with this particular bacteria in the sense that the more you're exposed to it, the more at risk you are for getting Parkinson's. But data from our labs over the last few years, we've found that, especially with the LRRK2 mutations, if you give the [00:05:00] pseudomonas aeruginosa to say a LRRK2 knock in preclinical mouse model of Parkinson's disease, the way that these mice and their immune system handle this bacteria it's fundamentally different to the way that I say a wild type black six mouse would respond to these bacteria. So we wanted to understand, okay, do we see these in patients as well?

Dr. Michele Matarazzo: Wow. So that's a lot of information. And so it's really interesting because you've been working your lab and Dr. Tansey's lab has been working on the immune system by giving the step also in not just looking at the inflammation, but how the immune cells respond to a challenge, an external challenge, and an external challenge that is present in our real world outside. Right? So I think that's very interesting approach. And now talk me about the background. Let's move let's move to the study itself. Let's talk about the design. So you included patients with LRRK2 mutations with GBA1 mutation, idiopathic [00:06:00] PD, healthy controls.

Before we go into the results, could you briefly explain how these groups were selected and what you were aiming to compare across them?

Dr. Rebecca Wallings: Yeah, no, absolutely. So obviously these individuals who volunteered to donate their PBMCs for this study were age and sex matched. 'cause we know that there is different immune responses at different ages and different between males versus females as well. These PBMCs in particular were sourced with a Michael J. Fox Foundation funded collaboration with Roy Alcalay at the University of Columbia and also Tel Aviv. And Alicia Garrido, who is at Barcelona, clinical hospital. These individuals were particularly sourced for a presence of either a LRRK2 mutation or a GBA mutation or individuals that have Parkinson's disease with no known mutation.

Certain exclusion criteria included individuals that had concomitant autoimmune diseases because obviously that would be a huge confound for our kind of study. And we [00:07:00] also had to exclude individuals that were on prescribed anti-inflammatory. Anything that would modulate the immune system we had to exclude them just 'cause obviously that would be a huge confound and inhibit our ability to interpret our data. This paper is really the result of a six year long process of us collaborating with these various different neurologists providing the patient PBMCs and us working in collaboration with Nicolas Dzamko at the University of Sydney, where through various different phases of this project we were optimizing the protocol to biobank these PBMCs, have them cryopreserved and then them be able to be shipped to either we were at the University of Florida when this paper was being carried out or University of Sydney so that we could then cryo recover these PBMCs and then do ex vivo assays on them. Which has been a, I guess like a rate limiting step for the field for a relatively long time. The PBMCs [00:08:00] don't always cryorecover very well, you don't really cryo preserve them very well sometimes. We had to optimize this protocol in order for us to cryo recover a high yield and high viability of PBMCs. And that in of itself actually took a couple years to get it right and make sure that we could run these assays in parallel across two different institutes and make sure that we were able to see the same phenotypes, we were able to replicate each other's findings. So this paper really was a labor of love.

Dr. Michele Matarazzo: Yeah. Now, before going into the findings, what did you expect? And mainly maybe the question is, did you expect to have the same results between LRRK2 and GBA or any difference that you already thought that you might be there.

Dr. Rebecca Wallings: That's a really good question. For context with these PBMCs, we plated them and then we treated them either with a vehicle or pseudomonas aeruginosa. But then we also co-treated with different compounds that will target either the. LRRK2 kinase domain or a certain compound that is known to [00:09:00] increase GCase activity.

'cause with these GBA mutations, you have a decrease in the in the GCase enzymatic activity that the GBA gene encodes. So one with regards to these different treatments whatever phenotypes we observe, we thought that these treatments would obviously ameliorate them. And what we were expecting to observe it's been reported in previous papers Matt Lavoie's group who's still at the University of Florida, as one of the groups that's reported this, that shows that LRRK2 mutations are able to modulate GCase activity.

So individuals that don't have GBA1 mutations but have a LRRK2 mutation it seems that there is altered GCase activity as well in these individuals. So there's definitely some kind of convergence and kind of crosstalk between these two proteins specifically at the lysosome. So we were fully anticipating that the treatments of these drugs would ameliorate phenotypes in the different patient groups as well.

Dr. Michele Matarazzo: And before we actually get into results, I'm staying a little bit on the methods here because it's very interesting. One of the things [00:10:00] that I saw is all the LRRK2 were the same mutation, G2019S, while in GBA1 there were different types of mutation.

Do you think that implies some variability also because those different mutations don't suppress the GCase activity at the same level.

Dr. Rebecca Wallings: Yeah. You hit the nail on the head. Absolutely. So like you said, with the LRRK2 mutations we were able to have all G2019S carriers. It is the more common mutation out of all the LRRK2 mutations. So it made sense for us to be able to have a lot of G2019S. Unfortunately when you're working with patient populations, you have to work with what you have.

And it would've been wonderful if we could have either stratified the GBA patients based on their specific GBA mutation. 'cause you are right, these GBA mutations do decrease GCase activity at different rates. And we did see a lot of variability in our GBA patients, and we do think it is driven primarily because of their different GBA mutations.

But unfortunately just with the end that we had of these patients, we weren't powered enough to stratify [00:11:00] per mutation. Absolutely ongoing research is gonna be looking at that 'cause we wanna understand a big focus of both mine and Malu's Lab is trying to understand how these different immune responses could be contributing to the heterogeneity that we observe in Parkinson's disease, in this clinical sense, in the pathological sense as well.

So we absolutely do want to understand what these different mutations are doing because we believe that there's gonna be different responses, especially in the immune system for sure.

Dr. Michele Matarazzo: Okay. We look forward to further studies published by you and your group. Now, let's go to the results of this one. What happened when you looked at GBA1 mutation and immune response?

Dr. Rebecca Wallings: I will try and keep it brief into the highlights. 'cause I'm sure as you went through this paper, there's a lot of findings. We have various different groups, various different treatments, various different immune cell types. We are looking at different readouts. It's a very results dense paper, so I'll keep it to the highlights.

One of the things that we found that was one of the less surprising [00:12:00] observations in particular the GBA Parkinson's PBMCs, when we treated them with the pseudomonas aeruginosa, they had increased secretion of cytokines into the medium. So they had a increased inflammatory response to the pseudomonas aeruginosa.

That wasn't the most surprising thing we observed because there's a lot of literatures to suggest that there is an increased pro-inflammatory response in Parkinson's disease. There's increased inflammation. It could be contributing to the neuroinflammation and subsequent neurodegeneration that we observe in the brain.

So that wasn't too surprising. What was somewhat more surprising is that we didn't observe too many cytokines secretion phenotypes in the LRRK2 patients. In fact, in some of the cytokines, so with TNF and IO 10, there was actually almost a decrease in the response that LRRK2s were actually secreting with their cytokines. Which, maybe I'll talk about that afterwards when you ask about the implications of of these [00:13:00] findings. So it seems like that the LRRK2 and the GBA were responding very differently, which in of itself was somewhat surprising given the fact that we know that LRRK2 and GBA converge on very similar pathways but they seem to not be responding the exact same way, at least to pseudomonas aeruginosa.

So that in of itself was very interesting. What was also a very unsurprising result was the compound that we were using to increase GCase activity didn't really actually seem to do much to the GBA patients. We saw these altered cytokine responses. We saw some altered GBA dependent phospho RAB10 which is a a kinase substrate of LRRK2.

So it seems like GBA is altering LRRK2 activity somehow in these immune cells. But when we hit them with the GCase activator, we didn't really see that much of a response. And one of the things that we discuss about why this could be happening is that there could potentially be a [00:14:00] compensatory response from other lysosomal enzymes to counteract the fact that there is lysosomal dysfunction caused by this GCase activity, and then by activating GCase, you almost hit a ceiling effect. That the lysosome have already started to compensate and keep themselves happier. So the presence of this GCase activator doesn't really do that much.

What was really interesting is that when we took the LRRK2 mutation, the LRRK2 mutant PBMCs, and we treated them with the GCase activator, we actually saw a fair amount of different responses. We saw increase in MHC2 expression on the plasma membrane of different monocyte subtypes.

And for those of listeners that might not know about MHC2 is the mouse gene, but HLA-DR in human. It's antigen presenting complex that's presented on antigen presenting cells. And we actually saw an increase in this when we treated the LRRK2 mutant carrier PBMCs with this GCase activator.

And we talk [00:15:00] about why that might be happening, due to the fact that there is lysosomal dysfunction in the LRRK2 patients, the presence of this GCase activator kind of kickstarts that system and allows these different antigens that are being processed in the lysosome that then subsequently gets shuttled up to the plasma membrane to be presented to T cells.

We think that kind of kickstarts that system. So the fact that we were able to see that in the LRRK2, but the GBA almost had this ceiling effect that we weren't able to observe, was really interesting. 'cause it shows that there's a very complex crosstalk between these two proteins and especially when they're mutated, that absolutely needs further dissection to understand where they're talking and why hitting LRRK2 with GCase activators does one thing, but it doesn't do that to GBA mutations that have less GCase.

It was an odd observation and we're still scratching our head over it.

Dr. Michele Matarazzo: Yeah. I think this opens the field to new ideas and new the hypothesis that will launch new projects. And actually you've been very [00:16:00] clearly explaining how GBA and LRRK2 are of the genes that are mostly related with Parkinson's disease, but from a clinical standpoint, they are on an individual level indistinguishable from idiopathic Parkinson's. But on a group level, they do have some differences. And for example, we know that GBA have a different non-motor phenotype. A LRRK2 it's basically the opposite from the non-motor point of view, they tend to do a little better.

So do you think that this differences that you see from immune and inflammatory standpoint have anything to do with the phenotype, on one hand. And also not only they're different from the phenomenological point of view, but they're also different if you look at the brain people with LRRK2, for example, they tend to have less or lower amount of alpha synuclein aggregation compared to idiopathic PD or to GBA-PD, where the synuclein is supposed to be the main [00:17:00] protein aggregated and if you look at the recent data with alpha synuclein seeding assay you will even have a little bit higher number than idiopathic Parkinson's disease and GBA mutated people. So you think the pathological and the phenomenology have anything to do here with what we see in the immune response.

Dr. Rebecca Wallings: Absolutely. Obviously right now, everything that I'm about to say is a hypothesis. It's speculative, obviously. So another paper that, first author of this paper was Dr. Julian Mark, who at the time was an MD PhD student in Malu's Lab who I had the honor of supervising as I was a postdoc with her.

He had a another paper that came out a little bit earlier than this paper. We didn't have any LRRK2 or GBA mutations in this paper. We had idiopathic Parkinson's patients as well as individuals that had RBD. So REM behavioral sleep disorder as a kind of prodromal group. And then neurologically healthy controls. And we did a similar kind of experimental paradigm in this experiment in [00:18:00] particular, we weren't using a bacteria, we were using cytokines to stimulate the immune system.

But what Julian did is he stratified the individuals that had Parkinson's, he stratified them based on whether they had early Parkinson's. So they'd been diagnosed within the last five years, or if they had what we were referring to as moderate Parkinson's disease. So they'd had Parkinson's for 10 plus years.

So they were just at a different stage of clinical progression. And what he found is, at least with the way that these immune cells were secreting cytokines, is there was a really nice correlation between the amount of cytokines that were being produced and these individuals UPDRS scores.

So it seemed like the more immune dysfunction that was being present, the more motor clinical symptoms that these individuals were presenting. And is that driven by differences in the immune system itself, or is it just because they are, slightly different ages. 'cause obviously the individuals that were in the moderate Parkinson's [00:19:00] disease were slightly older, obviously they're the ones that had early Parkinson's disease.

Was it a byproduct of just motor progression and neurogeneration and increased disease progression kind of feeding back onto the immune system? We're still not entirely sure, but it does seem that alterations in the immune response does have some kind of correlation with these clinical phenotypes that we observe. And we absolutely think that this will also be the case when we look at different patient mutations as well.

So if we have individuals that have GBA, LRRK2 mutations, we absolutely anticipate that we will see very different immune responses based on genetic status, disease status. And one of the other projects that we're gonna be starting in the next few months in my lab is we are trying to understand how markers of an aging immune system, an aging immune system sounds like a wishy-washy way of describing it, but essentially we're very interested in immunosenescence and immune cell exhaustion and this idea that [00:20:00] there's a suppressed immune response occurring in certain patients. And we are gonna be looking at these phenotypes in different LRRK2 mutations, GBA mutations, idiopathic Parkinson's disease, males versus females, to try and understand how these different immune phenotypes correlate with clinical and pathological progression as well to understand how it contributes to this heterogeneity.

Dr. Michele Matarazzo: Okay, so lots of work going on. Now one of the things the results you show that genetic mutation also shape how the immune system respond to a pathogen. What do you think this tells us about the role of infections or environmental exposures in Parkinson's?

Dr. Rebecca Wallings: I think it shows that there is absolutely a gene by environment interaction, especially when it comes to the immune system in Parkinson's disease. I think LRRK2 is a really great example to really emphasize that point where we know that LRRK2 mutations are not 100% penetrant.

And we know that LRRK2 [00:21:00] mutations are also associated with various different infections and other inflammatory associated diseases such as Crohn's disease and ulcerative colitis. It's been very well studied in the field of tuberculosis. We know that LRRK2 is functionally responsible for immune responses and responses to different infections. And I think that in of itself is a huge piece of evidence to suggest that there is a role of infections and these kind of environmental inflammatory triggers in Parkinson's disease.

But as well, I think if we just look at the epidemiological data, like the Spanish flu for example, where we see when that was occurring there was a huge uptick in individuals diagnosed with Parkinson's disease in the subsequent years, suggesting that the exposure to chronic infections and subsequent chronic inflammation really does predispose individuals who are already at risk, potentially genetically for Parkinson's disease, to then cross over that threshold to develop Parkinson's disease and get that clinical diagnosis.

Dr. Michele Matarazzo: [00:22:00] Great. Now, looking forward you already have unveiled some of the projects you're working on for the future. What would you say are the next key question that need to be addressed following this work?

Dr. Rebecca Wallings: No, absolutely. One of the things that my lab and Malu's lab as well are most focused on is how do we target the immune system and, also in who, and also when as well. I mentioned one of Julian's other papers where we looked at individuals who have RBD and compared them to individuals that have early or moderate Parkinson's disease.

And what we observed was so interesting and it really changed the way that I look at inflammation in Parkinson's disease. What we found is that individuals that have RBD they had this kind of hyper inflammatory immune response to different immune activators. But then as you went from RBD and then into looking at the early Parkinson's and moderate Parkinson's individuals and their PBMCs immune responses, it almost was this kind of [00:23:00] hyperactivation followed by this subsequent suppression in this kind of stepwise manner where you have this huge activation of the immune system in RBD.

But as you go from RBD into early Parkinson's into moderate Parkinson's it becomes more and more suppressed. And that was very surprising because we think a lot of the field views inflammation as something that is hyperactive. It is increased. It's something that needs to be suppressed.

It needs to be quashed. Which is why a lot of the clinical therapies that have made it to phase two clinical trials have taken this kind of suppressant, anti-inflammatory approach. But none of those clinical trials have yielded any promising results. I think, and my hypothesis of my entire lab is we've been looking at inflammation kind of the wrong way. It seems that in the kind of prodromal years leading up to Parkinson's, yes there is chronic systemic inflammation happening. There's potentially hyper inflammatory immune responses. But one thing that happens [00:24:00] in the human body, in the face of chronic inflammation in general. But then if you then combine that with an aging immune system, which naturally does come more suppressed as we age.

The immune system will adapt. It will always try and modify its own responses in order to stop damaging the host. 'cause that's its priority, is that it wants to protect the host, right? So if it's bombarded with this chronic inflammation, there's this hyperactivation for years and years, over time the immune system will adapt and it will compensate by suppressing itself.

And a lot of my research is a postdoc with Malu that I'm now working on in my own lab suggests that there actually might be suppression of the immune system happening in individuals with Parkinson's, which, when I say that, some people look at me a little bit funny and say, what do you mean?

That's not what the field has been saying for the last 10-20 years, but it seems like there is an inability of the immune system individuals with Parkinson's to actually resolve the inflammation, which is the problem. There seems to be this low grade chronic inflammation at baseline that is just always [00:25:00] present, but then if you hit them with an infection, cytokine activation, whatever the immune challenge is, it seems that there's a almost an inability to respond and resolve subsequent inflammation, which means that the chronic inflammation just stays there. For a long time. So instead of trying to suppress a potentially already suppressed immune system, my lab's approach and we're trying to understand if this is a therapeutically relevant way of targeting it in Parkinson's, but we actually wanna, I'm not gonna say activate the immune system, but rejuvenate the immune system.

Try to bring it back to a state of homeostasis where it is able to respond to immune challenges in a way that it once did when it was a lot younger, I guess is the word to describe it.

Dr. Michele Matarazzo: So instead of using that hyper inflammation phase as a window to suppress it is to avoid suppressing it later on.

Dr. Rebecca Wallings: Yes. So this is when the, when do [00:26:00] we target it and how do we target it comes into question. So the way the, and this is absolutely an oversimplified way of describing it, but the way that I think about it in my head is if we're thinking about targeting the immune system in individuals who are at risk for Parkinson's, so the prodromal stages where there is this hyperactivation that's causing this chronic inflammation.

In those individuals, it absolutely makes sense to try and suppress the immune system because we wanna get rid of that chronic inflammation. But it seems at least in the context of my research, right now most of my research focus has been on LRRK2 mutations. So whether this is relevant to other genetic mutations or idiopathic or sporadic Parkinson's, we're still not quite sure yet.

But it seems at least with the LRRK2 mutations that we've been studying in my lab, it seems that once Parkinson's disease there's onset and there's clinical symptoms and they've received that diagnosis, it seems that the immune systems actually has this kind of suppressive a phenotype.

So potentially, trying to use that suppressant [00:27:00] anti-inflammatory approach with those individuals wouldn't be appropriate, would have to try an alternative therapy to bring that immune system back to a well-functioning state by I don't wanna say increasing the activity. 'cause you don't wanna increase the activity and immune system that is already dysfunctional, support.

There we go. Yes.

Dr. Michele Matarazzo: Okay. Becky, thank you so much. If you had to pick one message that you want a clinician or researcher interested in this area to take away from your study and your work, what would that be?

Dr. Rebecca Wallings: I want people to understand that the peripheral immune system is a contributor to Parkinson's disease. So much of our research now suggests that it is systemic inflammation in Parkinson's and other neurogenerative diseases that is etiologically relevant. It is not just neuroinflammation within the CNS, it is also occurring outside the brain, which I think opens up a whole avenue of potential therapeutic implications.

The immune system outside the brain is far easier to [00:28:00] target. Rather than trying to target the immune system within the brain. So if we can try and understand how the peripheral immune system is contributing to these diseases and how we could potentially target it in the context of disease, I think will open up a lot more accessible therapies.

Dr. Michele Matarazzo: Well, Becky, thank you so much for joining me today. It was really a nice discussion on a very relevant and poorly addressed and understood topic. I would say. 

Dr. Rebecca Wallings: Thank you so much. It was a pleasure.

Dr. Michele Matarazzo: Okay, we've been discussed about the paper Parkinson's Linked LRRK2 and GBA1 mutations modulate the peripheral immune response to Pseudomonas aeruginosa published on the Movement Disorders Journal.

So I certainly invite you to go and take a look and thank you for listening. [00:29:00] [00:30:00]