Air pollutants promote dementia

From micro-up TV. This is twin this week in neuroscience episode number sixty five recorded on October 20, 20, 25 I'm Vincent Racken yellow and you're listening to the podcast all about the nervous system joining me today from New Orleans with your Morrison. Hey guys. How are you today? I'm doing very well. Thank you. Is the weather wonderful down there? It is so nice. It's cold. It's you know 78 degrees and it's cold. 78 degrees. Actually I have no idea what temperature it is but it's really nice. Here at 16C. Okay. Which I think is 61 60-ish. Also joining us from New York Tim Chung. Hello. Hi everyone. Yes. It's quite nice day today actually. Temperature is pretty good. Yeah. This weekend was very warm. Yeah. I thought we're supposed to be heading towards winter but I'm not complaining. Winter. New York doesn't have a real fall. You know you've seen those into Instagram memes when fall is a rapidly developing. It takes many many steps and then there's one day of fall and then you go right into winter. You have fake fall. You have it's very funny. Anyway, if you like these programs we'd love your support. We don't do advertising. We just depend on your donations to enable us to make these programs and we are micro TV is a nonprofit. So in the US your donations would be federal US tax deductible although for me it doesn't matter. You can have all the deductions you want. It's never more than the standard deduction but if you maybe you're better than I am but still we'd love you to donate just to support the dissemination of good science information. That's what we do on these programs. Go to micro.tv/contribute. Now it's very interesting that a theme on TWIV has been dementia and viruses. And in Friday we did a paper showing that reactivation of varicella-zostivirus is a risk factor for dementia or wool sorts. That being shingles and chicken pulp, right? Yeah. And if you get a shingricks help. Well, shingricks in the other one, the attenuated vaccine which is older that helps. So it really makes a difference. It reduces the incidence of dementia remarkably and it's a really big study and they control for all the confounding factors. And that's a problem with an observational study which we'll see today. You can never eliminate all the confounding factors. So in observational study you can never conclude that X causes Y. You can never conclude that Tylenol causes autism. Never. Even if you had great data. Well, you could say that there's an association and then you'd have to figure out another one. If there was, if there were one. If there were one. But of course, the case of autism in Tylenol, there is not. But so that's a good question. If you find no association, do confounding factors not play a role in it? Could they be driving the factor that they know? Confounding. Same way. Confounding a factor could obscure some true underlying effect, yeah. So it can work either way if you're looking for an association or not. Okay. But anyway, you can get around that. In this paper, they had multiple hypotheses that they tested and they're all internally consistent or you could also do experiments in the lab that could support. Yeah. Or if you're the president, you can get your population to try to do an experiment by telling them to stop taking Tylenol when pregnant. Yes. But I mean, if he had a bit more organ, if he was a bit more organized and did it seriously, he would actually get people to keep track of when they start taking Tylenol so that we can collect some data. Yes. But he just laid it out then. So I had two doses of shin bricks. So after the twiv, I was very happy. But now today, you're going to see that there's more than one cause of dementia. We're all screwed. We're all screwed. Yes. We should be listeners. That's it. Take away messages. We're all screwed. I thought the same thing when I read this paper, we're all screwed because maybe we should be hunter-gatherers, but we'd still be screwed because the lions would eat us, right? Yeah. You'd die of exposure. In the paper, they have a map where they show the air quality because it's what we're going to be talking about today. And it's really interesting. It makes sense. There's some parts of the country where it's like the air is pristine. Other parts, not so much. And then have to see how that overlaps with the condition that they're looking at is pretty interesting. And actually, it wasn't. If you looked at those maps, it didn't look like it was obvious. Anyway, the paper is going to be presented by Tim. Another paper about dementia. Okay. Should I start? That was your lead-in. Okay. So today, so I think we got a bit of setup already. So today, we're going to talk about this paper that came out in the Journal of Science quite recently in 2025, September. And it is called, the title is "Louis Body Dementia Promotion by Air pollutants." And let's go to the authors. So there are, I think, four first authors. And they are nearly in other areas. Yeah, a thousand authors here. The first authors are Zhang Liu Wu and Jia. Sorry about the pronunciation. I speak Cantonese and not really Mandarin. That's blame my parents. Don't actually don't blame my parents, blame me. And three last author, I believe. And that is Ted Dawson, Xu Zhonghan, and Xiao Bo Mao. And these authors are by and large, I think, from Johns Hopkins in the U.S. and also Columbia. And also Columbia. Yeah. I think that comes from New Orleans. There is. Oh, no. No, there is. And then I was worried that they, in the paper, they were going to say something about how New Orleans is like the worst place to be ever because of, you know, the answer. It does look, oh, there's that what it is. Yeah, there's a lot of chemical refineries and stuff. Okay, so there are actually, I see a lot of other places, like Georgia Tech, Texas Health Science, even in Pure College and University of Cambridge, Shoutout UK. So there's very much an international group of people, actually. As you can tell, they're going to gather data from around the world. But we're going to talk about, so two things that we're going to talk about, which is one, Louis body dementia. So I'll explain what that is. And the second one is air pollutants, which probably is self-explanatory. But the reason why I picked up on this paper, so a slight, like another intro is, so just for listeners, benefits, I actually do research on Parkinson's disease. So that's my day job. And in parking, if you're working Parkinson's disease, the one celebrity actor that is a champion on Parkinson's disease that everyone knows about is Michael J. Fox, aka Marty McFly, who got Parkinson's very, very young. I think he was like 29 below 30 when he got worst diagnosed, which is very atypical. And then since then, he's been, he has a Michael J. Fox Foundation, which is a foundation that both raises awareness for Parkinson's disease and also does funding and also do research grants for Parkinson's disease. So Michael J. Fox has been a huge champion to promote awareness about Parkinson's disease, also help research. But this may be, at least for me, the second person I know that comes to mind when we talk about Parkinson's disease, bit of a stretch is actually Robin Williams, which I don't know if it listeners, if they're a bit younger, they might, they might not be as well known to them. But to me, Robin Williams is Genie from Aladdin. And he's also Peter Pan from Hoke. Oh, such a good movie. Other listeners might know him as, I don't know what his name is in the film, but he's the, like, psychologist in Goodwill Hunting. He's also, he plays a lot of, like, so many things, like a lot of like teacher character. He's like, patch out of the doctrine, patch out him, um, play a woman in one movie. Oh, Mrs. Delphire. Delphire, right. He plays a dad across dressing dad. Um, and, uh, but the, the movie that actually, I have been aware of, but never watched until I decided to do this paper is this paper called Awakenings, um, which is based on a book by the neurologist Oliver Sachs, which, um, maybe some of the listeners have heard of. Um, so in Awakenings, um, Robin Williams plays a neurologist who came across, in the 60s, who came across all these patients, um, one of whom is played by Robert De Niro, and all these patients suffered from, um, this disease called encephalitis lethargicus. Um, so these patients suffered from brain inflammation in the past that make them lethargic, make them not move. And, uh, Robin Williams in this movie in this film kind of shows up to a, uh, neurologist, neurology ward and all these patients don't move. And they haven't been moving for 20 years. And then, uh, Robin Williams go around trying to being a new doctor, look for ways to help them. And then found this drug recently discovered drug called Leverdopa, which is a precursor to dopamine. It gets converted to dopamine in your brain. And he gave patients all the patients, L-dopa and all these patients went from kind of statuette, not moving to like becoming alive again. And then they were, you know, uh, dancing around, um, uh, on the dance floor, you know, playing baseball, all kinds of stuff. And it was a wonderful film. It is a little bit sad because it turned out the effect of Leverdopa was short lasting. It worked for one summer. And then for still a known reason went away. Um, but it's a very interesting movie to watch. I reckon that's a real story. It's a real story, like, um, and so the light has lethargicus is real. And people, um, we don't still don't know what causes it, which is scary because it can come back. It can reappear again. Um, but, um, according to all of us sacks, he thinks there's some evidence, which is still debated. That is an intro virus. So Vincent, you might be interested. Um, but, but there's no way of knowing, um, because we don't, um, I mean, maybe we can go to look at some, uh, autopsy samples and try to look for viral particle, but I think it's going to be hard. Um, so, um, it could be a hit and run too. And the virus is long gone. That's, yeah, exactly, exactly. Um, but it's very, um, it suggests that, you know, Parkinson's is Parkinson's, Sony and Parkinson's symptom symptoms, um, can come, can be caused by many things. But anyway, that was quite a, quite a digression, but I brought it up because Robin Williams, who plays a neurologist who is, who diagnosed these patients with kind of Parkinsonian symptoms. He himself, um, actually, it's a very sad story. He, um, took his own life, um, I think maybe around 10 years ago. And before he died, he suffered from, like, severe depression. And also, apparently, it very rapidly, um, lost his memory and suffered from a lot of anxiety attack and, like, just, like, deep, deep fear. And also, um, weird symptoms, like insomnia and, like, maybe slight tremor. And, and this, um, and he was also acting. He was working at that time. And he kept, he went from being able to do, like, Broadway shows, like seasons of Broadway shows with, like, remembering, like, an hour worth of lines to, like, forgetting single lines in a movie shoot that made him completely, like, if you can't even do that when a hundred people depend on you for a job, that's like, you can imagine how much of an emotional toe it took on him. So he suffered from severe depression and took his own life. And there's, um, so there's a letter submitted by his widow, um, Susan Schneider Williams to the journal neurology that details kind of the episode of his death. After Robin Williams died, the, um, uh, the pathologist did an autopsy on his brain. And they found in almost every neuron that they, or maybe even of every cell of the brain that they examined, I'm guessing it's neuron, every neuron that they examined in the brain, there was this pathology called, um, uh, Louis body. There's this clump of protein that stains for, um, the clumps off both protein and lipid that it stains positive for particular protein called alpha-synucleon. And they clump up into these, um, thing that neurologists have called Louis body inside neurons. And it is completely widespread in his brain. Apparently, it's one of the most severe cases of Louis body disease. The, the neurologists have ever seen. And it was like a semi-miracle that Robin Williams kind of held it together for, for that time. Um, and that explains pretty much all his symptoms before he died. So, how old was he? Sorry. How old was he? I can't remember. Um, I look it up. Yeah, it was just how long he survived. He, I'm guessing, maybe 60s and 60s perhaps. Yeah, 63. Yeah, it's quite, quite young. Um, so, uh, this is, really, um, so it was really hard for doctors to diagnose Louis body diseases, Louis body dementia. Um, uh, because you usually diagnose it after autopsy to confirm with staining, labelling against this thing called office nucleon. Um, so, uh, Louis body dementia is, uh, share something with Parkinson's disease. Actually, that's, um, which is another link to the, um, movie awakenings. Um, in both Parkinson's disease and in Louis body dementia, um, there's a tell tell sign in the brain, usually established after death in autopsy, which is, um, a proliferation of Louis body in the neurons. Um, but the main difference is this in Louis body dementia, the patient presents at the beginning with dementia. So memory loss, um, kind of maybe paranoid delusion, um, sometimes often hallucination, whereas in Parkinson's disease, the patient's usually first presents with motor symptoms. So, slowing of movement, um, and tremor, etc, etc. Um, and, um, uh, in Robin Williams case, it, apparently the doctors thought he had maybe early Parkinson's because he had a psych trauma, but as it turns out, because of what these Louis body symptoms, and also the, the dementia that he basically had because his memory was gone, it's likely Louis body dementia. Um, so that brings us to today's article. So we do not know, so for Parkinson's, both Parkinson's disease and Louis body dementia, we really don't know what the cause is. About 10% of Parkinson's disease has been linked to genetic mutation. But the ninth, the rest, the remaining 90% is what, uh, doctors clinicians caught idiopathic, it has no known origin. But we know that our risk factors out there, for example, um, for Parkinson's disease, um, I believe farmers are at a higher risk of getting it because of some of the pesticides that they use, such as rotin and rotinone and paraquat. Um, for Louis body disease and for, um, Alzheimer's in general. So for Alzheimer's, Vincent talked about it a little bit. It sounds like the emerging evidence suggesting herpes virus, the herpes family, I don't know species class, whichever, hope these viruses, uh, might be a risk factor for Alzheimer's dementia. So dementia from Alzheimer's is the number one cause for, for dementia. Um, the second most prevalent cause is Louis body dementia. And we broadly speaking, don't know what, um, what the, what some of the environmental or genetic course of Louis body dementia is. Um, but this paper that we are talking about today, um, kind of zoomed in on one potential culprit. And that is air pollution. So apparently, um, there has been ongoing, um, evidence, people are still collecting it. And in science, uh, evidence kind of kind of goes up in a Bayesian kind of way as you collect more evidence, you are more certain about your, your conclusion. Um, so there's ongoing evidence that a lot of dementia, including Alzheimer's dementia, dementia from Alzheimer's disease seem to correlate, uh, with, um, air pollution. And in this paper, they looked at the other type of dementia, which is Louis body dementia. And what they did was, so we're jumping into, we're, enough kind of, um, enough, uh, pre-talking, we're going into the paper now. So what they did, um, the first experiment they did is, uh, epidemiological study, um, uh, they took in U.S. So we grumble about U.S. being no, you know, very insurance-based, no public health infrastructure. But there is Medicare, um, and also Medicaid. So that, so after you reach, so call me if I'm wrong, but I believe after you retire, your employer no longer sponsor your healthcare and you go on to this thing that is government sponsored, called Medicare. Um, and because it's government sponsored, there's a centralized database. So, um, these researchers farmed, they collected and analyzed the previous, um, or the Medicare record from, I think like 2003 to 2014, something they're about, um, that encompasses about 56 million individuals, um, in the U.S. Um, with, across the entire U.S. mainland, I guess what you guys called, what is a contiguous, continental U.S. I don't know what you guys call it. Basically it's good. Uh-huh. Excluding how are you in Alaska? Yeah. Um, they looked at all the U.S. and they know based on the Medicare record, whether the, when the patient, if the patient, um, falls into these category, whether the patient had, um, Parkinson's disease. And if the patient has Parkinson's disease, they divide Parkinson's disease into two different subgroup. First subgroup is Parkinson's disease without dementia. And the second, second group is Parkinson's disease with dementia. And the fourth category is this thing called dementia with Louis body. Okay, this is a bit confusing and I would only say this one's for completeness, but this is super confusing. I apologize. This is how clinicians do that, do this stuff. Why we've been talking about this thing called Louis body dementia. Louis body dementia is an umbrella term that encompasses two different, two different things, dementia with Louis body and Parkinson's disease with dementia. So those two depends on whether you present with cognitive problems first or motor problems first. But for our, for simplicity, um, we will just lump them both together as Louis body disease. I think for majority of this paper. So when I say, sorry, Louis body dementia, even I've been getting it wrong. So when I say Louis body dementia, just think of it as having dementia. But again, um, the, so that's separate, that kind of Louis body dementia is kind of, um, separated from the other thing that we're going to contrast it with, which is Parkinson's disease without dementia. So Parkinson's disease without dementia is mainly motor symptoms, whereas Louis body dementia is mainly cognitive dementia symptoms. Um, one very quick stat, which is, uh, patients with Parkinson's disease are apparently around 80% of them eventually develop dementia symptoms as well. So this is something to bear in mind with is that a lot of, um, Parkinson's disease patient or PD patients, um, they might just not yet have dementia. But for the purpose of, um, this paper, it's PD without dementia versus Louis body dementia. So having all these, um, all these kind of diagnosis from all the patients are either healthy or you have Parkinson's disease. Um, if you have Parkinson's disease, maybe you have Parkinson's disease without dementia or alternatively, you have Louis body dementia, even though it's complicated, but we'll ignore it. Um, so they have that. They also have your zip code. So they know where you live in the US. And they also have things like your socioeconomic status. They know what your races and your age, they're all these kind of potential confounds that can correlate out. But the zip code is the most important because the second dataset that they got was your air quality based on apparently, they are like 2000 plus, um, air quality monitoring system in the US maintained by the environmental protection agency, the EPA. If that's to exist. Yeah. Yeah. Who knows if it's going to exist? Um, I've been destroyed. It's just scary. I actually don't know how much doge cut it or like whether, I mean, it's, there's a huge shutdown out. So there's no data collection ongoing. I'm sure. But, um, but there's been the, during the same time period, the same tenure, the EPA has collected all these air quality from thousands of days in around the US. So now you can have basically a plot of every single county in the US, um, how bad the air quality is. And by air quality, for this particular paper, they mean this, um, technical term called fine particulate matter, I believe is what they called, um, fine particulate matter called, and they, um, they, um, shorten that to PM 2.5, particulate matter 2.5. And the 2.5 stands for, this is basically an air pollution. That is smaller than 2.5 micron micrometer, um, in diameter. So just to just for scale, a human hairs roughly 100 micron wide. So this is like, maybe 140th of a human hair. So it's very, very small, um, but it exists and it's floating all around us. So as someone who lives in New York in Manhattan, right next to a power station and also one block away from FDR, from a giant highway, I read this paper with some dread. Um, this is all around us. And, um, but depending on whether your, um, your city or your house is kind of out in the woods or in the middle of the, the, the urban area, or next to kind of heavy pollution, um, you would have varying level of this PM 25 pollutant. And in the, in the map that they showed, you can see, um, in the east coast is particularly bad. So like around, ooh, I'm not good with US geography, but like around Pennsylvania and New Jersey, um, New York, it's super bad. And then as you go, some Michigan, um, yeah, as you go west, but, uh, but Maine looks good. Maine, New Hampshire for a month. I love trees. I love where I'm north where the maple leaves are. Looks great. Yeah. The rust belt, uh, by the, by the name, you can imagine is super bad. Like in the last part, like two or two, like last year and also colorful. For the worst place, the worst place is LA. Yeah, I'm really surprised. It's black. It's like black. You could not, like it's, it's saturated. California is super bad. I'm a little bit surprised. Um, what's really interesting? Like just a, well, it's also the geography of the place. Like the mountain. Like it's in a bowl. Yeah. Yeah. I mean, we know that the, the mountains can relate, um, change how the weather patterns are down there. So I wouldn't, uh, I think it's kind of a sink. But I guess LA is also famous for its smog from like traffic and stuff like that. But this also accounts for several, um, um, things that can emit this PM 2.5 air pollution are traffic, heavy industry, but also things like wildfires, which we've been having in New York, and also LA a lot as well. I remember like, was it last year or two years ago, the wildfire from Montreal, I think, like all the maple leaves, um, came, came down and then even inside the hospital, it's smelt like smoke inside the hospital. So that, you know, that can't be good. Um, so episodes like this can really drive up the PM 2.5 air pollution. So you have all these, um, you have a map of all the counties of how bad things are. And I will quickly point out is weird. Maybe you guys, you guys, uh, both of you American can tell me why the counties like on the east coast are all nice. They're like very, very tiny. And as you go to the west, they just get kind of bigger and bigger. It's almost like as people migrate to the west. They're just fewer people. They, I thought they just like, they just said, uh, whatever. We'll just have a big county. We can't be bothered to. Well, you know, maybe it has to do with like, uh, yeah, there's few, there are fewer people and then they're, maybe at the time, there was more farming. Okay. That makes places are ranch ranches. And so the, you know, I mean, well, this is all speculation. Anyway, it's just a huge one. It's like very obvious like, pattern. Anyway, um, so after with this zip code, um, geography based air pollution, um, pattern, they next to it, they plotted the, the incidence rate or the prevalence of Parkinson's disease, Parkinson's without dementia and also the, uh, uh, leery body dementia. And based on these data set, they can next try to, as Vincent said, correlate it, um, correlate one with the other. So this is an observational study. You have data, you, that you have collected in the past, that you can correlate and what the, um, the researchers found is that if you have, if you look at just Parkinson's disease, which clumps together, Parkinson's disease without dementia and also Parkinson's disease with dementia, um, there's actually a significant correlation between air pollution and your risk of dementia. Oh, sorry, your risk of getting the disease, which is Parkinson's disease. Sorry. So Parkinson's disease by itself is, uh, correlated with air pollution and they express it as a hazard ratio. So like, per, in per, you know, increase of five microgram of air pollution, you get, you have a 10% increase risk of getting Parkinson's, for example. Um, however, when they stratified this Parkinson's disease into Parkinson's disease without dementia versus Parkinson's disease with dementia, which is similar to the body dementia, um, they find something quite striking, which is when you, when you look at the risk of getting Parkinson's disease without dementia, it is actually smaller. Whereas if you look at the risk, uh, as a function of air pollution, whereas the risk of getting Parkinson's disease with dementia, as a function of air pollution, it's shot up. So it suggests, really, that the, the, most of the effect of air pollution is increasing the risk of, um, driving Parkinson's disease with dementia. Um, however, if you look at even Parkinson's disease without dementia, there is a significant effect of air pollution. So air pollution still contributes, still significantly correlate with Parkinson's disease, even without dementia, but it correlates much more strongly with Parkinson's disease with dementia and also with three body dementia. So, um, this kind of, um, cause the, uh, researchers to conclude that we probably should look, so first of all, they conclude that there is a correlation at least in the US between air pollution, specifically this PM 2.5, these small particulates, um, with, um, uh, Louie body dementia. Um, so most study would stop here and, uh, wrap up and publish a paper. And as Finston pointed out, this is, uh, this would be, uh, observational study, which is pure correlation. Um, but it doesn't tell you anything about causation. Um, so the researchers, uh, for this paper, they didn't, they didn't stop there. They started there. So this is figure one. So by the way, Tim, um, I was just thinking that it would be nice to look at the core, if there's a correlation between pollution and something else, but in fact, the PD without dementia is the control in a way because that's low. The correlation with pollution is low. And then when you just look at PD with dementia, then it goes way up the correlation. Ah, that's, that's true. But the control, uh, the control kind of, so they have a different disease to study, yeah. So they have a comparison. Um, I mean, the control would be people who didn't even get PD, like the healthy control, perhaps, but it will be on a gradient. So they're going to correlate. Well, that's in the data. There are people in the study who didn't get PD. So, um, that's where the correlation comes. I'm just curious if air pollution causes everything, you know, but I was going to say it's probably unless it's correlating. But that doesn't, um, rule out, there is a third factor that everything correlates with that actually explains everything. Right. So it could be something that correlates with air pollution that actually is driving everything. Um, so we, so to really establish air pollution itself as the driving, um, risk factor that causes things, ideally, the, the researchers would do something that we can't do because it's unethical, which is grab a bunch of people, separate them into two groups, and one of group of people, you give them this air pollution and the other group you don't, and then you observe them. Um, which you can't do. So they did maybe the next best thing. So which is they did exactly what I said in mice. So, uh, the researchers, what they did was they went to some of these, um, uh, kind of air collection devices like from the EPA, but they also actually went around the world. So around the world, every country has their own monitoring system. And all these pollutants get trapped in these air filters in these, um, in these machines. And what you can do is you can grab the air filters out. Um, well, what the, the researchers did anyway, they grabbed the air filters out and they trapped out all these, uh, PM 2.5 particular, uh, matter particles. They cut it up into tiny bits and then they, um, soaked them in organic solvent. Um, so a combination of methanol and dichloromythane, something like that. So it dissolves all the fine particular matter. And then they evaporate out all the solvent. So all you are left with is the pollution in like, uh, particle forms. Um, and then you dissolve your, um, all these pollutants in PBS, which is just a, uh, science water. Sorry. Sorry. Sorry. Sorry. Very good. So I call it science water. Sorry. How do we do? I always use like molecular, like pure, yeah. Uh, which is also science water. Science, salt, science, saltwater, science water. Um, they dissolve it in, uh, uh, science water. Um, and then they actually, uh, find mice, uh, while type mice, the first experiment, they looked at mice and they pipetted it into the mouse, the mice, mouse's nose every two days for 10 months. So instantly the first critique is what on earth are you guys doing? Why don't you just grab the polluted air and make the mice breathe it. But as it turns out logistically, it's very hard. You've got to go and grab like 10 months, 10 months long of air supply for these mice to breathe. So it's really difficult. Um, so the authors did kind of the next best thing, which is they forced, they pipetted into the mouse's nose. And that would kind of expose the mouse to, they admitted a much higher dose of the air pollution than people are normally exposed to. This is 10x higher. And it is once every two days. So it's kind of like a pulsatile treatment of high concentration air pollution as opposed to a chronic exposure of low, lower concentration. Um, so this is one drawback that they point. But because of logistic reasons, that's the best they can do. Um, maybe there are some researchers out there now who are leaving a cage of mice by the roadside and come back in 10 months and see what happens. But for this paper, they had to go this way. So this is definitely one of the criticism. But what happens? So that criticism says them aside, what happens if you kind of nasally irrigate the mouse with these air pollution, air pollutants for 10 months? What they did was this. They, after 10 months, they euthanize the mice and they look at the brain, kind of grows morphology, kind of structurally what does it look like? So they have two groups. They have the mice who got the control P treatment, which is just PBS, the science water in the nose. So these mice don't show any kind of weirdness in the brain. The experimental group, which got the PM 2.5 air pollution, air pollutant in liquid form into the into the nose for 10 months, they show huge holes in the brain where the ventricles massively expanded. It is actually. That's where I was shot. I too were shocked. So in the, in the kind of in the pictures they show these ventricles like blew up hugely and the cortex shrank majorly and like it seems like the amygdala might have completely gone and also some of the basal ganglia structure like the GPE. So what do you think about that Tim? And also with hippocampus volume also dramatically. Yeah, I didn't, I didn't see that though in the pictures. I don't know if like I have to trust them. I don't trust anybody. I trust no one. But according to that, that was shocking. According to them, the hippocampus volume also dropped. So it is what it is. If it's true and I mean, they collected the data, it's, it is what it is. And they're going to tell us maybe how it correlates with some of the symptoms, some of the molecular symptoms that we're going to go into. But I definitely was a little bit. I'm like surprise because these are quite big changes in the brain. Like, they're just huge holes. There's no two way of saying anything. Yeah. So they also in addition to looking at looking at gross morphology. Oh, actually, yeah, okay. So they showed this to be, so this is the gross morphology. They actually have a second experiment with mice that have a mutation that knocks out their alpha-synuclein. So this is a quick, I'm going to make a very quick digression to talk about alpha-synuclein. So alpha-synuclein is this protein that you make in your neurons, also in red blood cell apparently, but we'll ignore that. Your neurons make this protein got alpha-synuclein. And normally they exist in the cytoplasm and apparently in the nucleus. So sine nucleon is based on two words, synapse and nucleus. Because alpha-synuclein, the protein alpha-synuclein is usually most concentrated, according to people, in the synapses where neurons talk to each other and also in the nucleus in a soluble form. But when something goes wrong in Parkinson's disease, with or without dementia and also a lue body dementia, you see these alpha-synuclein start clumping up into lue body and lue body have both alpha-synuclein and also a bunch of lipids or glombed up together. And people, the hypothesis is that the lue body, actually the alpha-synuclein in the lue body, cannot be appropriately degraded by your cell's protein degradation machinery. So you're suffering from indigestion. And which is a similar kind of hypothesis, people kind of have with other protein aggregation diseases such as maybe amylode-beta, which is extracellular, and also maybe phosphorylated taut. I think from aggregate, although don't quote me on that, I know a lot less about it. So these alpha-synuclein, there's several things that are of interest to it to us for this particular paper. First of all, when you have diseases, especially with Parkinson's disease and also lue body dementia, and also at different disease called multiple systems atrophy, MSA, which we won't go into it because it's not related to this paper. People have increasing evidence that these alpha-synuclein proteins, they not only do they clump up together, they clump up together in such a way that they they causes more normal alpha-synuclein to form extra clumps. And these extra clumps can spread from neuron to neuron or maybe from mouse to mouse to themselves propagate these clumps. What I've just described is a pre-on disease. So these are malforming, kind of malmyshaped or malform aggregated protein that are clumping up together, that can that first of all cause disease, and secondly, can transmit. Right, but there, so just I was just thinking, I was like, oh my god, so if you stand next to somebody, no, they're talking, Tim, they're talking about, let me say, it's like communicable. It's like they are injecting the protein that's been isolated from a mouse into a mouse that didn't have the condition to begin with, right? It's not like one mouse coughed on another one and then, you know, so don't worry. So yeah, exactly. So this is a huge, I guess it's a we should take a few PSA now, which is this pre-on diseases as far as we can tell is not transmitted by air or even by touching people. So for example, the original pre-on disease discovered by, characterized by Stan Prusner, which got him of Nobel Prize, was in scrapies on Matt cows disease, basically. So that involved feeding cows to cows and also sheep to cows and in here mixing up two things, Tim. Am I? So there's scrapies. So the prisoner worked on scraping, which was just transmitted among sheep. Okay, but the Matt cow disease epidemic was caused by feeding sheep to cows, and then I'm 100 cows to cows. Maybe I don't know. It got messy. But it is caused by the same pathogenic agent, which is this protein called the major pre-on protein. And in human, and that got transmitted to humans when humans ate disease beef, basically. And the scary thing is you can cook your burger to as high as, well, maybe not as high as you want, but to a normal, well done phase, well done, cooked. I don't know what the culinary term is. Make it well done, but you still get the disease. You still get the disease because the protein remained misfolded and it would make your own protein misfold. So that's one of the, but you can only get it as far as we can tell by either eating the disease burger or by having some sort of perhaps kind of blood contact with the protein, with a mouth-on protein. So I know about this because there was a case, maybe a couple of years ago, where a French undergrad got Matt cows, quote unquote, Matt cows disease, like CJD, the human form of Matt cows disease when she was volunteering in a lab that was working on mice that had a mutation that made them have pre-on disease. And I think she cut herself on a crowd stat or something like one slicing brain she cut herself. And that turned into CJD, the Matt cows disease in like 10 years later. It was a complete tragedy. Yeah. Oh my god. So, so we have to be careful when working on these kind of, we do, we should be cognizant at least as newer scientists. Anyway. Oh my god. So one of the, one of the characteristic, so we're going to go, so let's go back to alpha-synuclein. So the first, the first protein that shows some of these kind of first of all mis-shaped clumping together property. And second of all transmissible property is in the major pre-on protein that Stan Prusina worked on. The second protein that shows this property discovered by Stan Prusina, I believe, like maybe 10 years ago, was alpha-synuclein. So what happens in alpha-synuclein is this. First of all, you can extract brain tissue, post-mortem brain tissue from humans that died of Parkinson's disease or Lewy body dementia and run Western blot on it. And which basically separates protein into different sizes. And then you stain for alpha-synuclein. What you see is in disease brains, the alpha-synucleins are half much high molecular weight compared to in normal brain, suggesting that they're forming maybe clumps, maybe polymers, oligomers. Secondly, when you subject to these proteins to proteinase K digestion, so proteinase K normally would just go into your protein and start chewing up all the peptide bonds and turn it into kind of peptide monomers. So what you see is, as you expose these proteins to proteinase K, before you run the Western blot, your signal goes down, because all the proteins, all your endogenic sites where the Western blot antibody label would recognize, they get eaten up, so you can't label for them anymore. Well, in disease brains, when you try to do the same thing, proteinase K doesn't work. It doesn't digest these clumps very well. It suggests that these proteins have clumped up in such a way that it makes it makes proteinase K really hard to access them. So they're forming misshaped clumps. And thirdly, another characteristic these things have is, if you get brain tissue from disease patients, or even the CSF, the cerebral spinal fluid, which you get from spinal tap, you can put in a bit of these patient samples with Alphysinucleon monomers. So these are Alphysinucleon that you get by recombinant molecule biochemistry. So you kind of make bacteria grow these Alphysinucleon proteins and then purify them. So that is just a pipette full of Alphysinucleon. And if you just add PBS to this pipette, or this tube, sorry, not pipette, this tube of Alphysinucleon, and let us sit around, nothing much happens. But if you add some patient samples to this tube of monomeric Alphysinucleon, what happens is they start forming clumps. So the hypothesis is that Alphysinucleon from patient samples, because they are, they themselves are misfolded and aggregated. They act as a seed, and they make the monomers in your tube that are normally dissolved. They start clumping up. And you can detect these clumps by kind of putting in a fluorescent label called THT, which is a Thylflavin Mark fluorescent Thylflavin marker, which does matter. But these labels are fluorescent when they kind of intercalate. So they wedge themselves into beta sheet structures. So beta sheets are the secondary protein structure structures that are found usually in protein aggregates that form in kind of this amyloid form that we see with amyloid beta, for example. But when protein comes up in this mishapen neuro degenerative form, they form these beta sheet. And therefore you can kind of detect them using this THT labeling. So patient samples can make a test tube worth of soluble Alphysinucleon, quote, unquote normal healthy Alphysinucleon, start forming these clumps, these mishapen clumps. And you can then pipet out some of these mishapen clumps and they themselves act as seed. So it is transmissible to, first of all, to a new test tube. So you can make a new test tube start forming clumps. Not only that, you can get some of these kind of amplification assay. So this acts a bit like PCR, but with proteins. So you can use these seeds to amplify, to get more seeds. And you can get these, and the result is what they caught up protein, a preformed protein fibros, PFF. And you can get some of these clumped up fibros and inject it to a mouse. So we discuss this previously in twin 30. You can inject these mishapen fibros that you get from a test tube into a mouse's gut. And researchers have found that these, this would cause your gut to have disease, kind of you can stain for Alphysinucleon phosphorylation, which correlates with the disease state. And this disease pathology would go actually up your vagus nerve to your brain. So it would go from the spread from the gut to the brain, which is one root that people hypothesize, how is how Parkinson's disease is acquired is that you can get it via your gut, which correlates with a lot of kind of early symptoms you get from Parkinson's disease like constipation and gut problem. And what is more is once you inject these kind of disease mishapen fibros that you get from a test tube into the gut. And once the disease spread to the brain, you can harvest, you can get the brain from these disease mice, and they would seat a new round of disease. So once again, it's transmissible. So that's whole cycle propagate based on mishapen misfolded aggregated Alphysinucleon. And the air pollution causes the aggregation to begin with, right? Exactly. So if all that is true, I'm actually running off time. I better speed up. But we're setting the groundwork, so we can speed up in a bit for the rest of the paper. But if that is true, then Alphysinucleon is the main kind of thing in the middle that propagates all these disease. It is almost a driver, one of the driver of the disease. And if that's true, going back to our mouse, which got pipetted, which got exposed to this PM2.5 pollutant, if Alphysinucleon is very important, then in an Alphysinucleon knockout mouse, they should not show any of these symptoms. So at least these symptoms should be much reduced. And that is exactly what they saw. So first of all, surprise, Alphysinucleon is actually not necessary, so we can knock it out. And the hypothesis is that there are other kind of sineucleon that can upregulate, that gets upregulated to compensate. So like beta and gamma sineucleon gets upregulated. But if you knock out Alphysinucleon, the mice show protection against the massively enlarged brain ventricle and also shrinking of the cortex in a hippocampus. The second thing, so this tells you that Alphysinucleon is very important, is probably kind of the one of the driver of all the pathogen. The second thing that the researchers looked at is phosphorylated Alphysinucleon, which is one of the markers of in disease brain, in these Louis body where all the Alphysinucleon clumps up, you see a lot of high per phosphorylation of Alphysinucleon at this site, a serine 129. So low and behold, when you expose mice to PM2.5 with to the air pollution, you see much increase of this phosphorylation. Whereas you don't see any in the Alphysinucleon knockout because the protein is not even there to begin with. And then not only that, in these air pollution exposed mice, they have increased phosphorylated towel, which is a marker of Alzheimer's disease, funnily enough. So it also correlates with dementia and also frontal temporal dementia, which is another type of dementia, also has increased phosphorytile. But interestingly, cow is hyperphosphorylated, similar to Alzheimer's disease. But dissimilar to Alzheimer's disease, there's no increase in beta amyloid. So that is another marker for Alzheimer's disease, is that you get these plaques, which is rich in this protein called amyloid beta, or beta amyloid, I can't remember which one is one of those. I think they're both used. But you don't see it in with air pollution. So that tells you it might be slightly different. There's a different format of brain degeneration. And addition, all the glio cells got activated by air pollution. So there's an increase in microglio activation, and you can tell by the morphology, normal microglio, quote unquote normal, Vivian might start, but quote unquote normal microglio show these a lot of kind of proliferation of like not new rights, but they have a lot of, I guess, processes. They spread the tentacles everywhere. But when you cause an injury, you can beam like a laser, shoot a laser in, or you give it some stress, like for example, air pollution. These tentacles kind of shrink, and they switch form into a different activated form. And that's what they see with PM 2.5 exposure. And this is protected in alpha-synucleon knockout. So and separately, astrocytes also get activated. So whenever there's any brain injury and neurons start dying, so if you get apoptosis of neurons, what you get is astrocytes start proliferating. We call this gliosis. And you see this with PM 2.5 exposure, you get many more astrocytes. And this is again protected in alpha-synucleon, Vivian. I just wanted to say that I think, yeah, so that the figures associated with those findings are in the supplement, I think, right? Yeah. And so the astrocyte standing is pretty remarkable. How's it? It's huge. Oh, like huge up regulation. Huge up regulation. Yeah. But I think it's, you know, it's difficult about GFAP. And I don't, what's difficult about, difficult about cytoplasmic stains. And if you're not doing a confocal, is that it's hard, if you have a very, a cell, it has a lot of different arms and branches. It's hard to tell. Like, oh, is that a cell or is it just like the arm from a knee from another cell? And so like, you can have the up regulations. It doesn't really matter. I guess for the takeaway message, but that, you know, it could be that it's massively upregulated. It is. And then it's possible also that the morphology and also the number of astrocytes is changing. But I just also thought that I wasn't really convinced very, very, I just, the microglia findings didn't make a ton of sense to me. Maybe it's just a problem where they just chose the wrong representative photo of their conditions. But it actually looked like there were fewer in the images they showed. Actually, the fewer microglia. I think they went with like how much, how, how branching each microglia specifically. Right. But, you know, that's just one measure. Yeah, that's right. And like oftentimes when you have, if a, if there's a micro or microglial population expansion after like in inflammatory conditions, especially if there's like an attempt to try to clear things or if there's like an inflammatory phenotype that pops up, then those cells proliferate. So, you know, maybe a future study would be to like dig into understanding what's happening with the glial cells. Since, you know, they're going to drive a lot of the kind of collateral damage that we see to neurons and neurodegenerative disorders. So, yeah. But I thought that was a, I thought it was a little weak. We will go back to the immune angle right at the end when they look at transcriptomics. But I guess I can't speak for the authors. But I don't know. You shouldn't, right? But the point here is, you know, but that's one, one potential kink is that apparently, yeah. So, something to watch out for is that maybe the microglio numbers are down, or they at least they didn't report it. But finally, what they, the last remaining glial cells they looked at was, oh, they go dendrocytes. And apparently the myelination up out, some of the markers of myelination might also be down. And this might make sense because if the neuron number down is down, then there are fewer neurons to myelinate. And in fact, neurons numbers are down as, did they show it? Maybe. I don't know whether they're labeled for new N, or come to my notes, not for this experiment. But they did, they labeled for double stranded DNA break. So, this is a one tunnel using at this thing called tunnel, I say, I wouldn't go into it. But this labels for when neurons, when cells undergo apoptosis, which is program cell death, the DNA gets fragmented. So, you have one way, you have a way of detecting it. And it's, you see more of it in air pollution exposed mice. And this is protected once again in alpha-synucleon knockout mice. And also, there's more oxidative stress, et cetera, et cetera. So, a wide host of kind of molecular and morphological changes in mice that are exposed to this air pollution for 10 months. So, this, and almost all of it, is protected by alpha-synucleon knockout. So, the next thing the authors looked at is, are there any deficits in behavior? Like, what happens when your brain, your missing half the brain, at least the ventricles are drastically enlarged? So, some of the things that they tested was nest building, which is, yeah, they say it's a, it's a cognitive. It definitely demands, we don't know what it, what it demands in a mouse, but it is impaired in mice that are exposed to air pollution for 10 months. I mean, it's also, it's, I think it's generally used as just like kind of like measure of well-being, because they'll also use it in like stroke studies or we've really toyed with the idea of using it in some, in as a measure of mouse well-being after infecting them with the virus. So, and I think it involves a lot of things that are kind of sub-cortical as well. It involves like hypotherlamic and stuff. Yeah, it involves many, many things. And I think like Rob Fromke in NYU showed that, like mice who don't have good oxytocin, who are not very good at parenting, they make very horrible nests. And in our hands, mice who are Parkinson's, that showed motor symptoms, they also make horrible nests. So, it's a lot of things. Yeah, the three XTG, the three XTG Alzheimer's mice also make terrible, they're just, they don't care. It's an umbrella thing of that, there's something wrong with these mice. But yeah, they're not very precise. Not super precise, something's wrong. But in, but these authors kind of zoomed in a little more, where they tested the mice on some memory task. So, they have this thing called Y-Mays recognition. So, they, the mice have to go into a Y-Mays. One of the arm is blocked. So, the mouse would explore just one of the arm. And then you remove the blocked arm. And you see if mice go and check out the new arms. And normal mice would check out the new arms. If you have memory impairment, maybe you forgot which arm you checked out previously. And you're more kind of indifferent. And that's what they saw with the pollution expose mice. And another thing they, the experiments looked at was this thing called novel object recognition, which is you just basically put in object A in a, in a box, show it to the mice, the mice investigate it. And then you take the object away. And then you put in two objects, the old object and a new object. And normal mice would spend more time investigating the new object. These mice, if they show deficit in memory, they would again be more indifferent. And that's what they saw. And this seems like there's some protection, but the stats is a little bit inconclusive. So, there's not significant increase from the, from the wild type mice. Sorry, protection by Alfred's going to signically knock out. So, it's not super strong. It's one of the criticism that they're going to have. Do they bring up the, the, they talk, I didn't look at their limitations yet, although I should. But do they talk about the fact that their knockout mice are on a different background than their wild type mice? That's a, that's a, that's pretty interesting. Yeah, it is, they're on a different, the knockouts are on a different background strain. But, and I only bring that up because, you know, I'm sure you guys have seen this too, that if you choose mice on different background states, they can have very different behaviors just at, like, rest, like, in home cage behaviors, really different. So then if they're starting at different places, it's, you almost can't, you can't compare between strains almost. It's more like you have to say, okay, an animal of this background that doesn't have the genetic mutation. How does it compare to an animal where whatever something has been modified? Yeah, they, they, I don't think they mentioned it too much in the limitation. They largely set that. It didn't seem to have a strong behavioral effect. But that would definitely be something to watch out for. Although, I would quickly point out that the Alphastics, they also have a different group where it is Alphastinucleid knockout who didn't get the air pollution exposure. And those guys look fairly similar to the wild type mice who didn't get the air pollution exposure. So that's another, another control group. Okay. Tim. You, you have to go. Yeah, I'm going to need to go. I'm sorry. Okay, well, no, it's okay. We spent a lot of time chatting at the beginning. So we will continue. You can, when you have to go Vivian, just leave and wait for it to upload. And then you can leave the room. Okay. Should I, should I just do that now since we're stopped? Or should I like say bye? We could go, we could say goodbye, Vivian Morrison from New Orleans. Thank you, Vivian. Thanks, guys. I'll see you next time. Bye. So hit, uh, leave and then wait. Hopefully, she'll wait. Okay. Okay. Go ahead. My bad. We spent a bit too long. No, I talked a lot at the beginning. But yeah. So, uh, so these mice definitely show both, um, uh, brain, uh, deficit, physiological deficit and also behavioral deficit. Um, so we know that air pollution. So this answers Vincent's earlier criticism of all these observational studies that you just don't know unless you do the experiment to look for causation. And this is what they did. And it caused both, um, neurological, uh, impairment and also behavioral impairment. So what they next did is, um, they, uh, went a little bit deeper. So you know, our first nucleensism is important because when you knocked out alpha-sonuclein, you protect, uh, against a lot of these, um, pathology. Uh, so next the authors actually transitioned, uh, a little bit. So this is a bit of a kind of a inside baseball. But as it turns out, the normal mice don't really suffer from, uh, alpha-sonuclein pathology a lot, mainly because they only live two years as opposed to humans, which live like, you know, 80 plus year and get a lot of these neurological degeneration, new degeneration diseases. So what they did is they actually moved to a new, uh, group of transgenic mice that have an alpha-sonuclein knocked in. And the alpha-sonuclein that they knocked in is from humans that have a mutation that give them early onset Parkinson's disease. So this mutation in, so they only kind of knocked in this alpha-sonuclein gene. And what they found is that normally if you just knock in these, um, if you just knock in this gene in normal mice after like one year, they start developing pathology. So they, um, so that this tells them that these mice are more susceptible to, um, to kind of alpha-sonucleinopathy, alpha-sonuclein related disease. So what they did is they exposed these mice to air pollution and they re-asked the question, what does it look like in the brain? Um, and not only that, they actually exposed, they, they spread out the, they kind of upgraded the, um, experiments a little bit. And they collected air samples from three different places around the world. So first one, they collected it from China. So me being, I just expect that to be massively polluted. It being China. The second they collected it in Georgia, in the US. And apparently this is a kind of like rather leafy residential area, but it does have some light traffic and it's close to an airport. And the place they collected from China apparently doesn't have any heavy industry, but has a lot of traffic nearby. And the third place they collected is from Europe, uh, from Prague in Czechia. And apparently this, uh, particular PM 20, so all these are PM 2.5 samples that they collected. And the last one that they collect from Prague, you can just buy from Sigma, which is a chemical company. I did not know that they sell them. Um, it's weird that you could just buy air pollution. Um, so they collected it around the world. And the goal is to, uh, to address the question, um, does, is this air pollution link only a US phenomenon? Because in the figure one that we talked about, it was US Medicare data. So they exposed these alpha-synucleon transgenic mice that are more prone to the disease to either PBS, which is the control signs water that doesn't cause much, um, uh, disease. So they, for these mice, because these mice are prone to getting the disease, the disease, they only expose them for two months, as opposed to 10 months before. So signs water control vehicle didn't cause any, uh, minimal pathology after two months. But in the remaining group, uh, whether the PM 2.5 is from China or from US or from Czechia, Czech Republic in Europe, they caused a very similar pattern of, uh, pathology, which in their case, they labeled for this phosph-hyper phosphorylation of alpha-synucleon. And this is especially prominent in the cortex and also in the hippocampus and also, uh, in the amygdala, uh, a bit in the midbrain. Uh, a bit, they say it's not in the midbrain, but you can see a little bit of it in the midbrain, also in the brain stem. Um, and also, also the olfactory, olfactory bowl. So one question, one interesting question is, um, uh, so I mentioned a bit earlier that, um, there is actually a hypothesis that you get Parkinson's disease through your gut by things that you ingest. So for example, pesticide, maybe farmers get it either by working, they, they will get it through the hands perhaps, but maybe they also unconsciously, like, eat it because of exposure. But a lot of, um, so there's some evidence, I wouldn't say a lot of, but there's increasing evidence that maybe Parkinson's can be, uh, transmitted first through the gut and then spread up to the brain. But there's a, separate, um, viewpoint that suggests, uh, these Louis body, um, diseases can be acquired through the nose because one of the early symptoms is actually, you lose your sense of smell, just like in COVID. So the early symptoms of having these Louis body related disease, which includes Parkinson's and Louis body dementia is, um, uh, conservation, loss of smell and, uh, sleep disorder in which you act out your dream called REM sleep disorder. And losing the sense of smell suggests that maybe you can acquire it. Um, maybe you see pathology first in the cortex, especially the olfactory bulb. And maybe you're somehow breathed in, which is, which would, um, square with the air pollution theme that we're talking about in this paper. So you see also in these gut, in these mice, there's like quite a lot of alpha-synucleon pathology, in the olfactory bulb, and also in the cortex, which suggests maybe they are more, uh, a nasal cortical cluster than a gut cluster, perhaps. Although these, uh, researchers also looked at just just to refute my, instantly refute my own point, they also looked at alpha-synucleon pathology in the gut and in the lungs, and they saw it in both places. So maybe, since they are tripping it onto the mouse's nose, some of it might get into the lungs and some of it would have been swallowed. So maybe that is causing pathology there too. Yep. Okay. So now we know that, um, it doesn't matter where in the world, uh, these, uh, these air pollution, uh, cause similar disease in the brain. Next, the researchers looked at what we talked about, uh, talked about a bit earlier, which is this pathological seeding of alpha-synucleon, um, kind of fibrils, of, kind of protein aggregate in the test tube. So what they did was this, they, um, they had mice that are, so these are, these, these are these transgenic mice that either had, um, no exposure to the PM2.5 pollution. So this way, the control group versus, uh, five months worth of exposure to PM2.5 air pollution. After that, they extract the brain and then they, um, homogenize the brain and they extract the insoluble fraction. So this is the fraction where all the clumpy proteins are going to sit and then they pipette out a fixed concentration and they put it in a test tube of monomers of alpha-synucleon. So the monomers of alpha-synucleon is going to act as kind of like in PCR would be like the ATCG. They act as the, uh, kind of the substrate of this seeding of these clumpy fibrils. And they're going to detect how much fibrils are formed using, uh, several assay. The first assay is this THT fluorescence assay that detects clumpy beta sheets. So what they found is, um, in the control group, which is, uh, alpha-synucleon transgenic mice, they actually show a little bit of symptoms. So I talked about a bit, a bit earlier, these mice are prone to developing a little bit of alpha-synucleon disease, mainly manifesting in like a more motor phenotype, like the dopamine cells are lost and they see like hyperphosphorylated alpha-synucleon. These guys, um, if you incubate them, if you incubate the brain, uh, have homogenous sample with these alpha-synucleon monomers for seven days in a test tube, they start forming these clumps and you can detect it as an increase in this THT fluorescence. That's fair enough, but what is interesting is that in the PM2.5 exposed mice. So these are the same transgenic mice, but you make them breathe, um, you don't breathe it. You make them exposed to this air pollution particles for five months and then you extract the brain and do everything else the same. You put them in the seeding assay. You get maybe close to twice as much seed, sorry, twice as much protein clumps, twice as much of these preform fibros. So the fluorescence is almost twice as bright. So this is first assay. The second assay is that they've got these preform fibros that have been clumped up and they ask, how resistant are them? Are these, are these clumps to proteinase K digestion? And what they found is that the clumps from the mice who are exposed to PM2.5 are much more resistant to proteinase K. So that tells them that these clumps both have more beta sheets and also are in a maybe different confirmation that make them more resistant to progenase K. And they did also a separate assay called circular dichroism spectra assay which uses light that tells you a little bit about the structure and they found that these air pollution gives you slight, it gives you a different structure alpha-synucleon um, uh, fibros. We won't go into that because that is the extent of our knowledge. Uh, so this, these experiments, I think I thought they were done with recombinant uh, synucleon. Correct. Yep. Right. So then they add the pollutants to that and in vitro they're misfolding, right? That is the next experiment. The first experiment, the first experiment, they actually, the first experiment had the mouse as the middle man. So they exposed the mice with the air pollution and then got the extracted the mouse brain and then put it in the test tube and they formed these clumps. So the next question is what Vincent got to? The researchers asked is the mouse necessary because you can imagine we talked a bit earlier about all the glial cells that I get activated. You can imagine these pollution, these pollutants can activate the glial cells. The glial cells may be secreting something that form all these, make all these alpha-synucleon clump up and then the neurons can't deal with the, can't digest the alpha-synucleon clump. The alternative hypothesis is that the pollutants themselves can directly somehow modulate the clumping of these alpha-synucleon monomers even outside of the cell, kind of in a vitro setting. So that's exactly what these researchers did, is that they ran another set of experiments where they just had alpha-synuc, recombinant alpha-synucleon monomers, soluble alpha-synucleon monomers and they just pipette it in some of these fine, particulate matters, all these pollutants, no cells, and then they shook it for like seven days, I think, so the same experiment. And they asked in the control group, they pipetted it in PBS science water. And they asked, first of all, do I get increased TNT fluorescence? Do I get more beta sheets? And second or four, is it also more resistant to protonase-K digestion? And the answer is yes in both cases, both cases. So what this tells the researchers is that the cells are not necessary, which is very weird. So that raises the possibility, is that these pollutants might themselves act as a catalyst to think the pollutants are actually getting into the neurons. So that's the next, that's the next question is that we do not know is how do like either the pollutants get into the neurons and start causing havoc or the alpha-synucleon have to get out of the neurons, clump up, and then get back in the neurons to cause havoc. Because there is a blood brain barrier, and these are pretty big particles, right? So they would have to make it pass a blood brain barrier. I do not know if they can squeeze in the blood brain barrier. That I don't know if the researchers address in this particular, but they do it experiment with with cultured neurons primary neuronal cultures and adding the pollutant doesn't do anything. Exactly. So Vincent got it completely correct. The next thing they did is they looked at culture neurons and they asked the question, can we just ask at the pollutants? And it didn't work strangely after 10, so you can add sprinkle in some pollutants for 10 days. So in mice they did it for 10 months. So it's possible you just have to wait 10 months, but at least in 10 days it did not work. However, if you sprinkle in the fibrils, so these would be the test tube where you put in some of these pollutants. So, okay, let's go back. The researchers now have these test tubes where they sprinkle in the pollutants and wait a 10 days. And the result is that the pollutants kind of quote unquote catalyze these fibrils. These fibrils have the properties of being having more beta sheets and are more resistant to proteinase K digestion. The researchers then got some of these fibrils and sprinkled it onto your cell, neuronal cell culture. And the control group is they sprinkled in either PBS, so basically no fibrils or fibrils from just alpha-synucleon monomers without any of the air pollution, any of the PM 2.5. So even alpha-synucleon by itself would form clums given long enough time. So if true, your cell is fighting a battle constantly to prevent this from happening. So they also sprinkled in these two types of fibrils. And the purpose is to show that the fibrils formed after exposure to the pollution is a slight is a different strain of fibrils, which they call the PM fibrils. And what they found is that the PM fibrils from the particulate matter caused a more caused stronger hyperphosphorylation of alpha-synucleon, which is a disease correlated phenotype, and more down-regulation of a neuromarker, suggesting that it's more neurotoxicity. So it tells you that this suggests that these preformed fibrils are important, and they might be different from your usual run-off-the-mill Parkinson's disease fibrils, like fibrils that are not exposed to any of these air pollution. So we are running long, so I'm going to very quickly summarize the rest of the study. I do apologize. The paper is, I think I uploaded the PMC public-central paper, so the draft of the paper is open access. The gist of the story is the researchers then injected these PM fibrils. So the fibrils that I exposed to just the air pollution into a live mouse. So they're moving back from in vitro to in vivo, and once again saw all this hyperphosphorylation of alpha-synucleon and also saw some cognitive deficit in these mice. So it tells you that it is affecting the brain as well. And let's see. Sorry, one second. Oh yeah, and they also, another thing, the next thing that they did, next experiment they did is they actually redid a lot of the in vitro experiment, but with patient sample. So now they've got brain, the brain homogeneate from patient. Actually, no, take it back. They actually got CSF, cerebral spinal fluid from patients. And it is actually recently, one of the recent breakthrough in all these alpha-synucleonopathy, disease with alpha-synucleon, that my PI, Dr. Unkang is also involved with, is using CSF, so cerebral spinal fluid, in patients who are still alive with us, to diagnose alpha-synucleon related disease. Because before you can't do it, you have to do it post-mortem. But now they can actually get CSF from patients and then do the seeding aggregation assay that we just talked about and show that, and they have shown that patients who suffer from Parkinson's disease and also MSA and potentially a lewy body dementia, lead to faster, stronger aggregation of this seeding aggregation assay with monomeric alpha-synucleon. So this is a brand new diagnosis that can potentially allow us to detect disease early. And this is important because you can detect disease early, you potentially can try to prevent it earlier. And with new degenerative disease, it's all about early prevention because one's neuron dies, very hard to get them to come back. So the researchers got CSF from patients with Parkinson's disease without dementia. So this is the one that is less related to air pollution versus CSF from lewy body dementia. This is the one that is probably, according to the first experiment they did, probably more correlated with air pollution. And the question is, do they also replicate what they saw in the previous study where the air pollution worsened or these seeding aggregation assay? And that's exactly what they found. Which is the lewy body dementia patient sample CSF led to stronger aggregation in this seeding assay, more fluorescence with the TNT, suggesting more beta sheets. And they're also more resistant due to protein kinase K digestion, suggesting more clumpy, different, more resistant clumpiness. So this whole story fits very nicely. So telling the, suggesting to us that the lewy body dementia, so the researchers are arguing for the case that the lewy body dementia is actually gives you an alpha-synuclean form. That is distinct from Parkinson's disease that are without dementia and that it is more susceptible to air pollution. So that is what they're claiming so far. Which I think we would probably discuss a little bit more would probably need further evidence, collaborating evidence from other labs. But very quickly we'll touch upon the final figure, which is they looked at transcriptomics from patients that has lewy body dementia versus Parkinson's disease without dementia versus I believe Parkinson's disease with dementia. So lewy body dementia versus Parkinson's disease versus healthy control. And then they come, they looked at all the genes that are different compared to healthy control for all these diseases. And they ask the question, how about if we go back to mice that are exposed to PM 2.5 to this air pollution. So people remember mice is the experiment where we can ethically do a causal experiment that exposed the mice to the air pollution. Do the mice who are exposed to the air pollution, do they have gene changes in the brain, specifically in the anterior single cortex, but for simplicity, we'll just say brain. The brain gene changes, does it look more like the humans that have lewy body dementia? And that is what they confirm is that the gene changes for the mice, the mice who are exposed to air pollution is more correlated. Compared to the control group of mice that are exposed, and also they had learned different group of mice that are injected with the pre-formed fibros that are exposed to the air pollution. That's also highly correlated with the lewy body disease patients. In contrast, the mice who are injected with just the pre-formed fibros. So that is maybe more of a Parkinson's disease without dementia phenotype. Those mice, the gene changes correlate less with the lewy body dementia gene changes. So that is further corroborating evidence that lewy body dementia is maybe more correlated with the air pollution and all the gene changes and pathology that's taking place in the brain after the air pollution, exposure. And specifically, researchers can look at off all the genes that change after exposure to air pollution, what family of gene does it kind of cluster into? And they found it is actually a lot of immune regulator genes. So immune system likely plays a factor in all this. So that is pretty much a whole story. Sorry for jumping through a little bit towards the end. So I'm really interested in what's in these particles. Chemically, I'm surprised that in three different locations throughout the world, they have the same effect, but I would think they're chemically very different. They might be different, but they are from roughly, supposedly they can be from perhaps similar source. A lot of it is from burning, I'm guessing hydrocarbon. So I wonder if you know, the particles, you inhale the particles and somehow they're metabolized and make something soluble which can get into neurons and cause the sinuclein misfolding. Although maybe non-soluble thing can also get into cells, I don't really know. Sure, it could be endosythose. It can be endosythose. And also the preformed fibros. Remember, those are very insoluble. One of the properties is that they are insoluble. So they probably somehow get endosythose. And some of the researchers on the paper have claimed to have found receptors that would end their responsible foundosythosing these misshaped and alpha sinuclein into the cells. As to the air pollutants, I think it's a work in progress of how they get into neurons in cells. Sure. Similar to other kind of environmental things that might be causing diseases nowadays, like microplastic, how they affect us. We just don't know. And it's an ongoing project to look at. Could also be that immune cells take up the particulates and they go into the, they are able to get in the brain and somehow they cause normal changes. Exactly. And that might explain how how come the immune regulated genes are upregulated. And so the two things to think keep in mind. One is the air pollution particles themselves can apparently cause this misshaping formation of alpha sinuclein. Yeah, that's true. The purified protein. Yeah, yeah. But whether there are potentially communications from immune cells to the neurons that can cause alpha sinuclein to be passed back and forth is an open question. So there are recent articles that suggest that microglia might communicate amongst themselves or maybe to neurons using these nano-chubes, quantum tunneling nano-chubes or whatever they call it. So this is ongoing active aerial research. Yeah. So I don't, seems unlikely we can do much about air pollution. But as you said, maybe we can diagnose this early enough to do some therapy, right? We may or may not be, so we may or may not be able to do much about air pollution because in certain parts of the world, they are electrifying their cars. And cars' traffic pollution from traffic is potentially one major course of these PM 2.5 fine, fine particulate matters. So it's a wait and see, but in addition to it, like burning off biomass can increase it. And even home cooking might be one way these things get released. So time will tell whether there's anything anyone can do. It's a question of public health, which is a very thorny subject as you probably know Vincent gets everyone riled up. So that's one thing I probably want to quickly touch upon, which is this paper gives, it kind of tells you one contributing factor to maybe Louis body dementia, which is air pollution. But the question is, is Louis body dementia caused by just one single thing? Just like is autism caused by just one single thing, like maybe Tylenol? Or more relevant to our podcast with Mauro that we did before, just, you know, your microbiome? Is it just one thing? It's unlikely for things that are complicated in the world. It's likely caused by multiple different things. Sure. Sure. And if you look, go back to figure one where they ask a question, how much how much increase risk you have when you increase air pollution by a bit, the increase risk is quite small. It's not like everyone who lives in LA that has very high exposure to PM 2.5, have Louis body dementia, even though I believe Robin Williams probably lived in LA. He's not representative of everyone in LA. So it is, so we probably a bit, we can't draw white, white, ranging conclusion based on just one single course is probably a multi-genic course for a lot of these difficult disease disease. But it doesn't say, it doesn't rule out that a public health intervention, which will affect millions of people, that's by definition what public health is, can improve the health of millions of people by raising each person's risk by a couple of percentage points. So these two things, I think, you know, have to keep in mind, terms of balancing, you know, outrageous claim versus still doing good, like public health and a measurable health benefit intervention. Yes, I'm worried in the US that the EPA is being dismantled and it will be regulation of air pollution as we see it makes a difference. And there is an actual cost. I mean, for one, on one side of the coin, we want to, it's good for the economy to keep burning oil, obviously, because electricity is a higher price thing. But it comes at a different cost later when if it causes health kind of health issues down the line. So it's one of those things that whoever's in charge have to think about, I guess. Well, so probably should not be burning coal anymore. And a lot of countries still are. Yeah, that's right. That's does that in its particles, even tires on the road makes particles. Yeah, yeah, exactly. Like why do people have to, why do people have to change their tires every two years? Or whatever? Like when do the tires go? It's going in my lung, living next to FDR. That's right. That's that. But it is, well, it's good that we're starting to identify some of these things because that comes with this lovely, it's a lovely study. I think it's really nice combination of observational and experimental approaches. Yeah, most study would have stopped at figure one, so they won. Yeah, very much. Lovely. Thank you, Tim. Thanks, Vincent. That's twin 65 show notes. So at micro.tv/twin, if you have any questions or comments, you can send them to twin at micro.tv. And if you enjoy these programs, please support us micro.tv/contribute Tim chunks at New York University. Thank you, Tim. Thanks, Vincent. And I'll just shout out to Awakening's the film by Robin Williams and Robert De Niro and amongst other people, go check it out. It's an interesting film if you're interested in new degenerative disease. And also, there's the letter from Robin Williams Widow that I think I'll send to Vincent to upload. It's open access and it's very touching, but also sad, recounting off the end of his life. Yeah, I'm Vincent Rackenello. You could find me at micro.tv. Wait, is there anything else I have to thank? I think about every thing. No, that's it. Yep. You've been listening to this week in neuroscience. Oh, thanks for joining. Maybe thanks for having me again. Oh, yes, Vivian Mars. Vivian Marsen is in New Orleans. Thanks, Vivian. We've been listening to this week in neuroscience. Thanks for joining us. We'll be back next month.