The Puglielli lab at the University of Wisconsin School of Medicine and Public Health has found a way to manipulate autophagy — a process where cells clean out damaged materials — to rid the brain of toxic proteins like amyloid and tau. Researchers hope to use the power of this process to develop future treatments for Alzheimer’s disease and other diseases of aging. Luigi Puglielli joins the podcast to discuss his team’s research over the past 15 years, why the scientific process can take years to turn ideas into possible treatments, and how he hopes this research can be used in the future.
Guest: Luigi Puglielli, MD, PhD, professor of medicine, University of Wisconsin School of Medicine and Public Health
7:47 Tell us about how you manipulated this process of autophagy. Why is this discovery so important?
15:14 What role does acetyl-CoA play in the brain?
19:58 What does the future look like for this research?
Read Dr. Puglielli’s recent paper “ATase inhibition rescues age-associated proteotoxicity of the secretory pathway,” published online on February 25, 2022 in “Communications Biology.”
Intro: I’m Dr. Nathaniel Chin, and you’re listening to Dementia Matters, a podcast about Alzheimer's disease. Dementia Matters is a production of the Wisconsin Alzheimer's Disease Research Center. Our goal is to educate listeners on the latest news in Alzheimer's disease research and caregiver strategies. Thanks for joining us.
Dr. Nathaniel Chin: Welcome back to Dementia Matters. I'm here with Dr. Luigi Puglielli, Professor of Medicine at the University of Wisconsin School of Medicine and Public Health, and a basic science researcher who studies age-related brain changes at the cellular level. He has published a series of papers on autophagy, which he describes as a natural garbage disposal within brain cells that removes bad proteins and sick organelles from the brain. When autophagy becomes disrupted, brain cells can clog with bad proteins such as is the case with Alzheimer's disease. In 2009, Dr.Puglielli identified 2 enzymes that regulate the induction of autophagy from the endoplasmic reticulum, an organelle that makes a lot of proteins. Since then he has studied how these proteins work and in 2016 he published a paper showing that the inhibition, or blocking, of these 2 enzymes can prevent Alzheimer's disease in the mouse. Last year, he published a paper further defining those enzymes and how they affect the autophagy process. More recently, Dr. Puglielli published a new paper that builds on his past discoveries and explains how restarting autophagy could lead to drug development for Alzheimer's disease. Luigi, welcome to Dementia Matters.
Dr. Luigi Puglielli: Thank you Nate! It's a pleasure to be here and thank you for the introduction.
Chin: You are welcome, and I am very excited to have you explain this very intricate process that you have discovered. You have a new paper out in Communications Biology, one of the prestigious nature journals. It's the culmination of more than a decade of research. So before you tell us about the paper itself, can you share with our listeners how your career evolved into Alzheimer's disease and aging research?
Puglielli: Well I've always been interested in aging and age associated diseases. In medical school I trained in geriatrics and as I moved from clinical research toward basic signs, I remained interested in the general concepts of physiology and the pathophysiology of aging. By studying the physiology of aging we can learn why aging leads to increased disease vulnerability; while by studying the path of physiology of aging, we can actually learn what happens in the disease that is highly associated with aging, such as Alzheimer’s Disease.
Chin: So with that in mind then tell us about your recent paper and along the way please explain for our audience any of those scientific terms that you're going to use such as endoplasmic reticulum, enzyme, this BACE’s 1 and 2 (β-Site amyloid precursor protein cleaving enzyme 1 and 2) and then acetylation.
Puglielli: Okay, in general, the cell continuously makes new proteins to replace old ones that have been removed or degraded. Or because they are needed for basic uses for example, a hormone or digestive enzymes. Or to simply perform normal activities that occur at the cell surface. For example, neurons that connect to each other to execute basic neuronal functions which could be making a new memory or recalling an old memory. So the endoplasmic reticulum is a very specialized organelle within the cell that makes a lot of proteins. These proteins are generally required to traffic through what is referred to as the secretory pathway, and this process and pathway is particularly important for neurons. Now, as in any process that requires building or assembly, some proteins will come out the right way, while others will come out the wrong way. The former, the proteins that came out the right way, need to be selected and shipped to their final destination. For example, to a synapse that is necessary to make a new memory or to recall an old memory. The latter, the products that came out the wrong way, need to be degraded or disposed of. If you let them accumulate within the cell they will become toxic. Imagine not being able to remove your regular trash every day or week. It will simply accumulate and at some point living in the house will be unbearable. Now to avoid these issues, the endoplasmic reticulum has a whole set of proteins whose job is to make sure that one, all new proteins are made the right way, two, that only good proteins go to their final destination, and three, that the wrong products are disposed of. Basically some sort of a quality control assembly line. The biochemical machinery that we have identified and that we study is meant to do exactly that; it labels the good proteins within an acetyl group and then ensures the activation of what we call radiculopathy which is literally autophagy from the endoplasmic reticulum. Now autophagy is a word that means self-feeding. It indicates the ability of the cell to digest and recycle unwanted material such as proteins that came out the wrong way and failed this quality control assembly line. So to answer your question, BASE 1 and BASE 2 are 2 enzymes that add an acetyl group to properly made proteins allowing them to leave the endoplasmic reticulum and go to the final destination. At the same time, these two enzymes ensure a balanced removal of toxic protein aggregates through the autophagy process. If this machinery works properly all is good. If this machine does not function properly, we run into trouble. So many chronic degenerative diseases that are typical of old age including Alzheimer's disease, are characterized by the abnormal or excessive accumulation of toxic protein aggregates. So if we help our cells and tissues to degrade these aggregates, we would be able to resolve these disease states. This is exactly what we show in our recent publication.
Chin: That was a really complete answer, so thank you Luigi. I'm going to ask some questions about it. So we have neurons and neurons need to move proteins in order to function properly and this endoplasmic reticulum makes many of those proteins. Nicely, it has its own sort of quality control system that, you know, there's efficiency. So if something isn't 100 % efficient you're going to have some bad proteins that are produced but it can take care of those. It can kind of mark them with acetylation and then it actually interestingly breaks it down itself and kind of gobbles it up and gets rid of it. That's sort of how a functioning neuron works and when it doesn't work, well you have this buildup and in the case of Alzheimer's disease, it would be this buildup of amyloid protein or tau protein and that leads to brain cells not functioning well. So you've identified two really key enzymes or other proteins that are meant to actually help with the quality control in flagging these bad proteins per se that build up, is that correct?
Puglielli: Yeah, correct.
Chin: Wow. Okay, and so well I feel like I'm learning a lot here for my biochemistry and my cellular biology understanding. But now in this most recent paper you actually manipulated this process right? So you were able to manipulate autophagy in the brains of mice and restart the process so that it could actually gobble up some of these bad proteins. This opens the door, as you put in your paper, to potentially changing the timeline of Alzheimer's disease and other age related neurodegenerative processes or diseases. Please explain that part of it and why this discovery is really so important in the scientific community.
Puglielli: Yes, so Alzheimer's disease is the leading cause of dementia and unfortunately there is no cure for the disease. So if we can harness the power of autophagy we would be able to prevent the disease or even mitigate this disease. Basically we would be helping the brain heal itself. The study that we published shows that we can actually do that at least in a mouse.
Chin: And I love the idea of allowing the brain to heal itself. You are using the natural machinery of our brains to heal itself, to get rid of the toxic protein. But it's really hard to study Alzheimer's disease or at least translate findings from mice to humans and we've seen that disconnect in the past. So why is that and is there a part of you that's concerned about the translation of what you're finding in mice to what potentially would happen in a human?
Puglielli: Yeah, well first of all, you're correct. It's difficult to study this disease. Human disease in mice. Mice do not naturally develop Alzheimer’s disease, they simply don't make those specific pro aggregates that you mentioned the beta-Amyloid or the neurofibrillary tangles that are the underlying cause of the disease in humans. So to circumvent this obstacle, we use transgenic mice that were engineered to express the human proteins and therefore make the same or almost the same toxic aggregates that we mentioned. So it's a valid tool to study the pathophysiology of disease but also try to identify machinery or mechanism that can allow us to delay the disease or even cure the disease in mice before eventually going to humans.
Chin: And now you've already explained to us the BACE proteins enzymes 1 and 2, but are these in humans and do they increase as a person gets older?
Puglielli: Yes the BACE1 and BACE2 are acetyltransferases or simply put, deacetylase proteins. They simply move an acetyl group from the Acetyl-CoA to a specific amino acid of the proteins. And as I mentioned these enzymes are very important for cells that require a very active secondary path such as neurons. They're very important for the brain. We know that in a normal brain they are regulated to adjust the secondary path in real time. So as we talk, I'm trying to understand you and figure out what you're trying to tell me, and you are trying to do the same thing. We also know that these two enzymes are completely dysregulated in the brain of patients with Alzheimer's disease. Something goes wrong as a function of age and as a function of the disease. They are simply higher and hyperactive and Alzheimer's disease causes the accumulation of these toxic proteins. So if we can shut them down or at least go back to normal, we would allow the brain to reset this machinery and dispose of these toxic protein aggregates.
Chin: I hope I'm not challenging your BACE too high Luigi. I hope I'm asking good questions of you. What I thought was really important in your paper too is that you talk about both of them BACE one and 2 but BACE 1 seems to be a really pivotal one. And it can switch on in some cells and I'm wondering how does that happen and what are things that might cause BACEs to switch on and decrease autophagy and lead to the accumulation of all these proteins.
Puglielli: We don't have the complete answer to that. We know that lower organisms have one BACE higher organism than mammals in general too, so we need both of them because we are a bit more complex and so we have discovered a mechanism to regulate these twins in a different fashion. For example, we know that BACE1 can respond to the inputs to Acetyl-CoA. The more Acetyl-CoA, the more activity. The less Acetyl-CoA, the less activity. BACE2 does not do that. They've been designed to perform similar functions but also different functions and so as we learn how to regulate these two enzymes, perhaps we'll be able to shut down only one enzyme and not the other one. For example, we know that BACE1 will be a better target for pathophysiology of age. So if we can identify inhibitor compounds that target only BAE1 and not BACE2, we can prevent disease and avoid any side effects.
Chin: I think that's a key part, because when I read your paper, you talk about the druggable properties of what you found and it seems like BACE1 is a druggable property, and that if we were to target that, it could potentially lead to clearance of Amyloid Protein or Tau protein.
Puglielli: Correct, correct. That's one of the main conclusions of these papers, that we have a pipeline to actually select appropriate inhibitors and target them only to one of the two enzymes. While studying the mouse, the mouse gets sick in two months and dies in four months, and so right there in only two or three months of work, we can actually test whether that compound is promising or not.
Chin: And I should clarify too since I didn't ask this earlier, it seems like you actually can eliminate both amyloid and tau protein in mice is that right?
Puglielli: Yes, that is correct. Yeah.
Chin: So the two proteins that we use to define Alzheimer's disease, you've been able in a mouse to allow the brain itself to clear out those toxic proteins? That seems like such a pivotal and important finding.
Puglielli: Yes, it is because there are compounds that can target one of these proteins but not the others and for the disease they're both equally important I would say and so we need to be able to resolve both of them.
Chin: Well then another question I have is, is there a negative consequence to doing something like this? So if you don't have Alzheimer's disease, let's say this is a normal mouse brain and you were to block these two enzymes, is something bad going to happen? I mean is there a purpose to having BACE1 and 2 do what they're doing?
Puglielli: Oh I'm sure there is a purpose to having both enzymes and not just one in the mouse. In the mouse we know that if you genetically shut down one of these two enzymes, the mouse is completely normal. Which means the mouse at least can live with only one of these enzymes now and there is no change in the lifespan. That just protects us from certain diseases in humans. We know that there are humans that have a mutation in one of these two enzymes and they are completely normal. The studies that we have are mutations that inactivate the proteins, but these genomic studies were done in people that are between the age of fifty and eighty which means they live basically most of their life without any disease manifestations; they are rare individuals. It would be nice to study them and to see what is actually happening, but this sort of a proof that we can live with just one of them.
Chin: And what if we don't have either of them though? Is there a problem if you don’t have either of them?
Puglielli: It would be a problem. We know in other organisms if you shut it down they die. So It's not viable, which means that we do need both enzymes and as far as I can tell there is no human being that has a block in both Enzymes. So I assume that they are essential.
Chin: Okay, and you know a very common supplement that people take is Acetyl-CoA. It's mentioned a lot in my memory clinic and so I'm wondering since you've talked about it and in your paper you talk about Acetyl-CoA. Is there a similarity there? Or what role does Acetyl-CoA play in the brain itself?
Puglielli: The Acetyl-CoA that I talk about is the Acetyl-CoA that is made exclusively within the cell and so it’s not the Acetyl-CoA that is coming from the diet. Of course the diet contributes to the pool of Acetyl-CoA that we make. It is fundamental for the brain. It is important for metabolism because the energy is the backbone for other metabolized neurotransmitters in the brain and is important for the acetylation of proteins in many compartments. Not just the endoplasmic reticulum, but also for example, the nucleus. For example, acetylation of certain products that are known as histones, regulates transcription. So it decides what protein we make or do not make so it has very complex functions that are extremely important.
Chin: Well thank you for answering that because that's a very common question of why some people take Acetyl-CoA. I know it's a supplement that’s not regulated and so I just wanted to get that answer out there. So thank you Luigi.
Puglielli: You're welcome.
Chin: How long have you in your lab been working on this line of research, this idea of autophagy and an endoplasmic reticulum and this whole process?
Puglielli: In short, since 2007. We came across titillation in the endoplasmic reticulum by accident, it was just an accident. But we immediately recognized the importance of this discovery and so we decided to go after the machinery that released the proteins because it was simply not known at that point and we discovered the entire machinery between 2007-2010. After that, we devoted our attention to biochemistry. In other words, how the machinery works. We also made the necessary mouse models because there was none, to study biology. The information that we collected suggests that these two enzymes, BACE1 and BACE2, were good targets for therapeutics. So in 2012 we ran a screen to identify inhibitors and we came out with a paper where we described the first eight inhibitors. In 2016 we were able to show that this inhibitor was effective to rescue from Alzheimer’s disease in the mouse. Then in 2018 we showed that the same inhibitors could delay aging in a mouse model of accelerated aging. So it went from 2012, when we identified the compound, to 2018 to know that it can actually be used in a model of aging, and now last year we showed that if you genetically eliminate one of these two enzymes, the mice are protected from Alzheimer's disease. Now we describe a pipeline that would allow us to identify additional inhibitors. We also show that these compounds can revert, not just prevent, revert some age associated diseases such as Alzheimer’s disease. You have to imagine that you know most of the patients will come to the clinic when they're having memory defects, so the process has already started. So we need to be able to catch the process as early as possible but also be able to revert and not just prevent it.
Chin: Wow, 2007, I mean we're talking fifteen years ago.
Puglielli: (Laughs) 15 years of my life. Yes, ah.
Chin: (Laughs) Luigi, one the reason I wanted you to start with that answer is because I wanted to ask you the question, well why does it take so long to go from bench to bedside? Many people ask this question of us both in clinic and in the scientific field and your answer to what you just said was part of it, but I would just like your thoughts for those who ask. Well why? Why have we not made so many steps? One I guess I would argue we have made huge steps but two, I just want to know from you. What do you say to the slowness of scientific progress?
Puglielli: Ah, because there are many things that need to be done before being actually able to test a specific hypothesis. As I mentioned a moment ago when discovering titillating in 2007, we didn't know the enzymes, so we have to identify the enzymes if we want to study them. Then we need to understand why we need acetylation in the endoplasmic reticulum. Why did nature decide to place the acetylation there? Then at that point we did not have mice that we could use so we had to make our own transgenic mice. And it takes time. In other words, there was a lot to discover before reaching this point and so in 2007 we published a series of 25 papers where we described all this work, and now that we know about mice, we need to move forward into humans. But that is a very long line of work and it is not easy.
Chin: Well I hope you have time Luigi because it sounds like there's a lot to do here, but it does make a lot of sense. It includes discovery but then invention; you created your own cell line and then more discovery and more explanation. So thank you for that answer because I think that helps at least touch on the idea that, in order to do the best science, it takes a very methodical rigorous way of doing it. So what does the future look like for this line of research you mentioned going into humans? How do you see this unfolding and perhaps leading to drug discovery that might stall or prevent Alzheimer's disease?
Puglielli: Yes, well I hope that the pipeline that we have described can help us identify more BACE inhibitors. Unfortunately 95% to 97% of all compounds in the working mice never reach the human stage.That's a lot, so something happens across the line from mouse to humans that permits them to be even tested. Therefore, if we wish to be successful and eventually reach the human stage, we need to then define many inhibitors; not just one, not just two, or three. Furthermore, we definitely need to understand the specific details of the underlying biology so that we can envision disease states where we can employ these inhibitors but also prevent possible side effects and so there is still a lot to be done in this direction.
Chin: Okay, well then Luigi, my last question for you is one that I like to ask all of my guests and I haven't had a basic scientist or basic science researcher on before. So I'm excited to ask your opinion on this because you study a range of diseases of aging at that cellular level. What do you incorporate in your own life, what do you do in your personal life to keep your body and your brain healthy?
Puglielli: Well, first of all, this line of work keeps my brain always under what I call learning stress, under pressure. I'm forced to learn new things every day, forcing your brain to learn new things. For example, a new language is very helpful to preserve your memory. Obviously I also exercise. Although aerobic exercise is excellent for your brain, if you think about i,t your body and brain is designed to interact with the environment. As we walk, run or bike we must look at things, we must hear things and we must adjust to walking, running or biking to respond to the environment. And the associated body stress, a bump on the road etc. This is what the brain was initially designed to do, to receive and process all this information that comes from the outside. Finally, I eat a lot of vegetables and fruit. But I do enjoy an occasional good meal.
Chin: Ah, well thank you Luigi for being on Dementia Matters, and we certainly hope to have you on again as your work progresses.
Puglielli: Thank you, it was a pleasure.
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Dementia Matters is brought to you by the Wisconsin Alzheimer's Disease Research Center. The Wisconsin Alzheimer's Disease Research Center combines academic, clinical, and research expertise from the University of Wisconsin School of Medicine and Public Health and the Geriatric Research Education and Clinical Center of the William S. Middleton Memorial Veterans Hospital in Madison, Wisconsin. It receives funding from private university, state, and national sources, including a grant from the National Institutes of Health for Alzheimer's Disease Centers.
This episode of Dementia Matters was produced by Rebecca Wasieleski and edited by Caoilfhinn Rauwerdink. Our musical jingle is "Cases to Rest" by Blue Dot Sessions.
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