Sleep, Mitochondrial Metabolism & Oxidative Stress | Gero Miesenbock | Episode 257

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Thank you for the invitation, Nick. Do you want to start off by just telling everyone a little bit about who you are and what your lab studies? Um I'm German Miesenberg. I'm uh a professor of physiology at the University of Oxford in England. Um we work on um neurobiology in general and more recently specifically on the neuronal control and biological function of sleep. And what what sort of creatures do you study?

Um fruit flies and um increasingly also mice because we want to be taken seriously. So why do why do you and why do s a lot of scientists study flies? What is For those of you for those that are not familiar with experimental research, fruit flies are very common. And sometimes when you're not familiar with that, that's that's puzzling at first. Why would you be interested in fruit flies? They have compact brains that are capable of quite remarkable cognitive feat.

Um as um Francis uh Crick and Seymour Benza um debated the relative merits of model organisms, Benza always says said to Crick. You know, they can do everything you can do, Francis, plus they can fly and land upside down on the ceiling, something you can't do. So there there's there's a behavioral richness there, um that's controlled by a very compact nervous system.

um that we have powerful genetic tools developed over more than a century um that we can use to dissect it. And more recently also a complete electron microscopy derived synaptic resolution wiring diagram. So it's a really, really, really fertile experimental system. But Um in um many cases, uh and this is why I only half jokingly say if we want to be taken seriously, uh people want to see that what you discover in an invertebrate.

is also relevant to the mammalian and especially the human brain. So um we we need to um show that there's generality to the kinds of things that we that we unravel. Mm-hmm. And and in in many cases there are, because, you know, at the end of the day, even though a fly is much smaller and simpler, they have many fewer neurons and so forth, they do, you know, fundamentally have a lot in common with a mammalian brain. They're they're built out of the same components.

And they do a lot of the same things. And so there are, you know, and I think part of what we'll talk about today are are some of the fundamentals that are uh the fundamental things that are happening in both vertebrate and invertebrate brain. Yes, you're you're preaching to the choir.

And so you a lot a lot of today will we'll talk about your work to study sleep and and what is actually going on under the hood in brains and nervous systems to to perform sleep and and why sleep needs to happen. What? So fruit flies sleep? They do. Yeah. Uh um it's been it's been known for uh about a quarter of a century.

Uh there were two papers published in 2000, one by Amita Sigal's lab and the other one by Giulio Tonnoni and Chiara Girelli's lab. Um, that showed for the first time that that That rest in flies is a sleep like state. And then from that, a lot of fundamental mechanistic work has evolved and derived, including our own.

Do do all animals seem to sleep where we've looked for it? And does that tell us that, you know, there's there's something something fundamental that's there and present in the nervous system, even of the simplest animals with the simplest nervous systems that make sleep necessary for the brain. Yes. Um in fact you don't even need a nervous system to require sleep.

Um there was a discovery reported by Leah John Toro at Caltech um in twenty seventeen that even jellyfish which don't have a centralized brain, they just have a distributed nerve net. um show a sleep like state, okay? Um and that I think that observation tells you something very, very fundamental. Um Есюно, а мій сліп із тим, убитос бев'я.

Whose function we don't understand, um and whose function we can't infer simply by observing the sleeping animal, right? I mean in contrast to say eating or drinking, where it's obvious what the action is good for. Sleep doesn't really give you a clue if you just observe a sleeping animal. So there's a huge variety of theories of what sleep might be good for. Including sophisticated cognitive functions such as hippocampal replay of newly acquired memories and so forth.

Um I would not argue that these functions don't exist. But I would argue that they can't be the primary ancient evolutionary driver wisely it was necessary Uh a telefish already requires sleep. And it obviously doesn't have a hippocampus and I'm not sure what its cognitive abilities are. So I think there has to be something really, really, really basic. Yeah. So so there has to be something basic because all of these creatures need to sleep, even creatures that don't have to

do spatial navigation like a mouse or even a fly can do. And so so basically what you're saying, and I think what we'll kind of unpack today is there must be some very fundamental things happening that probably have to do with cells and metabolism and stuff that even a jellyfish needs to take care of.

And then there are functions of sleep related to things like memory consolidation and synaptic homeostasis and all of this stuff, but these are probably things that came later and got stacked on top of this more basic and fundamental and ancient uh uh function. That's exactly what I what I how I think about the problem. Yeah.

Um, I mean to let the cat out of the bag. I think that that sleep has an ancient metabolic origin. It's specifically tied. To respiration, the enormous energy gains that you have when you use mitochondria to transfer nutrient derived electrons to oxygen as a terminal acceptor. That process has the advantage of of maximizing the free energy yield um of uh your your food intake, but it has uh it has side effects and we think those side effects

uh require sleep to offset them. But um but once we think evolution has sort of um come up with that solution of of putting animals to sleep for um a few hours, then of course these off periods were used for other things. uh such as synaptic homeostasis or memory consolidation.

uh or other kinds of things. So so so uh our our hypothesis is is is is inclusive rather than exclusive. Right, right, right. Yeah and that's kind of a common theme in evolution. If something evolves an adaptation or or or a mechanism gets put in place and it needs to be there, very often. um, it will get tinkered with or co opted into a new thing uh so that you preserve the old function and then you sort of start reusing it and retooling it for other things that that come later.

Exactly. Especially if if that um that that function has significant risks and costs. Sleep researcher and one of the founding members of the Chicago School of Sleep Research. Um Al Rechtschaffen um he he he quipped that if sleep didn't have an absolutely fundamental purpose, it would be um the biggest mistake the evolutionary process ever made. Um because of its risks and costs. Right. So there must be there must be some some really important benefit.

That offsets these risks and costs, but we don't really know what that benefit is. And of course, you know, we all sleep every day or almost every day most of the time. Um, and when when we don't, we we feel it. So we we have an intuitive understanding that there is a recuperative function to sleep.

Clearly has something to do with energy. It has it affects your memory, it affects your brain function. But you know, the the devil's in the details here, what exactly is going on there? That's what's been mysterious. So when we start thinking about the recuperative function of sleep. You know, you mentioned some of the theories that have been out there. You sort of you sort of mentioned the punchline in essence of what you think is going on. This has to do with

Oxygen based respiration and mitochondrial metabolism. So that so what we get from having mitochondria and needing oxygen to generate. uh energy is we get a a lot of energy. We can make a lot of ATP with our mitochondria, but you said there's side effects. I want to give people a sense for the basics of the electron transport chain that's in the mitochondria.

And h why that gives us so much energy, but also how the sort of side effect side of this works. So at a very high basic level, we don't need to be Uh comprehensive here. What is the electron transport chain in mitochondria and and how does oxygen fit into energy production? Why are we breathing this oxygen?

So the the y you can think of the electron transport chain in the inner mitochondrial membrane as just a wire that conducts electricity. Um except the wire is made out of proteins. large protein complexes, there's four of them that make up the electron transport chain, and they are encoded by two genomes. um by the nuclear genome and also certain subunits, and strikingly the ones that are most intimately involved with electron transfer are encoded by the mitochondrial genome.

So these two um genomes have to somehow interact with with angstrom scale precision and build um this wire that then conducts electrons. It's essentially it it passes a current from complex one down to complex four, where the electrons are transferred to oxygen. Now if you look at the redox potential difference that you can tap through this transfer, it's large. It's more than a volt.

This is why um oxygen tanks have um have danger labels, right? It's a very explosive reaction that that biology has learned to tame by breaking it down into three steps. So you have um you have three transfers of the electrons first from NADH, which comes out of the Krebs cycle in the mitochondrial matrix. to complex one, then to c to complex three, to complex four, and to oxygen. So so you you release the the the huge amount of energy in a in a in a gradual piecemeal fashion.

Um and um these electron transport complexes or or the current that flows through that wire. Then um enables proton pumping by this by these um um electron transport complexes. the the there's a proton gradient that's stored as in a battery across the inner mitochondrial membranes. And then the protons flow through complex five, which is the ATP synthase, which is a nanoscopic turbine that spins in the inner mitochondrial membrane, and that makes ATP.

Now the wonderful way of of doing um this chemiosmotic coupling, the the the the combination of electricity um that's that's central to to ATP synthesis is that it it frees you from the shackles of stoichiometry. If you did a chemical group transfer, you would always have integral reaction partner ratios, right?

Um but by by by interposing an electrochemical gradient, you can actually squeeze out the last fraction of of of energy that you can need because the stoichiometry doesn't have to be one to one or two to one or three to one. Right.

Right. Okay. Right. If we're so if we were doing things strict strictly in a a chemical fashion, you know, you would need a fixed number of ultimately particles from food to make one unit of energy. But because we have this flow of electrons creating this proton gradient.

and it's flowing through this little molecular turbine. It's just it's just a much more efficient way to generate as much cell uh molecular energy as as you possibly can basically. Exactly. And and the uh the the the the availability of oxygen um

uh to act as the terminal electron um acceptor, um then really increased the amount of energy that you could squeeze out a lot. And that's something that happened in in Earth's evolution around two and a half billion years ago because life it's thought evolved on an anoxic planet. There was no oxygen in the atmosphere. All the oxygen that's around was actually generated by life. Um

And and and and oxygen levels sort of rose gradually. Um, but then with two large stepwise increases. The first one about two and a half billion years ago. Uh and that's uh the time it was thought that then triggered um the symbiosis between two forms of bacteria that gave rise to eukaryotes containing mitochondria. And the second large increase in atmospheric oxygen levels

was about five hundred and seventy, five hundred eighty million years ago. And that's when multicellular life really exploded because you could just gain so much more energy. Um And uh and complex multicellular life became possible. So this this some people think was the spike that that lit the the cambrian explosion of multicellular life, uh the appearance of nervous systems or neuronal nets, and the appearance of of sleep. Okay.

So we've we've talked about all the advantages that that that electron transfer and oxygen has. What's the downside?

Um the problem is the electron transport chain in the mitochondria uses single unpaired electrons, okay, which is unusual for a redox reaction and something that oxygen loves because oxygen is a very peculiar gas. It has in its uh in its outer shell two orbitals. That have one electron each, one unpaired electron, and those

Spins of these two electrons point in the same direction. That means that if you want to engage oxygen in an electron transfer reaction, you need to add the electrons one at a time. So this is something that in a normal redox reaction, which always transfers electron pairs, uh that doesn't happen. So um so that has advantages. For example, this so-called spin restriction on the on the reactivity of oxygen.

prevents the spontaneous combustion of the atmosphere which is a good thing but it means that if you have unpaired electrons somewhere such as a mitochondrial electron transport chain their chance of leaking to oxygen is very high. Oxygen is like the magnet for these things. So basically the the the wire of the electron transport chain becomes poorly insulated in the presence of oxygen. Um once such an electron leak in the mitochondria happens, you form something called

reactive oxygen species. So uh a molecular oxygen that has only a single unpaired electron, um, and then we think the the that is a very, very dangerous thing to happen. So b you know basically You you want this electrical current flowing because it's enabling all of this amazing metabolic stuff to happen. But just like, you know, I have electrical wires in in in the home that make, you know, the lights and the TV and all of that possible.

You want this to be well insulated, you want everything to be protected, and you don't want the electricity leaking and sparking and lighting your house on fire. Exactly.

And and and the the leakiness also seems to be determined to some extent by how well the the mitochondrial And the nuclear encoded Electron transport chain proteins cooperate together. So every every individual um is different in that respect because um Because the the the mitochondria only come from the mom and the nuclear genome obviously from both mom and dad. So every individual is a new experiment.

that uh has to find out how these two components actually work together. And it's it's it's even thought I read somewhere. that that one of the of the uh of the most common reasons of of of spontaneous abortions in humans is actually mitonuclear conflict. So very early during during embryonic development um that the the the the match, the mitonuclear match is checked. And if the match is poor and you leak a lot of electrons, um the the the the the developing embryo at a very very early stage

And the goal's hypotosis. Yeah. So so depending on who mom and dad were, the amount of leakage of these electrons can be greater or lesser. But in everyone to some extent, there's always, you know, it's never perfect. And there's always gonna be some leakiness, there's gonna be some reactive oxygen species, and that is an inevitable consequence of this oxygen based metabolism.

Exactly. That's that's what we think um is what's going on, and that's what we also think is the root cause of the need for sleep in in animals that have um neurons that are Um the the energy you are power hungry. Mm-hmm. Mm-hmm. Yeah. So I mean s so this makes a lot of sense so far. If sleep is fundamental, meaning that we see it in everything from jellyfish to flies to mice to humans, it has to be

Tied to, it has to be serving some function that's also fundamental. And jellyfish don't have a hippocampus. Um, neither do flies for that matter. You know, that has to be something that all of these animals need and what they all have in common is this this metabolism, this oxygen based metabolism that uses mitochondria to create energy.

Exactly. And so let's talk a little bit more about the side effects of aerobic metabolism here. So we're using oxygen as an electron acceptor. Um, it's got advantages and disadvantages. When we have this leakiness in the mitochondria, when we generate reactive oxygen species. Why is that bad? Um, what do they what do they do? What are they lighting on fire um that creates your side effects or problems for ourselves? So reactive oxygen species, as the name says, are very, very reactive molecules.

And they target multiple cellular constituents from proteins to DNA to lipids. And cause either covalent modifications of proteins or lipid peroxidation.

And we think that lipid peroxidation is one of the key um events that happen um if you have a large mitochondrial electron leak. The reason is that lipids obviously are right next to the the electron transport complexes in the inner mitochondrial membrane, they are highly abundant and they are capable of entering a chain reaction once you produce one peroxidized lipid.

So if you have um say a membrane phospholipid with a polyunsaturated fatty acyl chain, and you hit that with a reactive oxygen species, you form a lipid uh peroxyl radical. And that can then uh propagate the chain by reacting with another lipid, sort of diffusing laterally in the membrane. There's estimates that suggest that um that one such hit. um can produce

hundreds of thousands of peroxidized lipids. So the reaction the reaction can really spread like wildfire. So literally we're talking about lighting fat on fire and one spark, like one oxygen uh radical can light hundreds of thousands potentially of lipids on fire. And so you can imagine, you know, literally it's like burning a hole in the wall of your house or something.

Yeah, i one could yes, that that's the way you can think about. I mean the the lipid peroxidation is is usually what makes fats rancid rather than lights them on fire, but uh the the the the metaphor of lighting the the membranes on fire is perfectly fine. Mm-hmm. And so so the membrane so mitochondria have two membranes. They have an outer membrane and an inner membrane. The membrane is is is made out of lipids largely, and those lipids can be

Uh, saturated, monounsaturated, and polyunsaturated. What is the difference between those fatty acids and what's what's the relationship between the saturation of the fatty acids and their ability to be uh to react with these reactive oxygen species? So it's usually the polyunsaturated lipids. Um they have uh the the the their their chemical structure, uh there you have something called a bisallylic carbon atom, that's the primary target for attack by um by uh by um an oxygen radical.

And that's how you start this peroxidate lipid peroxidation chain reaction. Now, interestingly, as this chain reaction now not um not spreading like wildfire, but looking at at the individual lipid molecule. As that progresses, the molecule undergoes a series of rearrangements and sessions. That ultimately then leads to the production of carbonyls, aldehydes, and ketones that are collectively also known as reactive electrophiles.

Um so melon dialdehyde is one of them, methyl glyoxyl. There's lots of lots of these reactive electrophiles that that are produced in cells. Now one of the findings that actually put us on um

on the scent of mitochondria as a p potential cause of sleep, was that we had begun by studying the biophysics of specific sleep control neurons in the brains of flies. And um And we we we we sought to discover how varying sleep needs regulates the electrical activity of these sleep inducing neurons. And that surge zeroed in on two potassium channels, one of which contained a subunit. that had sort of a mysterious structure. It looked like an enzyme.

Um whose endogenous substrate was unknown. And we found also relatively recently. That the most likely endogenous substrate of that channel subunit that regulates the induction of sleep. Are lipid peroxidation-derived carbonyls. So you essentially have a little sensor at the plasma membrane that registers. um what's happening in the mitochondria, how much electron leak you have, um how much lipid peroxidation you consequently have, and and that is then able to pick up

the accumulation of these longer lived lipid peroxidation products over time. So you generate a reaction reactive oxygen species as an inevitable consequence of Oxygen based mitochondrial metabolism. They can induce things like lipid peroxidation and they're gonna produce nasty chemicals that can break stuff in the cell.

You're basically saying there's a sensor, at least one sensor that can basically keep track of how many of these na nasty components you've you've made. And then you can immediately think of why that would be useful, right? The cell's gonna want to know if you've made a lot of these things or little or how long they've

uh been made and you can start to imagine how that could be hooked up to some kind of recuperative mechanism that might sort of shut things down so you can uh p you know fix things.

Exactly. So so we think that um uh and and the sensor is has has a very interesting property, uh uh and that is that it that it That it registers an encounter with one of these lipid derived carbon molecules, then changes its state. So each of these molecules can hold one bit of information, it flips from one state to the other, and then it stays stably in that other state.

until it is reset by electrical activity. So you can imagine if you have one of these sleep inducing neurons that has thousands of these channel and sensor molecules. And um if you obser if you could observe the cell and these molecules over time. As the mitochondria burn fuel and respire and use oxygen, and there's sort of a gradual electron leak and gradual levels of lipid per oxidation, these carbonyls appear one after the other. And once a carbonyl hits

one of these sensors, it flips its state. So can so the cell can literally basically count and remember. The past history of producing these nasty chemicals as a consequence of oxygen-based metabolism. And then at some point.

you reach a threshold or whatever and the neuron becomes active. And you're saying this is already a neuron that was known to induce sleep. So so essentially if we put these things together, you're saying the neurons naturally get turned on by a signal of how many of these nasty metabolic byproducts have been produced.

That's exactly what seems to be the case. So you can think of it The total amount you've spent awake, or what's called technically sleep pressure, your need to sleep, is sort of a continuous variable. and and you have these many of these digital binary devices. And so what what the cell does through this counting mechanism, it it quantizes the continuous variable into the discrete binary states of of this large population of of ion channels.

Um so it's like in a in a in an audio recording where you convert the analog waveform of music into the bits that you then store in digital form, right? Um And um and the cell does the same thing. It converts the analog appearance of these um uh of these lipid peroxidation products in in into a digital memory that's then read out. And um and induces sleep when when the fill level of the system reaches a certain level. And remind me, so this sensor

Is a channel and is it in the mitochondrial membrane or the cell membrane? It's in the cell membrane. Okay. Okay. And that also solves the the the we we've always scratching our heads. uh before we found this crucial intermediate of of lipid peroxidation products, how a signal that's short-lived, such as reactive oxygen species, um could could um could reach the the the the the electrical um impulse generating machinery in the cell membrane when it originates in the mitochondria.

Uh and the answer of course is that it's not the short lived reactive oxygen species that serves as a direct signal at the plasma membrane, but um the the lipid per oxidation products that are produced as a result of the electron leak. Got it. So so the reactive oxygen species are so ephemeral that they don't last long enough that they can kind of be detected and counted and kept track of.

But these lipid peroxidation products do. So on the one hand, they're a good signal to listen to because they're nasty. They're clearly indicating that like a lot of metabolism has happened and we need to know that. But they're not so ephemeral that you you you can't keep track of them.

Interesting. Okay. So so and and just to say it explicitly for people, you know, normally when we think about memory, we often think about, you know, the connections between neurons and things like this. But we're talking about a system here that's all within one cell. This this is one cell that's keeping track of its basic Basically, it's metabolic history by converting this analog signal into a digital signal.

Right. And and I mean the memory, um so so so memory obviously has a neurobiological and and a psychological sense, but it also it also has an engineering sense in in computer science. And um the memory that's that's in our computers and and in our cellphones uh um No known as DRAM or dynamic random access memory that was invented in in 1969 by Bob Denhard, who worked at IBM Research in in the Hudson Valley. And uh if you look at his patent.

Um the architecture of an elementary memory unit that can hold one bit of information precisely recapitulates. Um, the architecture of the ion channel that we've studied. Um, Denar's electronic memory is a two-component system. It contains a capacitor that stores one bit in the form of an electrical charge. And then a gate, an access transistor that determines

depending on its voltage, whether you can read or write memory to the capacitor. So you can charge or discharge the capacitor. In our case Um the memory unit is that ion channel subunit that flips from a reduced to an oxidized state depending on the availability of lipid peroxidation products. And whether it can be um uh read out is also determined by voltage, by the voltage across the plasma membrane. So so human beings dreamed up and built this type of system and they patented it.

And they had no idea about this biology and it turned out that that nature had invented more or less the a perfectly analogous system uh hundreds of millions of years ago. Yep. So let's go to the flies now. So so we've built sort of a a base here and we'll sort of connect the dots.

over the next little bit of the conversation. Let's talk a little bit about flies and sleep and what was already known about the neurobiology here. So so it sounded like you already knew that you had these very important sleep inducing neurons, neurons which were sufficient to to make flies sleep. You can turn them on and make flies sleep. Talk a little bit about the circuitry and the cells here that we already knew about that were really important for sleep and flies.

Um so the the the the neurons that we studied um are um cells that project to a particular layer or two layers of the dorsal fan shape body in the central complex. Um these neurons represent a small minority, there's thirty or thirty one cells. um among the hundred thousand or so neurons that make up a fly's brain.

Their role uh in the induction of sleep was discovered by a former postdoc of mine, Jeff Donnelly, when he was a graduate student with Paul Shaw um at Washington University in St. Louis. So this was part of his thesis work and then when Jeff came to work in my lab, um we began to um explore the function of these neurons and discovered that they are important For regulating sleep depending on sleep history. So they have nothing to do with the circadian clock.

effectors of sleep pressure and they as as such they must therefore also be equipped to sense sleep need. Um so so much of our work has been devoted to understanding how these neurons uh transduce. a perceived need for sleep

um into sleep inducing electrical activity. I think the the the really important conceptual um uh point to make here is that um it's it's been known for a very, very long time that sleep loss uh uh uh uh normal sleep-wake cycles I associate with numerous changes in the brain from electrical firing patterns to the changes in synaptic Strength to metabolite concentration changes to changes in gene expression. But if you just look anywhere in the brain, it's it's extremely difficult logically.

to to disentangle cause and effect. If you look at these dedicated sleep-inducing cells, you know that whatever is the fundamental driver of sleep. must connect in these neurons with the biophysical machinery that regulates the electrical activity. You knew these neurons were were important for

uh sensing sleep need at some level that they are sufficient to induce sleep. So whatever the natural signal is that corresponds to sleep pressure building must be sensed basically by these neurons. And what we were talking about before is getting at like what they're actually sensing. Exactly. So it's it's I I think it's also a a a a wonderful um example of something that I that I often say. So I early in my independent career, I I was involved in the invention of optogenetics.

And I always say that optogenetics is a beginning and not an end. So in in the case of these sleep-inducing neurons, what Jeff and I did, and Jeff and Paul, was to activate these neurons artificially and see that they can induce sleep. Um so you you you can establish the causal role in the induction and maintenance of sleep. But of course the interesting biology begins um when you understand what the natural signal is that normally turns these neurons on. Yeah.

And so so basically the idea we're going to is, you know, you're awake or flies awake for some time. Obviously the longer you're awake, the longer your cells have been doing what they do, using oxygen and mitochondria to create energy, um more Uh metabolites have been made. Um some of these lipid peroxides have been made.

Uh the cell is able to sense and remember these things. And at some point this sort of builds up in the cell and causes these neurons to turn on. And then that's that's basically what causes sleep. When you started doing some of the work that you just published, let's talk a little bit about how you sort of zero in in the fly brain on which neurons are important and and where things are happening.

That's going to take us to a technique that you use called transcriptomics. So what's transcriptomics and how did you use it to figure out like where to look in the fly brain here? Um so so transcriptomics is um a a a method that allows you to measure levels of gene expression genome-wide and in the in the version that we use. Which was single-cell transcriptomics, with the resolution of individual cells. So you can see which genes are turned on or off.

in individual cells under different conditions. So we looked at um the entire central brain of flies, were able to identify specific neurons and had flies that were either um fully rested um so collected at eight o'clock in the morning after a night of shutdai, or they had been uh sleep deprived the preceding night.

Um transcriptomics did not tell us where to look for gene expression changes. We already knew where to look based on based on these um biophysical experiments and optogenetics experiments that I had. um described before. We actually used um transcriptomic Um just to confirm in in an unbiased completely open minded way, the crucial role of mitochondria. We essentially we played ignorant. Yeah. Yeah. Yeah.

We already knew from our from our more mechanistic work that we had this sensor in the plasma membrane, that ion channel that responded to lipid peroxidation products. and that mitochondria was sort of the source of

of of the trigger that would that would spike the peroxidation reaction. So you have these you have these key cells in the fly brain that induce sleep. You already know that. You already know they're important. You've also independently discovered this this sort of lipid uh peroxide sensor that we talked about before. So then you can do transcriptomics on these cells and what that's doing. is it's allowing you to look in an unbiased fashion at what sheens are turned up and turned down.

when an animal is deprived of sleep. So you you basically prevent the flies from sleeping. You can isolate these neurons that we already know are crucial for sleep. And then just look at the whole genome and it's like what kind of genes are up and what kind of genes are down. Exactly. And the only genes that were up Um the mitochondrial. And it didn't need to be that way, right? That's that's kind of a striking result. Yes.

So this was this was this was a nice moment when we saw that all all the upregulated genes, uh almost without exception, I mean all of these measurements of course have have some level of noise. So there's there's there's other things in there. But if you if you try and group the the the the sort of the the genes collectively that are upregulated.

um the the the the data really rub your nose in the mitochondria as being as being crucially important. Yeah, and and there's a lot of genes that could have been up or down regulated here. So you know, a relatively small fraction of the genome really pops out here and you're saying just it all happens to be mitochondrial related genes. Yeah. Mostly. Yeah. Okay. So

Go ahead. The question of course became um what is this? Why why are the mitochondrial genes upregulated? Is this is this um a damage response? Um Is this is this just mitochondrial proliferation? What is it? So we decided to look at the mitochondria just in these 30 or so sleep inducing cells.

So we labeled them with a with a with a fluorophore in the living flies, and then we again um went through sleep deprivation versus rested, and we saw that sleep deprivation caused drastic morphological changes in the mitochondrial network some of the mitochondria became fragmented, very small, they attached to the endoplasmic reticulum, and part of the mitochondria also got taken up into lysosomes, a process called mitophagy. So it it it very much looked

Like a damage response. Um which is consistent again with the idea that um that that that you you you you really exercise your mitochondria, you have a significant electron leak, uh the electron leak creates all the downstream problems that we've discussed. um the mitochondria and their membranes get damaged as a result.

uh and so what you do is you you secrete the the damaged bits, you put them into lysosomes to discard them, you attach to the endoplasmic reticulum To reload your mitochondria with um with lipids that are synthesized in the ER and you upregulate. you upregulate mitochondrial gene expression to rebuild the organelles during recovery sleep. Yeah. And so so basically, right, the the longer that mitochondria have been working or turned on, let's say.

The more chances there are for them to become dysfunctional, to become damaged. They're generating these reactive oxygen species and these lipid peroxides. That's causing a problem. The longer you let that go without sort of resetting or replacing those mitochondria, the more the cell's gonna be in trouble overall.

And so that that's basically what we're talking about here. The animals are sleep deprived. They're not allowed to sleep. The mitochondria start to become dysfunctional. They look weird. They're aggregating in different parts of the cell. And the cell has to literally digest them and replace them. That's the bad idea. Yeah, yeah. Interesting. And so

One uh so so we we've talked about these lipid peroxides. We've talked about how polyunsaturated fatty acids are more prone to uh react with reactive oxygen species and produce these nasty chemicals. Talk a little bit more about the endoplasmic reticulum and how the new mitochondria get made. So it sounds like new mitochondria can get made. They kind of dock onto the ER and this is how they get loaded with like their lipids and the stuff that they're built from.

So this is one of the of the of of a really um important area in in in modern cell biology that has grown enormously over the past fifteen uh so years, um the study of of of interorganelle contact sites. So there's numerous such tightly regulated contact sites, including sites that that link the mitochondria to the endoplasmic reticulum. Um we now even have structures um In um in yeast that show the ER mitochondrial contact. And it's in fact a lipid channel. So you can import.

uh lipids from the ER into mitochondria. They can there's a hydrophobic tunnel and you can you can you can pass the lipids through. The reason why you need this, of course is that many lipid biosynthetic pathways are either exclusively concentrated in the ER or distributed between the ER and the mitochondria, so you have to import precursors from from the ER into mitochondria. Um so this again Finding the mitochondria attached to the ER

um suggest that that something is going on with with the lipids, as we've already inferred from studying um the the the ion channel. Now mitochondrial fission usually happens at these contact sites. Between mitochondria and ER. So the sequence that normally happens is the mitochondrion attaches to the ER and then recruits. um a dynamine related protein that sort of constricts it and um and and and divides the organelle.

Hm. Okay. So so mitochondria they can grow in size, they can fuse together, they can they can split apart, and you can you can sort of watch and and map all this out happening in the cell. And it tells you something about the health and the state uh and whether or not these mitochondria have become dysfunctional. Exactly. And there also seem to be clear relationships.

between mitochondrial morphology and mitochondrial function. So it's widely thought that if you have hyper fused mitochondria, so very large extended branch networks of mitochondria that allows particularly efficient ATP synthesis where is whereas if you have small, rounded, fragmented mitochondria, the efficiency of of of electron transport and ATP synthesis drops.

Got it. So in general, more smaller mitochondria is an indication of inefficient energy production and sort of bigger fused together mitochondria indicates the opposite, it seems. Right. Yeah.

And so you say in this paper that mitochondrial electron surplus induces sleep. What does that mean? And how did you measure that? So we we we use the the the the mitochondrial electron surplus as a as a as an alternative expression to the electron leak that we've been talking about. So if you if you feed more my uh electrons into the mitochondrial transport chain then you need to fuel ATP production if a mitochondrial electron surplus.

Um and um so it's it's like like everything um in in life, it's a it's a question of supply and demand, right? So we we we uh performed experiments where we either increased or decreased the supply of electrons or increased or decreased um the demand. So these were genetically targeted manipulations of the inner mitochondrial membrane. Where we changed this this relationship between supply and demand. Let me give you one example.

Um we talked before about how the electron flow down that wire um leads to the build up of a proton electrochemical gradient that is then tapped by the turbine to synthesize ATP. Um now there are there's a class of proteins called uncoupling proteins, which in fact short circuit that proton electrochemical gradients. They're little they're little um Proton selective pores in the inner mitochondrial membrane.

So y you you can imagine that if you if you incl if you if you punch a hole into the inner mitochondrial membrane, you dissipate that gradient in order to synthesize the same amount of ATP You need a larger supply of electrons to compensate for the whole, right? So this would be an example of reducing the mitochondrial electron surplus. And uh as predicted, if you over express such an uncoupling protein in the sleep-inducing neurons, you reduce the demand for sleep.

We've also done an opposite manipulation where we used a light driven proton pump. to build up the proton electrochemical gradient independent of the electrons that get um that get fed into the transport chain. So um in this way they have sort of hypercharged the mitochondrial battery, whatever electrons are fed in from the Krebs cycle are redundant.

Um that means that the electron surplus increases, the electron leak increases, and um sleep should increase, and that's exactly what happens. I see. So so if there's a greater electron surplus. And you manipulate the system in a way that that gives you that the animals sleep they they you you induce sleep basically. Yep.

Interesting. So why so so the cell has ways to couple or uncouple electron flow to ATP synthesis. How closely those things are matched is important here. You're you're basically saying You know, you could have the current flowing through this wire that's the inner mitochondrial membrane. If the current exactly matches the amount of ATP you need, things are kind of balanced. If you have more electrons than the ATP demands of the cell,

That's more electron leak. There's more chances to induce some of these metabolic byproducts that are not good for the cell, that's gonna increase sleep need because sleep is gonna sort of reset or recuperate the production of those things. The cell has things like uncoupling proteins. So the cell can decide and regulate how coupled the system is. Why, why does that exist? Why does the cell have the ability to tune and change how coupled the electron flow is to ATP synthesis?

Um it's it's not entirely clear, um, except in one context. So I think the best um understood role for these uncoupling proteins um is in thermogenesis in the generation of body heat in brown fat. That's how these proteins were actually discovered. Um so the the The brown fat cells use this uncoupling proteins to actually turn mitochondrial electron flow into heat rather than ATP.

This is one example. In other contexts, there is evidence that uncoupling proteins play a role in the central regulation of energy metabolism in the hypothalamus, for example. But how how widespread their uses in the nervous system to actually match electron supply and demand, um, I I am unaware of any detailed studies that address that. In general, it's thought

that all these forms of regulation are rather slow. So if you if you have a mismatch between electron supply and ATP demand, uh you can't correct that at the time scale of say action potentials. um which which may sort of lead us to to the deep mystery that have that that that has That that I've been obsessing about since we found that mitochondrial role, which is: okay, if if if the reason why we need to sleep. Is such a simple biochemical problem.

um such as electrons leaking from your mitochondria, right? Why do you need to lose consciousness? Mm. Fix that problem. Why can't you find a simple biochemical solution? And um I don't have an answer, but I hope to have one at some point. Mm-hmm. And so so you can artificially induce more electron leak than electron surplus that

that basically causes more sleep pressure and sleep need. That makes sense. And and so I guess the way to think about this in the natural state is the animal's awake. The longer it's awake for whatever reason, the more metabolism that's happened, the more uh excess electrons and reactive oxygen species and stuff that will have been produced, the more the the lipid peroxide sensor we talked about before will will be registering that that all of this has happened.

And the more inefficient the process will probably be as as as time goes on. And so these things are basically read out by the cell and you're you're coupling this readout of these metabolic byproducts. to the activity of special neurons and circuits in the brain that we know induce sleep when they become active in the right way.

Exactly. That's that's that's the way we picture the mechanism. Yes. And because we're talking about mitochondrial dynamics, ATP production, you know, this immediately is it it makes it very easy, I think, for me to think about like why you would also in other contexts, especially in so called higher organisms, why things like memory consolidation and synaptic protein synthesis and all of these things would

be correlated and tied with sleep because those are all energy intensive processes. And obviously if you can't reset and maintain the efficiency of the energy generation mechanism of the cell, then all of that other stuff downstream is not going to be possible either. Exactly it. And and that's true not only for high organism. Um flies also have serious memory deficits. If you sleep deprived them. So even even even even their memories um require sleep for consolidation. And so

So so so you think that this is basically this is probably basically why sleep evolved. This this It's tied to aerobic metabolism, the use of oxygen and mitochondria. That's why you see everything that you see in flies.

it's implies that this you know, this stuff sort of would have to be true in in vertebrates as well, because we have the same basic mitochondrial based metabolism, but also why something as seemingly uh simple as a jellyfish without a centralized brain would also have a sleep-like state. Yes, that's the way I think about it.

And so it needs to be it needs to be formally shown in in in mammals. I mean we we I think we all operate under the well founded assumption that that evolution is conservative. Um, but you still need to demonstrate that that similar mechanisms operate in the mammalian brain. Um and that's obviously another thing they are currently trying to do. And so to the extent that Sleep is a natural consequence of aerobic metabolism based in mitochondria.

This also ties into certain patterns that we see across the tree of life, which are really interesting. And I would love to get your comments on. So one of these patterns is that.

you know, smaller animal animals basically have faster metabolisms. They also don't live as long. And they sleep more. So when you look at say a mouse compared to a human, compared to a very large bodied animal, right, the mouse doesn't live nearly as long as we do and it also needs to sleep more hours per day than we do. Why is that and how does this maybe tie into the the oxygen part of the story here?

So so the relationship between um body size, mass specific energy consumption, and and sleep need um was first noted by Al Rechchaff, who I already mentioned in in in 1974. And then it was sort of uh formalized and I think um really really nailed in a paper by Van Savage and Jeffrey West. Of the Santa Fe Institute, um, which found that there is there's a power law that underlies this relationship. So if you measure mass-specific oxygen consumption, um So oxygen consumption per gram tissue.

per um per unit time and you plot that versus daily sleep amount uh on a on a on a bilogarithmic scale, um you you you get a straight line. Now, if you look at the exponent of that straight line, it's not a multiple of one-third, as you would expect from sort of classical Euclidean geometry, right? That it's sort of surface to volume ratio that matters. but the exponent is a multiple of one quarter and West had done a lot of work on sort of Scaling laws in various contexts.

and derive the idea that if you get that that you get quota power scaling based on the geometry of centralized research distribution networks, which is a fancy name for things like your cardiovascular or your respiratory system. Yeah. Effectively it's like there's an extra dimension here beyond the three dimensions of space.

Exactly. And um and it's it's sort of the it's the fractal dimension of these uh of these distribution networks that underpins that quarter power scaling. So and if you are a smaller mammal you have a higher capillary density of these distribution networks And um the distribution networks allocate more oxygen to each of your cells per unit time. Yeah. So you so a mouse can get more oxygen to more cells faster than our bodies can, than a blue whale can. And and and that has

Consequences for our entire organismal physiology. So if you are small Yeah. The smaller you are, the faster your heart beats usually, the shorter your life. And sort of tragically, the greater a fraction of your short life you you spend asleep. A mouse or a tree shrew, they sleep eighteen, twenty, twenty-two hours a day. An elephant can get away with like three, four hours of sleep a day. Yeah.

So yeah, so basically they're in some sense they're uh you know, they can use oxygen more readily. You know, you say in the paper, you know, a small animal can burn hotter, and there's certain advantages to this from like an energetic standpoint, but but the trade off is, and and you say this explicitly in the paper,

Uh, you know, the price you pay for being able to burn hotter and have this sort of uh more efficient distribution of oxygen to your cells is a shorter life and you spend more of it asleep. Exactly.'Cause you you ha your brain has to compensate for all of the the side effects of what you're actually using that oxygen for. The generation of the reactant oxygen species, the lipid peroxides, all of that stuff.

is is gonna come as an inevitable consequence of this oxygen that you're distributing to your cells. Yeah. And so um You know, we mentioned this before, but let's just sort of say it again for people and and put a bow on this from an evolutionary perspective. Right. So a very, very, very long time ago, there wasn't really much oxygen in in the earth.

but there was living things and then oxygen comes on the scene and we'll just call that the oxygen revolution. You basically say that a after this oxygen rev revolution, many, many millions of years ago, power hungry nervous systems appeared after that. And with them, apparently the need for sleep. And then although sleep since acquired additional functions like synaptic homeostasis and and memory related stuff, the

The power law that we just talked about relating to daily sleep need and mass-specific O2 consumption suggests that like this is the ancient purpose that evolved first. It had to do with the use of oxygen that was only enabled once there was oxygen on the earth.

And you you have this need because inevitably you know so so you're getting all of this ATP from the mitochondria, that's what you get, but inevitably you're producing these compounds that that lead to cell dysfunction and you need to be able to compensate for those and and sleep is appear apparently c serving that role. That's at least the way I see it. That's my that's my prejudice. I mean it's it's it's it's a not entirely unfounded prejudice anymore, but uh yes, that's that's how I

how I think about the problem. And then, you know, so so you're saying you're not saying sleep isn't also for memory consolidation, these other things, but those are, you know, those are basically roll they they stack on top of this one because they're all downstream of this energy metabolism part of the the equation here.

Or they could even be independent of it. Yeah. Um if if evolution just um managed to take advantage of the off periods that were required for metabolic reasons, right? I mean it doesn't mean that that the that the other functions of sleep

are a a directly downstream of metabolism. One of the things that you note is Including in humans, but Uh i uh a common symptom of mitochondrial diseases of various kinds is this overwhelming sense of tiredness. And it's actually independent of, you know, muscle fatigue and, you know, moving around that day. And that would make sense because If you can't officially use your mitochondria to make cellular ATP, you're naturally going to feel fatigued.

Now the interesting thing about these mitochondrial diseases is uh people have characterized these patients very, very carefully and they they tend to have normal ATP levels. Um they also have um uh have have in in many cases no problems with with with with with strength, with muscle, but they still have this overpowering sense of tiredness. And the way I think about it is

So so many of these patients have mutations in in the mitochondrial genome. So they they they have difficulty aligning the the the subunits of the electron transport chain precisely. So you could imagine It we we talked before about the wire uh through which you pass the electricity, right? You they so so these patients probably have a more resistive wire that that uh an electron transport chain that that opposes electron flow with a larger resistance. So what you need to do

to get the same amount of ATP synthesis, you you you sort of need to increase the voltage, the driving force with which you pass the electrons through that transport chain. So you need a higher supply of electrons. And there we are again. Um It's the it's the it's the it's the s it's it's the balance between electron supply and demand.

That determines the size of the electron leak. So my prediction would be that many of these patients have an increased electron leak and that that is the cause for their. Um sense of tiredness. Interesting. That that makes logical sense to me. You know, one thing I want to call out here too is it sounds like so we

How well matched things are in this wire in the mitochondria dictates how readily the current flows and and, you know, how how nicely all the pieces fit together and how efficient our energy metabolism is. We normally think about mitochondria in a maternal

centric way because we all inherit our mitochondria only from our mother. But it sounded like earlier you said some of the subunits of the machinery in the mitochondria are encoded in the nuclear genome. And so the nuclear and the mitochondrial genome both encode stuff stuff that needs to be put together inside the actual mitochondria. And that would imply that both both your mom and your dad sort of need to uh have pieces that match up well to have uh

you know, a a nice system here with all this, you know, that allows for good current flow and all of that stuff. So it's not strictly that mitochondrial function is entirely dictated by your mother, even though we inherit our mitochondria from the maternal side of our our lineage. That's absolutely correct. So so the the mitochondria they they they uh they are descendants from bacteria that were engulfed by another bacterium. Um

And and over time they have delegated much of their genome to the nucleus. So they they Um, but the vast majority of mitochondrial proteins are encoded in the nucleus. There's there's a sophisticated machinery that imports um these nuclear encoded gene products back into the mitochondria But um a small number

And these are exclusively electron transport chain components have remained resident in the in the mitochondria. And uh it's an interesting question why? Why why um have the mitochondria not outsourced these thirteen um proteins. I've looked um in the literature at at some of the answers that people have proposed. And and the one that that sort of strikes me as the as as as probably the most plausible. what the mitochondrial genome gives you is local Organelle restricted control. Right.

Um a a nuclear-encoded mitochondrial protein, and you get a signal that says make more of that um of that protein. You don't know which mitochondria actually quite requires the protein of the many hundreds to thousands in a large cell, mitochondria, right? So where where do you target the gene product? Whereas if you make the stuff locally, you can you can sort of fine-tune um

Your electron transfer chain depending on local demand. Yeah. It's just more it's yeah, you can fine tune things more because you don't have to wait for protein subunits to ship in from further away. Yeah. Yeah. And you can you can produce what you need only where you need it. Yeah, yeah. On demand so you you get the sort of nice

There there's a spatial and a temporal specificity there. If I want to make a little bit more of this subunit, but only in the mitochondria, like over in this part of the cell, you can do that. Yep. And so uh this is a little bit of a different topic, but it's it's worth calling out. So um

People might know, you know, so the name for the idea that mitochondria used to be free living bacteria that got engulfed by another one, that was Lynn Margulis' idea called the endosymbiotic hypothesis or endosymbiotic theory. And this is not purely just a theory. It's it the thing that you mentioned about the nucle the the nuclear genome containing uh genes that used to be in the mitochondria, where that c that's actually a key piece of evidence here that

Um, basically the way this works as I understand it is the mitochondria used to be bacteria. And so their their genes look bacteria like compared to eukaryotic genes like the one in our nuclear genome. Except you can actually see uh what are essentially bacteria like genes. In the nuclear genome. And that's actually the signature of what you mentioned, which is that over time, a lot of those mitochondrial, basically bacterial genes.

integrated into the nuclear genome. And so we can actually see sort of a genomic record of this integration, this very ancient integration. Um it's it's I I I would say that's probably the case, but I'm by no means an expert in sort of comparative um in in in genomic archaeology and tracing evolutionary history. So

Um, one one person I've had on the podcast that I'll point people to is Michael Lynch. So I did an episode with him about about evolution stuff. I would recommend that if if people are interested in it. Um I want to talk to you a little bit about sort of what comes next in your lab with this story about.

you know, cell metabolism and sleep and and how all of the things work out mechanistically here. What are you guys building on from this work and from the lipid peroxidation sensor work uh to to take the next step? Yeah. So there's I I think three really pressing questions that that we are desperate to answer. Um the first one is um Vi har fokuserat på de 30 eller så sleep control neurons. um a vanishingly small minority in the brain. And that's true.

um for most animals. So for instance, C. elegans has a sleep control circuit too. Out of its 300 something neurons, there's only one cell that is responsible for sleep induction. In in our brains it's probably A few tens of thousands of cells in the hypothalamus that do the same job. So we we think that these sleep-inducing cells. measure the state of their own mitochondria as

sort of a representation of what's going on in the rest of the brain. Um they are sort of like to to stay with an um with an electrical and engineering analogy, they are like circuit breakers or fuses. um they they see how well or how poorly their own mitochondria are doing and um and then blow the fuse before it gets too bad for the rest of the brain.

So the the we we are trying to test that hypothesis by changing the fuse in the brains of flies. So you could imagine that if you have a circuit in your house. That's rated for a current of two ampere, and then you put a ten ampere fuse in there. You uh you run into problems. You might set your house on fire. So what we are trying to do is equip the brains of flies with fuses that are overrated for the load that the brain is built for, and we will then try and investigate the consequences

um of chronic sleep deprivation or look for the consequences of of chronic sleep deprivations in the brains of these animals. And these consequences would be cognitive impairments, so learning and memory deficits. um and also a a shortened lifespan. So I think this is one pressing question to understand um what the relationship is between the sleep control cells and what's going on in the rest of the brain. Second and we've we've touched on that already.

is um does the same mechanism operate in different species? Is is this is this true across species, especially is it true also in mammals? And the third question, and we've also briefly touched on that, I think we are getting a a pretty decent sense of what the cause of sleep is. Um it's the mitochondrial electron leak, we think. But we don't know what the function of sleep is. We don't really know. what sleep does, what happens during sleep, does does

Does a sleeping brain have a smaller electron leak? Is it simply that the leak gets plugged somehow? And if it gets plugged, how? Yeah, yeah. Or is there some active repair processes that are being initiated uh while you take your brain offline? Um so that's another really, really A big important question to answer. Yeah. And you know, another thing I wanted to ask you about here is.

You know, we're we're talking about neurons in the brain and A couple a couple of things occur to me. So one, neurons In in a sense, neurons are sort of uniquely or especially sensitive to oxidative stress in the sense that

You can't replace most neurons in the brain. They're they're not a dividing cell. So they really need to track oxidative stress because you can't just make a new neuron in in whatever the mushroom body of the fly when when you need it. You can't just replace, you know, a key circuit in the hypothalamus of a mouse or a human. Um if those cells die from oxidative stress. The other thing is that um I know this is true at least in vertebrates. Maybe it's true in flies as well.

Um, I know that basically the the lipid membrane composition of neurons tends to be um more enriched than many other cells in things like polyunsaturated fatty acids. So that's another way that neurons might be sort of extra sensitive to oxidative stress and why they would really need to have mechanisms to track, you know, track all of the things that we've talked about.

What can you tell me there in terms of how uniquely sensitive to oxidative stress neurons are and what that has to do with things like the fatty acid composition of the membrane and what the animal's eating in terms of like if it's eating a lot of polyunsaturated fatty acids? Um so I I think that neurons have sort of three strikes against them that makes them especially vulnerable. And you've mentioned two of them. One is that they are not renewable.

Um two is that they have a lot of lipids, not just uh in terms of composition that they that they tend to have lipids that are uh highly polyunsaturated. Um but they also have a lot of lipid. They have very very extensive membrane systems. So their their surface to volume um ratio is very, very large because they have this vast extended processes. I mean, so there's there's a lot of of of of membrane that can get damaged in neurons. And um the third strike, of course.

Is the large amount of oxygen consumption. So in humans, um, the brain accounts for two percent of body mass. but consumes twenty percent of the oxygen we breathe. Um and that of course um also predisposes you to to to the risks of oxygen use, right? How the degree of polyunsaturation of your lipid membranes is controlled through diet.

Um I don't know. I mean there's still some influence because some of these polyunsaturated Fatty acids are essential, so you have to have them in your diet and and what you take in um will certainly influence your lipid membrane composition to some extent. Um but there's of course also um biosynthetic pathways that allow you to modify as long as you have enough of these essential components the the repertoire of lipids in your cells uh according to what they need.

Um but I think one one dietary variable that certainly matters is simply the total amount of calories that you consume. Um there is there is uh a large body of evidence, again ranging from flies to humans, that caloric restriction, so using your dietary intake of calories, um reduces sleep need quite substantially. That makes sense, right? The the less the less food components you're consuming that are gonna funnel through this electron transport chain, the less

or the less leak you're gonna get overall, and therefore the less um reactive oxygen species and lipid peroxides and stuff on average that you'll tend to form. It would seem to be the case. Yeah. Yeah. Yeah. Interesting. Um

Is there anything that you want to reiterate or any final thoughts you want to leave people with about this body of work that you do? This is, of course, not not all that you've done in your career, certainly, but when it comes to sleep and metabolism and this sort of deep connection, uh, what do you want people to take away from this? Um I think we've had a very wide ranging conversation. Um and um it's been thoroughly enjoyable. I hope it's been as enjoyable for the audience.

as it has been for me. Um I think the the the key message is that um that sleep um is at its root. um a biochemical uh the consequence of a biochemical problem um that um that that is that seems specific to the brain and that surprisingly the brain has found no way to solve. But to make us unconscious. Yeah.

Um well uh Professor Bisbach, thank you very much uh for your time and for joining me across the pond there. Uh this was fascinating. Um I'm gonna put a link to one of these papers in the episode description. Um I'll also call out, so you mentioned Jeffrey West's work earlier.

I've had him on a very early episode of the podcast and I recommend his work as well. I think that if you listen to that one and this one back to back, uh they go together actually in a very interesting way and and help explain how some of these things work. Um, he studied sort of the way that body size metabolism scale with with these networks in in biological uh animals.

Um so fascinating stuff. I look forward to following your work on this stuff. I I you know this this work that I'll link to, I think is this is very beautiful work. I'll just tell the listeners it's very hardcore. So if you want to go read these papers, it they're very, uh very hardcore, but very well done. They span neurobiology and biophysics and biochemistry. So uh thank you very much for for your time. Thank you.

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