Extending lifespan by rejuvenating mitochondrial membrane potential – Dr Brandon Berry

Extending lifespan by rejuvenating mitochondrial membrane potential - Dr Brandon Berry

And I would get asked kind of a lot um Did you test the lifespan in these worms I would say uh yeah we want to we’re Going to do it and uh eventually I did It and we we found it it seemed to be Living longer and I was like well I’ve Never done a lifespan experiment before I better do it a few times it seems to Be reproducible And at the same time as we got those Results I was getting more and more Interested in aging because of the Hypoxia literature and how different Protective paradigms and hypoxia seem to Not work with old age and I was thinking If we’re studying heart attack and Stroke using things that don’t work in Older animals like what are we even Doing so I wanted to learn more about Aging and that’s that’s when I started Talking with Matt caberline and uh I Showed them these optogenetic results With Mito on and he was happy to have me Come join the lab and figure out what Exactly was going on in these worms so Hello and welcome to the cheeky science Show where in this video I spoke with Dr Brandon Berry a current postdoctoral Fellow at the University of Washington Where he’s developing and using tools to Understand the function of mitochondria And their links with the season aging Here we discuss season trust father and The results from his latest publication

By the tool extended blam lifespan So hi friends and uh thank you for Joining me today and welcome to the Chica Science Show Thank you so much for having me I’m Really excited Great because I’m really interested to Hear more about your research Um and how you manipulate mitochondria And by for before we go there um it’s Kind of known that mitochondrial Dysfunction is Hallmark of Aging as I Was wondering if you could explain what Are mitochondria are and how does your Research relate to addressing the link Between mitochondrial dysfunction and Aging Yeah so mitochondria are the best Organelles ever Um they’re these evolutionarily ancient Uh they seems like they used to be Microbes free living and eukaryotic Cells Um engulfed them so they’ve been Evolving with life uh with as long as Life has been complex there’s been Mitochondria And what mitochondria do classically uh Everyone knows them as the PowerHouse of The cell and because that’s because they They make ATP so ATP is the cellular Energy currency and if you think about It everything that the cell does any Action that the cell takes is going to

Require energy and most of the time that Energy comes from mitochondria So that’s why we think that Mitochondrial dysfunction is like a core Hallmark of aging and we think it Probably underlies a lot of the other It’s not all of the other Hallmarks of AJ um just because when it comes down to It if there’s energy defects there’s Going to be problems Yeah both sides and so you’ll say one Thing that um is pretty important for What your research is is looking at Um the components that are found within The mitochondria the membrane and Obviously it’s linked with like aerobic Respiration and as you said the Generation of ATP so Um I think one thing we should probably Discuss in detail is how is that process Um and how does that process work and How does it involve proton transports Yeah yeah so The core part of mitochondrial function That I’m really interested in is the Membrane potential which is part of this Proton motive Force so mitochondria Function I I call it a battery charge It’s very similar to a battery Um so there are two membranes the Mitochondria outer membrane the inner Membrane inner membrane there’s an Electrochemical gradient so there’s a Proton gradient it’s positively charged

Hydrogen ions are separated across this Membrane So the positive charge on the protons is Responsible for most of that driving Force There’s a pH separation as well so just The pure concentration gradient adds Some of that potential energy as well But it’s it’s mostly membrane potential So the membrane potential is the charge And the pH potential is the Concentration of gradient so proton Motive force is both of them and it’s Kind of used interchangeably like people Will say membrane potential to mean the Whole gradient and I think that’s mostly Because the best ways to measure it You’re only really measuring the Electrical component but because that’s Most of it it’s kind of all people look At sometimes So that’s membrane potential And how that membrane potential gets Established is through some enzymes in The mitochondrial inner membrane called The electron transport chain and what Happens is is literally combustion so Just like in a in a power plant a carbon Source is burned to extract energy in Some way so the combustion that happens In mitochondria the carbon source is Food And the energy present is extracted Through different redox reactions in the

Electron transport Gene and what happens With there’s some energy in those redox Reactions that pumps protons from inside The mitochondria to outside and that Establishes this potential gradient more Protons outside than inside and that That’s where the membrane potential uh Comes from And so what the membrane potential then Does gets used to make ATP through the ATP synthase that’s also in the inner Membrane memory of mitochondria so There’s a chemical energy in food that Gets converted into this electrical Energy that gets converted back into a Chemical energy that the cell can Recognize and use Um so that’s the PowerHouse analogy but We should also say that um we really Know that mitochondria are a lot more Than just powerhouses they do provide ATP but there’s a lot of good articles Actually summarizing all of the other Stuff that mitochondria do Um they influence lots of different Things like hormone production immunity Um so they’re not just powerhouses and The the analogy that I like to use is Like Calling mitochondria Powerhouse of the Cell is like hauling a smartphone a Calculator Where it’s like that’s true but it kind Of misses the point of what they are and

What they do Um so yeah that’s that’s uh I like to Think about it No I like to think that’s a cool way to Think about it Um and so you’ve mentioned this person Makes a force and um how the persons get Put into the the space between the Membranes but how do you actually go About measuring what that uh potential Is and how does that potential change With age Yeah so measuring it can be pretty Difficult It’s really difficult if you want to Measure it in an animal kind of like how We’re doing in our animal model but Traditionally how this is done is using Isolated mitochondria so researchers can Isolate mitochondria from cells or Animals by grinding up tissue doing Different laboratory steps to just get Pure mitochondria and then you can Measure this gradient using small Molecules that are positively charged so The positive charge will be attracted to The inside of mitochondria because you Have positive charges outside negative Charges inside so there are these fatty Molecules that have a positive charge That will accumulate inside mitochondria And some of them are fluorescence you Could measure the fluorescence Accumulation

Um you can use it with electrodes there Are molecules that you can measure Electric fields and and determine how Much the mitochondria are taking up Um but then once you get into cells it Gets a little bit harder into whole Tissues it’s a little bit harder and That whole animals it’s really Really unclear what could be going on But we gave it a shot And so is that when any studies that I’ve looked at Um like how this membrane potential is Linked with different life diseases or Like educational diseases Yeah yeah so there have been early Indications in yeast models that it Seems like this uh potential declines With age the client’s pretty early with Age which is what we found in our model As well And it’s known that the the proton Motive force is implicated in lots of Different diseases so Mitochondria are involved in like any Disease you can think of there’s Energetic defect and mitochondria are Likely to be involved at some level Um so probably the earliest example of Looking at mitochondrial membrane Potential and disease is uh in Cardiovascular diseases like heart Attack and stroke where mitochondrial Membrane potential seems to fluctuate

And there’s lots of cool data out there I I studied this uh during my PhD I did Heart attack and stroke models And part of what we wanted to do was Look at how changes in mitochondrial Membrane potential would influence the Outcome from heart attack and stroke Models So it’s it’s one of those things that’s Such a fundamental process to Mitochondrial function where like once You start looking at it there are Changes happening but it’s usually Unclear what those changes mean or lead To Okay And so you sort of alluded to some of Your PhD work and obviously you’ve Continued uh some of that onto your pay Stalk and published the paper that came Out just last week and so Um you describe a tool called Mitochondria on and this is a way to Sort of manipulate or to increase this Proton meta Force so I was wondering if You could get some sort of like Backstory to how this will start it and How you develop the tool yeah so it all Started at the beginning of my PhD Actually so I was in a lab that used C Elegans which we can talk about uh some Reasons to use C elegans but uh this the Lab uh is uh led by Andrew watovich Um who is on the paper that we recently

Published as well we still work pretty Closely with Andrew and when I joined The lab Um he had this project so his lab uses Optogenetics to control many different Aspects of mitochondrial function And so optogenetics is this cool tool Kit that is used in Neuroscience a lot Uh where they use light sensitive Proteins to control some aspect of Physiology so usually they’re light Activated channels or pumps that are in Membranes so you can control Action Potentials and neurons and dissection or All circuits so being hardcore Mitochondrial biologists knowing that Proton motive force is uh the driving Force of mitochondrial function it kind Of followed that you could use a light Activated proton pump can we stick it in The inner membrane of mitochondria and Control that gradient So we actually ended up doing that in Both directions so we created tools Called mitochondria on or Mito on and Mitochondria off or Mito off so this is Just light activated proton pump facing In either direction you can do some Molecular tricks to flip the direction Of the proton pump and get the proton Pump in different directions So it took a long time for me to develop Uh the tool we tried a lot of different Strategies and eventually I got it to

Work and we’re very happy for that Um and so yeah I used it to study Hypoxia biology and see elegans worms And near the end of my PhD I got really Interested in aging Um I think a lot of us get interested in Aging because it kind of comes up in in The course of your research that things Start to change when animals get older And I just started to say like why is That and can we use these tools to study Aging itself That’s interesting and so Um like talk a bit more about the the Mechanism of uh this tool that you Developed how exactly like what is the Protein that you’ve used and did you Test different proteins because it’s um Obviously a proton Um it’s a light sensitive proton pump Right yep yeah Yeah so we tried um two different Versions actually there are a lot There’s probably way more now even so When I was early in my PhD there were Probably tens of different proton pumps People will go in and change different Residues to make them a little bit more Efficient or responsive to a different Color of light but for the most part They’re all related to the protein Bacteria rhodopsin so seven Transmembrane Alpha helices there’s a Cofactor in the protein called All Trans

Retinal or ATR so this is a cofactor That absorbs the energy from light and It does a rearrangement that forces the Protein to rearrange so proton so a Proton on one side of the membrane will Be able to get out on the other side of The membrane And as long as light is Illuminating the Protein this molecule will cycle and Protons will get pumped until the light Is turned off So we actually use the proton pump from A eukaryotic organism even though it’s Bacteria adoption related a eukaryotic Microbe that uses these proton pumps to Sense light and to move toward the light To do photosynthesis Um it’s one of the common ones that’s Used in optogenetics and we also used a Similar proton pump from a bacterial Organism and it just turned out that This one got expressed better first and We just ran with it Yeah it sounds like science um that’s Really interesting Um the song I was going to say Um yeah so you’ve mentioned that it uses Light to activate this proton pumping um Ability of the movement of protons but What sort of wavelength of light are you Using and Um do you control the wavelength of Light and does that have any Consequences with like heat transfer as

Well in these experiments yeah yeah so Luckily the light that these pumps Mostly use like I said there are some That shift the wavelengths a little bit Um it’s 590 nanometers which is yellow Greenish light Which is not the most damaging color for Mitochondrial Um proteins and for for biological Tissue like blue light can be pretty Damaging on its own and cause some Oxidation Um so it’s it’s not the worst color of Light But the really great thing is that in Our models do we use C elegans nematode Worms they actually don’t produce that Cofactor all trans retinal So we have to feed that cofactor to them So that’s a really great control for the Light exposure so we can have worms that Are expressing the protein we hit them With light But there’s going to be no optogenetic Activity so we know that the light Itself is not you know burning them up Or having some off-target effects so we Know the combination of the cofactor and The light is when the optogenetics is Active so we use that condition to Compare to all the other conditions Um so for that reason we really like Using the worms with optogenetics Because it’s internally controlled where

In mammalian tissue we have a synthetic Pathway for all trans retinals it’s Vitamin A Um so it gets a little bit harder to Control so I like using worms in Conjunction with other models to uh you Know be sure that we’re studying what We’re studying Yeah that’s a great control and um as You mentioned earlier so why is it Besides this point that you’re using Seattle guns obviously there’s a lot of Advantages but um one of the main ones Yeah yeah so C elegans are great for Optogenetics uh purely because they’re Transparent you can illuminate all of Their tissues you can see all of their Tissues you can do fluorescence Microscopy and see what’s going on You can see Um measurements of the mitochondrial Membrane potential using these Fluorescence dyes in the worms you can Stain the worms and kind of get an idea Of what the mitochondrial membrane Potential might be there’s some caveats With that Um but it’s pretty useful And then the reason for using worms in General Um especially for aging they have a very Short lifespan you can do whole Population lifespan studies in around a Month a little bit longer than a month

Um sometimes a couple weeks They have highly evolutionarily Conserved mitochondria Um kind of like I talked about at the Beginning but mitochondria are very Conserved because it seems to be that They were Evolutionarily ancient So a lot of what we actually know about Mitochondrion mammals comes from studies And see elegans early on and a lot of What we know about aging uh comes from Clans so I think it’s a it’s a great Model to nail down the fundamental Biology before Um chasing down things in more Complicated models Exactly and I guess one of the more Recent advances with working with worms As well is the the ability to sort of Genetically engineer them so I was Curious like how did you Um Express this mitochondrial on um tool Into the worms yeah so we did it a Couple different ways Um The first way is kind of the classic uh C elegans transgenesis where you can Just take plasmid DNA and inject it into Their gonad and then their progeny will Take up this plasmid as an extra Chromosomal array so if I have this Plasmid floating around and they’ll Express the protein from that

Um that was hard to do because it turns Out if you try sending a lot of this Mito on protein to mitochondria the Worms are pretty sick so we had to find Like the Goldilocks amount of DNA to Give them At the same time Andrew’s lab was Developing a crispr technique to In a defined way insert one copy of a Gene In one place in the genome where we knew Where it was So we ended up doing both of those Methods and the crispr strain Um Worked basically the same as the over Expression strain so we were very happy For that we had two different genetic Backgrounds to test it in and now we’re Just using the crispr strain Because it’s uh we know it’s in the Genome and you control the expression a Little bit better And so why I guess you are over Expressing it consecutively or was it I Guess you can control it Um obviously the activity at the light Level but in terms of personal Expression that was continuous yeah so It’s over expressed with the extra Chromosomal array because you don’t Really know how many copies of the array There is and in the crispr strain we Know there’s a single copy and we’re

Using an endogenous promoter so it’s Expressed under the eft3 promoter uh Just a random Pretty common worm promoter and then It’s also tagged with a fluorescence Protein so we can actually see that it’s Expressed in the worm and we can see That it’s in mitochondria in the world So yeah we think the expression is is Pretty um Low to medium expression it’s not super Highly expressed Okay and socially the major finding from Your recent paper is that by having this Activation of this Um mitochondrial on tool you were able To extend the lifespan of the worms so Was that a surprise in finding Yes yes it was very surprising um so Early in my PhD I I was focused on Cardiovascular stuff and hypoxia and I Would get asked kind of a lot um did you Test the lifespan in these worms I would Say yeah we want to we’re going to do it And uh eventually I did it and we we Found it it seemed to be living longer And I was like well I’ve never done a Lifespan experiment before I better do It a few times it seems to be Reproducible And at the same time as we got those Results I was getting more and more Interested in aging because of the Hypoxia literature and how different

Protective paradigms and hypoxia seem to Not work with old age and I was thinking If we’re studying heart attack and Stroke using things that don’t work in Older animals like what are we even Doing so I wanted to learn more about Aging and that’s that’s when I started Talking with Matt caberline and uh I Showed them these optogenetic results With Mito on and he was happy to have me Come join the lab and figure out what Exactly was going on in these worms Um so I did the lifespan and the crispr Strain so the first time we did it was In the over expression screen And I did the lifespan in the crispr Stream out here in Seattle and Andrew’s Lab back in New York Um was was doing more lifespans and kind Of surprisingly it worked every time and We Thought it was really cool didn’t know How to explain it kind of still don’t Know how to explain it which is exciting Uh because there’s a lot to do and There’s a lot of stuff that I wanted to Follow up on and I think Yeah when you study mitochondrial Membrane potential there’s so many Things Um to follow up with so that’s why I Like this stuff Um but yeah it was definitely unexpected Um and just the fact that we could see

The mitochondrial membrane potential Decline with age so early in Age Two They’re young adult worms and you can Already see their losing membrane Potential pretty fast and this is Um Supported by the yeast the early yeast Work as well they they lose mitochondria Membrane potential pretty fast Um so yeah it was all surprising and all Uh cool to do Yeah for sure I like I guess say like One rationale then is that if this is Decline in the pmf this person makes a Force with age that what the Mitochondrial one is doing is sort of Like rescuing the decline Yeah yeah that’s yeah we think it’s kind Of Slowing down the decline at least But again it can be hard to kind of put A timeline on it when it’s hard to Measure in Vivo but yeah we think it’s At least slowing down the decline Um kind of recharging the battery Uh technology And so another thing I was going to say Or like at least I think it’s worth Pointing out is that you started the Um the activation of the protein tool at Day one so that’s after the worms are Fully developed yeah did you test it in A developmental stage as well yeah so we Have some lifespan experiments during

Development um Andrew’s lab is more Focused on doing those experiments and I Think we have some some pretty solid Results so far Um But I really wanted to Study this during adulthood Um just because Even though we’re using the wear model I’m always trying to think Translationally and we we can’t really Do anything right now during development For aging but we could start to do Something when someone is already aging So I thought it was important to show That this is something we can do during Adulthood And I also thought that it was cool to Do that because Most if not all of the mitochondrial Interventions and worms have to happen During development to extend lifespan So you can do different things to mess Around with mitochondria and worms and They’ll get a long lifespan but if you Don’t do that during development they Won’t get the lifespan extension so That’s interesting it’s very interesting But maybe a little bit harder to Translate so I wanted to be sure that This was something We could uh do during adulthood Cool yeah and I think well some other Question I had was um you said at the

Start so you decided to pay with this Mitochondrial until but if you flip the Orientation you can also push the Protons back the other way and have Looks like Um to decrease the price I made to force Yeah if the G test lifespan in that State did it decreased the Um I guess that’d make the the ones very Sick Yeah so it did not make the worms very Sick Um the lifespan I think is still unclear We haven’t Um fully you know characterized it as Much as we have with Mito on with might Want like we have dozens of lifespan Experiments by now which is crazy to Think about so we haven’t fully Characterized it enough in the middle Off screen but we do know that like They’re not they don’t die instantly They’re not super sick When I first made the strain I thought For sure they were gonna die instantly I Was like we’re gonna turn off the Mitochondria and they’re gonna just die And they didn’t so I think that suggests That you know the proton pumps Are maybe not able to totally collapse In that brain potential which you know Makes sense because the mitochondria are Still intact they can make their own Rate module

Um also like there’s metabolic Compensation so like maybe the worms are Ramping up glycolysis to me So they don’t need their mitochondria Lots lots to figure out with that Straight as well Yeah no I reckon that’s like loads of Interesting questions you could address With that strain Um but say this is so far I guess uh one Thing I’m interested in and it’s like What do you think called the next steps Of taking this tool further obviously Like one thing would be interesting Would be to somehow translate it to mice I guess as you mentioned C elegans these Phones they’re transparent so it’s very Easy to manipulate yes and activate the Tool Yeah so we’ve actually Um we’ve got some preliminary data Published Um in HEX cells and in Mass T cells Um from a couple of years ago now Um so we we have this expressed in HEX Cells we’re working on expressing it in Other cell lines and we’re actually able To show in Mouse T cells Um we can make the cells more active so The idea is if you could for Immunotherapy car T cell replacement Therapy if you can give mitochondria a Boost in the tumor micro environment Where there’s not as much oxygen and it

Seems like we can make these cells Persist in a tumor micro micro Environment for a little bit longer than They normally would so this was our First indication that this is going to Do something in mammals and we’re Working on getting a mouse strain made At least to do tissue biology so you can You know expose tissue to light pretty Easily but there are pretty cool ways to Expose animals to light so this lab at University of Rochester that we Collaborated with it that did this T-cell project Um they do this cool technique they do a Bunch of different optogenetic Interventions in T cells and I think They use a skin cancer model and what They do is they’ll attach a little LED Light to the ear on a mouse and they can You know have uh light on both ears but The cells in in one or the other or Something like that Um so yeah we’re definitely thinking About mammalian translation and we’re Trying to get this Mouse made So very excited to have that in the near Future I hope Yeah for sure um and I think like Another interesting thing which you do Some sort of tests in this paper is like How Um you might be able to manipulate Lifespan further by combining this

Approach with other known like lifespan Um extension techniques so like one Thing you test in this paper is Um the ampk activation pathway Um which is yeah I mean would you like To elaborate on that yeah excuse me So yeah we wanted to know What is the mechanism Downstream so We’re like preventing this decline in Memory potential or giving them a boost Recharging the battery whatever That’s signaling through something Um or you know it could just be like They’re making more ATP and Powerhouse Of the cell that’s it Um I don’t suspect that’s it so we Wanted to test something that is kind of Just adjacent to that which the first Thing we you think to test is amp kinase So amp kinase Kind of maybe using the smartphone Analogy this is like the low power mode Um notification you get when when the Energy is low ampk comes on and tries to Do something about it can change how Energy Um is is made for the cell So we know from my previous Publications That both mighto on and Mido off can Change the phosphorylation status of Ampk under certain situations And we know that ampk activation extends Lifespan and worms and flies and in Other muscles

So we thought okay let’s cross the Mito On screen into the active ampk strain And if the lifespan is not additive then That’s some evidence that marijuana is Working through ampk signaling which Kind of made sense to us Worms were like way longer lived seemed Kind of additive maybe a little bit more Than additive Um so very cool again don’t know why So So more to follow up I think that it Suggests that the amp kinase lifespan Extension and Mito online extension are Working in different maybe parallel Pathways Um which is cool Um there are there’s not one way to Lifespan extension so if mito1 can be Used to boost the other ways to extend Lifespan that’s that’s great Um So yeah that’s kind of where where we Were for the amp kinase Um we wanted to get this finding Published so we were not the only ones Toiling away at these lifespanes anymore Um so we wanted to show We got this result we tried one Mechanism doesn’t seem like that was it Here you go everyone So um yeah we’re still following up with The ampy kinase and a bunch of other Pathways as well but again there’s so

Much work to do Um figuring out what’s going on that I Think it’s I I hope it’s going to be a Great resource for people to test their Favorite whatever with Yeah for sure and I think Um yeah there’s like so many potential Applications of it and again there’s Also I guess many ways you could combine It with different things like um Yeah color like the calorie restriction Um models but also yeah yeah there could Be like whole um like I don’t know Actually too much about C elegans and The extent of like Christopher uh Gene Editing approaches but can you do like Um knock down a different like Library Approaches to knock down different genes Yeah yeah I would love to do that I also Really want to get um like other kinds Of screens or just like looking at uh we Already have some uh proteomics in the Paper Um which is part of a larger data set That we’re working on uh just to see how How protein levels change we want to see How expression levels of different Things change just purely in the turn on Moto on or leave it off and see see What’s different Um crispr screen sounds awesome as well Um yeah I think it’s a great way to do That I I don’t know if worms would be a great

Place to do it but I think cell culture Would maybe be an easier and more Informative way place to do it and I’d Like I’d like to do that as well Um but yeah I think it’s it’s uh promise As a screening tool is is really cool Because you can What it does is basically makes Mitochondrial membrane potential an Independent variable Which before this technique is kind of Impossible to do in Vivo Because things are feeding into Mitochondrial function and things are Coming out of mitochondrial function so If you see a change in mitochondrial Membrane potential it’s like well is That causing something to change or did Something cause it to change or with These techniques you can say we’re Controlling it and then what happens so It’s it’s true like causal metabolism Which is Um going to be useful for a lot of stuff Yeah I’m sure it’s like good good way to Design experiments and so like as well Um with like further experiments Um and how the mitochondria is involved In like more than just being this Powerhouse of the cells you as you Mentioned I really do like that Smartphone analogy Um but like so the mitochondria are also Entirely linked with like Zelda from

Regulating apoptosis did you see like Because yeah Um was there any sort of uh analysis on Like cell deaf and how that was Influenced in your studies No we haven’t really looked in um Yeah I think there was a paper using a Different kind of Mido off so this was a Channel so it wasn’t a specific proton Pump but it was just a cation channel That they targeted to mitochondria so When they use blue light they would kind Of open a pore in the mitochondria it Kind of achieved the same thing it Turned down the membrane potential and They actually measured in I’m not gonna remember the cell type but Some some human cell line I think Um and they showed that it actually does Influence Um cell viability So it’s kind of interesting that in the Whole animals you know we didn’t see Like no more animals I mean more animals Survived longer Um with Mito on and with Mito off there Was nothing clear like 20 of the Population died instantly or whatever Which is kind of what I thought might Happen because of that so you know you Make a pour in the mitochondrial Membrane should that trigger apoptosis And yeah I don’t know But yeah so so there are other versions

Of the off Um that have been published and they did Look at that And another thing that I thought was Interesting from I think it was your 2020 paper where you do more of the tool Development was that you showed how like Obviously the current people suggest That having a high pmf this proton motor Force is good for extending lifespan but Is it in your opinion good to always Have a very high protein metaphorce Um or is there evidence that maybe Um intermittent times because Um I might be Um remembering your 2020 paper but there Were some suggestions that it was good To have a slight depletion in the pmf so That develop some sort of like response And stress response uh Yeah so the disclaimer here is that most Of what we know about pmf changes is From isolated mitochondrion cells so When it starts coming into like disease And aging the real answer is we don’t Really know and that’s like where The Cutting Edge is But based on all of the theory from Isolated experiments Having a high memory potential all the Time is probably not good Uh what what can happen is if the Mito Membrane potential is too high it can Cause a back pressure on the electron

Transport chain Kind of like you have a hose flowing you Just put your thumb on the end of the Hose and it’s going to like cause Pressure and maybe expose some leaks in The hose Um and and those leaks would be oxidant Production from mitochondria so oxidants Have kind of been a little bit of a Boogeyman in the field of aging and now We kind of know that it’s more nuanced Than that sometimes oxidants can be Pro Survival sometimes they’re damaging Um but we know if the mitochondrial Membrane potential is too high they will Make lots of oxidants Um so what I currently think based on Lots of different literature is that if You have a low A pathologically low membrane potential Your membrane potential is declining With age then it’s good to give it a bit Of a recharge I don’t know if it’s good to Charge it up so much that you’re Overcharged Um and I also think it depends on the Health of the electron transport chain Itself So a low membrane potential could be Adaptive so I don’t have any evidence For this but just in theory if you have Damaged electron transport chain that Just really wants to make damaging

Oxidants the cell could respond by Turning down the membrane potential to Kind of say don’t make any accidents Please so then if you make the membrane Potential higher it can force bad things To happen So this is this is the difficulty in Studying mitochondrial membrane Potential and trying to relate what we Know in isolation to what we know in Vivo and you know this is what I want my Lab to focus on in the future is What do we know about mitochondrion Isolation and what can we take from that Into a an animal and eventually into a Human Um can we achieve like through Mitochondrial medicine Because I think it’s kind of surprising That we know so much about how Mitochondria work like high school Thought about how mitochondria works and There’s nothing you can do if you go to A hospital with some condition There’s nothing you can do to directly Target your mitochondrial function very Little you can actually do it’s like why Is that why do we know so much but we Don’t have tons of ways to treat uh Mitochondria and I think there are lots Of promising ways but we need to Understand the fundamental biology a lot Better before that’s ever going to be Useful so that’s where I would like to

Have my lab work in that interface Yeah for sure that sounds awesome and I Think yeah obviously speaking um this Session like there’s so many questions That we sort of fully understand like I Teach to some extent the like the Election transport chain to students and I’m like the same ones who didn’t really Understand Jazz and that they’re like They sort of like learn it for rates but Um yeah this super interesting and they Actually brought up oxidants obviously There’s some literature supporting that In increasing antioxidants like Cohen’s And Q I believe declines of age Um there’s some evidence for that and so That is Um one of these sort of antioxidant Molecules and so do you think that Combining that with having Um a boosted Mighty on my uh like Synergize Yeah it definitely could I think it Could be easy to test especially in Worms it could be easy to test that Um and you know other redox metabolites Too I mean NAD is super hot right now in In aging and NAD is you know part of the Part of the process that leads to Membrane potential creation Um so I think all these things are kind Of keystones of metabolism happening in Mitochondria and how they could add with Each other will be really interesting to

Find out Yeah for sure and so um obviously Besides NAD is there any other like Areas in the longevity field at the Moment that you think is quite Interesting Yeah I mean I’m I only got into aging Like you know a few a few years ago and I have learned a lot uh getting into This field I am really sort of in awe of cellular Senescence and how mitochondria Seems like mitochondria are playing a Big role at cellular senescence you know Like everything else How are they well we don’t know and Maybe it’s oxidants you know I think Maybe it’s mitochondrial membrane Potential um Senescent cells their mitochondria can’t Seem to hold on to as much calcium Calcium is positively charged and Calcium is loaded into mitochondria According to the membrane potential that Points toward membrane potential so That’s you know my link between the Hallmarks of Aging And to me cellular senescence it seems Like Again it’s like something that’s at The Cutting Edge of human knowledge it’s Like what are senescent cells what is Aging really how are mitochondria Involved so I have this sort of

Mitochondrial bias to everything that I Think of but it opens the doors to uh Other cool areas Um so yeah I guess my biggest Um interested area would be cellular Senescence right now I’m glad to hear that Um yeah but that already made me think Of so many different things because Um yeah also one thing we’ve there’s Some slight I’ve not done it personally But I know people who are trying to look At the mitochondrial metabolism and how It changes in senescence Um and obviously like one of the Challenges is actually being able to Study mitochondria and like the the Tools Um that we developed are getting better I think um obviously I think even Applying this and inducing senescence in Like the car models yeah you could boost The pmf without Um change the decision process um Obviously I study p53 which sort of Seems to somehow decide between Senescence and cell death and obviously Then that links both them back to Apoptosis and I’m sorry the mitochondria Um So oh man you made me think of so many Interesting things yeah I mean if you Want a plasma let me know I’ll send it Out

Well after me when I’m writing my thesis But um maybe yeah Um and speaking of Academia how have you Found your process obviously you’ve did Your PC and your pay stock um but you Have any like tips or advice to other Students Yeah yeah for sure so I Um maybe this is becoming a rarer and Rarer occurrence but I had a great Academic experience it’s um Andrew was a great mentor Matt is a Great mentor I was able to have full Ownership over my projects like When Developing Mito on was like an unfunded Project in the lab Andrew let me He let me go for it you know and I just Uh was Able to do what I wanted with it which Was great and in Matt’s lab same thing I Was able to do what I wanted to do And that’s great It’s not it’s not the case a lot of Times in Academia that you can get that That much Freedom at those stages but my Advice would be Know that you really want to be doing What you’re doing if you want a PhD for A job Then you you have that reason If you have a p if you want to do your PhD because you just love learning about The topic that you’re learning about That’s a great reason

If you feel like you have to get a PhD For some reason that you don’t yet Understand I would say don’t do it it’s Not worth it you have to love it if you Don’t love it it will be very hard it’s Very hard when you do love it it’s a lot Of It’s a lot of work and not even in the Sense that I I have I have had and continue to have Great work-life balance like I’ve Figured out how to make that work for Myself but it’s still You know it wears on you having an Unanswered question like doing a PhD is Living with an unanswered question for Three four five six seven years that’s Hard to do Um the structure of Academia is also Hard to work with you have to you know Move for your postdoc move after your Postdoc move again potentially Um and that’s hard to deal with so yeah My advice is Know that you love what you’re doing and Then just go full into that that passion And that will help you Um get through it So that that being said Um I’ve really started to think less Um monolithically about Academia versus Industry I feel like there is this you Know Fight out there where industry people

Are like oh Academia sucks and it’s the Worst Academia people are like oh you Don’t you dare talk about going into Industry I think There are really good freedoms and Positives about Academia and there are Really good freedoms and positives about Industry and there are also really bad Negatives about both as well and it’s About finding what you want to actually Do and where does it make sense for you To do that So for me I kind of went into it Thinking like well you know I did a PhD I have to do a postdoc and that was Wrong Um but it worked out for me if it didn’t Work out for me you know that would have Been bad so I’m thankful that it did Work out So I would say before you make those Kind of decisions just know what you Want to do and talk to people and see Where can you realistically do what you Want to do and then do that and that’ll That’s a better way to find your path Yeah and I think that’s good advice and The other thing um because obviously I’m Very impressed if like how you sort of Have this like very logical approach With um your studies and how you had a Finding and this makes you think of this And then you do that and like I’m Definitely like sort of just

Um your basic sort of tips as like Dealing with experimental design or like How you go about approaching your Research questions Yeah so for experimental design it’s Been a long process to learn how to do That that’s that’s part of doing a PhD And like and still in the postdoc too is Learning how to do that Um Andrew can take some credit for teaching Me that Um I’ll say early early in my PhD I Wanted to do everything I I still want To do everything I talk about how cool It is to do everything and he would say Brandon just do this experiment stop Stop all of this and just do the Experiment and then once I did that I Was like oh my gosh it worked and now The next step is clear So part of the advice that I can pass on Is if you have a good experiment just do It and then decide what to do next Yeah no I definitely could have learned That faster than I did Um yeah Um I see just to sort of wrap up about Any sort of like Um besides like obviously the research That you’re doing and any sort of like Interesting Um like areas that you’re excited to see To follow up in the next couple of years

Yeah so I I mean I would really like to See more people incorporating our Optogenetic tools Um because I think It’s a way to kind of take mitochondria Out of the equation where where so often Like so many people are interested in Mitochondria now Um and Metabolism in general And I think it’s a great toolkit to just Say okay we’re going to make this an Independent variable so we can kind of Remove it from the equation So again in cellular senescence I really I’m learning more about how to uh study Cellular senescence in in different Small ways and I’m really excited to see How combining Metabolic approaches like this Uh with cellular senescence research Where that’s gonna go And other areas that I think are really Interesting in development Um I think there’s a lot of Imaging Approaches that are just so so cool and It’s changing very rapidly Um I think Brian glancy’s Lab at the NIH Has some awesome tools to look at Mitochondria in Vivo or in C2 C2 and Just seeing how In a very short period of time we went From seeing mitochondria as Street objects in the microscope so now

We can see the inner membrane and the Chris day and what it all looks like and We can see that the Chris Day have Different membrane Potentials in Different areas and how it looks in Muscles versus liver versus brain and I Think the Imaging is just progressing so Fast I’m really excited to see Um where that’s going to go is it gonna Plateau is going to get even better Yeah very exciting I mean speaking actually I was going to Ask you about Um like using microscopy to look at the Localization of um the mitochondrial on Protein because obviously I made the the Crystal is very like Um it’s highly stretched um uh Compartmentalized in terms of the ATV Synthase so if they form these timers And they sort of seem to be at the tips I believe and then yeah I guess we Always see this like linear pathway but Actually they’re kind of clustered Together and so I guess have you looked At Um any might um any images to see where Exactly the mitochondrial on proteins Getting um inserted So no we haven’t looked directly but we Know Um Pretty likely where they might have Where might I want is is likely to be

Because how I got the Mito on that the Proton pump Targeted to mitochondria So there are a whole bunch of different Mitochondrial targeting sequences so Like protein sequences that you can just Clone on to the beginning of your Favorite protein and it’ll send it into Mitochondria we had to go through a few Different ones to to actually get it to Work and the piece of the protein that We got to work is from the IMM T1 Protein it’s immt1 in humans and it is Part of the Christie Junction organizing Complex So we think that mightawan is Predominantly at Christy Junctions Because of that piece of protein we Don’t really know it could it could be All over because that leader sequence Might not be enough but Um Yeah so we think it’s at Christy Junctions which is probably right next To ATP synthesis which maybe would make It oh okay yeah That’s interesting yeah I think that’d Be super interesting to look into Um yeah yeah for sure So um why can people go to find out more About your research and to kind of Follow Your exciting journey into Mitochondrial uh like praise that makes The false discoveries

Um yeah so I I’m pretty active on Twitter at Mito Brandon that’s me Um and yeah I’m I’m here at University Of Washington in Matt cable Line’s lab For the next few months and I’m going to Be transitioning to finish off post-doc Stuff uh in David marcinix lab here at University of Washington Um where we’re going to be looking at Muscle energetics and muscle Mitochondria and uh trying to get this Optogenetic stuff going in some Sarcopenia models we’re really excited About so that’s where I’ll be for the Near future but always on Twitter I’ll I’ll be there Awesome so yeah thank you Brandon for Coming on today and like speaking so Eloquently through your research um it’s Been really interesting to hear about it And this exciting tool that you’ve Develop that I really do think will have Many applications in particular in Essence hopefully we’ll see Um so yeah um just basically Um best of luck with all your future Career plans um and it’s just been a Great conversation thank you so much Thank you that’s a great compliment and Thank you for having me this is awesome I’m very happy to have been on and yeah Good luck to you as well finishing off Thank you so I hope you’ve learned Something in this video

Thank you to my patreon supporters and Thank you for listening

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