Title: Immune and Metabolic Control of Nervous System Physiology and Repair
Date: Oct 24, 2023
Speaker: Prof. Simone DI GIOVANNI, James W. Hartnett Chair in Restorative Neuroscience, Imperial College London
Please go to our webpage for more IAS Events:
https://ias.hkust.edu.hk/
Again it’s my uh great pleasure uh to have uh Professor San uh the Giovani to um visit us and also give a lecture so um Simone did his uh uh MD and also PhD training in Italy then moved to uh us uh Georgetown for his PhD uh postto training then he
Started his uh independent career and uh at Germany uh tubingen then moved to U uh UK uh so he’s currently ahe and also the uh uh distinguished professor at Department of Neurology and division of Neuroscience in uh Imperial College of London and he’s he has done fantastic
Work in both the um accent regeneration and also the metabolic uh uh uh changes and interaction with immune system uh uh in different uh uh conditions including traumatic injury and also uh um uh diabetic Etc in the in the nervous system so um I hope he actually he a
Also a clinici so he’s a very unique background um both in the basic research and also the uh clinical practice hopefully will give us a very unique perspective in the uh um uh in the this uh control uh mechanism of the Aon regeneration uh from met metabolism to
NE immune uh interactions okay so uh without I hope you guys enjoyed the lecture and without further Ado I’ll give the floor to soney okay let’s have thank you very much Kai for the kind invitation and it’s great I’ve been in China for two weeks and uh it’s been
Very very interesting and the first time and and uh it’s it’s great ending it here before going back to London tomorrow um all right so I will start by showing you I guess you have from a diverse background so very brief introduction that I think will make the
Point clear this is an injured spanner cord of a mouse in this case you can see the sensory neurons that convey sensory information to the brain are uh damaged the accents are damag Dage they do not regrow similarly the motor neurons that come from the brain inovate the muscle
For muscle contraction are also damaged following SPAC Dinger they collapse and do not regrow trying to enhance the growth capacity of these accents is essential to allow Rec connectivity that will limit disability neurological disability hopefully enhance sory motor function the other condition that we’re interested in is actually equally
Dramatic and even more frequent the spod injury which is peripheral nerve damage this shown here is a sciatic n Crush in a mouse you see the following sciatic n crash you have some level of spontaneous axonal regeneration however it’s not dense enough and it’s not quick enough
To reach Target that can be the muscle or the skin or other their organs and therefore injuries in the peripheral nervous system also lead to permanent neurological uh disability there are many ways that one can uh approach the problem and what I will show you today is the mainly the
Cross to between immune cells and neurons and how that can affect their regenerative capacity additionally I will show you how some metabolic states can directly affect neuronal biology or indirectly affect neuronal regeneration via changes in the immune system in the last several years my lab has been interested in understanding environmentally environmental variables
Broadly speaking they include activity independent changes dietary changes and age because of course these are essential variables that our life has to deal with uh in the interest of time uh I will not talk about some published work but I will just talk about one recent published work and two un published
Stories that you will see they are interconnected conceptually although they are independent from one another however we Briefly summarize three modes of neuroimmune interaction the malab has defined the last few years with which I have all the common denominator to be focused on the Regeneration of sensory
Accents the first one that I want to briefly summ arize is um a paper that we published last year when we showed that cd8t cells that are typically cytotoxic cells but they can also be imuno regulatory are attracted to the dorsal rug ganglia which are the structures that convey sensory information from the
Periphery to the brain durine aging and they are attracted to the sensory ganglia because in neurons with aging there is the overexpression of a chemokine called cxl 13 which attracts cxr 5 the Lian ct8 positive T cells to the dorsal ranga after injury the neurons Express MHC class one which is
Recognized by these cells and as a consequence there is regenitive failure we were able to take advantage of a humanized monoclonal antibodies then antagonized 6xl 13 to promote regeneration and Recovery in these animals so this is a mode of interaction that’s a receptor Lian cdat cell dependent interaction that is
Directly links cdat cells with neurons that is reversible with monoclonal antibodies a second mode of interaction that we publish a few years back has to do with the extracellular vesicle mode of communication between macras that proliferate and are activated following a sciatic nerve injury these mcroof fases release extracellular vesicles that contain
Nadph oxidase which oxidizes specific proteins in the axons including pan Which is a very wellknown inhibitor of Axona regeneration P10 oxidation activates the Regeneration Pro PR of sensory neurons later we also shown that this overexpression of this enzyme in after spinal cord injury is able to promote regeneration and some
Level of recovery in the central nervous system the last mode of interaction which I will now actually uh discuss with you in more depth has to do with the secreted factors secreted factors that in this case are released by neutr that are activated you will see now by a
Gut metabolite called indol propionate to support Axona regeneration I will now tell you this story but before going there I have to tell you how things started how we got there and uh that story in a way started a few years back when we investigated whether Environmental enrichment that means
Putting animals in an enrich environment as you can see here versus an impoverishment which is standard environment which is kind of impoverished environment if you will uh because because it was known already that environmental enrichment enhances learning in memory and the synaptic plasticity in the brain since some of these
Mechanisms molecular level of re are recapitulated in Axona regenerative conditions we investigate whether environmental enrichment could enhance signaling in the sensory neurons that would support Axona regeneration long story short we published this paper a few years ago um we found basically that environmental enrichment activated neuronal activity activated a molecule
Called CBP which acetates his stones and promotes the Regeneration program we could then use a small molecule that activates this protein called CBP following SPAC injury and that promoted regeneration of sensory fibers and not shown here but published already also motor fibers and functional recovery all
Right so this is the background to say that environmental enrichment dependent mechanisms that contain as a major component exercise are shared when with dietary changes so that led to the hypothesis that if we put animals in a dietary regimen such as for example intermitted fasting maybe we could identify metabolic based mechanisms that
We could leverage to enhance the aona generative ability so to this end we did a series of very simple experiments where we put animals in a diet normal diet Al stands for a ad libitum or I have intermitted fasting this means for 10 days or five weeks initially the results were identical
Between 10 days and five weeks therefore we focus on 10 days because it just faster and then so we did feed no feed feed no food feed no food okay for 10 days then we did the sciatic ner crash and we waited three days one days or
Three days or longer but anyways now you will see three days as a model system and we measure regeneration and we found that intermitted fasting enhanced Axona regeneration the first thing we did then uh to try to understand why was metabolomics from the serum I thought it was a logical step because
Clearly intermitted fasting changes the metabolism and what we found was a number of metabolites that were differentially expressed between AD libitum and intermitted fasting and included some top metabolites that were either exclusively produced by gr positive bacteria or produced both by the indogen system and gr positive bacteria for example IPA in do
Propionate I will talk about it later is exclusively promot produced by Grand positive bacteria anyways overall the top metabolites suggested that intermitted fasting was altering the gut microbiome to have a first evidence that this was the case then we took the fces from the animal that were undergoing underwent intermitted fasting versus
That libitum we fed animals that had a libitum diet via gavage or fed them with animals that from the fces from intermitted fasting and we compare aona regeneration we found that if you give fces from intermitted fasting animals to ad libitum animals there was an enhance Axona regenerative capacity although
This was modest probably containing things that were potentially useful others that were less useful however this gave us a proof of principle that there was something in the gut macrobiota that could enhance aona regeneration when the animals were on intermitted fasting regimen since I told you Grand positive
Metabolites were in top of the list we depleted the gut microbiome posi gr positive microbiome with bomy for 5 days according to classical protocols then we put animals in intermitted fasting or at libitum versus control and again we measure aona regeneration following sciatic Crush you see in red intermitted fasting enhances aary
Regeneration vomisin in the other groups blocks the aona regenerative ability uh triggered by intermitted fasting then we did metabolomics on the serum again and this was a very useful experiment because the only metabolite that was blocked by by vomisin in both conditions and still of course induced by intermittent fastin as
Confirmed earlier was indul propionic acid here on the right you just see more targeted metabol metabolite measurement of inter of indol propionate that is more sensitive of course than just general metabolomics but these are essentially the same data okay what is indul propionate indul propionate is a metabolite of tryptophan produced almost exclusively
You see later piece of data that actually suggests it is true by Clum porogens which is a gr positive bacteria then uh I found in the literature that a group at Stanford had produced for other reasons Clum sporen that do LF fldc they have a metabolic impair pathway in the production of
Indul propionate so we received this bacteria either while type of mutant bacteria that do not produce IPA to test and we depleted the microbiome and we gavage this bacteria while type of mutant to test whether indeed the bacterial produced IPA supported Axona regeneration and you can see here that’s
What I was mentioning earlier the IPA in serum after you deplete the ground positive back back to microbiome is basically undetectable and when you give the wild type or the mutant again you see clearly the IPA in the serum comes only from this bacteria and this was done in collaboration with the whitesman
Institute that you know metabol the metabolite measurement was Carri out by them and uh and aona regeneration was reduced in case of uh lack of production of IPA so this suggested directly that IPA enhanc aary regeneration come comes from bacteria gut bacteria are in the gut however when
A metabolite is in the gut typically eventually goes also into the bloodstream so the question now was is IPA produced in the gut essential for regeneration or is it systemic presence important for regeneration in other words is it is this a indirect effect or is it direct effect from the serum that
Might then affect the dorsal ganglia neurons to this end we did two experiments we either gave IPA by Oral gavage so working through the gut or systemically and we found that in both cases IPA led to the similar exent of Axona regeneration I’ll just show you
One of the two examples this is gavage of IPA that enhances Axona regeneration this is the concentration of IPA following oral cavage therefore this suggested that bloodstream presence of IPA will mediate regeneration to understand Downstream mechanisms we did sequencing from dors ranga following IPA before and after an
Injury we can jump that and uh what we see here is that IPA delivery enhances neutr chemotaxis and followed injury again we have S response to interferon inflammatory response inflammatory response and so on so clear clearly the top categories that IPA dependent were related to immunity and the top where pro-inflammatory
Signaling including coming from neutrophils interferon gamma can come from both neutrophils and T cells and why we didn’t see any change IPA dependent in the sciatic nerve or in the dors ganglia in different immune cells we found the neutrophils where increased in the perivenular space following IPA in control conditions
IG and neutrophils were blocked when we use an antagonist of cxcr2 that we found overexpressed in our RNA sequencing screen and then then we found Express in the G neurons attracting neutr fils into the perivenular space of the drgs they don’t go into make making a contact with the
Ggs neutr remains in the perivenular space and they’re known of course to release a number of factors then of course in the same experimental design when we antagonize neutr chemotaxis down here we block the IPA dependent increasing axonal uh regeneration suggesting the neutrals are important for IPA dependent
Phenotype now in in the published paper if you’re interested you can look at some Downstream mechanism however I will in interest of time now move on to the last part of the story where we used IPA following aan sorry following sciatic nerve crush and what you see here is there is
An increase in epidermal ination of the sensory fibers I shown here which is quite significant there’s acceleration of recovery of sensory fibers the sense thermal sensitivity and why there is no difference in um in the bone Fray which detects Alodia which is a form of mechanical pain
So in summary IPA which is not the beer unfortunately uh enhances Axona regeneration in the peripheral nervous system I think actually Kai gave I did you give IPA into the optic nerve or we we we tried but yeah but there was no difference in only to injection right
Don’t know how much that can sustain yes yeah anyways maybe this does not work in the optic nerve and um however in the in in the peripheral nervous system uh had a an increase of aary geration mediated by neutres I mentioned the optic ner because it’s quite interesting because um a few years
Back there was a paper from Benjamin Seagal and Roman Gyer they showed the neutr fils and actually study neutr fils in more details than we did by single nuclear single s sequencing I believe um at least a subset of neutrophils can support aary regeneration in the optic
Nerve but I think in that case there was an inflammatory activation and so it’s possible that in the optic n maybe you need some combin combination of treatment to enhance IPA dependent regeneration what is attractive about IPA is that um it’s a metabolite that is physiologically present in the serum
Actually it’s the highest gut metabolite published in human serum which is quite interesting you can easily buy it and I’m not suggesting to take it but uh uh it could support clinical translation it’s not going to make the difference alone but could could be part of the of
The cocktail to enhance repair all right so you can imagine how intermitted fasting is a is a con is a situation where multiple mechanisms coexist and while we identify the bacterial dependent mechanisms we wanted to understand better understand what could be endogenous mechanism that would support the aona regenerative ability and
Perhaps we could learn more and leverage upon that to enhance axo regeneration to this end we did dorsal gangli in a sequencing the dors the whole DG so this is not single cell to look at the DG micro environment we did a few years ago probably now we would do single cell
Anyways in intermitted fasting vers of that libitum same identical uh experimental paradigm 10 days of AD libitum versus intermit fasten and when you look at the differentially expressed um classes these are induced induced genes you see lots of things that make sense hormone signaling lipid um including in Sensory
Neurons changes in metabolism metabolic processes and some of neuronal categories which are potentially involved with activity dependent growth if you look here are number of genes significance you see again lipid metabolic processes mainly if you look into that it’s lipoly so it’s lipid catabolism increasing cholesterol
Transport and there is a high also down here uh significance for circadian rhythm indeed it is known the intermitted fasting as opposed to fasting which is a very strong caloric dependent uh condition intermitted fasting changes the circadian clock in a way that aligns the Circadian rhythm and
It is known to potentially underline uh something that this data suggest what this data suggest is that several of these processes including circadian rhythm and lipid catabolic processes are underpinned by leptin signaling therefore we wondered whether letin signaling might be enhanced by intermitted fasting supporting perhaps Axona regeneration in Sensory neurons
Leptin is a hormone which is released by adipose tissue by adipocytes regulates our feeding Behavior by working through the specific nuclei in the brain by decreasing feeding behavior when leptin is up and it works in a counterbalance with another important hormone which is called adiponectin more more importantly perhaps for Axona
Regeneration is the Discovery for me at the time the leptin binds to leptin receptor which we found been expressed in Sensory neurons which it’s Auto phosphorilation in part and then Jack dependent phosphorilation leads to activation of stat three IR map can signaling C and pan akt M Tor so if you
Have any familiarity with Aon generation I would challenge you to find a signaling molecule that does all of this together it was quite stunning I must say and uh so we could not ignore this hypothesis I would say additionally evidence uh in other systems uh you see here in other organs
Have shown before the intermitted fasting although by an unclear mechanism enhances letin sensitivity in Target tissue nothing was known about neurons that are outside of the brain classical feeding Behavior dependent neurons okay so then we first looked whether intermitted fasting enhance signaling as I’ve shown you phosa 3 is a
Paradigmatic signaling molecule for leptin and before injury and after an injury following intermitted fasting I will show you just after injury here in the interest of time we found an increase in phosphat 3 in following intermitted fasting and we found the expression of leptin receptors in their gen neurons with
Apprpriate mutant controls we indeed found the leptin receptor is expressing these neurons next experiment we did we uh wanted to activate lepto receptor s sorry we want wanted to block leptin receptor signaling in intermittent fasting condition uh to see whether indeed intermittent fasting would trigger aona regeneration via leptin
Receptors via leptin signaling so you see here we either deleted with the cre AV virus letin receptor in letin receptor flux animals or we use control viruses we gave 10 days of AD limit to intermitted fasting perform s crash waited 3 days and bottom line regeneration index or that’s two
Different measurements here for aona regeneration sensor new in sensory aons you see the intermitted fasting promotes regeneration we knew this already and when you block Lapin signaling uh intermittent fast independent regeneration goes back to Baseline and we didn’t know this actually from a from a previous paper then next we gave leptin
Systemically mimicking AIT what a deposite release normally would do so now we overexpress the system and the idea is systemic leptin will bind to its receptor and sensory neurons and you can see that in control condition it promotes regeneration mimicking intermitted fast independent uh regen uh profile and again here we deleted leptin
Receptor in the same way as I showed you earlier and when you delete leptin receptor leptin mediator regeneration is blocked now the issue with leptin um when you give leptin systemically although it’s I think it’s a nice experiment is the leptin receptor is expressed in immune cells and um immune cells play an
Important role to control regeneration of course in particular in the peripheral nervous system that do make a difference to Regeneration so so we wanted to see whether neuronal leptin could promote Axona regeneration neuronal leptin signaling now leptin is released by cells and binds in autocrine or paracrine manner to its own receptor
So we could take advantage of this uh knowledge by overexpressing leptin in neurons and activating neurons themselves by autoc crime mechanism and uh okay sorry this just to show you the leptin delivering does activate the signaling and when you have leptin receptor which is deleted you have inition of the
Signal um so what we did now is um give leptin by V varus over expression in the sensory neurons versus a control and you can this is just to show you leptin expression gfp expression and leptin and you see that Axona regeneration is enhanced when you give uh overexpress
Leptin in neurons now to prove the mechanism oh before that I’ll tell you more when you overexpress leptin you have an increas in stat three signal within neurons and when you wait also um this is probably three weeks following injury several weeks I’m not positive it
Is three but I think about three weeks we can also see an enhancement of epidermal Innovation so the effect is quite robust then we went to more difficult conditions which is in terms of growth which is panod injury these are sensory neurons spanac Cod injury does not allow Sensory neurons to
Invade the lesion site and when we get overexpress leptin we saw quite a substantial uh regeneration of the sory neurons across as City bridges in this crash model of spanac injury and I honestly I’ve rarely seen this nice regeneration of sensory neurons um before probably again underpinning the
Fact that leptin as you’ve seen from the signaling yeah it’s probably quite a powerful uh um signaling molecule good now I come to what I I was thinking to tell you uh earlier and yes good so what we did now we wanted to prove uh the mechanism the autocrine mechanism so
While uh when we tried to measure leptin from from the serum it was very diluted so it was it was quite difficult we did try um we were able I think to any way uh prove the principle by delivering by overexpressing leptin in Vivo culturing endorser ranga neurons either gfp or
Expression of leptin and uh that promotes uh regenerative growth as expected in either wild type animals or leptin receptor DB n neurons when that happens when we give leptin receptor n neurons jfp and um and uh leptin gfp do not change in terms of regenerative growth suggesting that leptin which is given
Into neurons Works through its own receptor and so it must come out to work through its own receptor and then bind its receptor again how does leptin exert its regenerative effect so again one step back systematic approach again in the usual uh experimental design We performed a neuronal enrichment it’s a
Technique that we set up in the lab hopefully we’ll publish the methodology soon where we have a strong enrichment of neurons versus other drg cells that as as opposed to Fox sorting it’s less traumatic and allows many more neurons to be recovered and uh what we did we
Did that of course to try to have an enrich population of leptin ere expressed neurons as opposed to the whole drgs and this sequencing was a very fortunate sequencing because as you can see it’s very monothematic meaning that you have a strong enhancement of transcription and a phosphorilation which is expected by leptin
Overexpression if you see that and of course those phosphorilation Pathways have been shown to be to impinge upon leptin of course they are expected to enhance transcription and uh then we went to do a fishal test to see correlation between overexpression of uh leptin versus intermitted fastin at libitum see this
Means that overe expressional leptin mimics intermitted fasting mimics sciatic nerve injury but does not recapitulate a non regenerative condition like dorsal column axotomy in the spinal cord then since we saw that transcription was strongly activated the effect lasted for apparently anatomically for a few weeks we looked at classical epigenetic marks
That we previously shown to be associated with reg generative ability such as k27 that was enhanced after leptin overe expression in the neurons we also looked at the activation of phosphoserine 2 on RNA polymerase 2 specifically neurons that Express leptin that’s important important um for transcription and also that was
Increased finally the last piece of data before I tell you briefly last story uh was to prove if indeed transcription was important for leptin dependent mechanism so we use a classical approach that others have used before which is by in overexpressing either gfp or uh what is it or leptin
Again we proved again the leptin of expression promotes uh where is it uh here promotes growth in control conditions or with atomizing the blocks uh transcription and you indeed see the leptin overexpression does not promote regenerative growth with actinomycin suggesting it needs transcription and then we we measure again a
Transcriptional readout in neurons phosphoserine 2 on RNA polymeris 2 this is in line with the leptin activation uh that needs suggested needs transcription and uh this is in summary what I think I’ve shown you that leptin binds leptin receptors activates transcription to sustain um Axona regeneration what I think is uh
Happening uh this is just a summary of what I told you but what I think is happening is the leptin basically uncouples leptin signally uncouples feeding Behavior because leptin signaling stops you from eating from axonal regeneration from repair repair mechanism probably because you need to have some compartmentalization of energy demanding
Tasks but this is of course just an hypothesis because I kept on wondering why that is the case now if you think along these lines you will probably appreciate the last part of my talk it will be quite easy I think for you to follow because again
It’s the same kind of experiments which is again now in this case focus on peripheral nerve sciatic nerve Crush but this question we wanted to ask is over the years we’ve done lots of RNA sequen in different conditions so now it was natural to think is there anything underpinning these different conditions
That are in blue Pro regenerative in red non regenerative across injury conditions peripher of injury non injury conditions so priming conditions such environmental enrichment or intermittent fasting or a combination of environmental enrichment peripheral nerve injury versus versus conditions where regeneration is either strongly reduced with aging or doesn’t happen
With the cental spod Dinger and then we perform very simply an enrichment analysis of Pathways across these data sets and what came out is the circadian clock was you will see now probably something that is in phasee with the regenerative conditions and antiphase with aging and SPAC Cod injury what you
Can see here is regenerative conditions and in white DCA non regenerative conditions circadian rhythms are at the top of the list well you look it’s quite interesting because we did these things in independently and then after a few years things come together lipid and stero are up here and these are leptin
Dependent and leptin aligns the Circadian rhythm so actually it was nice to see this that’s why I’m telling you conceptually things are quite connected what we did here we looked at a number of clock core genes into the RG neurons to see whether the NG Sensory neurons were following the expected rhythmicity
In the clock I think you all know familiar with the clock you all know right that there is a circadian dependent oscillation in signaling in all cells not only the central clock although in the aurons nobody think had shown it before probably this is not too unexpected in my view because all cells
Have a circadian clock just for you as a reminder this is basic cell biology but just as a reminder the clock is a self-regulating oscillatory control system relies on two genes clock and protees and Bea bind to ebox sequences activate per and cry one that in in Reverse inhibit Beal and
Clock so it’s a self sustaining inhibiting mechanism there are of course other genes that contribute but the only critical indispensable nonredundant Gene is Bal one and you will see why subsequent experiments focus on conditional deletion of Bal one all right what Franchesco did in the lab and
With a couple of other people they did that AAL sciatic Crush injuries every 4 hours throughout the 24 hours then they waited either one day or three days and measure axonal regeneration what they found is that the all different time points there was not much difference in aona
Regeneration but at zit gabber 20 this is a technical terminology it means 300 a.m. which is 300 p.m. for you humans okay 3:00 a.m. for Animals we have maximum aona regeneration then in subsequent experiments we always use the the opposite time 3:00 a.m. versus 300 p.m. because of course they couldn’t do the
Whole set with for all experiments uh it was we didn’t have at the time you can have these Chambers where you can invert the Cycles but we didn’t have them so they actually had to go through the night and um all right so now we know that 3. is
Enhances aona regeneration we did sequencing again from enriched neuronal population at these two time points following an injury and you can see there’s a very strong separation three biological replicates at 3 p.m. four biological replicates at 3:00 a.m. and you can see circadian rhythms are not very top but they’re significantly disr transcription
Phosphorilation signaling probably not surprising but it was very good to see this classes that were differentially regulated and then you see a number of regeneration Associated genes that are also disregulated and a number of core and uh and Associated core genes all right so this suggested that
Indeed uh 3:00 a.m had an enhancement in a number of regeneration Associated mechanism supporting the anatomical data next okay so before going next I just want to show you the nice separation of the of the two groups between 3:00 a.m. 3: p.m. despite both having an injury so
This was partially surprising I would say again fish te test there were with the odds ratio here that I quite like to do because it’s a kind of an unbiased gives you an unbiased broad view of tendencies in terms of gene expression changes of course we have all is more
Information that you need when you do sequencing and you see here that a circadian regenerative gain 3:00 a.m. versus 3 p.m. is associated significantly associated with several regen conditions not all for example in Rich environment does not have injury in itself but intermitted fasting is also without injury but it’s very strongly
Associated with circadian rhythm and we know the Circadian rhythm aligns intermitted fasting I stress this thing thinking about in way the previous story so this was nice to see and there was no association with no regenerative injury now next what we did we conditionally deleted uh BML one in
Sensory neurons and then we looked at Sona regeneration at after doing the injury at 300 p.m versus 3:00 a.m. but basically what this shows two different way of measuring it is that BMA one conditional deletion block regeneration only when the Regeneration is circadian dependent apparently at 300 p.m. but not at 3:00 a.m.
Suggesting that it is not a general disruption of the molecular transcriptional mechanism but seems to be circadian specific and so I think it was nice to see this time dependent inhibition of aary Generation Um next again we looked for weeks later when we did injury at 3:00
A.m. versus 3 p.m. in control versus bal1 conditionally delete at animals and this was the most surprising data because four weeks later we still have differences in epidermal innervation 300 p.m. 3:00 a.m. you have more BMA 1 deletion blocks the benefit then we did retrograde tracing to support the data with
CTB the in distal to the leion side it’s very simple experiment but it’s quite useful and robust where you can count retrograde labeled axons the mark regenerating neurons in the again in the four different paradigms and you see similar results as epidermal in innervation suggesting longterm effects
Of the circadian clock which is Beal dependent on aary genitive ability here just to show you the overe expression of classical regeneration Associated genes at 3:00 a.m. that were also found in the RNA sequencing but independently from RNA sequencing these were published before and these changes are are
Significantly um stunted reduced in case of B One n uh animals um conditionally delated animals I’m now getting to the end of my story just show you at the protein level in gfp uh expressing control or Creed deleted bal1 neurons you see here the expression of fos for S6 which is
Downstream mtor signaling or I think it’s phosphor crab here uh showing that this important signaling mechanisms at the protein level are also circadian dependent bal1 dependent the last experiment I want to show you is quite early it’s it’s I would say it’s a um how do you call it
Pilot experiment if you if you will at this stage to prove whether it is possible to use a drug that is known to align the clock and amplify the Rhythm by activating certain clock genes it’s been shown in cell lines in cancer cells in other conditions which is called
Lithium it’s a very wellknown uh drug that of course has got multifactorial effects interesting lithium is a gsk3 inhibitor mainly gsk3 beta if you Google it it’s part of the secondary genes that regulate the clock so the two things are actually connected uh and lithium promoted a three days following injury some level
Of aary generation it’s not particularly exciting that’s why I call it like a pilot proof of principle experiment what was good to see was that in condition I deleted bal1 the lithium effect was blocked also we use cry one2 it’s a different and kind of circadian clock
Genes and also it was blocked so suggesting at least in these experimental settings lithium promoter regeneration um in a clock dependent way more experiments are needed uh and I think probably better specific specific clock G drugs are needed to perhaps use this as a therapeutic intervention and this is just the
Summary I think I told you this I can just mention this might have implications for restorative intervention such as time neuro Rehabilitation and uh as I mentioned Chrono active drugs could be used on a less exciting more prosaic level this also warns us uh although I think we knew it kind of
Already that we should really be very careful when we do our experiments because some of the variability that we see which is of course due to probably many factors might also be contributed to by doing experiments at very different times and um on the positive side when the paper
Will come out you can have a look at the usual number of supplementary figures you can see all the different time points on the positive side there’s a huge um um a huge um time span there’s not so much difference in Aon regeneration so you can be kind of quite
Flexible still and uh so I’m going to thank this work is done in collaboration by several people in the lab but Jessica leted the leptin story Tom in Rich environment was published if you a few years ago now he’s in um in Switzerland Elizabeth drove the IPA story Franchesco
The Circadian one he’s now also in Switzerland um I need to thank Dylan dot for um IPA collaboration IPA work gregar for suon the rich environment experiments in rats and uh Alexander for measuring IPA and uh ma for some of the bioinformatic analysis and these are the the founders and yeah thank
You by the way I’ve been reminded if you’re interested in post do send me an email okay uh that’s was great uh any uh questions uh from the audience y okay fine I’m scared thank you Professor for your talk and I’m wondering have you done any functional analysis on the regenerated
Neurons and see uh if they are a functional and which mode of um for example the IPA uh um guided or the Lapin guided regeneration yielded more functional neurons you mean in terms of uh neurological recovery yeah uh yes or like IPA yes I I showed it to you uh IP
A enhanced accelerated sensory recovery at least of some modalities uh with leptin we didn’t measure that oh okay and have you thought of um for example a cocktail um uh like uh treatments that combine leptin and IPA for regeneration uh yes but I decided not to do
It because I prefer to do something else oh but that’s the honest answer but yes it’s it’s a good point but I have to choose so I decided yeah to do something different but um it’s of course a good point yeah thank you okay maybe I oh
Okay thank you very much for the interesting talk I want to uh H so the uh last part regarding the saring clock so the assay of the Regeneration has been done four weeks later than injury and then that what matters is the the time of the injury right so uh in maybe
Uh for example uh when while they are sleeping like ZD 12 is much more uh capable to regenerate rather than GTA so which means that uh like or sleep state or less locomotive activity allows them to regenerate more yeah yes it’s very good question that I always get meaning that
It’s a good question and uh I can answer I can answer in this simple way in both cases so Locomotion locomotive activity so I guess you’re thinking that animals during the night they have more locomotive activity right compared to the day so is locomotive activity influen circadia dependent regeneration very
Good point when we did Bal one conditional um dele in sensor neurons and we measure locomotive activity we didn’t see any difference this is one line of evidence that suggest the probably that doesn’t make a big difference additionally in the other three hours or no four hours
Yeah yeah during the there a bit there about 12 hours whether when locomoter activity is quite High during the for the animals versus the other 12 hours and the other time points we don’t see any difference so anyways in conclusion we don’t think locomotor activity has an
Influence on that sleep it’s a different story we did not sleep deprive the animals so invert the cycle it would have been a very good experiment I was waiting reviewer to ask they didn’t so I didn’t do it but I would expect I would expect that if you would be actually
Very good experiment um that if you disrupt the cycle by sleep deprivation inverting the sleep then you we should not see a 3:00 a.m. specific increase in exonary regeneration maybe at some other time I guess or maybe not so we didn’t do that but it’s a very
Good point if it’s uh some kind of sleep dependent or solely C and clock dependent what is the actual mechanism you can think of I was thinking about uh to try to connect reptin story but uh perhaps because of the uh like feeding State sagarian clock will be reset yes
Maybe reping and the uh feeding and regeneration or sleep even maybe all coupled I agree 100% because and again it was nice because for us independently we develop kind of these stories and then my conclusion earlier after leptin is based also upon the Circadian work so basically yes intermitted fasting promoting
Regeneration I do think has a circadian clock dependent mechanism we should do intermitted fasting bal1 deletion I guess right and see if indeed that is the case to prove this the point but yes in summer I agree with you problem is feeding time of the day and sleep they
All contribute to control I think tissue repair probably broadly because I cannot imagine that nature just focuses on nerve regeneration right so yeah and and I think it makes sense also because any anything you do in your life is quite compartmentalized you don’t typically go to the bathroom read
And dat at the same time and talk at the same time and you know and so on you can do a couple of things maybe but not all together so things need to be compartmentalized in an energy dependent probably uh Manner and also I think that’s important to a comparation task
For to have enough time to escape from predators or I don’t know there are number we can discuss at length I will stop here but I think it’s quite interesting okay broadly speaking oh interesting thank you very much okay okay hi thanks for the very U nice talk
And have two naive questions the first one is um uh could this intermedial fasting affect the the microbiota in the gut the second one is how does IPA affect the neutrals functions thank you sorry den and the last question the which function ah neutr okay first question is very
Easy so we published that so it’s again supplementary figure 12 and uh we did look at the gut microbiome following intermittent fasting there are changes in the gut microbiome uh they are not dramatic changes but there are significant changes in specific bacterial species including the ones that belong to idium
That Clum spens belong to so so you can have a look if you like and um the second question neutr activity okay so we did not study neutr mechanism in a systematic way following IPA however what we found is the neutr dependent interfer on gamma production enhances sever regeneration
Associated genes that depend on interfering receptor expression in Sensory neurons following and this is IPA dependent did you check other types of immune cells like tea cells it can also Secret in from yes we did check again we looked at all different type I mean the major types of immune cells
Innate adaptive immunity we looked at their um presence in the the injury site and the numbers and uh at the drg site at the cell body site unlike neutr Fields the increase the numbers in the drgs we didn’t see any differences however that does not mean that maybe their
Signaling might still be affected but those are the experiments we did we didn’t see a like neutr fils any changes in their presence and activ general activation state in the leion site and the dors thank you okay maybe I have a couple questions so the a the leptin signal leptin part
So you have to give using a then you can see the very strong regeneration but how about the systematic leptin would that have reached the level to see a a significant regeneration yeah so we gave leptin and I’ve shown that systemically IP and that promotes regeneration we we look regeration of the sciatic
Nerve yeah yeah we did not do that experiment at spod injury the main reason is uh because I wanted to look at specific mechanism and uh and if you do give LEP of course it’s it’s a good experiment it’s just maybe more translational experiment yes if you give
Systemic leptin after spod injury it’s uh it’s difficult to understand what what is going on actually leptin is been given after spanac cord injury by the way uh in the past and they showed some neuroprotection some positive effect I don’t remember anymore it was not a particular exciting paper anyway but you
Know there is some literature there and and that in a way was for us a dis incentive to do it and also we understood that yeah we would have probably not learned too much but and plus I think if you go down the route it’s uh yeah it’s a lot of
Work cuz you have to do different ideally different concentrations because it’s difficult to control and uh yeah but yeah of course it wouldn’t be bad okay so the BM1 knockout you showed you only did it in a conditional knockout right in drg not a systematic knockout what happened to systematic B1 yeah yeah
BM1 B one so in b the C has been disrupted disrupted everywhere so we didn’t do that because I think that probably would not give too much information but we thought about but again we didn’t do it nobody asked would be interesting to do bile one um conditional deletion take condition you
Know deleted miles in the central clock and see if you disrupt maybe that’s what you were thinking about so if you disrupt the central clock only and not the peripheral clock do we still do we see a difference and so suggesting may be more contribution of metabolic right rather than within be
Quite a cool experiment and would be interesting yes the last part actually the last question is very is amazing you showed if you do the lesion at let’s say uh uh uh 3 right but you see the effect like for we last four weeks so what exactly happens let’s say uh is because
Of right after the injury those those initiation of axons very fast let’s say because usually we know in inv Vivo take about a day right for the ACC start to initiate to grow seems like in those mutant in the time you do it they they can initiate faster would that actually
The major reason why you can see a functional recovery even later on I mean or some other reasons right to see I I don’t know that’s a good point but you know to know that one should do a bit of a Time course to see if they
Continue have this difference or only at very initi know we know that the 24 hours they already have an advantage and the 3 days they keep on having Advantage but as you know that in the periphery it’s very later on it’s if you measure the ax become too dense it’s difficult
To see later on but we know about those two time points so it’s possible they have an advantage that the others don’t catch up with um in terms of molecular mechanism what I can imagine is that sensory neurons are pre are primed already time dependent time tuned
Is primed at 3:00 a.m. and then injury comes and the signals come systemically and retrograde signals come to the dorsal ganglia and that priming mechanism that probably relies on epigenetic I think change es that we didn’t study systematically allows then the long-term advantage that’s how I can
Imagine it so because the St three is high right the time right so so like a stronger and faster transcriptional change to sustain a relative yes I think I think that’s the most likely explanation yeah it would be nice to see look at what happened to the injury
Let’s say injury signal we know is also very critical so at the time of injury whether locally there’s some kind of change yeah we looked at that of course of course and that that will will be published in in a supplementary figure where uh again yes we looked at all the
Different immune classical immune cells and their activation state after injury at the different times we couldn’t see a difference oh but I’m also aware and that if you look at the in the injury site is in extremely extremely rich with immune cells so I think can be a bit tricky to
Observe differences at the region site anatomically morphologically uh while because there are so many because it’s so so dense you know with those cells so that’s so better way that we didn’t do I think would be to sequence you know the different cell populations different time points and see whether there’s a
Difference I would be surprised if there is no difference yeah so there must be a difference regardless when we conditionally delete bal1 in Sensory neurons we block the phenotype of course that does not exclude that there is an upstream signaling communicated with neurons and then we block it by be one conditional
Delion because that’s a core clock mechanism so I think actually the two mechanisms are not mutually exclusive despite what we will show in the supplementary that there’s no difference in immune sell yeah okay any other questions okay go ahead may add a question so I’m curious to know whether
The Regeneration speed itself is also associated with the Circadian that you have described like for example if does it grow in the morning but just stop or just pause in the afternoon is there a correlation between this speed and secan itself yeah well typically when you look
At um Sensory neurons in the peripher after stin a crush you look at speed meaning that you know they grow slower or faster okay so then you can have sometimes you know the density is potentially another Factor but typically what people have shown is the speed so I
Think what we are seeing in our data is actually different speeds at different times okay but I think that may be a overall result for multiple days that you have watched so do you carefully look at the the specific time points like uh just about half a day what will
Be the speed change yeah so you think that’s a speed difference I’m just curious to know I think so yeah I think so I think it is a speed difference yeah okay thank difference in speed yeah you need to look at basically slope right so that will
Give yeah thank you you could actually yes I mean we don’t we could potentially graph I guess yes the the three time points that we have you know in a way in in slope kind of yeah yeah we didn’t do it but I guess the data is there yeah
One can do it yeah oh I have a question about the B1 uh as you mentioned uh every cell has a cin uh it’s B moment is a DI specific or uh do check the B working any other cellot types or any other neurons in other regions so Bal one is ubiquitously
Expressed but in this paper I didn’t have time to show you you you can look I think we did a specific experiment where we cultured the G neurons and then we um use a reporter with Lucifer is okay so a reporter Gene uh which is a pair anyway
It doesn’t matter which is a target of Beal okay Beal one and we see that there is a specific oscillation throughout the day the day of the signal in the G neurons now when we delete Beal this oscillation is blocked in the G neurons then we do a rescue experiment we put
Beal back in and the the oscillation circadian oscillation is restored plus the invivo experiment that I show you with conditional deletion of Beal in neurons show that our mechanism depends upon Beal one in Sensory neurons okay but as now discussed with Kai we don’t know whether if you disrupt
B Mal one which again is expressed in all cells only in the central clock which is in specific nuclear in the brain also disrupt the aary generative ability of sensor neurons we didn’t do that experiment we don’t know that but what we do know is that disruption of bal1
Specifically in Sensory neurons is sufficient to disrupt the clock dependent regenerative ability does it make sense yeah I think it’s her another question her approach maybe another spec let’s say what if to the spinal cord injury or the yes for example op nerve you would expect similar kind of yes yes
So SP spinal code injury does not regenerate typically so we didn’t do Beal one condition delion after Central spanac cord injury because it is a no regenerative condition however however one could do for example soal conditioning lesion where you do have some some improvement in sensor regeneration or actually now that I know
Hope you know one could also do leptin would be great experiment actually would be to do leptin over expression in the G neurons that we also know aligns the clock God when I say it I hope yeah the reviewers are this is po yeah the other
One will not ask me but that would be actually great exp right letin over expression Sensory neurons and delete condition delete BML one and see if that disrupts regeneration in the SP cord because yeah anyways we didn’t do it but that would be I guess probably the best
Experiment okay any other great I mean so this is uh smone give us very comprehensive understanding how the not only the neuron self and also not only the so-call injury inhibitor there’s a very in you know you know um environmental interaction including immune system or other factors interact
With the uh neurons and effect regeneration okay let’s give another round of applause and after that thank