"Exploring the Crosstalk Between Neurodevelopmental Disorders and Circadian Clocks" Jonathan O Lipton (04.14.2021)

April 26, 2021

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00:15Alright everybody,
00:16I think we're going to get started.
00:18Hello and welcome I'm Lauren Tobias
00:20and I'd like to welcome you to our
00:22Yell Sleep seminar this afternoon.
00:24A few brief announcements before I turn
00:26it over to Doctor Heckman to introduce
00:29today's speaker so please feel free.
00:31Please take a moment to
00:33make sure that you're muted.
00:35In order to receive CME
00:36credit for attendance,
00:37you can see the chat room for instructions,
00:40and there's a unique idea that you
00:42can text up until 3:15 Eastern Time if
00:44you're not already registered DLC ME,
00:46you'll need to do that first.
00:48If you have any questions during
00:50the presentation, I encourage you.
00:52Thank you for the chat rooms
00:54throughout the hour,
00:54and we will also invite people
00:56to unmute themselves at the end.
00:58We do have recorded versions of these
01:00lectures that will be available on line
01:03within two weeks at the link provided.
01:05In the chat and feel free to share
01:08our announcements for this weekly
01:10lecture series to anyone else
01:11who you think may be interested,
01:14they can contact Debbie Lovejoy
01:16directly at her email address.
01:17I also want to just let everybody
01:20know that we're going to be
01:22holding our annual sleep.
01:24Yale Sleep Research Symposium on Friday,
01:26April 30th that's from 10:00
01:28o'clock in the morning until
01:302:00 o'clock in the afternoon,
01:31and it's going to feature
01:33talks by Sam Cuna Upenn.
01:35He's going to speak about Sleep Medicine
01:38after the pandemic as well as Theresa
01:40Ward at the University of Washington.
01:42Who's going to speak about sleep
01:44health in pediatric populations
01:45with chronic conditions?
01:46So I'm going to post the link to register
01:49for this free event in the chat and
01:52please feel free to join us for that.
01:54So with that,
01:56I'll turn it over to Doctor Eric Heckman.
02:00Good afternoon everyone.
02:01I have the pleasure of introducing
02:03Jonathan Lipton today so he is joining us
02:06from Boston Children's Hospital below.
02:09Background on Doctor Lipton,
02:11he did his undergraduate at Brown
02:14followed by his MD and PhD at
02:16Albert Einstein in New York City.
02:19And following that,
02:20completed neurology training at
02:22Boston Children's Hospital as
02:24well as his sleep training both
02:27at Boston Children's and Beth
02:29Israel Deaconess Medical Center.
02:31Since then,
02:31he's continued to work at
02:34Boston Children's Hospital,
02:35as well as being an assistant
02:38professor at Harvard Medical School.
02:40He has had funded research
02:42for over a decade now.
02:44Looking into this circene Clock,
02:46an often it's overlap with
02:49neurodevelopmental disorders,
02:50I still talk about today and he has been
02:53awarded the Young Investigator Award
02:55from Sleep Research Society as well
02:59as many publications and being on the.
03:02Review Board for Sleep Advances Journal so.
03:07Doctor Lipson thank you very much
03:08for preparing for today and we all
03:10look forward to hearing from you.
03:13OK, thank you Eric and thank you
03:15for inviting me and having me today.
03:18The talk today is going to be very
03:21science heavy, so I apologize to those
03:23of you who are not that interested
03:25in in the underlying biology,
03:28but I'm going to sort of try and
03:31contextualize what we're trying to
03:32do and what I see as some of the
03:35opportunities in this very exciting field.
03:38My talk is really about the crosstalk between
03:41developmental disorders and circadian clocks,
03:43and I think that word is
03:45very important because.
03:47As I'll show you that what I've
03:49learned from my own work is that by
03:52studying developmental disorders.
03:53We've learned at by studying development
03:55disorders and circadian rhythms.
03:57We've learned something new about
03:59neurodevelopmental disorders themselves,
04:00and certain specific ones,
04:01and also we've learned new
04:03things about the circadian Clock,
04:05and so I think these these two
04:08processes are important to one another,
04:10and I think they you know we.
04:13It's an important point to make.
04:17So let me just make sure I can advance
04:19here so I have no disclosures,
04:21so get that over with.
04:23So let me dive right in and start talking
04:26to you about clocks and circadian rhythms.
04:28And obviously you understand them from the
04:31perspective of their their role in sleep,
04:33and I'm sure you see many patients
04:35with circadian disruption,
04:36so I won't spend too much time introducing
04:39the Clock and dive right in one of
04:42the real questions is you know why?
04:44Why are clocks so ubiquitous?
04:46Why?
04:46If you go to any city in Europe or any place?
04:50In the world really,
04:51you can go to the center of the
04:54town and you'll see a Clock tower
04:57o'clock in the center of town,
04:59and the reason is is because we we,
05:02we use clocks as prediction tools.
05:04The most fundamental aspect of
05:06our life on this planet is that
05:08the besides gravity maybe is that
05:10the planet rotates and it rotates,
05:12creating a 24 hour predictable and
05:15iterative geophysical oscillation.
05:17And that we experience with the
05:19light dark cycle and plants.
05:21And as you see in the middle there
05:23is the classic flowering plant,
05:26a flowering Clock.
05:27A plants are no exception to this
05:29an our entire metabolism is rooted
05:32ultimately in photosynthesis which is
05:34gated by the light dark cycle and so are we.
05:37And so I love this slide because it
05:40sort of points out just the importance
05:43of clocks to both as prediction tools
05:46and also even for our mental health so.
05:49This is,
05:49uh,
05:49this is a picture from a famous movie by
05:52Ingmar Bergman called Wild Strawberries,
05:54and it's about this older gentleman
05:57who goes to sleep and he starts
05:59to dream and wakes up.
06:01Wakes up in his hometown and looks
06:03up at the Clock tower and the Clock
06:05has no hands and this triggers a
06:08existential dilemma through which
06:09the whole movie is about.
06:11I won't get into all that,
06:13but basically it illustrates the
06:15idea that without time without
06:17a sense of time, we lose our bearings.
06:19We our sense of our our sense of context.
06:23So, circadian rhythms are the cellular
06:26mechanism that synchronizes cellular function
06:29and ultimately organismal function with
06:32this iterative oscillation of the planet,
06:35and it allows the cells of our
06:39body to anticipate the needs.
06:42Anticipate their own needs and guide
06:45animal behavior to optimize those needs
06:48as a function of time of day, and so.
06:51Because of this fundamental nature,
06:53it's probably not because of
06:55their underlying cellular basis.
06:57It's probably not that surprising
06:59that you can find circadian rhythms in
07:02pretty much all aspects of biology and
07:05certainly all aspects of our biology,
07:07including many aspects of behavior,
07:09physiological control, metabolism,
07:10and even on more molecular basis
07:13gene expression.
07:14And the reason for that is that,
07:17as I mentioned,
07:17the Clock is truly a multi
07:19scaled organizing principle.
07:21By that I mean you can see circadian
07:23rhythms at the level of the
07:25chromatin opening and closing genes,
07:27turning on and off modifications of proteins.
07:29How proteins get in and out
07:31of different organelles,
07:32you conceive circadian rhythms as
07:34they interact between different
07:35tissues of the body.
07:36And then of course,
07:38larger things like you know behavior
07:39and organization of behavior
07:41between different social groups.
07:42Even so, it's really this.
07:44Organization principle for which you
07:46can use almost as a lens to unpack.
07:48You know this rhythmic biology that we
07:51can now unpacking multiple levels and
07:53study at multiple levels by looking
07:54at essentially the same output,
07:56which is this rhythmicity which
07:58I I find very exciting,
08:00because as someone who's interested in
08:02understanding what's the molecular basis
08:04for behavior and how that molecular
08:05basis gets disrupted in disease,
08:07the circadian Clock provides this
08:09beautiful example of how we can
08:11use this rhythmic output as a way
08:12to unpack molecular mechanisms.
08:14And also.
08:15Build them all the way up to
08:17understand how behavior is organized.
08:19So this talk is really about developmental
08:21disorders and I think it's important
08:22to realize that circadian rhythms,
08:24like all biological systems,
08:25develop.
08:25And this is from this is from a
08:27nice review paper by Seth Blackshaw,
08:29and when it is former graduate students
08:31where they talk about the development
08:33of the Super Chiasmatic nucleus,
08:34which as you guys know,
08:35is the central circadian oscillator,
08:37I just wanted to put this up
08:39to show that even before birth,
08:40so I hope you guys can see my
08:42pointer even before birth.
08:44Even there's like seven days before birth,
08:46so this is like the late trimester
08:48in a mouse.
08:49You can see circadian rhythms of
08:51oscillation in the early SCN,
08:52so circadian rhythms are becoming
08:54rhythmic in the brain very early on.
08:56We actually know very little about
08:58how those are organized and how those
09:01organized function in the early brain.
09:03This is a classic actor Graham from a
09:06from the from an old old old paper.
09:09You know,
09:10a 75 year old paper looking at circadian
09:13rhythms in a in a in a human infant,
09:16and, as you'll notice,
09:17is that the circadian oscillations
09:19of behavior are gated behavior
09:21really doesn't develop until
09:23around three to four months of age,
09:25and before that it's really much
09:27more much more poorly organized.
09:29And it's not because the
09:31underlying clocks are not there.
09:33It's probably we don't
09:35know the reason exactly,
09:36but it's probably because
09:38until this point around here,
09:40the infant is just feeding every 2-3
09:42hours and so feeding will Trump this
09:46organization of sleep wake cycles.
09:49Obviously,
09:49circadian circadian rhythms as they develop,
09:51have a huge impact on sleep architecture.
09:54Without getting too much into it,
09:57we know that so in early infants,
10:00the sleep architecture has
10:01a very rapid and frequent.
10:07Changes are changes between
10:09RAM and non REM like sleep.
10:11These become more ensconce in the
10:13child and then are really like
10:15laid down in the in the adult.
10:17As we know our as our best
10:20understanding is really that what
10:21keeps us sleep late into the night
10:24is this underlying circadian Clock,
10:26which like galvanizes our REM sleep.
10:28How that circadian Clock works in early
10:30infant to galvanise architecture,
10:32I would say at this point
10:35is completely unknown.
10:36Importantly,
10:37sleep ontogeny parallels brain ontogeny
10:40so I would refer you to this little
10:44paper that we wrote and really.
10:46The only reason I put this in here
10:48is just to remind you that sleep and
10:50circadian function is very important
10:52for the development of the brain and
10:54what we think are the development
10:56of these fundamental sculpting.
10:58Both synaptogenesis refinement and
11:00pruning of synapses that we think are
11:02essential to the normal development
11:04of human behavior and probably are
11:07contribute in many different ways
11:09to neurodevelopmental disorders.
11:10Again,
11:11the idea being here that sleep and
11:13sleep dysfunction are probably
11:15very important not just as outputs
11:17of neurodevelopmental problems,
11:19but maybe even underlying them.
11:22And again,
11:22classical work going back to you know
11:24again 30-40 years ago showing this
11:26these dramatic changes in sleep architecture.
11:29So I always talk about sleep as this
11:31one of the most developmentally
11:33regulated behaviors.
11:34And for those fellows who are
11:36listening or those people who are
11:38listening or interested in research.
11:40I think this is one of the most exciting
11:43and untapped areas of sleep research.
11:45We really know very little in general
11:47about how the mechanisms underlying sleep,
11:50sleep in development,
11:51and why Slean why sleep is
11:53so important to development.
11:55I'll skip this so sleep dysfunction
11:58and neurodevelopmental disorders
12:00are what I refer to as common
12:02bedfellows and the reason what I
12:04mean by that is that there is an
12:06incredible overlap in individuals
12:08that suffer from neurodevelopmental
12:09disorders and sleep dysfunction.
12:11I'm sure all of us who do Pediatrics have
12:15experienced this in our clinics where.
12:18Very often you'll and I experience
12:20a lot as a neurology resident.
12:22I would see patients for autism
12:24and epilepsy and diagnostics,
12:25but really what they wanted to
12:27talk about in clinic was the fact
12:30that they don't sleep out.
12:31The kids don't sleep and so you know
12:34it has a huge impact on quality
12:36of life and indeed a huge impact
12:38on underlying biology.
12:40So sleep dysfunction as most
12:41of you probably know,
12:43is associated with behavioral dyscontrol,
12:44lower seizure thresholds,
12:45mood disruption, metabolic disease,
12:47potentially even obesity,
12:48diminished quality of life measures.
12:50And obviously, we could spend many,
12:52many days discussing each of these.
12:53I don't have time for that.
12:55I want to dive into some of our actual work,
12:59but just this is a more of a reminder
13:01to you guys that this is these.
13:04These there's a.
13:05There's a potent interaction between
13:06sleep dysfunction or developmental disorders.
13:08Some of the specific examples,
13:10of course, would be autism,
13:11Fragile X syndrome, tuberculosis,
13:13complex Angelmann syndrome,
13:14many, many others.
13:15And I just wanted to raise this
13:17idea that I think what we can see by
13:19looking at animal models and even in
13:22humans with these disorders is that.
13:24The rhythmic dysfunction is often
13:26so fundamental to their clinical
13:28presentations and possibly
13:29even the disease progression.
13:31I think it bears asking the
13:33question whether these diseases
13:35are fundamentally rhythm opathy's,
13:37and I know this is a little
13:39bit of acute term,
13:41but I think it's important
13:43just to to think about that.
13:46Maybe the rhythmic,
13:47the dysrhythmias in these disorders is
13:49actually fundamental to their progression
13:52and maybe even for diagnostics.
13:54So the question is when you have
13:56these complex, multifaceted and
13:58multi factorial interactions,
13:59how do you start to unpack it?
14:01From a scientific standpoint?
14:02And obviously there's
14:03no right answer to that,
14:05so one would be you.
14:06You take the system,
14:07you take a working system and you perturb it.
14:10Another would be you studying
14:12already perturbed system.
14:13So really what I'm trying to say is you
14:16could either take like a normal animal,
14:18let's say or a normal model of a
14:20normal or typically functioning
14:22system and you can muck around with.
14:24Sleep or muck around with molecules
14:26that you know are involved in
14:27your developmental disorders.
14:29Or you can take a newer developmental
14:31model and then study clocks.
14:32And that's really how my work started.
14:34But I think what you'll see is
14:36that it started with these.
14:38This very sort of almost naive
14:39approach and then we got into
14:41some very very deep biology.
14:43So I started this really because
14:45actually what I was a resident
14:46and I was doing my ICU rotation.
14:48I was my clinical.
14:49My clinical tending was stuff so
14:51he knew became one of my primary
14:52mentors and his focus of his lab was
14:55really understanding the underlying
14:56biology of this newer developmental
14:57syndrome called Too Brisk.
14:58Larose is complex and we were
15:00just chatting about.
15:01You know what I was going to do
15:02with my career and whatever.
15:04And I said, oh, you know,
15:06I think I'm going to study sleep and
15:08he's just set off the couple you know,
15:10kids with TSC don't sleep,
15:11they have terrible sleep problems
15:13like I didn't. I didn't know that.
15:15I never even know we don't study sleep.
15:17No one ever mentioned sleep during
15:19our clinical training 'cause we're
15:21always in the hospital taking care of.
15:23Like you know, patients who are very,
15:25very sick. So I started looking into this so.
15:29Well,
15:30I'll tell you what I'll tell you this story.
15:33So too is chlorosis to remind you
15:35guys is AutoZone will dominant
15:37or neurogenic neurogenetic and
15:38your developmental syndrome.
15:40It presents with epilepsy intellectual
15:41disability about 30 to 50% of patients
15:44have sort of classical features of
15:46autism and then very very frequently
15:48have 30 to 50% of these kids also
15:51have sleep disorders disorder.
15:52The disease is characterized by
15:54these pathognomonic tubers which you
15:57can see here on these Mris.
15:58So he's like. Areas of.
16:03In this case.
16:06Do you want me to abnormality?
16:08And it's really a disconnection syndrome
16:10and it's caused by so it causes these
16:13very abnormal white matter connections.
16:15It's caused by mutations in one or two,
16:18one of two genes, either TSC, one or TSC.
16:212 which form a complex and just
16:24to give you an idea,
16:26there is now an appreciation that in addition
16:28to these sort of character characteristics
16:30of intellectual disability and autism,
16:33there's a whole syndrome of
16:35neuro psychiatric dysfunction.
16:36Into risk losses,
16:36which is referred to as the TSC
16:38Neuro psychiatric disorder of
16:39which sleep is one of the primary.
16:44Symptomatology So what is the biology of
16:47TSC and why is it so appealing to study?
16:51So TSC is what's referred to
16:53as an mtor opathy emptores.
16:56The mechanistic target of rapamycin.
16:57This is a protein kinase which is present
17:00in all cells of the body and it is a
17:04core regulator of nutritive status.
17:06It basically is a decision point in all
17:09cells about whether to grow or to not grow,
17:13whether to break up to make
17:15protein or break protein.
17:16Whether to make mitochondria or
17:19not make mitochondria and many,
17:21many other things,
17:22and it does so by integrating upstream
17:25pathways that include growth,
17:27growth factors,
17:28nutrients such as amino acids and stress.
17:31So changes in oxygen tension
17:33or other other stressors,
17:35and the TSC complex,
17:37which is the cause of tubers chlorosis.
17:40It's right in smack in the middle
17:43of this cascade, and its basic
17:46function is to suppress so block.
17:48Amateur,
17:49so when TSC is blocked when TC
17:52is lost you lose this inhibition.
17:54And mtor is high in one way of
17:57blocking TSC is by using certain drugs,
18:01including where how was actually originally.
18:03This pathway is originally discovered
18:05which is wrapping my Sonoran.
18:07In clinical terms would be sirolimus
18:10or everolimus.
18:11Our raffle logs and that's where
18:13this protein got its name.
18:15Actually they found the drug first in.
18:18On rapper New Ian Rappa Nui is
18:20Easter Island in Polynesian and
18:22they found this drug in a bunch
18:24of bacteria and they wanted to
18:26study with the what this drug did.
18:28They found that it blocked cell division.
18:30They started using it as an immune
18:32regulator and eventually they figured
18:33out that the way rapper Mysonne works
18:35is by blocking mtor and that's how
18:37the whole field started now and I
18:39showed you here a pared down cartoon.
18:41Mtor signaling is much more complicated
18:43as you can imagine as all these
18:45pathways are I won't go through all of this.
18:47This is what a former mentor of
18:49my often referred to as Chinese.
18:51Well,
18:51where you basically have hundreds of
18:53pathways interacting with each other,
18:55and of course any crucial homeostatic
18:56pathway is going to be incredibly
18:59complicated,
18:59because even like the circadian Clock,
19:01it's incredibly redundant.
19:02It's built not to break,
19:04it's built to sort of regulate,
19:05but not fall apart, and so it has many,
19:08many interactions and complexities to it,
19:10which we don't have time to really
19:13get into today.
19:14I just wanted to point out that
19:16as you can see,
19:17the TSC complex sits literally
19:19in the middle of this.
19:21Literally, in the middle of this figure,
19:23because it's again,
19:24it's an integration point for
19:25the regulation event or so when
19:26it's dysregulated.
19:27You can imagine all sorts of
19:29havoc is wreaked on a cell.
19:31So just to remind you again,
19:33mtor basically regulates the making of
19:35protein and the breaking of protein.
19:37So while it's making protein,
19:39it also is suppressing the breaking
19:41of protein.
19:41When mtor is inhibited,
19:43it'll start breaking protein down and
19:45stop making it so it's sort of
19:47literally this little seesaw.
19:48So again, see styles are very appealing
19:51to people who study circadian
19:53rhythms because we like to study
19:56seesaws that oscillate with 24 hours.
19:58So there's actually pretty crummy papers
20:01on directly looking at TSC clinical,
20:03clinical, clinical,
20:04clinical dysfunction of sleep in TSC.
20:06There are a few and they show
20:09sort of fragmentation and some
20:11evidence of circadian dysfunction.
20:13Some circadian phase delay,
20:15sometimes advanced circadian rhythms.
20:16It's really a bit of a mish
20:19mosh to be honest,
20:21but there is strong evidence and certainly
20:24anecdotal evidence that sleep is a
20:27major problem for patients with TSC.
20:29I can just say as an aside,
20:31this is not scientific information,
20:33but I could just say the first time
20:35I spoke at a TS Alliance meeting
20:37and I gave a talk about sleep and
20:40these meetings are both scientific
20:42and for patients,
20:43and I can tell you that the room was
20:46completely totally jam packed to the rafters.
20:49And it's not because I
20:51was speaking about nobody,
20:52but it's because there's such desperation
20:54in this community to understand why
20:56their children sleep is so dysfunctional.
20:58So there's some evidence.
20:59When I started my work,
21:01there was already some evidence that
21:03in Drosophila and even in mouse
21:05models that are tubers process pathway,
21:07my impact circadian rhythms.
21:08I won't take you through all
21:10the complexity of this slide,
21:11but I just wanted to point out this
21:14is work done by Anita Sehgal's lab
21:16that when when you block the function of TSC,
21:19one in Clock cells in the in the
21:21flies you lose this normal gating
21:23of weight rest activity cycles.
21:25So suggested that if you block
21:27the TSC function in this case,
21:28it's TSC,
21:29one you can disrupt circadian rhythms.
21:31Around the same time,
21:33roofing Cole was working in Carlow.
21:35Britain's lab started looking at
21:36kinase kinase pathways in the Super
21:38chiasmatic nucleus and how they
21:40impact rhythmic behavior and he
21:42basically found that application
21:43of rapper Meissen could change
21:45the phase of freerunning rhythms,
21:47again suggesting that the M Tor
21:49pathway is regulating the light and
21:51then a light light sensitivity and
21:53also the underlying rhythmicity of
21:55the of the Clock and you can see
21:58here that rapper mice and causes
22:00a blockade of the normal.
22:02Phase changes that you can see
22:04impacted by light.
22:05OK,
22:05so this suggested that the M Tor
22:07pathway is required for normal
22:09circadian function,
22:10so we sought to study this in
22:12a model of tourists corrosive.
22:14So we have two two mouse models
22:16that we studied.
22:17This in first is a heterozygote model
22:20where you lose one copy of the TSC 2 gene.
22:23We have to study it this way because if
22:25you lose a both copies in an animal,
22:28its embryonic lethal and the animal will die,
22:31but the heterozygous.
22:34Survives and has various problems,
22:35including various cognitive problems
22:37and various of problems with synaptic
22:39plasticity and excitability,
22:40and to make a Long story short
22:42where we basically
22:43found is that there is a
22:46significant shortening,
22:47a free running period in these animals.
22:49So what we're looking at here is we
22:52are running so you can see the mice in
22:55train normally to a light dark cycle.
22:58I'll remind you that mice that
23:00we study are nocturnal, so there.
23:02Active in the dark and then they
23:05basically as soon as the lights
23:06come on they could be taken apps
23:09and then if you put them in darkness
23:11which will do is they you uncover
23:13the underlying rhythmicity of the
23:15free running oscillator dictated
23:16by the suprachiasmatic nucleus and
23:18the mice will run.
23:19And the reason why you see this graph
23:21sort of move this way is because
23:23the underlying periodicity of a
23:25mouse is usually less than 24 hours,
23:27at least of this strain of mouse.
23:30And so we were able to compare
23:32these these periods.
23:33Between Gina types and so we did
23:35that you can see that there's a
23:37significant shortening in the in
23:39the in the mutant,
23:40and then if we apply rappa mice
23:42and again remember Rep,
23:44my Son is going to now block
23:46the function of M Tor,
23:47so it should rescue some of these phenotypes.
23:50We were able to completely
23:51block this abnormality.
23:52We then use the more severe model
23:54and this model lacks completely
23:56knocks out one copy of this case,
23:58TSC one and I'll just mention that TSC
24:01one and TSC 2 have largely overlapping.
24:03Functions that's not entirely
24:05fair to 100% true,
24:06but we can think of them for this
24:08talk is having overlapping functions,
24:10and in this case what we did
24:13is we knocked TSC,
24:14one out of all post mitotic neurons
24:16using a synapse incread driver.
24:18So this is a transgenic animal
24:20that is expressing this.
24:21This double transgenic,
24:22and so all TSC one is lossed
24:24from post mitotic neurons.
24:26What we did is because these
24:28animals get quite sick after birth.
24:30We treated them with rapper Meissen
24:32until they reached adulthood.
24:34Enemy in the meantime,
24:35we implanted them with the data logger,
24:37so we're able to follow their temperature
24:40rhythms and to make a Long story short,
24:42we really see is that in the mutant
24:44there's a complete disruption
24:46of this rhythmicity under free
24:48running conditions when we now
24:50apply a light dark cycle,
24:51they can actually regain their rhythmicity,
24:53but it suggests that the underlying
24:55oscillations in the SCN,
24:57and potentially in their outputs,
24:58is fundamentally dysfunctional.
24:59Without TSC. Without normal mtor function.
25:03So we were really interested.
25:05Now we had this this mouse phenotype.
25:07We were really interested in sort of
25:10delving into what's the underlying
25:11biology that might underlie it and
25:14so remember, TSE is blocking mtor.
25:15We were really wondering about now
25:17what is the relationship between
25:19mtor dysfunction and the fundamental
25:21Clock mechanism itself?
25:22So again, to remind you guys,
25:24the Clock is present in all cells of
25:27the body, at least for the most part,
25:30and it is built on a negative feedback loop.
25:33Which was described by over over several
25:36decades and in 2017 was awarded the Nobel
25:39Prize for Understanding this mechanism
25:41to briefly sum it up for you guys,
25:43you have be Model 1 and this is in mammals.
25:47You have female one.
25:48In Clock they form a partnership and
25:51they bind to DNA and regulate the
25:53rhythmic expression of thousands of
25:55genes including their own inhibitors.
25:57And in this case it's the period
26:00jeans and the cryptochrome genes.
26:02Whose products go out into the cytoplasm
26:05and come back in and block the function?
26:07Have email 1:00 o'clock and so you have
26:10this iterative feedback loop that by which
26:12a system is driving its own inhibition,
26:14and that the loop itself
26:16takes about 24 hours.
26:17Now obviously this is
26:19extraordinarily pared down.
26:20It's way more complicated,
26:21and this is a slightly more detailed version,
26:24and it's even more complicated than this,
26:26but we don't have time to
26:28dive into all of that today,
26:30but I'll go back for a moment.
26:32Most of my work is really been
26:34about this single protein,
26:36bmal one because we found direct links.
26:38Between TSC dysfunction of the mtor
26:41pathway and bmal one and so I'm going
26:43to show you now all that all that data.
26:46So wide email,
26:47one email.
26:48One of the reason our it was our focus
26:50is because without the email one,
26:53you lose almost all circadian rhythmicity.
26:54So this is a female one knockout mouse.
26:57Here you have these nice ensconce
26:59circadian rhythms.
26:59That's all lost in the female knockout.
27:02Here you have cells expressing circadian
27:04Reporter without the Mail in the black.
27:06You lose those oscillations completely,
27:07so the take home message is you
27:10need to be mailed to have a rhythm.
27:13So we initially started this work by
27:16looking at TSC cells that lacked TSC 2.
27:19So we took cells that either had the
27:21gene or lack the gene completely and we
27:24just started doing some investigations
27:25and we found this is just a Western blot.
27:29So for those of you not familiar,
27:31basically the black lines represent the
27:33black smudges here represent specific
27:35proteins and don't worry about it.
27:37For those of you who are not
27:39familiar with it,
27:40don't worry about the the
27:42technique or the underlying.
27:44Looking at the plot,
27:45even I'll just type to give
27:47you the take home message,
27:49which is that females elevated
27:51so in cells that lack TSC 2,
27:53there's more female OK and in the
27:56brain you can see that in a wild
27:59type rain or normal brain we can see
28:01this rhythm of female in the cortex
28:04worth peaks around early end of the.
28:07Sleep period and then in the mutant brain.
28:10This rhythm is largely disrupted such
28:12that it's just kind of high all the time,
28:15OK?
28:16We then did this very sophisticated
28:19biochemical assay that don't
28:20worry about the details,
28:22but the details basically show that
28:24the amount of email that's being made
28:26or the bounce of protein synthesis of
28:29female is elevated about 50% when
28:31you lose the tubers grossis complex.
28:33So the idea is that without
28:35regulated interactivity,
28:36you have exuberant protein synthesis,
28:38and one of the proteins that get
28:40that gets over produced is be male,
28:43and so that was important because
28:45that could explain why we have.
28:48More bemelen these cells. OK.
28:51So yeah, so there there's more
28:54female here in the mutant than
28:56there is in the wild type.
28:58OK, so in addition,
29:00the other thing that regulates
29:02how much protein there is in a
29:04cell is it how much is produced
29:06and how much gets broken down.
29:08And we knew from work for many
29:10of many many groups that female
29:12is actually under regulated
29:14proteostasis or regulated degradation,
29:16and so we sought to understand whether
29:18or not that degradation was also
29:20disrupted in our mutant background.
29:22So very interesting, Lee.
29:23The the gene that regulates the
29:25degradation of female excitingly
29:27happens to be this ubiquitin ligase,
29:29this enzyme called UBE 3A and to
29:31those of you who are in the know,
29:34you'll know that you be 3 is
29:35thought to be the causative protein
29:37for another newer developmental
29:39syndrome called Angelmann syndrome.
29:40So already there's kind of this really
29:43from the perspective of a pediatric
29:45neurologist at there's a lot of
29:46excitement here because you know,
29:48we're starting to understand how there's
29:50two risk arose as complex through M,
29:52Tor and now we have.
29:54Investigation of the,
29:55UH,
29:55the this Clock protein is
29:57being dysregulated and we know
29:58it's also regulated by another
30:00neurodevelopmental syndrome protein.
30:02So you start to sort of imagine
30:04how the Clock can be integrating
30:06a lot of these different neural
30:08pathways that are very relevant
30:10to neurodevelopmental diseases.
30:12Interestingly,
30:12in addition to this protein that can
30:15promote the degradation of female,
30:17there also enzymes that can
30:19block the degradation of email,
30:21and they're called deubiquitinase is,
30:23and one that's been described in
30:25the literature is called USP 9X.
30:27And wouldn't you know it is also
30:30responsible for a excellent **
30:32linked intellectual disability and
30:34highly linked to synaptogenesis
30:35and fundamental synaptic function?
30:37OK,
30:38so our hypothesis was that this
30:40degradation of the Mail might be
30:42disrupted in the tubers chlorosis
30:44background where you have exuberant
30:46production and we have disrupted
30:48proteostasis in the cell.
30:50So this is the sort of fundamental
30:53seesaw now you have you be 3 driving
30:55the degradation and you have ESPN
30:58X USP 9X blocking the degradation.
31:00So first of all,
31:01don't worry about all the details here,
31:03but I'll just show you is if you do
31:05a degradation assay for bnymellon,
31:07you compare a wild type cells
31:09to mutant cells,
31:10which you can see is as the female protein
31:13degrades overtime in the wild type.
31:14It doesn't degrade in the mutant.
31:16In fact it doesn't degrade it almost.
31:18And if you do that using alive
31:20Reporter you can sort of report.
31:22They almost in real time this
31:24degradation and you can see that
31:25the decorative the half life
31:27of degradation is
31:28markedly elevated in the mutants,
31:29suggesting that there's a real problem
31:31not only with the production of email.
31:33But with the the degradation of female.
31:36And so this is a very busy slide.
31:38I won't spend too much time on it,
31:40but just to show you that basically the.
31:44We think the reason for this
31:46degradation I'll you know I'll skip
31:49all the Western blots 'cause it's
31:51probably painful is a disrupted.
31:53Disrupted balance of the Association
31:55of female with UV3A and USP,
31:579X so affectively.
31:58More of the female is being protected
32:01from degradation so there being
32:03more produced and not enough is
32:05being degraded and that's the take
32:07home message of this of this slide
32:10we used a drug that can block this
32:12deubiquitinase so we can we can enhance
32:15the ubiquitination and doing so.
32:17We are now trying to test this in
32:20animal models of TSC to see if it can
32:23actually rescue some of the phenotypes.
32:26And I'll show you data in a moment
32:28to see why that actually might
32:30be a reasonable thing to try.
32:32So if we block USP 9X,
32:34we can completely suppress
32:35circadian amplitude.
32:36So this is like higher and
32:37higher doses of the drug.
32:39This drug that blocks USP 9X,
32:41which is now going to
32:42enhance females degradation.
32:43And remember,
32:44I told you that if you don't have the email,
32:47you're not going to have a Clock,
32:49and that's what this would support.
32:51So as you degrade the email
32:53you degrade the Clock.
32:54So that's sort of proof
32:56of principle that this is.
32:57Mechanistically sound idea.
32:59So this is really where we are
33:02at this part of the talk.
33:04We have the TSC pathway which regulates mtor.
33:07It regulates then be Mal and and
33:10the degradation of email through
33:11the relative involvement of either
33:13ubiquitin ligase or a deubiquitinase
33:16which are working in opposition to
33:18one another to balance the amount
33:20of email that you have and what we
33:23have in the TSC mutant background
33:25is we have exuberant mtor and we
33:28basically have an upregulation of
33:30the amount of email that's made.
33:32And too little of it being thrown
33:35in the trash,
33:35so there's almost like 2 problems
33:37that are being that are being sort
33:40of working in cahoots to corrupt
33:41the Clock here by just making
33:43the Mail all the time.
33:45And that's what we think
33:46is part of the phenotype.
33:48And I'll show you data to support that.
33:52OK,
33:52so you have this seesaw and
33:53the see saw is imbalanced so
33:55that there's too much female.
33:56That's basically the message.
33:58Don't worry about all the Westerns
34:00and all that other all the IPS and
34:01all these liquid in assays we do.
34:03We do all these like assays that are
34:05that take a long time to explain it.
34:07I would love to do so if those
34:09of you want to hear about it.
34:11I'm happy to talk about it,
34:13but I think from a messaging
34:14standpoint this is the
34:15message abnormal mtor abnormal
34:17amounts of email, disrupted Clock.
34:19So this is a friend of mine is a
34:22very well known artist and so she
34:24designed this for my for my lab.
34:26This is like a nice alarm Clock where
34:28you know be Mail in the shadow of TSC.
34:31Bmal is running away with the
34:33the the Clock it's causing havoc.
34:34These little guys are making a mess.
34:37OK so this is.
34:39I, for those of you hating this talk so far.
34:42I apologize 'cause this is just the tip
34:45of the iceberg because there's all.
34:47There's more. There's more details.
34:48As you might imagine,
34:50but indeed, this is a you know,
34:52with an iceberg.
34:53Of course,
34:54there's this whole underlying biology,
34:55and there's a lot more here to unpack,
34:58and I'll show you another.
34:59Another wrinkle to this story,
35:01which I think is interesting.
35:03So the next part of the story is how
35:05studying tubers chlorosis actually
35:06taught us something new about
35:08what the circadian clocks doing.
35:10What I've shown you so far.
35:12Is how the Clock is disrupted in
35:14a model of TSC and now what I'm
35:16going to show you is based on
35:18those those findings and thinking
35:19a little more deeply about what
35:21we actually were showing here.
35:23We were able to find something new about
35:25the circadian Clock and an actual show.
35:27You some new data from my
35:29lab that shows something.
35:30I think it's really, really interesting.
35:32So again to remind you,
35:34the mtor pathway is is this
35:36crucial regulator of growth,
35:38and it's disrupted in TSC and one
35:40of the main things that M Tor does.
35:43It regulates growth through making protein,
35:46and so we thought a lot about if if
35:49emptores dysregulating bmal couldn't
35:50be doing so by what it usually does,
35:54which is by phosphorylating proteins,
35:55it's a kainic,
35:56so kindly phosphorylates proteins
35:58and one of the core regulators,
36:00or one of the core outputs rather.
36:03Event or is this Chinese called
36:05S6K1 and S6K1 phosphorylates many
36:08translation factors and to summarize
36:10six years of my life in one slide.
36:12What we found is we found that
36:15S6K1 phosphorylates female,
36:16so the Mail is a substrate
36:19of the mtor pathway.
36:20So not only is mtor regulating
36:23the production of email,
36:24and not only is it regulating
36:27the degradation of email,
36:28it's actually modulating the email
36:30itself through this S6K1 and also other.
36:33One other kinase as well,
36:35but it it it basically phosphorylating
36:37this protein and what it does
36:39is the phosphorylation of bmal,
36:41then mediates be males interaction with
36:43the protein synthesis machinery itself.
36:45So remember, be males,
36:46a transcription factor that
36:48spends most of its life in the
36:50nucleus and it's been studied as a
36:53transcription factor for two decades.
36:54So this was a little bit of
36:57heresy to sort of,
36:58start proposing that a Clock
37:00transcription factor has this role in a
37:03fundamental process in the cytoplasm,
37:04namely.
37:05Protein synthesis.
37:07And so to make a Long story short,
37:10we found that female interacts with this
37:12whole translation machinery in the cytoplasm,
37:14and these are just like
37:16immunoprecipitations showing like
37:17individual translational regulators.
37:18So this whole initiation complexes,
37:19which you probably learned in biochemistry
37:21and we're very happy to forget,
37:23but are very important in
37:25the production of protein.
37:26We found that female can actually
37:29associate with those proteins both
37:31in cells and in liver and brain,
37:32and we found actually that it
37:34can do so in a rhythmic manner,
37:37so females Association.
37:38With the translation machinery actually
37:41demonstrates just circadian oscillation.
37:43And.
37:43What the important part of this
37:46graph is really just to show,
37:48So what you're looking at here
37:50is the interaction of female with
37:52these different translation factors.
37:54So everywhere you see this black band,
37:56you're saying that the female is
37:58actually pulling down this protein when
38:00we mutated that phosphorylation site,
38:02so that one single amino acid
38:04where mtor phosphorylates it.
38:05If we mutate that site so
38:07it can't get phosphorylated,
38:09none of these proteins interact,
38:11so now none of the translation
38:13machinery can interact with female.
38:15And when we add the Mou into
38:17cells and look at the amount of
38:19protein that's being made,
38:21the more bmal you add,
38:22the more protein you make.
38:24But if you make this single point mutation,
38:26nothing happens.
38:27You can add as much as you want
38:29and you'll never get more protein.
38:32So this basically nominated this
38:33transcription factor in the Clock as
38:35a translation factor as a regulator of
38:37protein synthesis through the mtor pathway.
38:39And this is showing if we now take cells
38:42and we synchronize them in a dish.
38:45We can actually see a rhythm.
38:47This is every four hours.
38:48We can see a rhythm of high,
38:51low,
38:51high low protein synthesis that
38:52where we lose by email.
38:54You can see there's this rhythm
38:56but it starts to degrade by the
38:582nd cycle so it can't maintain the
39:00oscillation without having female
39:01in the cell.
39:02To get this phosphorylation signal to
39:05tell it to make protein at the right time.
39:08So that's summarized here and
39:09I just want to say again,
39:11this is 5 1/2 years of work that
39:14I'm summarizing in in 20 seconds.
39:16But the point I wanted to make is
39:18that the email is this critical
39:20component of the circadian Clock that
39:22undergoes rhythmic phosphorylation
39:23Association with translation machinery,
39:25and in so doing contributes to an
39:28oscillation in protein synthesis.
39:29So we learn something new about
39:32the Clock from starting with this
39:34clinical question about TSC.
39:36So now what I'm going to show you
39:38is just a couple of pieces of
39:40data where we now asked, OK,
39:42well this protein gets phosphorylated.
39:43What does that mean? What does it do?
39:46Does it do anything like that?
39:47When I showed you all this stuff is in
39:50cells and and cell lines in cell culture,
39:52does this thing actually do
39:54anything in the in the brain?
39:55And this is new work from my lab and because
39:58of the function of female in the cytoplasm,
40:00we started looking at neurons neurons.
40:02As you know, are these incredibly
40:04beautiful nuclei with these
40:05incredibly elaborated cytoplasm.
40:06And the cytoplasm is where I would argue
40:08alot of the interesting stuff happens.
40:10You have all the synaptic
40:12connections and synaptic.
40:13Transmission and also the the
40:15interaction of cells with one
40:17another and for a variety of reasons
40:20we started looking at the mound,
40:22the cytoplasm and what we've
40:23discovered since this work is work,
40:25we're about to send out for
40:27publication is we discovered
40:29that female is actually present,
40:31not just in the cytoplasm,
40:33But actually act synapses and so all
40:35these white dots that you see here
40:37in the hippocampus with the mouse
40:39are actually places where female
40:41colocalizes with defined synapses,
40:43and we found that the phosphorylated
40:45form of the protein does so as well,
40:48and this is looking at females
40:50colocalization with synapses in
40:52in hippocampal neurons in a dish.
40:54We also did this by looking
40:56at the ultrastructure,
40:57so in this case we used
40:59immunogold to basically,
41:00which uses a gold particle that's later,
41:02that's connected to an antibody,
41:04and in this case the
41:06antibody is against female,
41:07and then you can penetrate mouse tissue
41:09or any any any any tissue that you
41:12can do this in an and would you then
41:14do is by doing electron microscopy.
41:16You can look at the Indian Gold label as
41:19a way of seeing the female molecules and
41:22so we can see here is that these fuzzy.
41:25Fuzzy shapes here are actual synapses.
41:26That's the postsynaptic density
41:27in the presynaptic side where you
41:29can see the synaptic vesicles,
41:30and once you'll notice is that there's
41:32lots of female at these presynaptic
41:34vesicles and this is the knockout,
41:35just to show that the antibody
41:37is specific to be melon,
41:38not just labeling some garbage in
41:40the in the in the in the brain.
41:43Uhm?
41:43OK,
41:44I think I am running short on time,
41:47so I'm going to skip that so we so
41:49in order to study what this thing,
41:51what this phosphorylation system is doing,
41:52we made a mouse using CRISPR.
41:54So we knocked out this single.
41:57We know that we made a change
41:59in the single amino acids,
42:01so the protein cannot get phosphorylated
42:03and consistent with our work in cells.
42:05We found that the phosphorylation had no
42:06effect on the transcriptional oscillation
42:08in circadian rhythms and as a result,
42:10circadian behavior as driven by the Super
42:13chiasmatic nucleus seems to be normal.
42:14So here you can see like
42:16the free running period.
42:17I didn't show the quantification,
42:19but there's no difference between a cohort
42:21of wild type and of mutant animals,
42:23so this was not surprising to us.
42:25We weren't really expecting
42:26to see a global change.
42:28In circadian behavior,
42:29but it got interesting is we started
42:32to delve into the neurobiology
42:33and there's a lot on this slide,
42:36so I'm just going to summarize it quickly.
42:39Basically,
42:39what we found is that in mutant
42:41animals that
42:42lack this phosphorylation site,
42:44they reduce the amount of neurotransmitter
42:46that they can release in hippocampus,
42:48and they have evidence of presynaptic
42:50dysfunction, which this is an asset
42:53that basically measures the probability
42:54of release and a a increased increased
42:57dip here is basically effective.
42:59Of a impaired release of neurotransmitter,
43:01there's no change in the actual synapse
43:04number between the wildtype in the mutant,
43:07yet the network dysfunction in these
43:09animals is very dysfunctional,
43:11so this is long term potentiation where you
43:14use a stimulation to see how long can they.
43:18A network maintain dysfunction,
43:19and what you can see is
43:22that it can maintain that.
43:24Maintain the signature of of potentiation.
43:26It just does so much lower level.
43:29We think because it's not releasing
43:32as many vesicles.
43:33And this is the interesting part
43:35that I I hope will make some sense.
43:38Well, we found as though,
43:40even though there's no change in
43:42the global rhythm of the animal,
43:44we found that there is a a a loss of
43:47the synaptic vesicle accumulation.
43:49So in wild type animals we see that
43:52there is a diurnal change in the number
43:54of synaptic vesicles we actually counted
43:57by hand over 85,000 vesicles from 40
43:59to 50 micrographs from different animals,
44:02so we are very sure about this data we spent.
44:05Many months are counting this and
44:07what we see is that there's a
44:09diagonal difference in the amount
44:11and the number of vesicles that's
44:13completely lost in our mutant.
44:15So even though the mutants have
44:17normal global circadian rhythms,
44:19they lose this.
44:20What we're calling a local rhythm at the
44:22level of the synapse and the key question is,
44:25does this mean anything if you know the
44:28global circadian behavior is normal?
44:30What about other behaviors?
44:31So we don't.
44:32We put these mice through a battery
44:35of different cognitive behaviors.
44:36And what's really interesting is that they
44:39seem to have a relatively specific defect
44:41defect in hippocampal related memory,
44:44so this is just showing that the
44:46mice do not remember the context in
44:49which they've been been delivered
44:51a a paired stimulus.
44:53So this is a classical measurement
44:55for hippocampal memory,
44:57and these mice are very dysfunctional in
44:59this in this regard, and it actually,
45:02this is correlated very nicely
45:05with this change in.
45:06In the amount of potentiation,
45:08because these are often connected
45:10to each other,
45:11the other Physiology connected
45:12to the behavior OK?
45:14So I've told you a lot of
45:17different things and which is.
45:19This is sort of like the word salad
45:21of my my professional life and I'll
45:24just show you a two more quick things.
45:27So again, just to reiterate,
45:29we found that the TSC to risk
45:31LAROSA'S pathway regulates mtor,
45:33and in so doing it,
45:35dis regulates circadian rhythms.
45:36But we think by disrupting
45:38the function of the Mail,
45:40and it does so by over phosphorylating it,
45:43producing too much of it.
45:45And disrupting its proteostasis.
45:46And we think this has a disruption of both.
45:50Has stands to disrupt both
45:52global circadian dynamics.
45:53If there's enough disruption of mtor
45:56and potentially even local circuit,
45:58local synaptic rhythms through this
46:00phosphorylation mechanism and.
46:02I think some of you might be
46:04wondering if what I told you is true.
46:07Then one question is as well.
46:09If the email is high in models of tubers,
46:12sclerosis, and models of TSC loss,
46:14what happens if we lowered email?
46:16Can we make a difference on these
46:18mikes and the answer is yes remarkably
46:21so with regard to circadian rhythms,
46:23we found that in our mouse model,
46:25not only do they have a period defect,
46:28but they have this jet lagged effect.
46:31So the the mutant animals.
46:32Respond to a period shift much more rapidly,
46:35almost like an arrow that's
46:36been pulled too tight,
46:38and when we lower the amount of email,
46:40so this is a little counter
46:42intuitive because I told you
46:44you need the email for o'clock,
46:46but you need the right amount of email,
46:48so if you lower one copy of email,
46:51we could entirely rescue this phenotype.
46:53Similarly,
46:54we were able to rescue the free
46:56running period that I showed you
46:58in the very beginning of the talk.
47:00In the in the other model,
47:01and this is still preliminary,
47:03but we are starting to believe it.
47:05If you have this model that I
47:06showed you where you knock TSC one
47:08out of all post mitotic neurons.
47:10These animals shown here will die.
47:13And this is the control both for the
47:15crian the flocks strain, so they don't.
47:18They live normalized.
47:19If we knock be mild down in.
47:21In that background,
47:22we can extend the life span almost 50%.
47:24We don't know why exactly,
47:26but we know we can.
47:28And this again is consistent with
47:30the idea that email is one of the key
47:33downstream regulators of the TSC pathway.
47:35OK,
47:35so summary tuberculosis mouse models
47:37demonstrate abnormal circadian rhythms,
47:39which we think is related to
47:41a defective balance of female
47:43translation and degradation.
47:44That ESC pathway regulates circadian
47:46rhythms of protein synthesis in cells.
47:49We think through the phosphorylation
47:50of the male and female phosphorylation
47:53in data that we haven't published yet.
47:56We're about to send out.
47:58We think we've identified a novel
48:00role for the local control of synaptic
48:03function by the circadian Clock.
48:05And the important thing is that
48:07the Clock might be a point of
48:10convergence between multiple pathways.
48:12So remember,
48:12I told you that TSC is causing
48:15dysregulation o'clock through bmal,
48:16but females being regulated by other
48:18proteins that are also responsible
48:20for neurodegenerative syndrome,
48:22neurodevelopmental syndromes that
48:23have differences but overlap with
48:25TSC and so it becomes an exciting
48:28idea to start to think of the Clock
48:30as a capacitor for these different
48:33neurodevelopmental syndromes,
48:33almost like a common.
48:35Like a final common pathway that
48:38we can target.
48:39And so this is mainly for the
48:40trainees to say that I just wanted
48:42to sort of put put you through again,
48:44sort of the the arc of this part
48:46of my
48:47my professional life is really I
48:49started with this critical question
48:50that came out of a rotation and we
48:52went to an animal model of cellular
48:54model and behavioral analysis and into
48:56the cell biology and the signaling
48:58pathways and that took us to this novel
48:59by calling not novel ideas about what
49:01the Clock might be might be doing,
49:03and even the breath of what the
49:05clocks functions are in the brain.
49:06And so we've now come back.
49:08Circle and really the ultimate
49:10goal for this work,
49:12is to find ways to use this
49:15system to improve sleep,
49:17but also to maybe even mitigate underlying
49:20causes of neurodevelopmental disease.
49:22So we have lots of future questions.
49:26Babies up here, yeah?
49:27Happy to look at and then of course
49:30I have to thank all the people who
49:32did the work so members of my lab,
49:35former members of my lab and
49:37my collaborators,
49:37Alex Rundberg is Sasha Memori
49:39and members of their lab.
49:40And then of course my my friend and
49:42colleague Peter Society,
49:44Southwestern,
49:44my former mentor stuff to him for a
49:47lot of the work I showed was done
49:49when I was a postdoc in his lab,
49:51and of course all of our funding
49:53throughout which none of this
49:55would have ever happened.
49:56OK,
49:56thank you so much.
50:06Thank you Jonathan very
50:07much that was fascinating.
50:09I'll open the floor for any
50:11questions anyone may have.
50:15Have a question? Jonathan
50:16can you hear me? Yep yeah.
50:18So what happens if you give
50:21certain limits to healthy mouse like?
50:24Does it change anything?
50:27Well, well, so so.
50:30Now in 2010, and even before that,
50:33roofing cows showed that rapper myosin
50:36can block light induced phase shifts.
50:38So and that came out of work,
50:42showing that light actually
50:43induced mtor activity in the SCN.
50:46So first they showed that light
50:49can actually potentiates mtor,
50:51and then they basically showed
50:53that if you time if you time rappa
50:57mice into different points in the.
51:00Circadian Clock, you can effectively
51:02block the effect of light,
51:04so the take home message
51:05would be that REPL, mison.
51:08By itself can potentially
51:10impair phase shifts,
51:11and it might be consistent with
51:13what we see with the TSC model,
51:16where we see very rapid phase shifts
51:18with emptoris sort of exuberance.
51:23So could it?
51:24Could it possibly be of benefit if there is
51:27too rapid a phase shift? Like it could be
51:31only problem is of course rapper myosin
51:33has so many so many effects, right?
51:35So it's a little hard to make
51:38the argument that you're going
51:40to use recognizing for sleep.
51:42By itself, I don't think anyone would.
51:44Would would do that,
51:45but I think it's part of the reason why we.
51:48I mean, it's really part
51:49of the reason we want.
51:50We want to do our work is
51:52because we're hoping to be able
51:54to identify things that can.
51:55You know if sleep is the main problem,
51:58we want to be able to sort of target
52:00that without affecting like all the 3000
52:02other things that emptores doing so you know,
52:05you know recognizing is
52:06well tolerated in general,
52:07but it isn't immune suppressant and
52:08it can have lots of side effects,
52:10so it's probably not like the best sleep.
52:13Sleep modulator.
52:14You know what I mean?
52:16I was thinking of non 24 because it.
52:19Goes diving out. Maybe it'll have at least. I
52:22think that's an interesting.
52:23That's an interesting idea.
52:25I mean, maybe, maybe not REPL Meissen,
52:27but maybe something something that you
52:30know could target this mechanism that
52:32that that that would be interesting.
52:36You don't think you could find some
52:37teenager that would take rapper mice,
52:38and if they can play their
52:39video games later at night.
52:42I was actually going to similar
52:44question that meaning is there.
52:45Is there any?
52:47Evidence that people who are on
52:49mtor inhibitors have increased
52:51problems with sleep.
52:52Yeah, we don't
52:53really know. Honestly, it really is
52:56something I've wondered about a lot.
52:59We we don't know.
53:00I mean, one thing that seems to be true,
53:03which is that patients with
53:05that there have been trials.
53:07Now for using like sirolimus and
53:09everolimus in NTSC and the primary
53:11outcomes have been, you know,
53:13sort of a mixed bag a little bit.
53:16They haven't measured sleep directly,
53:18but from what I understand anecdotally.
53:22I think he paid patients generally
53:24feel better so he could again be like.
53:28You know it could again be
53:30in in the setting of TSC.
53:31You're kind of normalizing things.
53:33I think the question becomes in a
53:35setting where you're taking rappa
53:36Meissen for another indication.
53:38It's really different question,
53:39because then you're potentially
53:40suppressing it already.
53:41Normal baseline of M,
53:42Tor and I think if I had to say one
53:45message is that with anything in biology,
53:48and especially with any homeostatic pathway,
53:49you don't want to have too much.
53:52You don't want too little and
53:53it's the same thing with M Tor
53:55over exuberant or causes cancer.
53:57Lack of mtor called his death.
53:59So it's like you know you you
54:01you you don't want to have you
54:03know no protein being made.
54:04You also don't want too much being
54:06made and that's a simplification.
54:07But the idea is that these all these
54:09homeostatic systems have to be regulated,
54:11and I think this is exactly the message.
54:13This is a message from the
54:15Clock world as well, right?
54:26What happens to these mice as they get older?
54:31Mike, yes mice. They're hitting
54:33hitters like us as they get older.
54:38So I actually don't know about,
54:40you know whether they have like
54:41clearly like age dependent phenotypes.
54:43Most of the work has been
54:45done and like you know,
54:46young Ish or like sort of.
54:50Middle of adulthood.
54:52There's certainly a lot of work
54:54showing that in the more severe models,
54:56there are critical periods of
54:57intervention during development,
54:58which is sort of the opposite of your
55:00question to definitely like critical
55:01periods during which you know TST
55:03is probably misshaping cortical
55:04circuits and things like that.
55:06So there are points where you
55:08have to intervene or before which
55:11you have to intervene.
55:12With regard to like long-term phenotypes,
55:15to be honest, I'm not.
55:17I'm not really sure. I mean,
55:20one might imagine with over exuberant,
55:22or that you would have a.
55:25You know,
55:26potentially shorten lifespan.
55:27There's a lot of evidence that showing
55:30that mtor suppression prolongs prolongs life,
55:33probably by regulating caloric,
55:35you know,
55:36by regulating the amount of
55:39oxidative stress that's produced
55:41from the turnover of.
55:42Biomolecules basically.
55:51Any other questions?
55:55I'll just if there are no other questions.
55:58I'll just chime in just to let folks
56:01know what our talk is for next week.
56:04So we're going to be hearing from Dennis
56:06Wang from Kaiser who's going to be
56:09thinking about automation, big data,
56:11and artificial intelligence in the
56:13management of obstructive sleep apnea
56:15for future and current implications.
56:16So please join us for that.
56:20Thanks everybody, have a great week.
56:22Thank you, thank you Jonathan.
56:24Thanks everybody.