73 – Linking SYNGAP1 with Human-Specific Mechanisms of Neuronal Development

Pierre Vanderhaeghen, MD, PhD

Please refer to Dr. Vanderhaeghen’s paper here

These are our introductory remarks

Our presentation today is ‘Linking SYNGAP1 with human-specific mechanisms of neuronal development.’ I have the pleasure to introduce today’s speaker Dr. Pierre Vanderhaeghen.  Dr. Vanderhaeghen is Full Professor at the Department of Neurosciences in the Faculty of Medicine at Katholique University of Leuven, in Leuven, Belgium.  He also serves as Group Leader at the VIB-KU Leuven Center of Brain & Disease Research, VIB Leuven. Dr. Vanderhaeghen received his MD in June 1992 from the ULB Medical School and his PhD in Biomedical Sciences in 1996, also at the Universite Libre de Bruxelles.  He completed his Post-doctoral Fellow at Dept. of Cell Biology at Harvard Medical School from 1996 to 2000.

THIS IS A TRANSCRIPT ONLY:

0:07hello everyone and welcome to today’s webinar my name is Olga Bodie and I’m a single parent and volunteer here at

0:14syngap research fund and our presentation today is linking syngap one with human specific

0:20mechanisms of neuronal development and I have the pleasure uh to introduce

0:26today’s speaker Dr Pierre vanderhagen Dr vanderhagen is a full professor at the

0:31department of neurosciences in the faculty of medicine at the University of living in Belgium

0:37he also serves as a group leader at the Vib KU Living Center of brain and

0:44disease research Dr vanderhagen received his MD in June

0:49of 1992 from the ulb medical school and his PhD in biomedical Sciences in 1996.

0:57also at the University of ulb he completed his postdoctoral fellow at

1:04the Department of Cell Biology at Harvard Medical School in where he attended from 96 to 2000.

1:13a recorded version of the webinar will be available on the srf website under

1:18webinars on the family menu and by the end of this presentation you will have the opportunity to get the answer to

1:24your questions we’d love to hear from you please write your questions in the Q a below

1:30and for those of you just joining us welcome and again our speaker today is Dr Pierre vanderhagen

1:37this presentation today is linking syngap1 with human specific mechanisms of neuronal development

1:44it’s now my pleasure to turn things over to Dr vanderhagen thank you

1:51thank you so much Olga I hope everybody can hear me

1:56um and it’d be useful for me to make sure that everybody can also see my presentation can you confirm that maybe

2:03Olga that you see here yes okay do you see my mouse as well yes yes

2:10all right fantastic okay so um well thanks a lot for the invitation

2:17it’s a real pleasure for me to to be able to to speak to you and I guess it’s

2:22quite uh quite remarkable I guess that new technologies enabled us to do this

2:27only a few years ago it would have been probably basically impossible

2:33um but um despite yes I think the fact that we all prefer a live presentation I think

2:39this is a wonderful opportunity I hope to share with you some of the of the work

2:44we’re doing and I would be very interested also to hear uh your feedback and your questions so

2:51um I realize that I’m talking here to an audience who’s very interested in

2:57um disease associated with Singaporean deficiency um this is not the focus of the main

3:04focus anyway of of our lab and in fact the focus on our lab is uh neither

3:10neurodevelopmental diseases it is actually more a very basic question

3:16um that has to do with the origin of of what makes our brain human so a a

3:24question very much related basically to the development and to the evolution of

3:30the brain in our species uh but while I try to show you today is that there are in fact

3:36um quite intriguing links between on the one hand the mechanisms that somehow

3:41link development and evolution of the human brain and on the other hand that link

3:46these with neurodevelopmental diseases such as the one caused by mutation in the

3:52Singaporean Gene and I think Beyond let’s say the Curiosity driven aspect

3:58that I will show you as a scientist I think you will find out hopefully that

4:04there are interesting uh potentially interesting implications for a better understanding of neurodevelopmental

4:11diseases including Singaporean Patty so to speak so without further Ado let’s

4:19start with a short introduction what I like to to remind or explain to some of

4:24you um what we know basically and and what we want to understand about what makes

4:31our brain human so there are multiple levels of specificities of the human

4:36brain when you compare it with the brain of other species including the chimpanzee so our closest uh cousin on

4:43this planet um and among other other things there is of course the fact that the human brain

4:48is much expanded in size and this is mostly linked to the increasing size of

4:53the cerebral cortex that contains many more neurons but also not any type of neuron but

5:00mostly those neurons here that populate the surface of the cortex the upper layers and that are specialized actually

5:06in what we call corticocortical connectivity that is basically intra-cortical circuitry that is thought

5:13to underlie a lot of the higher cognitive function that are characteristic of our species

5:20there are a number of things that we could spend hours discussing what what makes the the brain human in terms of

5:27anatomical molecular and physiological differences but the one I really want to

5:32focus today upon is the fact that it is characterized the human brain by a very

5:37prolounged development which is also called human brain neoting and what I

5:43mean by that is Illustrated to you here on the left side it’s the fact that basically it takes up to two decades for

5:52the normal human brain to develop and you can see here morphologically some some very striking images where you can

5:59see on the one hand how the brain of a one month old is increased in size over uh in this

6:07case up to six years and this is not linked to an increase in the number of neurons because the neurons were already

6:13generated before birth this is mostly linked basically to the growth in size

6:19and complexity of the neurons themselves in particular growing dendrites axone synapses and so on and you can see here

6:25for instance that in the prefrontal cortex up to 15 years is probably needed

6:31to get to the maximum amount of synapses that can be generated you can see here the comparison with the mouse

6:38we are basically the same process that we know in the human takes up to two decades takes about six weeks so that’s

6:45what nail Techni is about is the fact that there is a sort of out of scale prolongation uh of the development of uh

6:55of of the human brain and most strikingly of the other super cortex and you may wonder is that good for anything

7:01well it’s actually good for many things potentially and in fact it’s been recognized uh for decades if not more I

7:09think even Darwin mentioned this that it’s probably a selected trait as we say in evolution so something that was

7:15somehow selected in the evolution of Harmony needs and then prejamin hominins and and the human species because

7:24um basically the pro-launch development of neurons after birth and for many years a loss basically for a longer

7:30period of what I would call to simplify an optimization of the neural circuits in response to the environment so this

7:37prolonged development basically makes our brain uniquely sensitive for a very long period to the environment social

7:45interactions interactions with the outside world and so on and this is indeed as we all know a critical feature

7:51of human brain development now on the other hand even though uh we

7:57would recognize that at least for the evolution this is a very important process there’s very little known about

8:02it so it’s still very little that is understood about how is it that human

8:07brain development is so much prolounged and on the other hand and and so that’s

8:13one first thing that we’re very interested in our lab is to try to understand what are the mechanisms molecular cellular genetic that somehow

8:21enable to understand how come the human cortex is so nailed that it makes so

8:26much time to develop and on the other hand what we’re also very interested in is what are the implications of this nail

8:32tenu for normal brain function and then of course also for brain diseases because if something is really important

8:38for normal brain function it is reasonable to hypothesize that when it is disrupted it may be linked it may be

8:45cause actually impaired brain function disease brain function and so these are

8:51the two aspects that we study a lot in the lab and where as you guess uh this is basically what brought us to to study

8:57to get interested into into singap one but before we get there I want to First tell you a little bit about how we got

9:04interested into the mechanisms underlying neotony and what what we know about it at this point so the way we got

9:12interested in this is first a number of years ago when we and others invented

9:17ways basically to make cortical neurons so make neurons of the cerebral cortex artificially in a dish from pluripotent

9:25stem cells and I’m sure many of you are familiar with that type of Technology but what we did basically to try to

9:31probe basically the function of these human neurons is to transplant them in the mouse to basically test to which extent found

9:39in the context of an individual brain these neurons generated artificially could develop so it was really to start

9:46with just a sort of technological tool and that’s just to show you a few images

9:51of these human neurons that we inject in The ventricle of the brain and then we can see them migrate here these are the

9:59transplantation are only neonatal mice so young very young peps and then if we wait a few weeks you can see now that

10:07the mouse cortex is stuffed literally with a lot of human neurons that you see here in Grain and that these are the the

10:14neurons that we call xenotransplanted because they come from another species in this case the human species and they

10:21go into the mouse now uh what’s good about this despite the fact that it’s an

10:26artificial system in fact it revealed to us something quite relevant to what I was telling you about the neotany and

10:33that’s what I’m showing you now what it revealed to us is basically quickly the following if we take Mouse pluripotent

10:41stem cells and differentiate them in cortical neurons in addition transplant them in the mouse well it takes them

10:47about four weeks as you can see here to become in the mouse relatively mature cortical neurons if we

10:55do the same experiment with human neurons transplanted in the mouse that’s what shown to you here you see that it

11:01takes many months in fact it takes more than a year to get

11:07neurons that that start to be mature and so that’s strikingly reminiscent basically of the very prolonged timing

11:13of neuronal development and we and others since then have done many experiments taking for instance neurons

11:20derived from stem cells of different species and transplanting in the mouth and and the bottom line is what I show

11:26you here which is that the developmental timeline of cortical neuron following

11:32transplantation from any species into the mouse is remarkable similar to the

11:38time it takes for the neurons of that species in the brain of these species so

11:44in for instance the chimpanzee the human the macaque or the mouse so strongly suggesting that basically there is

11:52probably a sort of a clock or hourglass as I prefer to say that is intrinsic to

11:58the neurons and that controls the developmental tempo of the cortical neurons another question is what could

12:04be this developmental Tempo um that and what is this hourglass or this clock that’s in the neurons and so

12:11[Music] um I will get that into into a moment but before that I want to show you one last

12:18uh important aspect and that make up your mind and which is that maybe these

12:23human neurons here develop very slowly in the mouse not so much because they

12:28follow an intrinsic uh developmental clock but because they’re just not in the right place and so that they are

12:35wrongly developing and because of that they just take that they just slow down and maybe stalled in some kind of

12:41aberrant developmental path but in fact we spent many years to test that and the

12:47answer is no these human neurons actually seem to develop quite normally and I think the best proof we have so

12:53far along these lines is shown to you here what we’ve done basically in the

12:58lab is to transplant those cells and then wait many months for the neurons to develop and then probe the function of

13:05these neurons using in Vivo Imaging and in this case the experiment that I’m showing you is basically an experiment

13:12of microscopy multifoton Imaging of the mouse cortex of a live mouse that is

13:18awake and you can see here that we can image individual human neurons in that

13:24Mouse and we aren’t here located in the visual cortex and the mouse at that

13:30moment is watching visual stimulation and we can look at the response of the

13:35human neurons that’s what shown to you here you can see on the upper left what the mouse are watching what’s the mouse

13:42what the mouse is watching and you can see the individual neurons and I hope you can you can see two things first

13:48that the neurons respond to the visual stimulation and second that they don’t respond all at the same time they are

13:55all specifically responsive to specific stimuli corresponding to different Direct questions or orientation of the

14:04visual stimulus and that is a characteristic feature of the visual cortex so to us this strongly indicates

14:10that these human neurons despite the fact that they develop very slowly are actually in the end acquiring very

14:18mature and sophisticated circuit features and you will see that this is providing us novel tools to study human

14:25neurons in Vivo and understand what could be going on during normal development as well as in disease

14:31so to to summarize the first part the there is an intrinsic hourglass of

14:37development of human neurons if we transplant the human neurons in in the mouse they develop at their own pace

14:44for that reason yet they can achieve substantial maturation and functionality and so uh one question that we’re

14:52interested then in the lab as I told you before is what are the mechanisms that regulate this timing and then on the

14:57other hand what could be the implication for normal or abnormal development in the human brain there’s a third

15:03implication that I will not have time to discuss with you but that I’m happy to discuss later on in the Q a and which is

15:09the perspectives that this type of work is offering for potential uh therapy

15:15leading to brain plasticity or repair now the mechanisms I’m going to go

15:20quickly here because this is really hardcore developmental biology but basically you could imagine two main

15:25mechanism on the one hand uh you could imagine that the genes that control the

15:32development of the neurons in the human and the mouse are following different timelines or are actually different

15:38genes and this is why it’s taking a different time in the end alternatively there could be Global

15:44changes in the neurons that somehow influence the same genes along a

15:51different speed so that’s what I mean by changes within the gene regulatory Network or outside

15:56of it and what we found in the lab is that the two hypotheses are true the two

16:01types of mechanism cool exist and in fact work together to control The Hourglass of the development of timing

16:09of the neurons what we found a few years um I’m sorry a few years ago yes but

16:15that we published a few weeks ago is that for instance the basic metabolism of the neuron is very important as a

16:22global controller if you’re interested you can check our paper that came out recently in science on this but there

16:29are also Gene regulatory mechanisms and new genes that are at stake and that’s

16:34what I’ll be telling you about now in particular of a new Gene family meaning

16:39a human specific Gene family that we think is a very important controller now

16:44what do I mean by new genes so many decades ago developmental biologists in particular

16:51as well as geneticist found out the very surprising thing that I’m sure you will

16:56know and which is that the genomes of many different species contain the same amount of genes and are very similar to

17:03one another this is true vastly meaning that for instance between human and chimpai and Z

17:09most of the genes are common but there are a few genes that are different and these basically come from what we call

17:15Gene duplication meaning one gene can become another one through a copy paste mechanism and then

17:22change through evolution of function and structure and these genes that we call human or

17:30hominid specific there are many of them and a few years ago we identified several dozens of them that are

17:36expressed in human cortex development and in particular among them some genes

17:41that you can see here that are selectively upregulated during human cortex development in cortic

17:53to now what are these genes these genes were actually discovered by or colleague

17:58and a friend actually Frank palu works at Columbia University what he discovered a couple of years ago now is

18:06that there is a gene that is common to all mammals so also present in the mouth

18:11that’s called srgap2a and then in recent Evolution a few million years ago in our

18:17ancestors this Gene was duplicated into other genes called asagap2bnc

18:23which we found to be expressed heavily in cortical development and what Frank and his team also found

18:30and that you will see is very relevant to what we’re discussing today is that if you over Express the human specific

18:38Gene in the mouse it results in a delayed maturation of the mouse cortical

18:44neurons so a kind of neotony of course not transforming the mouse

18:49into the mouse neurons into human cortical neurons but nevertheless decreasing very substantially the speed

18:55of their development and interestingly it’s also important for what I’ll be telling you about they saw the same

19:01thing if they were removing one copy of the SR Gap to a gene the ancestor Gene

19:08so suggesting that these human specific Gene act as inhibitor of the gene from which they derived and that is present

19:14in all manners now this was a number of years ago what we set up to to do then

19:20is to study whether um this this is a sargap2c gene present

19:26only in the human genome to understand what they could be doing in human neurons and that’s the work of a very

19:34talented postdoc in the lab Batista so how did we do this well we first used

19:39our system starting from stem cells transforming them in cortical neurons and then infecting them with these

19:45constructs that lead to down regulation in this case selectively of the sagap2cg

19:51you can see here this area 2C and how it goes down following this knockdown and then the setup is basically to First

19:58knock down the gene then transplant the human neurons that no longer have the human specific genocide yet to see and

20:05then ask how they develop in the mouse in Vivo and looking in particular in this case

20:11at the morphological development look at these dendritic spines that reflect synapse formation and then also look at

20:18their physiological activity again reflecting in this case synapse formation as well as maturation and what

20:25we found is a very striking observation I think so you can see here basically how these dendritic spines develop over

20:31time from very few spines to many in control situations you you see text here

20:37up to 18 months to get the plateau of development of the dendritic spine so so

20:42reflecting of all the neotony I was telling you about and now here is the situation following the loss of the

20:49human specific Gene you see that the dramatic increase in the number of

20:54dendritic spines over time and that’s what shown to you here so here is the normal pace of development

21:02of this of the dendritic spine increase and you can see how it’s accelerated following the loss of the human specific

21:09Gene conversely if we remove the ancestral Gene the gene that’s present in Old mammals and sargap2a it’s the

21:16opposite phenotype we see fewer dendritic spines which fits with what fraud Polo initially uh suggested that

21:25these genes and sargaptor and sagap2 see our antagonist with one another and that

21:30the human specific genes that we see here are actually required for the normal neotany of the human cortical

21:36neurons in Vivo we confirm this using physiology and again same thing I’m going to go quickly here but you can see

21:43increase in the frequency of the synapses in the amplitude of the

21:48synaptic currents and the frequency of the synaptic uh potential sorry and their amplitude

21:56and you can see here also changes in the ratio company in the air currents that

22:01reflects basically increased maturations of these synapses so overall there’s an acceleration in the formation and in the

22:10maturation of the synapses in the cortical neurons that lack the human specific genes

22:16so that was already quite interesting for us because it was the very first demonstration that a human specific

22:23genes were Gene present only in our species and no other species is actually required for something that we think is

22:30quite important because it’s partly basically setting the very slow tempo of

22:36human cortical neurons of course this is just one gene among probably many others that contribute to it but it was a first

22:44important step to try to move on and so to try to understand which other genes

22:49could be involved in this we we as basically this classical question in developmental biology which is what are

22:57the downstream mechanisms which are the other molecular players that together with the sargap2c could control the the

23:04neote and you will see uh so how we did this the first approach that we use is

23:10basically again doing a knockdown of the sagap2c human specific Gene of the human

23:16cortical neurons generated from the stem cells but then staying in the petri dish for practical reasons then look at the

23:24composition of the synapses of these neurons and what we first found as you can see is that overall the compositions

23:31of these synapses is fairly similar at the molecular level with this

23:37very gross approach whether we um knock down sagap2c or sagap2a and by

23:46the way I’m sorry I should have started with this you can see here the level of srgap2a so the ancestral protein

23:54and you can see that the level of this protein is of course going down when we use a knock down to knock down a

24:00sargap2a but you can see interestingly that it goes up when we knock down a sargap2c and this

24:06was actually sort of expected there is data from other labs that have gone

24:12beyond the observation that this agap2c in a sargap2a seem to uh that well that

24:19Sagat 2C seems to inhibit the sagap2a it seems that this inhibition goes through

24:24a diminution of the amount of srgap2a meaning that if you remove a sargap2c

24:31well then you increase the level of Sr gap2a okay now how about now other types

24:39of proteins and this is where I guess you guessed it we stumble upon singap one

24:45um that turned out sorry I’m going a bit quick here that turned out to be actually

24:51dramatically changed following knockdown of srgap2c so you can see the normal levels of singap one in the cortical

24:58neurons and you can see how it’s decreasing following a removal of the SR

25:04gap2c human specific Gene and you can also see already here we’ll get back to that later that the levels of singap one

25:11seem to increase on the other hand when we remove a sargap2a so since C is

25:17blocking a we had this model here suggesting that c

25:22is blocking a and a is maybe diminishing the levels of singap one and this model

25:30would explain basically what uh what you see what you see here so that to us was

25:36very interesting and so we asked the question of course of what could be the

25:42relevance of the biology of uh of singap one in in human neurons and now we get

25:49you know territory that I think many of you are much more familiar and maybe to some extent more familiar than me

25:55basically of of the wealth of knowledge about syngap one that on the one hand from a basic perspective is a very

26:02abundant personality protein involved in synapse development and function uh the mutation of which can lead to

26:10intellectual deficiency epilepsy autism spectrum disorders and interestingly uh the work of Gary

26:19Rambo and colleagues has shown a number of years ago mimicking the mutations found in the patients but in the mouth

26:27that it can be associated with accelerated development of the synapse

26:32in the mouse so a little bit conceptually similar to what I showed you with removal of the human specific

26:39genes in the human cortical neuron so we thought that this was a maybe more than a coincidence and so we looked now uh in

26:48parallel I would say to the study that we did on the human specific Gene we looked now at the Singaporean Gene but

26:54in the same model in human cortical neurons and this is a work that we did in the lab thanks to benvelmark a very

27:01talented postdoc as well as a vasobena a visual physiologist because you will see

27:08that we went actually when it comes to singap one all the way to visual function and how the human can be

27:15implicated in that in normal and Singapore mutated conditions so first we generated singap one mutant

27:23pluripotent stem cells and particular neurons using conventional crispr gas technology you can see here normal

27:31neurons and the level of singap one here what we would call knockout neurons so

27:36we know a protein at all and here singap one affluence efficient neurons as I

27:42will call them so when one copy is deficient while the other one is normal which mimics basically the disease and

27:50what we’ve done then is to prepare cortical neurons of each of these three

27:55genotypes and then the French differentiate them in cortical neurons

28:00transplant them in the mouse and compare them in terms of morphological and synaptic

28:05development as well as looking at them at the level of the circuit in the visual cortex

28:12so um that’s just to show you basically the type of Imaging that we can do in

28:18the mouth of the human neurons but this in this case looking at other dendritic

28:23spines and one important thing is that since we can image those neurons in live

28:31animals we can repeatedly image the same neurons over time and really have an idea of the dynamic of development of

28:37these neurons which is really critical for the question we’re interested in of Developmental timing so you can see here

28:43similar neurons over time and you can see in normal conditions how the spines

28:49increase in density and you can see in the singap one heterozygotes and

28:55homozygote mutants how the dendritic spine basically formation is accelerated

29:01you can see here a first detail quantification and here a synthesis of

29:07that where you can see that the the knuckles in gap when neurons basically are already very High very early on a

29:14few months after transplantation and you can see that the upload insufficient neurons are also actually increased in

29:21speed quite importantly compared with the control so there’s a striking acceleration of dendritic spine

29:27formation in the human cortical neurons in Vivo and this is actually quite consistent with the data at a given

29:33Rambo and colleagues that found in the mouse with one important thing to bear in mind is that in this case what we

29:40found is basically that the mutant human neurons are basically months ahead in development compared with the control

29:46while in the mouse because of their very fast speed of development the difference

29:51of time was only a few days and I think it’s something important to bear in mind now this is for the morphology this is

29:58now for the synaptic development and you can see here again that there is an increase in the number of the synapses

30:04as well as in their maturation following the loss of one copy of syngap one so in

30:11the upload Instagram efficient condition so consistent with the fact that synapses are formed at a faster speed

30:20and also they mature faster in the singap one applying sufficient neurons

30:27finally the third thing we we looked at in the singap one uh mutant neurons is

30:33potential consequence is at the circuit level and I think that’s a very important uh level of course especially

30:39when wants to understand brain function and disease because we are not just uh

30:44made of our brain is not not just made of a couple of neurons it’s made actually of networks of these neurons

30:50and these are the ones that subserve specific functions so if we want to understand the links between a

30:56particular genetic defect and a disease phenotypes and and go to diagnosis and

31:03treatment we really need to basically study this circuit level so that’s what we did using the approach I told you

31:09before of using mice that were transplanted and where the human neurons are infected and transduced with genes

31:17that enable in Vivo functional Imaging in live mice that are then stimulated

31:23with specific visual stimuli and ask how the human neural don’t respond human

31:29neurons with the normal genotype or that are displaying singaporeana coins

31:35efficiency and we made two uh observations that I’m sharing with you now the first important observation is

31:42here when the mice are not looking at visual stimulus so they’re basically stimulated with a gray screen so that

31:50there’s not much stimulation of the visual cortex and you can see there’s a first important difference between the

31:57singap one apple insufficient and control neurons which is that their level of activity is increased and I

32:03think that may have very important pathological implications obviously you

32:09can see the quantifications so this suggests already probably because of the increased speed of development and

32:16maturation of the synapses that these neurons are more active than they should at that stage of development after seven

32:23months post-transplantation on the other hand and perhaps even more strikingly we looked then at how these

32:30human neurons are able to respond to visual stimuli and I’m not going to go into the details but you can see and

32:37guess here that we can look at various neurons and see how they respond to particular stimuli that again correspond

32:43to specific directions or orientation of visual stimuli that’s the way visual

32:50physiology uses and classifies the neurons and and we can see basically that the syngap one upload insufficient

32:57neurons respond actually much faster in time meaning that while at two months

33:05post transplantation there are very few cells in normal conditions that can respond that’s because they’re neopenic

33:12they’re very slow to develop we can already see that singap one uploads sufficient neurons a substantial

33:19fraction of them can respond and this goes on with time so suggesting that what I would call the acquisition vision

33:25of visual responses is also accelerated in the human Apple insufficient neurons

33:32and we are exploring this further now to look at further consequences of this but I think this is all consistent with what

33:39I’ve been telling you uh in the beginning that the human neurons follow intrinsically their own pace of

33:47development depending on their species and so in that case the human neurons develop very

33:52slowly but it also depends then on uh the genome composition of of these

33:59neurons and if syngap1 is uh if one copy of of Singapore is missing well these

34:06neurons now start to develop faster again in an intrinsic fashion uh

34:12independently of of the environment you can also see another important thing here which is that here what we call for

34:18instance orientation selectivity index which is basically an idea of the Precision of these neurons to recognize

34:25specific stimuli is actually similar between the two the neurons of the two

34:30genotypes so suggesting that it’s really the speed of development of the neurons that is specifically altered in these

34:38conditions in the human neurons at that early stage of development now let’s go back to the molecular

34:44mechanism and and this will be the the last part of the talk where I will try

34:49to basically link again the observation I showed you before that sigap2cs this

34:55human specific Gene involved in negative human neurons and then seeing Gap one which I showed you that it’s also

35:02involved in the nail Technic speed of development of human neurons and and as

35:07I get to a way that could be potentially in between from the biochemical data I showed you here so we did another

35:14experiment and in fact I’m showing it to you now and that experiment is to look

35:20now at the molecular composition of the synapses in the neurons that are blue

35:26insufficient for singap one first and what you can see here is that the levels

35:31of singap one are lower that’s expected there’s one copy missing but you can see something else here

35:38which is that the levels of a sagap2a are increased when we remove one copy of

35:45syngap1 so that means that the model I showed you here is actually only partially true or let’s say it’s only

35:52part of the truth in fact if you put everything together it means that sagapped way somehow is down regulating

36:00the level of Singapore but what that is shown to you here is that syngap1 is done regulating the level of a saga

36:06two-way so it’s as if there was a sort of tug of war if you will between the two proteins to be located at the

36:13synapse and somehow each one is inhibiting the other one with sagap2bc

36:18being Upstream of that and tipping potentially the balance towards

36:26um one of the two sides and the second interesting experiment

36:32that we did at least I think the result is interesting is that in the scene Gap

36:38went upload sufficient neurons we then removed srgap2a and reasoning that this should have an

36:44impact on scene Gap one and indeed it has and you can see it the impact is quite dramatic so in the syngap one

36:51mutant neurons where Singapore is not reduced in quantity in the synapses when

36:57we also remove a gap two-way you can see that now it goes to levels that are

37:02almost normal and so that’s um what uh trigger then or question that

37:12I showed to you the next question that I’m I’m showing you here and which is basically whether

37:18srgap2 down regulation could somehow rescue the singap one synaptic

37:24phenotypes I was showing you and so uh to address that oops I’m going

37:30a bit fast yeah what we did is to take a cortical

37:36neurons that are deficient in singap one or normal for Singaporean levels and

37:42knock down the levels of Sr gap2 a and ask what’s the outcome now in

37:49terms of morphological and physiological development and here is the first type

37:54of experiment so this is to remind you again what I showed you before which is that the density of the dendritic spines

38:02is increased when we remove Singaporean but now here is what’s going on when we

38:08remove a sagap2 at the same time you can see that basically those levels go down

38:13to almost normal levels that’s what’s shown to you here also you can see the levels going up following singap one

38:20mutation and how they go down following the double removal if you will of singap

38:27one on the one hand and as I get two-way on the other I think it’s even more striking if you look at the physiology

38:32where you can see again normal Singapore neurons and heterozygotes neurons and

38:39you can see that basically the singap one uh normal neurons show an increase in

38:49the synaptic frequency and amplitude following uh the upload sufficiency of

38:54Singapore and this is basically going back to what seems like normal values or

39:00at least much reduced and closer to normal values following the knockdown of

39:06srgap2a and again the same is true for the Empire and MDA ratio so suggesting

39:12that the increase in the speed of the dendritic spine formation and maturation and of the synapse formation and

39:19maturation um is is basically decelerated when both

39:25of the genes are reduced and try to to conclude on this on the

39:33one hand we have this molecular model here that I show you where there is uh those three proteins interacting with

39:42one another in a very Dynamic way in the human cortical neurons and that’s

39:47basically to show you the way we think it’s going on so on the one hand in non-human neurons in the mouse

39:54neurons for instance Singapore and srgap2 are involved in a tug of war

39:59where they inhibit one another and that somehow leads to a relatively fast speed

40:06of maturation of the neurons in the normal human neurons the human

40:13specific genus are gap2c is actually tipping the balance by inhibiting a

40:18sargap2a to reduce um those levels and thereby basically uh

40:27increasing the neotenes slowing down the speed of

40:33the human cortical neurons why in the case of singaporeana pro insufficiency

40:38where the levels of singap one are now lower well this type of balance

40:44basically is going the other way around so that the development of the cortic of the cortical neurons the human cortical

40:51neurons is now accelerated in an abnormal fashion

40:57so if you want to wrap up in uh in a more conceptual way what I showed you

41:02today is on the one hand that the Temple of synaptic development is controlled by

41:08a balance of srgap-2 and Singaporean genes that this balance is somehow

41:14tipped by a gene that’s only present in our genome and then that two are surprised and I

41:21think it’s interesting uh obviously for uh the understanding of the mechanisms underlying uh disease caused by

41:28Singapore insufficiency Singapore seems to be directly linked to this mechanism

41:34that controls the speed of development of the human cortical neurons and of

41:39these human specific uh neote so with that I will uh I will stop and

41:47thank you again to the people involved in this work I mentioned a Baptist and

41:53Ben as well as Vasa I would like to mention also here who has a driving

41:59force of many of the experiments that were shown to you today and I forgot to

42:04mention that many of the experiments that we did on the srgap2c human specific Gene were actually done in

42:11collaboration with a long-standing collaborator from polu and his former

42:16postdocsicilian or in Paris who discovered originally this Gene

42:23um in the in the human genome so with that I will stop and I will be happy to take uh any questions or hear your

42:29comments thanks a lot thank you thank you very much that was absolutely fascinating

42:37um these webinars generally we have a few parents and a few scientists join but

42:42then parents watch them later so what I try to do is pepper you with as many questions as I can anticipate

42:50um and I and I know we have other parents and and people on so if you have questions please put them in a q a or

42:56raise your hand if you want to talk but I mean the obvious question is great in human hats can we knock down

43:04srgap 2A and recover the phenotype which I think as to your third point about juveniles

43:09and plasticity that you didn’t have time to touch on so if that’s if that’s an accurate sort of

43:16simple hypothesis what are the risks of not been knocking down an Sr Gap to it I mean is that actually feasible in a

43:21living creature what else would go wrong if you did that in a syngap hat human and then

43:27did you could you tease a little bit what you didn’t talk to today which is the the rest the rescue in the

43:32plasticity because those of us who are parents of of human hats are living you know with waiting with

43:38baited breath for Asos or Gene therapies to upregulate syngap and then the question is okay what what is that then

43:45we’re going to create an entire new class of disease right zero to age X haploid sufficient recovered via gene

43:52therapy what do those brains look like were you reintroducing Gap later in life so just two softballs for you two easy

43:59questions these are yeah two fascinating uh two very important questions on two

44:05fascinating topics uh obviously you know I don’t have straightforward answers for you on the one hand I think what would

44:12be expected in development if you knock down a sargap2a is the opposite phenotype then if you if you knock down

44:19symbi Point meaning that in your in this case uh things could go

44:25too slowly meaning that the synapses and the circuit in general would develop

44:30let’s say instead of a if if I mean this is very speculative but it will mean

44:36that you know instead of taking two years it might take four years to get to the same result or in the mouse from six

44:42weeks it could take many more weeks so and and obviously as always in biology balance is really fundamental and too

44:50much of anything is not good so I don’t think at least in early development it

44:55seems like a valid option to you know just say let’s reduce the sargap2a and everything will be fine it’s not like

45:01there’s a good Gene and a bad Gene it’s really the balance between those two that’s interesting but I think on the

45:06other hand it’s it’s really important to think that we found here probably uh you

45:14know a sort of symmetrical regulator of synapse of singap one you know a sort of

45:21a twin brother or you call it as you as you will or the yin and yang a brother

45:27of the synapse formation and I think when it comes to therapy it’s usually you know in many other aspects where we

45:34have made much more progress you know like Endocrinology Cardiology and so on it’s actually really crucial to know

45:40about the two aspects to see on which one it’s it we can work on so I don’t

45:45know to which extent the sargapped way will become let’s say a pharmacological Target to be specific but I think it’s

45:51certainly very important to understand that Singapore is not just on its own here but that is our Gap two ways next

45:57to it and on top of it that there is a very special type of Gene that’s only

46:03present in our species as sagap2c that you cannot study in the mouse and

46:09that I think justifies a lot of the experiments that we do that I think to understand fully what goes on in the

46:15human neurons at least in this disease we also need to look at human neurons in

46:20in various specific ways to to really make sure that we fully understand uh the the the picture to to go back to

46:27your second question so um about the plasticity and the and the repair

46:33so there is actually so I I showed you several times uh those human neurons

46:39that seem to be able to respond to visual stimulation in those mice now okay you may say okay this is very nice

46:47you know this is this is like a cool Gadget and and that’s it um well I would argue on the one hand I

46:54think it’s a very interesting tool to study human disease and I hope that I convinced you of that because we are

47:00able now to actually study human neurons deficient for Singapore in action and

47:05understand what’s going on so I think that’s that’s quite unique and it’s the early days of course

47:11um but there’s a second uh thought I think that may have come to you looking

47:16at this is that as I showed you that these human neurons take many months in the mouse to develop this it means that

47:24they develop this capacity to integrate in the visual circuit and become functional in Old adult my

47:33so that means that they’re able to work in that circuit after and long after it

47:39was established and so I think this has very interesting implications for the prospect of basically for instance

47:46rejuvenating cells or transplanting new cells in a disease brain and ask what’s

47:52going on no this may not be relevant for neurodevelopmental disease like Singapore and Patty but you can think

47:58for instance of neurodegenerative diseases stroke and other types of disease where Parkinson’s disease where

48:04as you know a lot of researchers and conditions are thinking of uh hoping to

48:12to basically slow down the course of disease by adding new types of neurons

48:18in a circuit that is losing neurons and and so this I think is very interesting

48:23because it shows us that juvenile neurons are able to integrate and become functional despite the fact they are in

48:29a circuit that is already functional and to my knowledge this is actually the first time this is clearly demonstrated

48:35like this on top of that I didn’t have time to show you but we have started now

48:40new types of experiments completely unpublished where we are testing now the

48:45plasticity of the transplanted neurons so basically the way we’ve been doing this is to look at what we call Visual

48:52plasticity so we change basically the visual stimulus in the mice and we ask

48:57other human neurons respond to this and interestingly what we find so far is

49:03that the human neurons display what I would call juvenile properties so they’re still very plastic despite the

49:10fact that they are in a mouse adult cortex that does not show this plasticity anymore because they’re

49:16adults so suggesting that indeed if

49:22um young neurons whether from transplantation or genetic manipulation or pharmacological manipulation are

49:28found in the context of an adult circuit it could actually lead to

49:34changes in the plasticity of of the circuit but again this is the early days

49:39and we’ll have to see here how it goes I hope that uh sort of answered in part

49:45your questions it does it did thank you it’s utterly fascinating so we have two questions in the Q a and I’ve allowed

49:52both people to speak Dr Courtney do you want to ask your question uh yes so I was really interested in

49:58those very large changes you showed in synaptic syngap levels after the knockdown uh is that also the case for

50:06the total level of syngap in the neurons or is it about the translocation of syngat one into the synapses yeah great

50:14question so we see these changes in synaptosone fractions these are the data that I showed you and we don’t see that

50:21in the total levels so we think that it’s likely to be linked more to the

50:27regulation of the synaptic levels and potentially in the translocation and you

50:33probably are you you know that that indeed Singaporean can change dramatically of sub cellular

50:38localization at the synapse or outside the synapse depending on plasticity paradigms for instance so I think it’s

50:45very likely that this toggle of War so to speak is really happening only at the level of the synapse and perhaps in

50:50response to activity and so that’s a very interesting question indeed and was the antibodies specific to any isoform

50:57or was it a pound finger yeah that’s another great question this one it was actually recognizing all of them and we

51:04should definitely look into the various size of forms you’re probably so obviously you you know about this so you

51:12probably noticed on the western blood that there are several bands so that would suggest actually that there’s more

51:18than one ISO form we’ve tried to look whether one would change more than another but I frankly

51:24we have no evidence for that uh I think we would need actually specific antibodies to address that that question

51:30yeah it could be phosphorus or something else and so on as well yes yeah yeah okay thank you yeah you’re welcome

51:44Mike you’re muted I was gonna I was just saying Dr Courtney works in Finland on and does

51:50some does some research on some Gap mutations so we’re very lucky to have him

51:55um Jr you had a question yeah thank you um what great research

52:01I’m I’m so interested in all this I have a sort of a question not exactly about

52:06the data you showed but I’m interested in your visual stimulus assay and why you chose that in particular

52:13um are so our sun Gap children seem to have a strong visual system

52:19so they have like a good visual memory they you know vision is really strong for them whereas like listening sort of

52:26audio input is a lot harder um and I was wondering if you tried

52:32other stimuli besides fish oil or if that is that just like something pretty easy to do is that why you did it

52:38or did you try other things that didn’t work as well I see yeah yeah so um yeah I was not

52:43aware actually of what you were saying about the stronger visual system that’s quite interesting

52:49um so no we chose basically the visual system I would say for very practical reasons that this is a a system that’s

52:58relatively easy to to to stimulate with the the mice and where

53:05we can actually stimulate the mice with complex patterns so it while for instance with somatosensory or auditory

53:11it would be much more complicated to do so it’s really mostly a practical reasons and for the same reason actually

53:16we have not checked other types of uh of of stimuli so that’s why we uh we chose

53:23uh the visual stimulus there there’s actually another reason which is that

53:29the human cortical neurons that we generate here from pluripotent stem cells they tend to so you know that

53:36there are many areas in the cortex visual auditory somatosensory and so on

53:41and each of them has a different molecular identity and the neurons that we generate in the dish they tend to be

53:47more visual like so they they tend to be basically more potentially at least more

53:54adapted to the visual cortex environment so that’s the other reason why we we uh

53:59we focus on visual stimulation that obviously it would be quite interesting

54:04to to look at others well since these neurons uh for our sun

54:10Gap patients seem to uh sort of grow up too soon I wonder if that’s

54:15I wonder if they’re visual uh you know abilities have to do with those are the

54:22neurons that develop first right so with those neurons develop well they were

54:27able to develop properly before they uh matured right whereas if

54:33uh yeah that’s an interesting idea yes another idea I didn’t have actually uh yeah I mean so the it’s in fact the

54:41prefrontal cortex so the very front of the of the cortex that is the last to develop

54:46and the visual cortex tends to develop at earlier stages so I guess that would

54:51sort of fit with your hypothesis um I think it goes back but I’m sure

54:57you’ve been studying I mean you’ve been talking about this together and that clinicians are uh studying this I think

55:04it it it it raises at least to me the question basically of the the precise

55:12properties of of the sensory uh abilities in uh in in singap one

55:20patients indeed I I think it really raises the question uh is there better

55:26or worse capacity in terms of

55:31responsiveness to particular types of stimuli and so on and basically how do the sensory system develop in general

55:39considering what I showed which is that some aspects may not develop at the

55:44right base and yet as I showed you at least at the particular time point where we looked at the capacity of individual

55:51neurons to respond to at least to discriminate different orientations and

55:56Direction was actually quite normal I mean actually completely normal so I think that there it is again more to

56:03understand uh and and where I think physiology and and psychophysic work uh

56:09with clinicians and patients would be very interesting to confront with the findings in the very artificial system

56:16that we’ve been looking at the question yeah thank you so much I

56:23have a second question that’s completely unrelated um so I loved your protein blood so it

56:28was just so beautiful uh with the and I noticed that the psd95 was staying at the same level the whole time right so

56:35yeah um so syngap was really going up and down and it had you know dramatically and uh

56:42and it had you had some Upstream effectors that changed that have you

56:48found other are you studying any other genes like is syngap going to kind of be

56:53uh like a bottleneck for other um maybe

56:59um Channel ION channel jeans or other you know other things that might be Upstream is that is this going to be a

57:06bottleneck where if you actually increased in gap you could change more than one um

57:11yeah that’s that’s another great question so as you as you probably uh so

57:18why we I think we were very lucky in a way that we looked at very few proteins

57:23at the synapse in singap one turned out to change that dramatically and uh in

57:29fact to be completely honest with you we were already but you probably guessed that looking at singap one in human

57:35cortical neurons and wondering you know what they could do with developmental timing and somehow this is why we we you

57:43know we had our attention let’s say triggered and that’s why we tested them

57:49in the indoor sagap2 context but then we got I think really lucky to see this dramatic result while as you notice many

57:55of the proteins don’t seem to change that’s it and I think that could be interesting as well there is work that

58:03is actually also uh published as a I mean a pre-printed on bioarchive that

58:08you may want to check from the group of so the the group I mentioned so she was

58:13a postdoc in frankfurlough’s lab she’s now at Ernest in Paris and what Cecil did first using Mouse

58:20work looking for interactors of sigap2 is on the one hand

58:27like us if you will but completely independently from another perspective she also found syngap 1 as the

58:34potentially interesting interactor and another one so that’s to answer your question you know what else could there

58:39be there at the synapse level that would be very important and the other very important protein very interesting

58:45protein that she found is encoded by uh well it’s called basically

58:50a katinin Delta II um so ctnn D2

58:57which when mutated on on one alley so Apple

59:02insufficiency of that Gene is actually responsible for this syndrome called credusha that leads to intellectual

59:09disability and another cognitive phenotypes it’s of course a different

59:15disease than the one caused by singaporeana sufficiency but it’s also a

59:21neurodevelopmental disease and a disease that the synapse and it’s very likely that we have not tested yet but that it

59:28could also as a result impair the neotany of of the human neuron and it

59:34will be very interesting in the future to see how much in the very simplistic model that I showed you with only three

59:41proteins as I get through CSI get 2acing Gap one whether other proteins like this

59:47one CTL and D2 actually work together or against to to find regulate basically

59:54the speed at which those synapses developed and and function but I think we are really only at the beginning here

1:00:01I think another very important experiment that we should try to do in the future but that’s difficult is to

1:00:07really do a less biased approach I would say where we would try to look at all

1:00:13the protein interactors of Singapore and srgap2 but in human synapses in cortical

1:00:19neurons in development so this has been done in the mouse but never to my knowledge in the human and given the

1:00:24results we got with the Saga 2C I think it would be very interesting and important to know if there are let’s say

1:00:31human specific parts of the interactome as we call it so the all the proteins interacting one

1:00:38another and that somehow regulated the synapses I think that that’s a very

1:00:43it’s a it’s it’s it’s a it’s a big technical challenge but it’s possible given that we have an experimental model

1:00:48and I think that’s something that we we should definitely try to do in the future

1:00:54wow well your um your experiments I I was amazed at how long some of them take with 18 months time and stuff so thank

1:01:01you very much for working hard on this we’re our kids obviously develop over many many years so sure I will I will

1:01:08definitely relay that you know to actually the people who do the real work in particular Batista who’s been really

1:01:13amazing to get this experience going uh they are indeed thank you for acknowledging that they are actually

1:01:19quite challenging technically yeah I think um so I just shared in the chat

1:01:25for for any of the parents who are on a link to your paper on bio archive I hope I got it right and I also shared a link

1:01:31to a paper that came out of Montreal last year where um I think a Spanish postdoc did some work

1:01:37on audio simulation which might just be interesting since that came up as far as the interactive and protein protein

1:01:44interactions I don’t know if you’re aware of Marcelo Coba but um Professor Koba is or Dr Koba is at USC and he does

1:01:53a lot of proteomics and has a number of um patient-derived cell lines in his lab so he might be someone to yeah I think

1:02:01he uh he actually got in touch with me and uh if I’m not wrong and I think we should definitely talk yes because he

1:02:08he’s got all those spaghetti grams with all the jeans that he thinks about yeah he’s constantly talking about genes I’ve never heard of

1:02:14um which is great it’s great yeah yeah of course please go ahead no no it was

1:02:21just mentioning but I mean it’s a so that’s the difficulty of the type of research we do right is that we try to

1:02:29go very much in depth in looking at one thing but but the price to pay for that is we cannot look at everything and so

1:02:36indeed as you saw it take it takes years basically to look at one type of neuron with one deficiency

1:02:42um and I think the challenge we have is that you know unlike let’s say experiments in flies that takes one week

1:02:48and where you can look at many genes at the same time and get a lot of information here we get actually information that I think is as close as

1:02:55it can be maybe to to normal and diseased human brain but at the same time it’s you know very low throughput

1:03:01and and very slow so I think it will be interesting in the future to look at different mutations to look at the

1:03:08various Azure forms also as somebody mentioned um but yeah we will we will need much

1:03:14more data I guess to to to to get into that but um I I wish we had even more actually to

1:03:21to more time to to be able to do it faster but it will be important in the future indeed because we’re only looking

1:03:27here at a very small part let’s say of of of of of of the picture

1:03:34one of the things I think about so when you know when we get diagnosed as families we don’t think of ourselves as

1:03:40in a in the genome we think of ourselves as genetic epilepsies and neurodevelopmental diseases

1:03:45and the big ones like is basically Dravet scn1a that’s an

1:03:51ion Channel and when I’m watching your your presentation and seeing how you think about okay this Gene this Gene

1:03:56actually they antagonize each other and it’s about balance I feel like the synaptop of these

1:04:02shank three singap one stxp1 are almost more complicated than the

1:04:08channelopathies because the channelopathy is like if you can just figure out the channel and maybe I’m making that up because it’s a bit

1:04:13self-serving but we’re actually the opposite of self-serving but the the balance in the in and the network and

1:04:19the interaction of all these genes over the life of a human is just so complicated and that’s that’s

1:04:25what you’re starting to untangle right yeah yeah yeah yeah I agree with you although uh

1:04:33I think general practice are quite complicated as well uh and uh that

1:04:38they’re actually a very fascinating Avenue of Investigation to understand

1:04:43the specificity of the human brain development and function because I mean

1:04:50the reason why I’m saying this is that you know the textbook view let’s say of Channel work is that you know this is

1:04:57really like the hardcore uh machine of the neuron so this is going to be the same in every neuron you look at and

1:05:03this is going to be the same in every species and so on right and it’s more defined tuning like at the synapse level

1:05:09and so on the things are going to be different um what we found actually recently well so what other groups have found recently

1:05:15actually is that when you do electrophysiological recordings of human cortical slices coming from surgery

1:05:22resection following epilepsy for instance this is the work uh uh for instance from the Allen brain Institute

1:05:28you’ll find actually interesting intrinsic differences in the properties of the neurons in the

1:05:34human versus other species suggesting actually changes in the channels and we

1:05:39recently came across another family of human specific genes that’s

1:05:45called lrc 37 that’s a another preprint actually that that that’s online that we

1:05:51found is a regulator of voltage sodium channels so like the you know the the

1:05:57most famous the most concerned of all the most important one that makes you

1:06:03fire action potential it’s actually regulated in human neurons in a different way than the other species

1:06:09because there’s this Gene rc37 that acts on them

1:06:14um again I think it’s Food For Thought to understand human neurodevelopmental diseases because what we take for

1:06:20granted as being purely conserved may actually turn out to to display um differences uh depending on the on

1:06:28the context of the of the species that that you’re looking at so I think in that sense unfortunately uh nothing is

1:06:36simple even even the channel no no nothing is simple but I mean for instance one of the problems we have right 80 of our diagnosed population

1:06:43have protein truncating variants yeah if you look at predictive models like I think it’s Lopez Riviera 2019 the

1:06:50predicted mutations you would expect to find are majority missense minority protein truncating yeah right and so why are we

1:06:58not getting more Mission patients diagnosed maybe the phenotype is so severe it’s lethal maybe it’s so mild

1:07:05they’re not presenting disease or maybe people have no idea what to do with those mutations and their and their the

1:07:11patients are getting identified tested and diagnosed with variants of Uncertain significance and therefore we’re not

1:07:16finding them because the patient the families aren’t being told which actually happens and so the question becomes how do we do an assay on all of

1:07:23these missense mutants that we found in humans but we don’t know if they’re disease

1:07:29costs are they indeed nulls and therefore creating heads or are they dominant negative in which case we

1:07:35shouldn’t give them up regulating medicines right so this is a this is the wall I’ve been banging my head against and and where ion channels I I totally

1:07:44agree with everything you said where ion channels feel simple to me is people say well if it was an ion Channel we just

1:07:50throw it in Excel do some electrophysiology we don’t we have an assay very nice I see what you mean yeah that’s very true we have no way of

1:07:57figuring it out so since you seem to think deeply about these things I just want to plant the seed yeah yeah I’m

1:08:02happening to think of an assay we can do to test different missense mutations please let us know yeah okay no no I got

1:08:09what you mean yeah no no I understand better that’s totally true yeah in fact and indeed with this Gene we’ve been

1:08:16studying it’s been it’s been fairly easy to to do molecular assays because it’s

1:08:22patching and so yeah it did it’s it’s it’s way easier than look at um I I

1:08:28think uh uh yeah I I I totally see what you mean and indeed

1:08:34well we’ll think more about it please do and uh but I fully agree with you and I

1:08:39mean you know since the beginning that we got interested in the in the in in Singapore and we were very struck by the

1:08:47complexity of the protein the fact that all these eyes are formed that seem to have different and sometimes completely

1:08:52opposite effects uh I think it it makes the interpretation of of the genetics uh

1:08:59much more complex and uh yeah if you ask me I think I think you know more people

1:09:04should study and so on and uh I hope that you know

1:09:10putting it in the map of human evolution if you will uh is going to attract also

1:09:16uh more people to understand more what is going on I mean I think the fact that it’s linked to a sagap2a was a real real

1:09:22surprise to us uh you know despite the fact that so many things are already unknown already known

1:09:29um so I think in addition let’s stay to the type of readout you were talking about so trying to have like higher

1:09:36throughput I think that that’s very true um but I think another thing that we need to understand better is within the

1:09:43synapses you know what are the molecular Partners the most critical ones of of

1:09:49Singapore engineering development and so on and there are new tools like bioid for instance proximity labeling uh that

1:09:57I think would be a very interesting targets for these types of proteins where you can basically uh I don’t know

1:10:05if you know this technology but basically labels the proteins that are not necessarily binding directly but

1:10:10rather in the neighborhood and that that can be purified on that basis I think this is a new powerful way to um

1:10:17understand how protein function because at the end how they function depends on

1:10:23what what what are the proteins that they interact with and so this is really a new way to look at it where I think uh

1:10:29for proteins I guess I got two in singap one probably a important discoveries

1:10:35could be made amazing Professor Dr Courtney has a question too uh

1:10:42coming in from Finland do you want to ask a question Dr Courtney yeah just related to the point by Mike you were

1:10:49making uh about being able to evaluate different uh misens mutants for example so uh yeah the the assay you were

1:10:57describing with the transplantation seems to as you said it’s a kind of cell autonomous Behavior Uh and the cells I don’t know

1:11:04if they’re interacting with one another or that you know there’s so many Mouse neurons there that there can’t be that much of a direct interaction between the

1:11:11different human neurons who transplanted maybe so does that mean you could run a kind of pooled screen for multiple

1:11:18different mutants at the same time in the same brain if you would label them a different fluorescent proteins for

1:11:23example you’d be able to instead of having to do one movement for every

1:11:29brain you could actually test 10 or 20 or 100 so is that at all feasible or is it I see what you mean yeah yeah so

1:11:36um yeah so um in principle it could be of course so to answer your question so we we have

1:11:42looked at that in the past and so we have indeed a lot of evidence that suggests that the main interactor

1:11:50synaptic interactors of each human neuron are Mouse neurons yeah and

1:11:56actually that’s what that’s what makes them so functional is that they are embedded in a fully developed circuit

1:12:01and so on so that that’s what makes them work in a very different way that if they were let’s say in an organoid or in

1:12:08a in a in a Petri dish where you would have all kinds of neurons uh that are

1:12:13quite immature interacting with one another while here they’re sort of helped if you will by the host now we

1:12:19have been trying actually to do a very simplified uh uh setup of what you suggest so

1:12:27basically taking normal uh control human neurons versus mutated

1:12:34uh appoints efficient neurons and Transplant them with a different fluorescent protein and so that can work

1:12:40the problem is that there are not so many neurons and so it you know you diminish basically the number of neurons

1:12:46that you can analyze and send so if you were doing it the way you suggest it you

1:12:52know with 10 different ones I’m afraid you may end up with only a handful of neurons on which you would need to have

1:12:59a readout uh from a screen so I think unfortunately uh I don’t I don’t think

1:13:04the the in that sense I don’t think that the the system that I described is a is

1:13:11a valid uh or let’s say um realistic way to to to to to to to to screen I think

1:13:18it would be on the other hand an interesting system and I think a valid system to

1:13:25confirm or test in depth a particular hit let’s say of a screen that would

1:13:32have occurred in vitro and in fact this is this is something we’re doing for other neurodevelopmental disease where

1:13:38we have collaboration with um some

1:13:43some groups basically involved in finding you know small molecules that

1:13:48that can change the outcome of particular phenotypes in vitro and where there we can use our model to look at

1:13:55whether these molecules have the same impact on human neurons in Vivo so as a

1:14:00sort of pre-translational test if you will but but I think to to to find new

1:14:06targets to find new hits um I think that would just not be feasible given the relatively low Trope

1:14:12throughput of the experiments and also of the number of neurons we have in each

1:14:18in each Mouse unfortunately yeah okay thanks a lot thank you very much thank you very much

1:14:25very much this was a fascinating presentation I’m grateful for you thank you thank you thank you for the

1:14:30invitation I really enjoyed it very much I hope this was clear enough for everyone and

1:14:36um yeah we’ll uh we’ll keep uh we’ll keep posted we keep each other in touch to see how things move on with with the

1:14:44field please do again we’re so grateful thank you for your time all right