84 – Zebrafish SYNGAP1 model hyperactivity may be explained by elevated arousal in low arousal environments

Julia Dallman, PhD

Dr. Dallman’s Bio

Julia Dallman is an Associate Professor of Biology at the University of Miami. Julia has thirty years of experience using animal models (sea squirt, fly, and zebrafish) to Understand neurodevelopmental processes. She did her doctoral training in electrophysiology and developmental neuroscience with William Moody at the University of Washington, and post-doctoral training in molecular and chromatin biology with Gail Mandel and in zebrafish genetics and physiology with Paul Brehm at Stony Brook University. Her research seeks to understand how genetic mutations impact the development of circuits in both gut and brain to produce behavioral phenotypes in zebrafish autism models. More recently, she has worked with clinicians, public health experts, and mobile application developers on objective measures of gut function that families can conduct at home.


0:07hello everyone and welcome to today’s webinar I’m Lauren Perry I’m operations

0:12manager at syab research fund and mom to a 13-year-old with singap one and I have

0:18the pleasure to introduce today’s speaker Dr Julia Dalman Dr Dalman is an

0:23associate professor of biology at the University of Miami she has 30 years of

0:28experience using animal models including sea squirt fly and zebra fish to

0:34understand neural developmental processes she did her doctoral training in electrophysiology and developmental

0:40Neuroscience with William Moody at the University of Washington and post-doctoral training in molecular and

0:46chromatin biology with Gail mandal and in zebra fish genetics and Physiology with Paul Bram at Stony Brook

0:54University her research seeks to understand how genetic mutations impact the development of circuits in both gut

1:02and brain to produce behavioral phenotypes and zebra fish and autism models more recently she’s worked with

1:08clinicians Public Health experts and mobile application developers on objective measures of gut function that

1:15families can conduct at home um a recorded version of this webinar will be available on our website under webinars

1:21on the family menu and at the end of Dr Do’s presentation you will have the opportunity to ask questions and we’d

1:28love to hear from you so please write your questions in the Q&A and it’s now my pleasure to turn things over to Dr

1:34Julia dolman thank you so much for the introduction and for inviting me to speak yeah I will open up the

1:43slides um I’m going to tell you today about a study that we just completed that looks at at um behaviors in our s

1:51Gap one model um zebrafish model and what we find is that we think that um

1:58elevated arousal in low arousal environments may explain some of the hyperactivity we see in this model and

2:05I’m speaking on behalf of the authors of this study so sereni Sumi Pala um this

2:11was her PhD dissertation Su hakan um Robert Kel Yaki sham Sayad and Richard

2:21henir so this is a picture of a synapse and as um you are all very likely aware

2:29many of the genes linked to neurodevelopmental disorders are enriched at this synapse and this

2:36includes syap and shank 3 both genes that are of

2:42great interest in my laboratory and that we’ve generated zebra fish models

2:47for both singap and shank 3 also share

2:52um strong behavioral manifestations so this is a um a paper from lean Warren at

2:59all 20 22 that shows scores on sensory and

3:04behavioral um symptoms where green is um

3:09where the Maj majority of uh typically developing responders score and uh s Gap

3:17and Fel mcdermid syndrome are out here to the right um and

3:22so these conditions are associated with altered sensory processing um and so

3:29that’s something that we can really study in detail in zebra fish so um that’s what we set out to

3:35do so the central question is how do syap one mutations impact nervous system development and

3:41Behavior Uh and what we’re kind of trying to understand is the suite of behaviors um that that manifest and and

3:49in humans with singap related non- syndromic intellectual disability uh some of the behaviors um

3:57include uh epilepsy that can be intractable hyperactivity and or

4:04hypoactivity disrupted sleep increased R risk-taking and sensory seeking so um

4:11descriptions of of really enjoying flowing water or perpetual motion um are

4:17some of the of the descriptions of people with s Gap that come from these

4:27Publications so um we zebra fish in part because uh they have pretty high Gene

4:33conservation with humans um you can do high throughput behavioral assays so

4:38these red lines are actually tracks of zebra fish where there’s one zebra fish larva per well of this 96 well plate you

4:46can see right into their GI traps um and

4:51um you know GI distress is also a common symptom in both sin Gap and um shank 3

5:01and um they have very accessible early development and it’s possible to do whole brain Imaging so these this

5:09collection of traits um makes them a really nice model in which to try to

5:14understand how symptoms arise symptoms phenotypes arise so um I’ll start off

5:21talking about what we’ve learned you know sort of validating the model that that we use um and so here is an a

5:30diagram of the singap protein from humans and it had there are um four

5:36conserved domains the plexin homology in cyan the C2 domain in pink The rasap

5:43Domain in in mustard and the Quil coil domain in Orange and you can see that in

5:49Mouse and zebra fish um these domains are

5:54conserved and you may notice that zebra fish have two sing gaps so there’s a s Gap 1A and a sing Gap 1B Gene um both

6:03with conserved domains and if we look at domain conservation um as percent of

6:09amino acid identity you can see the mouse has considerably higher conservation than the zebra fish and

6:16that um s Gap 1 a and 1B kind of alternate on Which is higher in which

6:21domain so in C2 B might be a little higher in the Quil coil domain a might

6:28be a little higher and so um so they both they both show high

6:36homology in melon s Gap it’s very well known that there are lots of different

6:41isoforms and this just this slide just um remind you of that showing the Four

6:48well-characterized C terminal isoforms so here you can see um that at the um

6:56five Prime end you’ve got three alternative SP spce uh start sites and at the C terminal end you’ve got four

7:03alternative C terminal ends um one of these encodes this

7:10uh alpha 1 protein that includes a pdz liand so pdz is um a a protein

7:20interaction domain that is characteristic of pdz 95 which scaffolds

7:26that glutamatergic post synaptic density and so this alpha 1 is the one that’s

7:31most very highly enriched at that postseptic density by its pdz interaction

7:37domain so when we were setting out to do these studies we wanted to understand how these isoforms related to these

7:44duplicates and so to do that we um collected all of the uh syap 1 a and 1B

7:52proteins from the ncbi database identified unique exons that they

7:58encoded and then then searched those exons against RNA databases to see which

8:04ones there was evidence that that those those forms were expressed and that led us to identify um

8:13several isoforms in syap 1A you can see that there are three alternative splice sites here there’s a very small

8:21alternative um splice that is less than three amino acids and and you see that

8:27type of splice also in humans and then these different C terminal ends and it gets much more complicated in s

8:34Gap 1B where there are 10 different isoforms with m a lot of differential

8:40splicing at the C terminal end and one of these isoforms encodes this pdz

8:45interacting domain so that um is interesting to us because it means these

8:51duplicates aren’t serving identical functions only syap 1B encodes this

8:57Alpha 1 I form so

9:04um knowing those things we set out to um create

9:10stoploss um mutations in the singap one gene to try to model um s Gap one

9:18related um intellectual disability and this is a a slide of different patient

9:27um variants from GL camp at all

9:322019 to do this we used crisper cast 9 and this is work that was done by Rob

9:37coal when he was in the lab um and we inject guide rnas that Target the syap

9:44Gene and create um stop codons in the

9:50the end terminal region of the protein we validate that these create

9:55loss of function both in laral zebra fish where we collect RNA and we look

10:01for the level of expression of the syap 1 a and syap 1B genes and you can see that in both heads and homozygotes those

10:08transcripts are downregulated indicating that there’s nonsense mediated Decay also um we sent some fish brains

10:17from homozygous and wild type fish to um

10:23yoichi araki in Richard huger’s lab and he ran uh Western with his is um they

10:30have a lot of different s Gap one antibodies and you can see that the wild type Ze fish have a band of the same

10:37size that’s found in Mouse and the mutants lack expression of

10:44that band even though they have lots of tubulin which is the internal loading

10:50control so that means that we’ve created a loss of function um model in both s

10:56Gap 1 A and S Gap 1 B and unlike Mouse are the the homozygous model um is

11:04viable so so we can get adults that are um lacking both s Gap 1 A and S Gap 1

11:11B and so to study behavior one of the things that’s that’s um really well uh

11:19documented in zebra fish are these arousal States so I wanted to talk a little bit about arousal before jumping

11:26into the data and I think one of the um clearest demonstrations of changing

11:31arousal States during the day is is our cycles of being awake and asleep same

11:38nervous system but very different behaviors depending on the time of day and this um gets much more granular I

11:45think we all know that that we um need a boost at certain times of day or we get sleepy at certain times of day um highs

11:54and lows and these are regulated by these arousal Pathways that are conserved between humans and fish and

12:00include noradrenaline serotonin histamine hypocretin which is also known as ereen and dopamine among many many

12:09others and so these fine tune some of our outputs um arousal is associated

12:15with increased Locomotion and so Locomotion is one of the ways of of um

12:21looking at arousal state so here what we um have taken

12:28advantage of is different environments that evoke different states of arousal so in the light the zebra fish are very

12:35visual animals and they’re um fairly relaxed and they don’t move a ton so

12:41each dot represents um a set of five larv and so this is graphing the

12:47proportion of a of many many larv that move in the light and you can see um

12:53that it’s nowhere close to 100% if you move those larv to the dark there’s this

12:59dark response where movement increases dramatically and that’s associated with

13:05increased arousal it turns out that um just as as with us if if you take away

13:12our sense of of vision um you you go and look for a place where you can turn on

13:18the lights again the fish do the same thing and then another thing that increases arousal is vibration or tap um

13:27also known as the acoustic start response so you can see that um the fish

13:32also increase Locomotion a lot to this vibrational stimulus and so dark and tap are both

13:40states that are associated with elevated arousal um but they’re a little bit

13:45different and you can tell that if you try to anesthetize these behaviors so these are increasing this

13:53is the the amount sorry amount of anesthesia required to stop movement to

14:00light in light conditions in dark conditions and during tap and you can see that tap takes a lot more anesthesia

14:08to suppress that movement in part because it’s an aversive stimulus it’s

14:13it’s something unpleasant and so it takes more anesthesia to suppress that than either of the behaviors that are

14:19going on in light and dark and so you can um rank level of arousal from light

14:28being low level to Dark Being higher to tap being aversive and and and very

14:37high so to look at so we started with high we started with the tap and to to

14:43measure the tap response we put the fish in this um Nest Dano Vision chamber um

14:50that has a a tap device I put a hammer here to represent it um that can deliver

14:58different strength strengths of vibration um which is uh can elicit the

15:04acoustic startle response and then we load this chamber with Offspring from

15:09Wild type and syap homozygous um wild type females and homozygous singap males um and load this

15:18dish both with wild type and singap hats and then see how they respond to this

15:24this tap and we do um a so this this is a

15:31behavioral assay um that was developed by Kurt Marsden which is where sereni is now doing a postto and it involves 10

15:39low intensity Taps followed by 10 high-intensity Taps all both of these are separated by

15:4710 uh sorry 20 seconds um and then it goes into a phase of Rapid high

15:54intensity TAPS in Rapid succession so only a second between each tap and this allows us to measure how well the

16:01nervous system adapts to this stimulus or um otherwise known as short-term

16:08habituation then there’s a recovery phase and then um another set of

16:13high-intensity taps with a 20 second interval and so what you can see is um

16:20here is in the black line is the wild type response and in the purple line is the singap Hat response and basically to

16:28tap um there’s no significant difference across the whole behavioral assay um the

16:35both lines have less of a move move um

16:41slower in response to these uh lower intensity Taps move further in response

16:48to the high intensity Taps habituate with repeated highfrequency stimuli and

16:54then come back to this Baseline level after a rest um and so if we calculate a habituation

17:02index which is basically this movement minus this movement you can see there’s

17:07no difference between um while type and syap heads so this is the the um habituation

17:20index we looked a little bit um closer at uh the difference between the

17:27different phases um depending on genotype and you can see that this low intensity tap in Phase One

17:35and high-intensity TAPS in phases two and five the response of wild type is

17:41not significantly different across these different intensity Taps but singap has

17:46another gear um so it’s at a lower response um to the low intensity Taps

17:53and then has a higher response significantly higher um or faster response to the high intensity

18:00Taps if we look at percentage of response we also see that s Gap is a

18:05little bit more responsive than wild type um and that sin Gap increases its

18:12proportion that are responding as the Taps get

18:20stronger so that is so so um in terms of our Rive stimulus our tap stimulus there

18:28isn’t really a dramatic difference um in between s Gap and its wild type siblings

18:35um other than a a increase in dynamic range so so um so what about these light

18:44and dark behaviors we also measure the light and dark behaviors using this nus

18:50chamber and we do four Cycles so first we adapt the fish to dark and then we do

18:58four um cycles of lights on to lights off and um and the reason we do this is

19:06that it evokes a beautiful um visual motor response so

19:12here you can see that response um what’s plotted here is um the speed of movement

19:19so millimeters per 30 seconds during these five minutes of light followed by

19:24five minutes of dark and you can see that in the light the speed of movement is low um relative to the dark and that

19:33it’s a very robust response to dark so um you can also see so graph are um wild

19:42type response and then again hats are in purple and homozygotes are in Pink So

19:49during the light um the homozygotes are most most hyper most active and during

19:56the dark the hets are are most most active and if oops and if we um plot

20:02these numbers across individuals you can see that in light there’s a dose

20:09dependent um increase in hyperactivity um as you go from Wild type to het to

20:18homozygote in the dark uh there’s a much um subtler difference with the hets

20:26being most most different from the wild

20:31type but we really wanted to know so this this level of analysis is pretty

20:38coarse um because this repres five minutes

20:43represents a lot of bouts of activity for a zebra fish and so we wanted to

20:49know um whether this observed hyperactivity may be due to more

20:55frequent movements which would indicate descending commands from the brain or

21:02increased movement per bout which could be increased recruitment of motor

21:09outputs and so what we did was we generated an idealized larvae and the way we did this is we had a very um

21:18large n so sereni collected data on a lot of animals and we we um if you if

21:26you collect if you sort of pull um the the um distribution of

21:34displacements the the size of each bout in Wild type wild type had we we had a

21:40total of 11 19,1 35 bouts across all over our 173

21:47larvey um and by plotting these bout distributions and

21:53dividing by the number of larv we get behavior for an idealized larva normally

21:59behavior is so noisy that you don’t get these smooth curves you only do this if you do this this um in a lot of numbers

22:07so that you have representation even in in these large movements that where there aren’t very many of those

22:13movements and so what you see here is that the S Gap are moving much

22:19more frequently in the light um so on the y- axis is the number of events per

22:25size of displacement and

22:31uh the you know here wild type are at 500 singap hets and homozygotes are at a

22:38thousand um for the smallest displacement size and in lights off the

22:43differences are much more subtle um you can see as indicated by the data on the

22:49previous slide hets are most have the largest number of bouts followed by homozygotes and then wild

22:56type so in terms of this question we definitely have more frequent

23:03movements but seeing that Stark difference between the the um s Gap

23:10behavior in the light we wanted to know a little bit more about what was going on there and so um

23:20so what’s happening in light versus dark and this is data from um um from AIC

23:27htics horick from when he was in Harry burgess’s lab and they showed that this

23:33hyperactivity in dark is due to the fish trying to return to the light um in

23:40other words the activity in the dark is produced by light seeking

23:45Behavior so what does our light Behavior look like compared to our dark Behavior

23:51well this is the probability of a behavior that has of of

23:57a particular dwell time which means the time in between different bouts of behavior and you can see in the

24:04dark um there are lots of short dwell times meaning they don’t sit still for very long they’re mostly on the Move

24:12whereas in the light there are more of these long dwell times they’re they’re more um they spend

24:20more of their time not moving and you can see this um also if you subtract

24:26these probabilities from each other so it’s dark minus light so when you’re

24:31in positive um dark is you have more um of

24:38these short dwell times in dark more of these long dwell times in in light if

24:43you look at displacement the size of displacement in light you have lots of short displacements so you have short

24:50displacements and long dwell times and in dark you have um much larger

24:59displacements so the nice thing about collecting these statistics about

25:04movement characteristics in light and dark is it lets us interpret what we’re seeing in

25:10syap and so here’s the sying Gap data so in light um we have the wild type in the

25:19in the black circles and S Gap in the purple and pink and you can see that if

25:25you um subtract singap from wild type you see a pattern that looks very much

25:30like when you subtract um dark light from dark so s Gap in the light are

25:39behaving like wild type in the dark they’re producing these kind of seeking behaviors that have um shorter dwell

25:48times and larger displacements whereas in the dark there

25:53really um aren’t differences that that go along with the dose of of the syap

26:03gene so in addition to more frequent movements we have increased movement

26:10size in the light that we’re seeing from singap and so it it seems that singap

26:17are participating in the sensory seeking in the

26:22light um and so what you know what the way we’re

26:28interpreting our data are that we’re seeing these high arousal behaviors in low arousal environments sorry environments is is

26:36twice there so um here we have our um

26:43arousal uh wedge and syap hyperactivity is most pronounced where arousal is

26:51lowest so what we find is that in in response to these aversive stimuli we

26:57see a larger dynamic range in syap but not um not a very pronounced

27:04hyperactivity during the dark transition we see a greater frequency of movements but not in a way that depends on syap

27:11het or homozygous status and in light we see pronounced hyperactivity where singap are moving

27:18like wild type do in the dark and the effect is greatest in the

27:23homozygotes so what what might this mean well

27:28um looking at some of the literature on other things that affect that produce

27:35these types of symptoms um or phenotypes elevated cortisol or stress

27:41hormone can produce a larger dynamic range but it also produces freezing in

27:47the light so we don’t think anything is going on with with elevated

27:52cortisol our data are consistent with increased go directed seeking behavior

27:59in the light um it’s been shown that increasing these

28:07arousal peptides cgrp or cck can produce increased activity in the light another

28:16um arousal pathway cocaine amphetamine related transcript um can produce a

28:21larger dynamic range this peptide hormone hypocretin and ereen can both

28:27produce higher dynamic range and um and increase behavior in light and

28:35serotonin is also associated with this increased

28:41seeking but these are only a subset of um the arousal Pathways that

28:50are known to influence transitions between different behavioral States so

28:55here are some others and this also is not a complete

29:01list so our central question was how do s Gap 1 mutations impact nervous system development and behavior and our data

29:08really point to a role for arousal Pathways but I think the outstanding

29:13questions um persist in that we have this picture of a post synaptic density

29:20that would be found throughout the brain um so how is it that we have um so

29:27there’s some circuits that seem quite significantly impacted and others that that seem to be working um fairly well

29:35and so what makes some circuits vulnerable to reduced copies of sin Gap

29:40While others are not is still uh an open question and one that that we’re interested in in

29:48addressing so I want to acknowledge the people who contributed to this work uh Dr sureni sumala got her dissertation

29:56working on syap and and and singap really inspired her to continue um

30:02working as a scientist so she’s now pursuing a postto at North Carolina State University with Kurt

30:09maren uh this work couldn’t have been done without Ricardo sepa who keeps our zebra fish in good shape suak KH has

30:16moved on to um Ellen Hoffman’s Lab at Yale uh Robert kosel is now seeking a

30:21faculty position sham sad is um from um physics and he’s the one who talked to

30:27into um analyzing all those behaviors those 119,000 behaviors so individual

30:33bouts um icki did the the Western in Richard huganir lab and we thank them

30:38for their support and then this work was funded by the um grants from National

30:44Institute of Mental Health and National Institute for child health and human development and a safari pilot award and

30:51um institutional support from University of Miami and John’s Hopkins and with that thank you for your attention and I

30:58will stop sharing that was awesome

31:05um so for the there I know who’s watching from looking at Facebook and

31:10from this and for the for the newly diagnosed parents you just now you understand why we get so excited about

31:16models because we just saw what happens when you can go from Models to results to interpretations so what’s for the

31:22kids and I think that’s important I have I have some technical questions and I have some translational questions and

31:29then I’m I’m happy to take questions from the audience and I don’t know Jr if you’re on the call but I’m sure you

31:34probably have questions because you did my you did fish once but so translational

31:42serotonin anything you can expand on there because there are both sort of

31:48within the singap community a lot of people have had results both with gut motility and

31:53mood HTP and then within the broader epilepsy community I think I was just at

31:58an epilepsy meeting talking to the CSO of the D syndrome foundation and there’s four different serotonin drugs right now

32:05in trials for d one of which is with longboard which is um open by the way to all patients with n Dees so serotonin is

32:13something everybody sort of perks up when you say that and I and I was hoping before I go into a bunch of hopefully

32:19geekier questions you could elaborate on what what this tells us about serotonin

32:24and how we would think about it yeah so um serotonin has a really interesting

32:30tradeoff with cortisol in a geeky way in that at least in zebra fish in zebra fish um serotonin

32:39is associated with this sensory seeking Behavior where like they look for

32:44flowing water and the serotonergic rafy are important for that um and in the case of of so so how

32:55that relates to it being an anti-seizure medication um do you know which

33:00receptors they’re targeting for the anti-seizure medication B and it’s different there’s there there’s

33:06different drugs going after I mean I think the longboard is the C but I’m not

33:11looking at it strictly through the lens of seizure like I mean I what when those of us who are in the community were giving our kids five HTP religiously

33:19we’re doing it to affect to improve motility and to improve mood yeah yeah

33:25so I mean it’s a nice to right because because um it’s you know it’s linked to

33:32these dorsal Rafi which are really important for for kind of engaging with your

33:37surroundings in a way that is less fearful let’s say so that’s in the brain

33:44right and then in the gut serotonin most 90% of the serotonin in your body is in

33:51your gut and is important both for communicating with the brain

33:57and for for keeping the gut moving so it’s not surprising that

34:03serotonin is um is something that people

34:08are looking to so I’m taking you a little off topic here because this is about arousal not about gut motility but

34:14I just want to double click on that I have quoted you many times to people being like 90% of our seratonin is in

34:20our gut yeah like one of my favorite sound bites but if so and then it we give kids

34:275 HTP some gut symptoms get better mood gets better so we’re replacing serotonin

34:33so does so but then if I think about it a deeper our our singap head our human

34:40singap hats and for the people on the who are new to science speak that’ss means you have one good copy one bad

34:46copy and Homo zgat have two bad copies which are viable means they’re they can live in fish but not in human so the

34:54humans are all heads right so if 5htp helps the human heads then does

35:02that mean that singap makes serotonin and that’s why they’re deficient or that singap pets are desperately using up all

35:09their serotonin and therefore they’re deficient and therefore supplementation is helping like what sense don’t know

35:16and the thing is I mean 5hdp is the precursor for serotonin but

35:22it’s it’s it’s tryptophan right I mean it’s it’s the building blocks yeah

35:29you’re getting the building blocks for serotonin so it could be building blocks go into other Pathways as well as

35:36serotonin so I I’m sorry to say this but we having a beneficial effect from 5htp

35:44and tryptophan um could be through through a bunch of different Pathways and it’s

35:49something that we’re interested in for sure because because of our gut work and

35:55and also because we’re really interested in in that gut brain

36:01axis but um but you just you just don’t know that’s a really good point which

36:08arm yeah no I’m glad you said that we don’t know there are some plausible hypotheses there is a connection to

36:14serotonin because it’s the precursor but it also affects other things okay better answer cool also I mean like the the the

36:22bacteria break it down and make indle and talk to the talk to the gut that way

36:29and can induce peristalsis that way so that’s an independent route to increased

36:34movement in the gut that could be via the the microbes HTP 5 HTTP plus bacteria equals

36:41indol equals gut works so have nothing to do with serotonin could could bypass

36:47serotonin whatever it works that’s all I know that’s right no I’m I’m just saying we don’t know yeah no it’s it’s fair

36:53it’s fair I’ll stop saying okay I get it so can we go back to the fish now so I I just as I was sharing with you before

36:59the the webinar I just came from a meeting where a lot of people were talking about fish and

37:06um the another lab that’s done a lot of work with fish has decided that the 1B variant is is the best model for singap

37:14y whereas all your slides had AB hats and ab homozygotes so does that mean

37:19that in the hats you knocked out one copy of a one copy of b y the home exet you knocked out both copies of both y

37:27so you’re basic so the is is is the thinking behind having a hat that’s got one and one is that these two have

37:32always worked together and as you pointed out beautifully they have different isopor so basically just mash them together and yep okay treat them is

37:39one because the idea is that they’re subdividing the job that that one does in us which is validated by that slide

37:47you showed with the different isop forms how they validated by that and and I the in data I didn’t show yeah um it is nuts

37:55how um it turns out you have to have them in balanced

38:01numbers or you don’t get

38:08Survival so if B if B alals outnumber a alals yeah you don’t recover as many if

38:15you do a hetet incross right I mean that’s kind of nitty-gritty but but

38:21there’s there’s an interaction between those two and I there’s data from Mouse as well if you just knock out alpha 1

38:30that’s a really hard hit for syap like that’s worse than knocking out

38:35the whole Gene and I think our data are consistent it’s a different it’s a

38:41different hit if that makes sense so that yeah yeah it makes sense and it takes me in two different directions and

38:47then we’ll come back to the title of your talk the first Direction it takes me in

38:53is if you were doing a drug screen with zebra fish based on what you know and

38:59you know a lot about singap Z you probably know more about singap zra fish than anyone

39:05what what would model would you use would you use an AB hat yeah okay and then I want to double

39:13click on what you said about alpha one because for the and for the audience we like where is Mike going with this there

39:19are different therapeutic approaches for singap that people are talking about at the genetic level one of them is a

39:25something you have a good copy of bad copy one of them is ways to make the good copy work harder and make more the

39:30isopor problem goes away there because the body decides which isopor the good copy will turn into other people are

39:36talking about replacing a specific isopor and some of these arguments are well look we know alpha 1 does a lot so

39:42let’s just throw an alpha one and and some people are like well you don’t know what the other 10 are doing that’s where

39:49I sit that could screw things up and so maybe we should think harder about that now if I think about what you just said

39:55that sort of validates this be careful picking a horse and so you know what the other horses are doing argument right

40:01yeah interesting very much so and then you could also take what you

40:08said and spin and argue the other way right you said if we take out Alpha One it’s worse than taking out the whole Gene that’s right could say well maybe

40:14the converse is true maybe we just put in Alpha One we’re fine you could argue that way but but I’m telling you the

40:20balance is important yeah the the survival data is very they’re very clear and and honestly

40:30um you know syap 1B mutants clearly survive fine if that’s the only horse in

40:36the game if that’s the only fish in the game it’s fine but if there are other other genotypes vying for

40:44resources with that syap 1B singap 1B homozygotes like we didn’t recover any

40:51from that hitting cross two H we and we had like an n of 131 right and none no

40:59syap 1B homozygotes with syap 1 a wild type we’re viable we’re

41:06viable so so it’s just um it yeah it that’s one of the reasons

41:14that we think and and you you know I mean I I

41:20actually haven’t looked at it seems like a lot of the the um patient mutation are

41:26are happening in the middle of the gene the part that isn’t alternatively spliced right so that’s going to affect

41:34all isoforms and there’s at least two then and and if you’re going to go down that

41:41rabbit hole like it said in vas camp and we’ve seen it in the patients people with mutations in the first four exons

41:47don’t seem to be as they’re disabled for life but they’re not as severe right it’s yeah step function similarly

41:54there’s this other mutation at the end uh see in the intron where um in another

42:00model it is a hat but the balance of isoforms is different and the human

42:06version of that at least the younger ones have another step function increase in function right so there there’s a lot

42:13going on with the splicing but you’re right anyone in the Middle with a with a protein truncating variant in the middle

42:18is probably aead and and and what’s weird about in the clinical world we used to get diagnosed post seizure right

42:25yeah so our kids had seizures now thank God doctors are seeing delayed kids in

42:31sequencing and so I have the unfortunate role with I’ve been on the phone with uh parents of ones and two year olds and

42:40Mom is holding this beautiful little child where the phenotype isn’t very obvious yet saying well maybe we’re GNA get lucky and I’m looking at the genetic

42:46report and I’m like I hate to say this but I doubt it like you need to

42:53know what an obson seizure looks like what in Mya CL Chic seizure looks like and be ready because got a protein truncating variant

43:00right in the middle of Gene it’s coming baby and the sooner you identify it treat it the better so that’s the other

43:08thing that is um you know to bring it back to arousal

43:15yeah please um this you know Sullivan kadam there are papers that

43:22have looked at seizure and those seizures tend to happen happen at State transitions you know between REM and

43:30non-rem and between wake and sleep so and that’s those are those are

43:37arousal transitions hi this is John hi John tends to demand

43:46my attention sometimes I’m very familiar with the with the Sullivan paper and John you’re embarrassing yourself on video and

43:54um that’s a really good point that’s I thought of it I’ve always talked about that paper in terms of wake sleep and

44:00sleep wake but you’re talking about it now in arousal States and I thought the two things you had on that slide on that

44:06presentation that stuck with me were the two wedges so with low with

44:12low low sensory input they’re seeking it more right y that makes sense so it

44:19suggests they need a lot but then if you go to the tapping when you give them too much they tend to get tweaked out well

44:25there’s just not there’s just the there the the difference between with wild type is much less pronounced it’s just

44:32not pronounced yes understood and John be

44:39chill and then the other other thing you said was it I liked it I liked it when you connected the curve between singap

44:46minus wild type to light to dark minus light right that was like a okay that

44:53makes sense so and that’s the title of the and that’s y John um apparently ate something bad

45:00yesterday threw up so school won’t let him come he’s completely fine he’s making me nuts but I can’t send her to

45:05school right now so that’s John is with us in case anyone was wondering

45:12um so what but what can you can so like is the takeaway light dark

45:21minus dark minus light equals singap minus head means what singap minus wild

45:28type it means that singap is is so in dark the fish are seeking

45:35light so what we think it suggests is that in light singap are still seeking

45:42they’re still doing this sort of sensory seeking Behavior even though they’re in in a context where normally that would

45:51have fish just being chill and not doing much so they behaving as if they’re in

45:56dark which and behaving as if they’re in dark is behaving like you’re seeking sensory input you’re seeking you’re

46:03seeking you’re searching for something I think we should study zebra fish siblings of scapens and see if they

46:09learn to create more stimulation as a result is that what happens yeah I think that might be a viable hypothesis but

46:17but does it does it suggest that okay here’s my last question does

46:22it suggest that more stimulation will satisfy them or does it just suggest that they are insatiable that they

46:29cannot process that sensory input without some kind of chemical Aid that’s

46:35a good question and and we we need to look at

46:42that it’s so so the data make it look like the hyperactivity is most

46:49pronounced in the first in the first period so they’re doing this seeking seeking seeking seeking but you repeat

46:56you repeat you repeat and finally it goes down so we need to look at that a little

47:02bit better and and what we’d like to do I mean you know when you asked what we’d

47:09like to do is test a bunch of the seizure meds and a bunch of the gut meds

47:16against this range of phenotypes so gut and arousal

47:22and um and see how whether there there’s something that does do what the

47:28serotonin Pathways have done on a on a sort of Case by case basis that can


47:40both just bribed John’s complicity for just one more minute then I only but um

47:46so so two thoughts on that the first is we are we have done a drug screen with Clement Chow in flies

47:54that data is publicly available and we’re happy to share it with you if you’re thinking about drug screens sure

47:59also doing a drug screen with rare base where um that is not that is protected

48:04by various agreements but there are there is a short list of molecules and a couple of classes there uhuh but might

48:12be um might be worth testing in a model that we can’t talk about publicly but we

48:18probably talk about privately so if you’re heading in that direction it would be really interesting

48:25to to look at those I think um I love you I

48:31love you too John because it it um I mean and I’m going off topic and

48:39then I want to defer and then last call to the audience I do see we have um Professor Courtney in the audience Mulia

48:45and and Mara and others so if anyone’s got a a comment please ask it or text me

48:51but I’m glad to hear you’re thinking about testing drugs against phenotype in fish and you know I always say this

48:59please call us if we can help support that work because

49:05um I think it I think it’s really important and and what I what I’m starting to appreciate when I started

49:10srf is we need a genetic therapy and now you know guess what we’re blessed we have companies publicly and privately

49:17working on really cool technology yeah that’s awesome but we’re cursed in that the FDA is still figuring out how to

49:23approve this stuff and companies are still how to reply and it’s it’s going to happen but it’s going to take too

49:29long so my my passion and focus on repurpose drugs is is only grows with time and I’m

49:38I’m really I’m really excited to talk to you about um anything we can do to

49:43validate yeah no I think I’m very excited about it too just in just I mean like you talked to so I am right down

49:51the street from Andre Jimenez Gomez and you know so talking about the

49:57seizure meds and the pathways they work through it’s really like it’s all over

50:03the map right and and having a way to

50:08empirically test them against the idiosyncrasies of a genetic condition I

50:14think is really helpful so so I’d love I will look up The Clement Chow

50:19stuff and and um I’d love to get your your

50:25take on those things because we are now now that we’ve now that we’ve looked at

50:30a number of different outcomes right we’ve got ways of looking at the gut we’ve got ways of looking at these

50:36arousal Pathways I think we’re ready to screen we can interpret what we get if

50:42that makes sense no it it does make sense and Chow will be talking at our meeting so if

50:48there’s any way you can make it to Orlando that day it would be great he he will be there person thank you so much for doing this