45.f – 3rd Annual Synapse Roundtable, Sensorimotor integration in gut and brain of zebrafish Syngap1 and Shank3 models

Event Time

December 3, 2021

THIS IS FOR TRANSCRIPT ONLY:

0:10good morning everybody thank you for

0:12coming to the third annual synapse

0:13roundtable my name is michael gralia i

0:15am the managing director of the singap

0:17research fund and it is such a thrill to

0:20welcome our incredible speaker here

0:22today

0:23and over to dr dalman who is a

0:25neurologist a neuroscientist i should

0:27say at the university of miami and like

0:30every all of our speakers today dr

0:32dahlman is working on a couple of our

0:34genes so um in the interest of time i

0:36will let you take it away thank you so

0:38much for being here

0:39thank you so much for inviting me to

0:41participate in this round table and i

0:44was really

0:46i’m i’m glad i’m following

0:48terry jo i i use animal models um but

0:52we’re trying to do this work

0:55um in the context of also

0:58being cognizant of natural history and

1:01um and so

1:03i hope to

1:05tell you a little bit about our

1:06integrated approach

1:08um

1:09looking we’ve started with

1:11one and shank three models of

1:15zebrafish models okay so here’s a

1:18quick rundown of what i’m gonna go

1:21through i’m gonna give you a brief

1:22introduction

1:24talk about zebrafish models talk about

1:27integrating the clinical and basic

1:29studies and then give a little summary

1:31in future directions snapshot

1:34so

1:35when um as someone who works in an

1:37animal model and not even a rodent model

1:39a zebrafish model

1:41we focus on

1:43symptoms that we feel like we can make

1:47mechanistic insights on and where the

1:49mechanistic insights might

1:52inform treatment strategies and so the

1:55two that we’ve focused on are altered

1:57sensory processing and gastrointestinal

2:00distress

2:01both

2:02i’d say symptoms that that we know a

2:04little less about

2:07uh and then

2:09one of the things that that i’ve learned

2:12in my foray into this field is that

2:15the gut itself um is an organ that

2:19functions through

2:20sensing information

2:24nutrients in the lumen and producing a

2:27behavior

2:28peristaltic movements to aid in

2:30digestion

2:31so this this idea of sensory processing

2:34being an

2:36important um symptom that has big impact

2:39on quality of life

2:41this altered sensory processing could

2:43play out in the gut as well

2:45and as all of you know the speed of

2:51identification

2:53of genetic forms of neurodevelopmental

2:55disorders has been very rapid

2:58and so

3:00this is just a slide from sadder stream

3:02at all 2020 that shows

3:05some of the

3:07single genes of large effect that have

3:09been linked to neurodevelopmental

3:11symptoms

3:13and i’m circling shank 3 and syngap

3:17i think

3:18i’ve i’ve put in the the year in which

3:22um

3:23variants in those genes were linked to

3:27um

3:28neurological conditions in humans and

3:32you can see that singap was linked much

3:35more recently than shank 3 and as such

3:38the natural history that’s known for

3:41different conditions is very

3:43different just in part based on when

3:45that linkage occurred

3:48okay so my lab focuses on on

3:51the sensory motor

3:54symptoms

3:55and

3:56in particular we’ve we’ve taken a deep

3:59dive into gi symptoms in part because so

4:01little is known about

4:03how those symptoms arise

4:05and actually um in attending felon

4:08mcdermott family foundation meeting and

4:10talking to families these gi symptoms

4:13were

4:14symptoms of a big concern

4:17and so

4:19so that’s how we got into looking at

4:21these um and

4:23so this this circle here are molecularly

4:27defined forms of asd with documented gi

4:29distress and

4:31um this is an incomplete list uh i

4:35before

4:37agreeing or not before the meeting this

4:39morning i looked at stx bp1 and they’re

4:42also gi symptoms described

4:46in in that

4:49condition even though it’s not listed on

4:51this this set

4:53of these

4:55genetic forms of neurodevelopmental

4:59disorders

5:00six have

5:01in animal models

5:04of six of these forms have shown that

5:06there’s reduced motility reduced

5:09movement in the gi tract

5:11and

5:12that movement is critical to

5:15digestion and gut function

5:18because it’s part of a reflex that when

5:21the gut senses nutrients

5:24it induces these contractions it induces

5:26mucus secretion and and um

5:29and hormone secretion that aids in

5:32digestion coordinates with the brain

5:34coordinates behavior

5:36so the one of the hypotheses that we’re

5:40interested in testing is that this

5:41reduced motility is a common aspect of

5:43asd linked gi distress

5:46and then um you can see that three of

5:49these animal models are zebrafish models

5:51i’ll show you why zebrafish

5:54are

5:55useful for looking at gi movement

5:58but there are a lot of

6:01genetic models in zebrafish that haven’t

6:03been examined for gi motility

6:07so zebrafish are

6:10a good model for this because by six

6:13days after fertilization

6:15they have these relatively translucent

6:18larvae that are eating on their own um

6:22and

6:23you can actually watch this is a movie

6:26of a larva that’s been fed and the food

6:29includes six micron beads so you can

6:32actually watch

6:33these peristaltic the part of the reflex

6:36that i was talking about um

6:39in vivo

6:41and that makes it quite

6:43um straightforward to quantify what’s

6:45going on

6:46different from us right where digestion

6:49is happening inside and it’s not visible

6:53in addition we’ve been looking at um the

6:57response of the nervous system to

6:59external stimuli like light um and and

7:03as i said we’re starting to think about

7:05this sensory motor integration both at

7:07levels of gut

7:08and brain

7:11okay so what have we found um well so

7:15this is

7:16again these peristaltic movements uh um

7:20that our response to feeding in wild

7:22type

7:23this is our shank three model and

7:25believe it or not the movie is going so

7:27there’s a little twitch that happens

7:29here every now and then

7:31and this is a fed syngap

7:33model larva you can see that there’s

7:36actually quite a bit of motility in the

7:39gut but it’s not coordinated from a ross

7:43anterior to posterior

7:45direction

7:47um and so we can quantify this this um

7:51behavior um and we can show that so for

7:54instance

7:55in um

7:57in wild type there’s a particular rhythm

8:00that is also seen in singap but is

8:02slower in shank three

8:04uh

8:05and so

8:06this

8:07echoes um

8:09this need to kind of understand

8:14these studies suggest that so yes both

8:17are showing

8:18gi

8:19symptoms

8:21both

8:22genes seem linked to these gi symptoms

8:24but

8:25manifest in slightly different ways that

8:27that

8:29that are important to understand

8:32so the other test that we do in the

8:34zebrafish is whole gut transit and what

8:37happens when you in in both shank 3 and

8:41syngap larvae so what’s happening here

8:43is we fed them beads in wild type and in

8:45wild type what happens is the beads are

8:47consumed and then make

8:49their anterior posterior

8:51um

8:53through the gi tract to be expelled

8:57uh and this usually happens within 12

8:59hours of consumption the first expulsion

9:02whereas in shank 3

9:04at this intermediate time the beads all

9:07accumulate at this junction between the

9:09upper the the intestinal bulb which is

9:12like the stomach and the upper intestine

9:14and only by 24 hours do you get

9:17expulsion

9:19this is true in syngap as well and this

9:21is quantified here

9:24so

9:25what are the cells that mediate this gi

9:27reflex well it turns out that your gut

9:29is lined it’s sprinkled with these

9:32um endocrine sensory endocrine cells

9:34called enteroendocrine cells in light

9:37blue

9:38and those actually make most of the

9:40serotonin in your body

9:42in response to either mechanical or

9:45chemical stimulation in the lumen of the

9:47gi tract those are activated and talk to

9:51enteric neurons that activate the smooth

9:53muscle cells

9:54so

9:55this circuit is really important for

9:58taking food in the lumen and inducing

10:01the

10:02reflex that is mucous secretion and

10:04contractility in the gut

10:06and um to

10:08go deeper into this circuit um we’ve

10:10been collaborating with liu and john

10:13rawls at duke university

10:17the other um thing that points us to

10:19these enter endocrine cells as being

10:21important is that if when we quantify

10:24enteric neurons and anterior endocrine

10:26cells which are important for motility

10:29um both syngap and shank 3 mutants have

10:32reduced and numbers of enteroendocrine

10:34cells

10:36which could mean that they’re less

10:37sensitive to luminal stimuli and

10:40therefore don’t produce the normal

10:43response

10:45and i think these um you know both from

10:48the therapeutic perspective

10:50and from um

10:52um

10:54animal model studies most of the

10:56attention

10:57um with these genes that are linked to

11:01neurodevelopmental disorders it’s been

11:02focused on the central nervous system

11:04but the gut also has a

11:06a significant nervous system through the

11:09enteric neurons

11:10and

11:12um

11:13you know

11:14in the collaboration with with john

11:16rawls he does this single cell

11:18sequencing of the intestine

11:22and here

11:23is so what they do is they

11:26take the intestinal cells

11:28um and do rna sequencing on the single

11:31cells

11:32and what’s shown here are the different

11:35types of intestinal cells

11:37and then on the this this axis

11:41are genes that have been linked to these

11:42severe neurodevelopmental

11:45disorders

11:46and and where these genes are expressed

11:49in the intestine

11:50and these first five rows are the entire

11:53endocrine cells so you can see that

11:55shank 3 catnap norexin

11:58cdkl5

12:00scn are all enriched in these sensory

12:03endocrine cells

12:04indicating that they could be really

12:06functionally important to the gi

12:07distress that’s impacting

12:09um these

12:12individuals

12:13you can see that other genes linked to

12:15asd are more generally expressed

12:16throughout the intestine and syngap is

12:19not expressed in the intestine of the

12:21zebrafish but isn’t expressed in the

12:24enteric neurons

12:26and so this um

12:29this raises the

12:31possibility that

12:33that

12:35therapies will have to be cognizant of

12:37those cells in the gut that are also

12:40that will also need

12:44to address the genetic the genetics of

12:46those those cells

12:50okay in in addition to

12:52um gut

12:54uh sensory motor integration we also

12:57look have looked

12:58conducted studies of um responses to

13:02visual stimuli in the shank

13:04in the in our shank model

13:07and what we find

13:08uh so this is an assay that is done in

13:11zebrafish where you have light to dark

13:14transitions and any time you turn off

13:16the lights there’s a lot more swimming

13:18and that response is dampened in

13:20um

13:22in

13:24both in two different models of shank

13:27three with where the mutations fall

13:29either in the n-terminus or the

13:30c-terminus

13:32and what you can see if you do

13:34if you map the responses in the brain

13:37um

13:38the responses to that that

13:40turning off the lights are that the

13:42pineal gland

13:43turns on

13:44and the hindbrain and spinal cord turn

13:46on corresponding to this increased

13:48movement whereas in the shank models

13:51the pineal turns on

13:54but it fails to activate this hind brain

13:56region

13:58which is associated with the movement

14:01so this this suggests that the sensory

14:03processing deficit

14:05is in the hind brain

14:09so to test that possibility we

14:12transplanted cells from a wild type

14:14animal

14:15into a shank 3 mutant animal here you

14:19can see at one day post fertilization

14:21these cells have populated the hindbrain

14:24and this is at five days or actually six

14:26days when we do the behavioral assays

14:29now these these

14:31shank 3 model has a wild type hindbrain

14:35and that actually rescues their response

14:38to the light stimuli

14:40so that

14:41this shows that

14:43behind brain is actually shank three in

14:45the hind brain is really crucial

14:48at least in the zebrafish for responses

14:50to light stimuli

14:52okay um

14:53i know i’m i’m

14:55losing time so i wanted to tell you that

14:58we’re also trying to

15:00contribute to better understanding gi

15:02symptoms

15:03in um

15:05in

15:06neurodevelopmental disorders um

15:09and

15:11uh

15:12one way is

15:13as

15:14mentioned um by the previous by terry

15:17it’s really important to have objective

15:19measures and so it turns out that you

15:21can measure whole gut transit time by

15:23eating a muffin with a blue dye in it

15:26and this was actually done on a

15:27epidemiological scale in england um but

15:31not with with um

15:34kids with neurodevelopmental disorders

15:36and we also have um in collaboration

15:40with bahraikh moshiri who’s at atrium

15:43health we developed a blue muffin that

15:45that copies the the tests that are used

15:49um

15:51that involve x-ray and um

15:55to to measure whole gut transit with the

15:57idea that if we could use the blue dye

15:59and look for a rival of a blue poop um

16:02that would provide whole gut transit

16:04that could be done at home and without

16:06intervention

16:07and this is um

16:10uh ready we we have seen whether this is

16:14a child with a syngap

16:16um

16:17mutation and

16:19she’s

16:20willing

16:28yeah

16:30it’s edible huh

16:32okay so we’ve also developed an app to

16:35try to do in a prospective manner gather

16:37these symptoms related to gi distress

16:41and

16:43what we’re hoping to do is

16:45use these measures

16:47in

16:49patient populations at the same time as

16:52we’re looking for mechanisms of gi

16:54phenotypes and zebrafish models to

16:57develop strategies for these gi symptoms

17:01more generally and this is the wonderful

17:03group that has supported this research

17:05it’s been a huge team effort

17:08and so i think i have a teeny amount of

17:10time for questions if anyone has them

17:25um dr dalman do you see the question in

17:28the q a panel yeah i do um and i think

17:31um it is possible that increasing so

17:34there are you can

17:36yes

17:37this is something that we’re planning to

17:38test

17:40it’s likely that it

17:42um

17:43that it would work in in the model that

17:47has problems with the entire endocrine

17:48cells but we have to test that

18:01you