44 – Investigating the functional single cell biology of SYNGAP1 pathways

Event Time

November 4, 2021 at 5:00 pm

Description

Here are our introductory comments: 

Hello everyone, and welcome to today’s webinar. My name is Sandra Apler; I’m a Syngap parent and the founder of the European Syngap organisation Leon and Friends. We are very excited to have this webinar with our sister organization in the US, Syngap Research Fund. Our talk for today is “Investigating the functional single-cell biology of SynGAP1 pathways”I have the pleasure to introduce today’s speaker, Dr. Michael Courtney.

Michael Courtney is a Senior Research Fellow at the Turku Bioscience Centre in Finland. The main focus of his work has been to develop and apply optical methods to investigate neuronal responsivity and the underlying signalling networks. His research group initially focused on the roles of the JNK and p38 cell stress signalling pathways in models of neurological diseases. His team identified novel druggability of protein scaffolding mechanisms mediating NMDA receptor-evoked pathological signalling pathways acting via p38 MAP kinase, by the development of first-in-class inhibitor tool compounds targeting NOS1AP. These showed efficacy in multiple neurological disease models. Meanwhile, protein-protein interaction methods were developed to help identify additional opportunities for druggability and to facilitate compound screening. Population snapshot omic data acquired from disease models and their responsiveness to compounds demonstrated the need to quantify the dynamics of signaling networks at the single cell level.

To address this, his lab developed a minimally invasive optical perturbation and measurement pipeline implemented on high-throughput automation platforms. The pipeline allows simultaneous monitoring and optogenetic control of signalling circuit dynamics in individual cells in an interacting network, as well as in situ measurement of pathway kinetics. Single-cell omic analysis algorithms can be adapting to analyse the multiplexed dynamic datasets acquired. This identifies the distinct responsiveness profiles of individual cells. This insight can help interpret the consequences of disease-associated protein and signalling network disruption, and the potential druggability of specific targets, aberrant pathways and cellular phenotypes.

His lab recently began applying this approach to investigate pathways that are either regulating or regulated by SynGAP1, together with the impact of drugs of relevance to SynGAP syndrome. One ongoing project, funded by Leon and friends e.V., is to identify the endogenous RasGEF that counteracts SynGAP1 function, as a potential drug target for treatment of SynGAP1 hypofunction. Funding is being sought for additional projects to help understand the normal and aberrant activities of wild type and missense mutants of SynGAP1.

We would like to take this opportunity again to thank you for your commitment to our Syngap1 children! A recorded version of this webinar will be available on the SRF website under Webinars on the Family menu, and on the Leon and Friends website.

Webinar Overview

Dr. Michael Courtney is a researcher at the Neuronal Signaling Lab and Turku Screening Unit at the Turku Bioscience Center in Finland. He starts off the talk with the goals of his lab: identifying protein signaling networks and druggable sites within these networks, as well as developing methods for precise measurement and manipulation of single cells over time. Dr. Courtney explains how cell signaling works and how single cell responses are heterogenous based on cell type.

He then talks about the effects of statins, old drugs which can inhibit small GTPases like Ras which are prevalent in SYNGAP1. In neurons, statins reduce ERK, extracellular signal-regulated kinase, and calcium responses, but unexpectedly activate JNK signaling, a kinase which is associated with negative consequences.

He closes the webinar explaining split protein complementation methods, including fluorescence complementation and proximity labeling. These methods can be used to investigate the effects of sequence variants on SYNGAP1 localization and stability, as well as to identify the protein dynamics associated with SYNGAP1 variants. In the future, this information could help determine locations for drug sites.

Other Relevant Publications by Dr. Courtney

Spatial quantification of the synaptic activity phenotype across large populations of neurons with Markov random fields

Unexpected Heterodivalent Recruitment of NOS1AP to nNOS Reveals Multiple Sites for Pharmacological Intervention in Neuronal Disease Models

THIS IS FOR TRANSCRIPT ONLY:

very happy to introduce you now to

0:06another incredible scientist Dr Helen

0:10[Applause]

0:15wilsy I’m Helen wizy um I’m an assistant

0:18professor at ucsa and investigator with

0:20the Chan Zuckerberg

0:22biohub um my lab studies um over a

0:26hundred genes that are associated with

0:28autism and epilepsy

0:30one of which is is singap one um and and

0:34my lab actually uses frogs to do all of

0:37this so in a second you’ll see um a

0:39picture there we go of what this looks

0:41like um and so because we want to study

0:43a 100 genes at once we need a model

0:45organism that’s very high throughput and

0:48amendable to studying you know dozens of

0:51genes in parallel but the story I’m

0:52going to tell you today is about s Gap

0:55and a function um that’s beyond the

0:57synapse um so first let me give you just

0:59an introduction to

1:00frogs um so the species I use is zenopus

1:04tropicalis um this is a diploid species

1:08of zenopus and so unlike zebra fish

1:11which have a duplicated genome uh

1:13zenopus tropicalis is very simple

1:15diploid genome that’s much more orthol

1:17orthologous to the human genome so um

1:20well conserved um at the DNA level one

1:23pair of frogs will lay over 4,000

1:27embryos in a day so we have a lot of

1:31animals to work with On Any Given

1:33afternoon they develop very rapidly um

1:36within and so yeah that big dish there

1:39of embryos on the bottom left um that’s

1:42a 15 cm dish that’s not a petri dish so

1:45that’s how many animals we have and they

1:47um develop very rapidly in um under a

1:50week you have a free swimming behaving

1:52tab pole with complex behavior and can

1:54learn um they’re coste effective uh my

1:57animal care costs are orders of magnet

1:59magnitude smaller than rodents they’re

2:02great for drug screening you can just

2:03put the drugs in the water with the Tad

2:06poles and they will go straight into the

2:07brain but if you remember one thing

2:09about frogs let it be

2:12this we can make half mutant tadpoles so

2:16what we do is we take we catch those

2:18embryos right at the two cell stage of

2:21of life right after that first cell

2:23division after fertilization and we can

2:25micro inject crisper cast N9 reagents

2:28into just one of those two cell cells

2:30effectively making a tadpole 5 days

2:32later where only the right half of the

2:35animal carries our mutation of interest

2:38and then you can compare what happens on

2:40that right half of the animal to the

2:42contralateral control side to understand

2:45what is the function of that Gene like I

2:47said as fast as five days H we can make

2:49a guide RNA in in two days um so you

2:52give me a gene we can knock it out and

2:54look and see what it looks like in the

2:55brain in a vertebrate uh in under a week

2:57and for 25 bucks so it’s it’s high

3:00throughput it’s scalable it’s cheap and

3:02this is a typical afternoon in the lab

3:04these are neurula stage embryos where we

3:06can make thousands of half mutant um Tad

3:09poles and then take a look and see what

3:11happened this is our first proof of

3:14principle that we did back when I was at

3:15postto where we can mutate a a gene

3:19required for eye pigmentation on just

3:22the right half and so here you can see

3:24um on the right half of this tadpull has

3:26an albino eye it’s lost pigmentation you

3:29can compare that to the contralateral

3:31control side that still has pigment and

3:33you can infer that the function of that

3:35Gene is an eye

3:37pigmentation okay so we did this um for

3:40quite a few genes um during my post talk

3:43and uh one of them was

3:45singap um and so when we did that um we

3:48I thought there would be no obvious

3:51phenotype and then we would do a lot of

3:53electrophysiology and be thinking about

3:56brain activity and but we had the

3:58surprising result that in these animals

4:01on the injected side which in this

4:02animal is the right half um their telen

4:05seylon region or their forbrain region

4:08of their brain was very small um it

4:10seemed to be specific to the forbrain

4:13the midbrain High brain looked okay and

4:15and so you can see in that inset there

4:17that is the four brain region uh of the

4:20Frog and on the edited side you can see

4:22a smaller brain compared to the

4:24contralateral control so this was s

4:26surprising since the you know historical

4:29and beautiful function that’s been

4:30described for singap one is in um is at

4:33the synapse um so you would imagine that

4:36maybe you wouldn’t see these structural

4:37changes um early on and this implies

4:40that sting Gap is required for the

4:42process of neurogenesis and actually

4:44building the cells um to make the brain

4:47now this isn’t a weird frog thing um and

4:50you’ll see I think later today Marcel’s

4:52talk she has a beautiful paper that just

4:54came out looking at something similar in

4:56human organoids and she sees the same

4:58thing right that that singap one plays a

5:01role early on in brain development um

5:04far before synapses are even

5:08formed who does this have to talk to

5:10there we go so this led us to the key

5:13question here is does singap one have a

5:15function beyond the synapse um and we

5:18were encouraged to pursue this question

5:20once we started to look into um clinical

5:24uh findings about singap and and so we

5:27looked in the citizen data so thank you

5:29to all the families who participated in

5:31generating this

5:33data um and as I’m sure everyone is

5:37familiar there’s obviously a very high

5:39incidence of epilepsy and autism and you

5:42might say okay that is those could both

5:44be explained by a synapse only mechanism

5:47but when we look deeper we also found

5:50quite a few other conditions that I

5:54would argue are difficult to explain by

5:56a synapse only mechanism so 39% % of the

6:00partici participating patients had

6:02skeletal defects like scoliosis club

6:05foot things that are difficult to uh

6:08assign to a synapse function dysmorphic

6:11features um so the the face bones are

6:16formed from cranial neural crust those

6:17are not firing neurons with action

6:20potentials those are migrating neural

6:22crust cells that form the face um

6:24respiratory issues a lot of asthma and

6:27heart defects that are structural so a

6:29lot of heart murmur uh cardiomegaly and

6:32some aortic dissections so this isn’t um

6:36uh I would argue not consistent with a

6:38synapse only

6:41mechanism okay so what else could it be

6:43doing first thing we did was take a look

6:45at publicly available RN seek single

6:47cell data and that’s been coated by the

6:50human protein Atlas and said well where

6:51is singap

6:53expressed as you might imagine all these

6:55red bars are different neuronal cell

6:57types so this makes sense it’s certainly

7:00at the synapse and you would imagine it

7:01be highly expressed in neurons also

7:03expressed in asites but there’s one more

7:07bar there ciliated cells cyia might be a

7:11New Concept to some people in this

7:12audience and I’ll dig deeper into what

7:14cyia are and why you should care about

7:16them but this was a very interesting

7:17finding that this other kind of cell

7:19type ciliated cells and are coming up

7:21here we looked into a different data set

7:24to see if this was robust and indeed

7:27what we found was that sing up one

7:28expression

7:29is highly correlated with core

7:32cyia uh markers Fox J1 which is a

7:35transcription Factor That’s essential

7:37for building the celium and RL 13B which

7:40is another classic cyia marker and so

7:42what you can see from the single cell

7:43data um and is statistically significant

7:46is that same Gap one expression is

7:48correlated with both of these cyia

7:51markers and if we look back uh the

7:54citizen data what you’ll see is those

7:57conditions that are hard to explain by a

7:59synapse only mechanism are

8:01classic cyar Related Disorders so for

8:05all of these things it’s well known that

8:08cyia defects can cause

8:10these all right what are cyia all right

8:12cyia come in two flavors on the left are

8:15motile cyia so this is one cell and it

8:18puts up about 150 cyia and they actually

8:22beat and move the other flavor of cyia

8:25are non-motile or primary cyia um this

8:29the right is a human neural progenitor

8:31cell so you can see that little small

8:35magenta thing is the

8:37psyllium and all cells in the body

8:39except for red blood cells I mean

8:41they’re weird they don’t even have a

8:42nucleus all cells in the body other than

8:44red blood cells have ayia including

8:46neurons so all neurons have them

8:49too these motile cyia um have a more

8:53restricted localization they’re really

8:55in the ventricles of the brain to move

8:57cerebos spinal fluid they’re also

8:59aligning the respiratory tract to move

9:01mucus they’re also um in the

9:03reproductive system both in the

9:05Fallopian tubes and females and sperm

9:07and Mal so sperm tail is

9:10ayum the other kinds and all other cells

9:13in the body have a non-motile celium

9:16also known as the cells antenna um and

9:19so they function in sending and

9:21receiving signals they’re kind of like

9:23the original synapse uh even algae have

9:27ayum and for a long time they were

9:28thought to be a vestigial structure

9:31until until it became very clear in the

9:3390s that cyia are incredibly important

9:35for organ development both in the kidney

9:38the heart and the brain and

9:40developmental signaling Pathways often

9:41rely on the celium to signal so for

9:44example Sonic Hedgehog signaling cannot

9:46happen properly without a functional

9:48celium and they’re also really important

9:50for neural neural Transmissions so some

9:52serotonin receptors only localized to

9:54the celium dopamine receptors trip

9:57channels to sense Heat and spice and

10:01many gpcrs are specifically localized to

10:03the celium and here you can see it’s

10:05very small it’s usually right close to

10:07the nucleus it’s that small magenta

10:09thing there and this is a human ipsd

10:11derived excitatory

10:13neuron and it’s well established that

10:15cyia defects affect many different organ

10:17systems so this is a review just talking

10:19about if there is a cyia defect all of

10:22the different organ systems that can be

10:24affected and you might recognize a lot

10:27of these symptoms and singap uh affected

10:31people so a taxia epilepsy mental

10:35disability facial

10:37anomalies uh some conal heart defects

10:40skeletal defects hearing loss um so we

10:43wanted to P pursue this does sing up

10:45would have a function at

10:48theum uh as chance would be zenopus is a

10:51great model for studying um cyia in a

10:54sense we didn’t so much uh expect that

10:58zenopus would turn out this way for us

11:00but silly you’re beautiful in zenopus

11:02this is a two-day old zenopus embryo 48

11:05Hours um uses eye on the left side tail

11:09on the right and it’s an embryo if you

11:11zoom in on those little toughs they have

11:13this regularly spaced beautiful

11:16epithelium with motile cyia um each one

11:19of those little clusters is one cell

11:21that puts up 150 cyia that beat to move

11:24um mucus and you can see here here’s a

11:27movie Alina made of the CIA beating so

11:30these kinds of cells are in the

11:31ventricles of of one’s brain and then

11:33also in the respiratory

11:35tract um to see if this could be a good

11:37model for studying the function of

11:38singap and cyia we first wanted to

11:40establish that singap was expressed in

11:41the right place at the right time and

11:43just like humans singap um in frogs is

11:46expressed in tissues beyond the brain

11:48it’s also expressed in these cranial

11:50neural crust cells so those are the

11:52migrating neural crust that form the

11:54facial structures it’s in the heart it’s

11:56in the embryonic epidermis muscles

11:59if you take a closer look at one of

12:01those multiciliated cells in the

12:02epidermis and by Florent

12:06rnac R C2 hybrization you can see

12:09expression in those particular cells

12:11it’s in the right place at the right

12:12time to study it and then Alina who is

12:14here a graduate student in my lab took a

12:17closer look to see if singap was on cyia

12:20and so indeed but um she did this in two

12:22ways this is the first one which is an

12:24antibody staining and so you can see

12:26those magenta things are the cyia on the

12:29surface of the of the epidermis you see

12:32singap and Puna along the ciliary axin

12:36and then if you go deeper into the cell

12:38right at the surface of the cell where

12:40the base of the celium is which are

12:42called basil bodies which are modified

12:43centr zomes you also see singap there on

12:46the croomes so this is one way it’s an

12:48antibody we don’t love

12:51antibodies been fooled once and so

12:54instead she also gfp tagged the human

12:57version of singap BL and put it into the

12:59frogs which is Trivial and said where

13:02does it localize and indeed in

13:04multiciliated cells human singap one

13:07localizes again to that basil body the

13:10modified croome and also to the axin of

13:14the celum and especially strong right at

13:17the surface um of the celium of the cell

13:20right at the base of the celium where

13:22there’s high levels of polyglutamated

13:24tubulin to stabilize the

13:27psyllium um with this this is now a nice

13:29platform to start thinking about um

13:32patient variants the first two we looked

13:34at um are here they’re close to the pH

13:37domain and we wanted to ask do these

13:39missant variants change the localization

13:42of singap one at Celia so what I’m

13:45showing you so far is what you’ve

13:46already seen is the is the localization

13:48of the reference sequence and when we

13:50introduce this first missense variant um

13:54what you see is a Mis localization to

13:56the membrane and so while in in the

14:00reference case uh there’s no membrane

14:02localization now you can see this

14:04membrane all around the multiciliated

14:06cell that’s um sequestering singap away

14:09from the celium into the membrane and

14:12this other misinference was different

14:14instead of going taking the protein to

14:16the membrane it actually caused

14:18aggregation into these big

14:20blobs um and but again taking protein

14:23away from the psyllium and then

14:26aggregating and so we think that this

14:28could be a powerful strategy going

14:29forward to understanding um and

14:32dissecting some of the missons

14:34variant okay and so then obviously the

14:37the experiment to do is to try to knock

14:39down singap one and see if it affects

14:41cilia we can do our cute trick in

14:43zenopus where one side is our control

14:45and the other side of the animal has

14:47been mutated so on the left side here

14:49you can see the uninjected side two

14:51different guide rnas on the left side

14:53they have their beautiful tough ofilia

14:55and on the right side you get these big

14:56bald spots where the Gene has been

14:59edited where you actually lose cilia um

15:01Al together if we take a zoomed in look

15:04at one of those patches they look like

15:07this on the left is a control crisper

15:09where we have mutated a gene it’s just a

15:12gene that’s not involved um in any

15:15relevant process it’s involved in

15:17pigmentation those cyia look great

15:19150ish cyia per cell coming out of the

15:22cell however when we manipulate singap

15:25one either by crisper or with what we

15:27call a Morphin these days is called an

15:29anti-sense

15:31oligonucleotide it’s a more fly know um

15:34uh by these two different methods we see

15:36the same thing um some cells have no

15:38cyia at all but then you can find these

15:40cells that are just partially affected

15:42where they’re trying to make cyia but

15:44they just can’t quite make them um and

15:47importantly and this is preliminary but

15:48we believe in it is that when Alina adds

15:51back the that same human plasmid with

15:54the gfp tagged singap you see a rescue

15:57of this phenotype so this could also be

15:59a powerful platform now for studying

16:02variants to see if they can also rescue

16:04this phenotype or not and assign

16:06functionality to various um variants all

16:09right everything I Shang to part is

16:11frogs and I always people always say

16:14this this just frogs so let’s let’s nip

16:16that in the bud okay so what we also did

16:19was I I have a buddy next door David

16:21casner who has rats and so I got some

16:23wild type Rats from him and looked um I

16:26told you that um in mamalian brains uh

16:30uh along the ventricles of the brain

16:32there are these multiciliated cells and

16:34so we wanted to see if we could take a

16:35look and see singap

16:37there and indeed this is now those

16:40multi- ciliated cells in The ventricle

16:42of the brain and are WRA um and what you

16:44can see is that syap is on those cyia

16:47what’s cool here is like it’s really

16:48kind of on the tips of the cyia which I

16:50think is really fascinating um we can

16:53talk more about that if you interested

16:54in that um but we don’t think this is a

16:57frog thing we think this is going to

16:58trans translate to to Maman Ms as well

17:00and we’re expanding this work now in the

17:02in the rats and thanks to support from

17:06srf okay I told you there were two kind

17:08of flavors so far we’ve only talked

17:09about the motile cyia and but I also

17:12want to give some time to non-motile

17:13cyia since those are the ones in neurons

17:17um and so we wanted to ask if it was

17:18also on those kinds of Celia and so we

17:21looked in two different human cell

17:23culture models hack 293 t- cells there

17:25are our embryonic kidney cell line and

17:27here we’re over expressing the the

17:30singap one gfp tag construct and we see

17:32it um throughout the celium in that

17:34cells height um and then an rp1 cells

17:39which are a um a retinal epithelial cell

17:42type from humans um again we’re

17:44overexpressing zap and we see it again

17:46at the tip of the cium um but then also

17:49at the base and this work and this work

17:51was done by Elina who is here so make

17:53sure you grab Elina and chat with her um

17:55and this was independently replicated by

17:58um our colleagues in Mark vasher’s lab

18:00in another cell type imcd3 cells um from

18:04rodents all right so what I’ve sh you

18:06today is that singap when patients have

18:08Celia related conditions that are hard

18:10to explain by a synapse only mechanism S

18:14one in humans and frogs is highly

18:16expressed in ciliated cells um and a

18:20wide variety of cilas and GAP localizes

18:23there and patient Drive variants cause

18:25singap when to Mis localize at the selam

18:29and loss of singap in frogs at least

18:32leads to Celia defects and so we think

18:34the take-home Point here really is that

18:36singap one has functions beyond the

18:38synapse one of the which is at the

18:40celium and uh alen’s got a preprint

18:43coming soon so you can check out all the

18:45details um once that’s online okay my

18:48last plug is for um any families who are

18:51here we are trying to work on biomarkers

18:54for these cyia defects um so if the cyia

18:58are not pro properly functioning in the

19:00nose the nose doesn’t make the right

19:01amount of nitric oxide a gas um and so

19:05James is here find James this is his

19:07email address if you’re interested in

19:09participating in this research it’s a 30

19:12second non-invasive test you put this

19:14just superficially in your nose it

19:16measures the amount of nitric oxide who

19:19knows if this will work it’s a lowrisk

19:21high reward kind of thing um so if

19:23you’re interested we can do patience

19:25there’s James find James find him in or

19:28email him he’s got a table out in the

19:30lobby um it’s a 30second test um uh

19:35great we would love to have people

19:36participate we can test any patients

19:38over two years old and we can also test

19:39siblings or parents um okay great and so

19:43with that you know first and foremost I

19:45want to thank singap families for

19:46participating in research and for being

19:48here and for being wonderful and I want

19:51to thank honorary members of the lab Jr

19:53and Mike um whose discussions have been

19:56incredibly helpful for thinking about

19:59this new function of singap um members

20:02of the laboratory especially Elina who’s

20:04leading the singap work she is here go

20:06find her um and James who’s doing the

20:09nitric oxide to see if that could be

20:10helpful as well and I think our sources

20:12is offending and and of course the srf

20:15uh so thanks for your attention if I

20:17have time for questions I’d love to take

20:23some how do I do on time you are on time

20:27you don’t have time for questions

20:31I just want to emphasize James is here

20:34he’s out here today and tomorrow family

20:37members kids if your if your kid is here

20:40let’s get a reading on them if their

20:41siblings are here let’s get a reading on

20:43them what you just saw is someone go

20:45from frogs to potential implications for

20:48therapies to here’s how we can do it

20:51clinically absolutely amazing and we are

20:54really excited to support her

20:57work